Handbook of Pathogens and Diseases in Cephalopods

Camino Gestal · Santiago Pascual ·
Ángel Guerra · Graziano Fiorito ·

Juan M. Vieites Editors
Handbook of
Pathogens and
Diseases in
Cephalopods
Handbook of Pathogens and Diseases
in Cephalopods
Octopus vulgaris and Sepia officinalis (by Jorge H. Urcera); Loligo vulgaris (by Felipe
Escolano)
Camino Gestal • Santiago Pascual •
Ángel Guerra • Graziano Fiorito •
Juan M. Vieites
Editors
Handbook of Pathogens
and Diseases in Cephalopods
In Cooperation with:
Editors
Camino Gestal Santiago Pascual
Institute of Marine Research Institute of Marine Research
Spanish National Research Council (CSIC) Spanish National Research Council (CSIC)
Vigo, Pontevedra, Spain Vigo, Pontevedra, Spain
Ángel Guerra Graziano Fiorito

Institute of Marine Research Association for Cephalopod Research (CephRes)
Spanish National Research Council (CSIC) Naples, Italy
Vigo, Pontevedra, Spain
Juan M. Vieites
ANFACO-CECOPESCA
ISBN 978-3-030-11329-2 ISBN 978-3-030-11330-8 (eBook)

https://doi.org/10.1007/978-3-030-11330-8
Library of Congress Control Number: 2018966850
© The Editor(s) (if applicable) and The Author(s) 2019. This book is an open access publication.
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Dedicated to
Frederich G. Hochberg,
Roger T. Hanlon and
John W. Forsythe
Preface
Octopuses, cuttlefish, squids, and nautiluses compose a diverse and ancient class of Mollusca,
the Cephalopoda. They play an important role in the trophic structure of marine ecosystems
and are a valuable fishery resource sought in lucrative European and Asian markets.
Cephalopods have evolved highly advanced sensory systems with large, very sophisticated
eyes and the most complex brain of the invertebrates. They have a demonstrated capacity for
both short- and long-term memory and are able to perform impressive high-order cognitive
tasks on a par with some of the lower vertebrates.
They also are distinguished by special anatomical and biological features. Octopuses, for
example, have muscular arms capable of performing a range of delicate tasks. Their suckers
are equipped with sensitive chemoreceptors that ‘taste’ minute changes in their immediate
environment. Squid and cuttlefish, for their part, have a unique dual mode of locomotion:
Pulsed jetting drives bursts of speed, and fin flapping allows high-precision maneuvering.
Cephalopods’ delicate skin has peculiarities that contribute to their evolutionary success and
add to their general air of intrigue. The epidermis is formed by a single-celled epithelium over
a dermal layer of connective tissue. The latter has light-reflecting cells (iridophores and
leucophores) and a remarkable array of pigmented cells, chromatophores, which are under
direct control of the brain. With the exception of Nautilus, this allows the dramatic changes
in skin color, pattern, and even texture that impress specialists and non-specialists alike and
that make cephalopods the masters of marine camouflage.
Owing to these and other distinctive features, cephalopods have a long tradition as valuable
experimental models in neurobiology and related disciplines. As might be expected, they also
have great appeal as ornamental aquarium species. More recently, their short life cycles, high
ratio of production to biomass, high protein content, and high market value have drawn
attention to their aquaculture potential, and this has become an area of active research.
Because of their advanced sensory discrimination, extraordinary ability to learn and perform
complex tasks, and their overall behavioral complexity, cephalopods are the only invertebrates
recognized in European Union Directive 2010/63/EU that sets out welfare standards for
animals used in scientific research. This directive applies to cephalopods used in laboratory
studies, maintained in aquaria, and raised for aquaculture.
The increasing importance of cephalopods in scientific and commercial activities motivates the
need to learn more about potential threats to their health, namely pathogens and disease
vectors. Accurate disease identification and effective treatment are, in fact, essential for any
program that may have an impact on cephalopod health and welfare. We presently are on the
early part of that learning curve and would benefit from a user-friendly guide to cephalopod
diseases. Such a guide would summarize important anatomical and histological structures
required for necropsy, describe assertive histopathological analyses, and discuss the diagnosis
of infectious and non-infectious diseases along with their pathologies.
That is, in fact, the aim of the timely Handbook of Pathogens and Diseases in Cephalopods.
The Handbook provides the reader with current knowledge of cephalopod disease etiologies,
diagnoses, and pathologies. It brings into the light many important facts that contribute to our
understanding of cephalopod pathology that readers will find of great practical use, including
vii
viii Preface
accurate assessment of parasites and pathogens, and the effects of the cultural environment on
the health and welfare of these truly captivating invertebrates. The Handbook of Pathogens
and Diseases in Cephalopods is an essential reference for everyone in the field.
As one example, those who work in the field know very well that cephalopod skin is very
fragile and prone to damage. Thus, physical contact during capture or any subsequent han-
dling, and especially abrasions inflicted when scraping against a tank wall, can lead to the
invasion of pathogens that result in serious infections. The highly active swimming habits of
squid and cuttlefish thus impose strict design considerations to minimize skin and fin abra-
sions. To complicate matters further, the synergic effects of stressors while in captivity—such
as high density or sub-optimal water quality—may adversely affect the immune system and so
make disease outbreaks all the more likely. This common problem alone is a strong argument
for studying the diagnosis and treatment of cephalopod pathologies covered so thoroughly in
the Handbook.
Jointly written by 40 authors from research groups distributed over 3 continents, 18 chapters
of the Handbook of Pathogens and Diseases in Cephalopods are a detailed and up-to-date
reference that will prove to be useful in many disciplines. It comprises two parts. The first lays
the foundation of accurate necropsy and histopathological analyses. It also describes the
tissues of early life stages and adults of the more important European species: Octopus
vulgaris, Loligo vulgaris, and Sepia officinalis.
The second part is a broad and thorough assessment of parasites, pathogens, and diseases
found mainly in European cephalopods. Among other topics, it covers conditions caused by
fungi and Labyrinthulomycetes, viruses and bacteria, Protists (coccidians and ciliates),
Dicyemids, and Metazoans. For completeness, there also is a chapter on the pathogens and
diseases in non-European cephalopods.
Other topics include valuable information on aquarium maintenance and a section on the
cephalopods’ remarkable ability to regenerate lost or damaged tissue. An additional chapter
discusses senescence, the process by which cephalopods cease eating and live solely on their
stored reserves during a period of self-imposed starvation.
Improving the health, maintenance, and survival of cephalopods in captivity is essential for
advancing the field, and this is inextricably linked to the quick and accurate diagnosis and
treatment of cephalopod diseases. In this very important respect, the cephalopod research
community will welcome the Handbook as an authoritative reference that will play a critical
part in furthering our knowledge of these enigmatic and ecologically important animals.
Dr. Erica A. G. Vidal

President, Cephalopod International Advisory Council
Acknowledgements
The authors would like to thank ANFACO-CECOPESCA and Regional Ministry for Maritime
Affairs, Xunta de Galicia, for their collaboration in supporting the edition of this publication.
We also thank the Spanish National Research Council (CSIC) and Institute of Marine
Research (IIM-CSIC) for their support and facilities. We thank Graham Pierce for their
comments and advice on specific sections of this book. In addition, we appreciate the help of
Manuel E. Garci (IIM-CSIC) for photographic assistance of fresh specimens during necrop-
sies, José Manuel Antonio Durán (IIM-CSIC) for his technical assistance in tissue processing
for histological analysis of part of the material, and Lucía Sánchez (IIM-CSIC) for her
technical assistance in image analysis and edition.
This work benefited from networking activities carried out under the COST Action FA1301
and is considered a contribution to the COST (European Cooperation on Science and Tech-
nology) Action FA1301 “A network for improvement of cephalopod welfare and husbandry in
research, aquaculture and fisheries” (http://www.cephsinaction.org/).
ix
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Camino Gestal, Santiago Pascual, Ángel Guerra, Graziano Fiorito,
and Juan M. Vieites
2 Importance of Cephalopod Health and Welfare for the Commercial
Sector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Juan M. Vieites, Carlos S. Ruiz, Felicidad Fernández, and Roberto C. Alonso
Part I Functional Anatomy and Histology

3 Functional Anatomy: Macroscopic Anatomy and Post-mortem
Examination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Ángel Guerra
4 Functional Histology: The Tissues of Common Coleoid Cephalopods . . . . . . 39
Ramón Anadón
5 Tissues of Paralarvae and Juvenile Cephalopods . . . . . . . . . . . . . . . . . . . . . . 87
Raquel Fernández-Gago, Pilar Molist, and Ramón Anadón
Part II Pathogens and Related Diseases

6 Cephalopod Diseases Caused by Fungi and Labyrinthulomycetes . . . . . . . . . 113
Jane L. Polglase
7 Virus and Virus-like Particles Affecting Cephalopods . . . . . . . . . . . . . . . . . . 123
María Prado-Álvarez and Pablo García-Fernández
8 Bacteria-Affecting Cephalopods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Rosa Farto, Gianluca Fichi, Camino Gestal, Santiago Pascual,
and Teresa Pérez Nieto
9 Protist (Coccidia) and Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Sheila Castellanos-Martínez, Camino Gestal, Santiago Pascual, Ivona Mladineo,
and Carlos Azevedo
10 Protist (Ciliates) and Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Dhikra Souidenne and Hidetaka Furuya
11 Dicyemids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Hidetaka Furuya and Dhikra Souidenne
12 Metazoa and Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Santiago Pascual, Elvira Abollo, Ivona Mladineo, and Camino Gestal
13 Aquarium Maintenance Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Antonio V. Sykes, Kerry Perkins, Panos Grigoriou, and Eduardo Almansa
14 Regeneration and Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Letizia Zullo and Pamela Imperadore
xi
xii Contents
15 Other Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Camino Gestal, Santiago Pascual, and Sarah Culloty
16 Cephalopod Senescence and Parasitology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Katina Roumbedakis and Ángel Guerra
17 Pathogens and Related Diseases in Non-European Cephalopods:
Central and South America . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Yanis Cruz-Quintana, Jonathan Fabricio Lucas Demera,
Leonela Griselda Muñoz-Chumo, Ana María Santana-Piñeros,
Sheila Castellanos-Martínez, and Ma. Leopoldina Aguirre-Macedo
18 Pathogens and Related Diseases in Non-European Cephalopods:
Asia. A Preliminary Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Jing Ren, Xiaodong Zheng, Yaosen Qian, and Qingqi Zhang
Contributors
Elvira Abollo Centro Tecnológico del Mar, Fundación CETMAR, Vigo, Pontevedra, Spain
Ma. Leopoldina Aguirre-Macedo Laboratorio de Patología Acuática y Parasitología,
CINVESTAV Unidad Mérida, Mérida, Yucatán, Mexico
Eduardo Almansa Centro Oceanográfico de Canarias, Instituto Español de Oceanografía,
Santa Cruz de Tenerife, Canary Islands, Spain
Roberto C. Alonso ANFACO-CECOPESCA, Ctra. Colexio Universitario, Vigo, Ponteve-
dra, Spain
Ramón Anadón Department of Functional Biology, University of Santiago de Compostela,
Campus Vida, Santiago de Compostela, Spain
Carlos Azevedo Laboratory of Cell Biology, Institute of Biomedical Sciences (ICBAS/uP),
University of Porto, Porto, Portugal
Sheila Castellanos-Martínez Instituto de Investigaciones Oceanológicas, UABC, Ensenada,
Mexico
Yanis Cruz-Quintana Grupo de Investigación en Sanidad Acuícola, Inocuidad y Salud
Ambiental, Escuela de Acuicultura y Pesquería, Facultad de Ciencias Veterinarias, Univer-
sidad Técnica de Manabí, Bahía de Caráquez, Ecuador
Sarah Culloty School of Biological, Earth and Environmental Sciences, Aquaculture and
Fisheries Development Center, University College Cork, Cork, Ireland
Rosa Farto Marine Research Centre (CIM-UVIGO), University of Vigo, Vigo, Spain
Felicidad Fernández ANFACO-CECOPESCA, Ctra. Colexio Universitario, Vigo, Pon-
tevedra, Spain
Raquel Fernández-Gago Department of Ecology and Animal Biology, University of Vigo,
Lagoas-Marcosende, Vigo, Spain
Gianluca Fichi Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Pisa,
Italy
Graziano Fiorito Association for Cephalopod Research (CephRes), Naples, Italy
Hidetaka Furuya Department of Biology, Graduate School of Science, Osaka University,
Toyonaka, Osaka, Japan
Pablo García-Fernández Aquatic Molecular Pathobiology Group, Institute of Marine
Research, Spanish National Research Council (CSIC), Vigo, Pontevedra, Spain
Camino Gestal Aquatic Molecular Pathobiology Group, Institute of Marine Research,
Spanish National Research Council (CSIC), Vigo, Pontevedra, Spain
Panos Grigoriou HCMR, Gournes Pediados, Irakleion, Crete, Greece
xiii
xiv Contributors
Ángel Guerra Ecology and Biodiversity Department, Institute of Marine Research, Spanish
National Research Council (CSIC), Vigo, Pontevedra, Spain
Pamela Imperadore Association for Cephalopod Research (CephRes), Naples, Italy
Stazione Zoologica Anton Dohrn, Biology and Evolution of Marine Organisms, Naples, Italy
Jonathan Fabricio Lucas Demera Grupo de Investigación en Sanidad Acuícola, Inocuidad
y Salud Ambiental, Escuela de Acuicultura y Pesquería, Facultad de Ciencias Veterinarias,
Universidad Técnica de Manabí, Bahía de Caráquez, Ecuador
Ivona Mladineo Institute of Oceanography and Fisheries, Split, Croatia
Pilar Molist Department of Functional Biology and Health Sciences, University of Vigo,
Leonela Griselda Muñoz-Chumo Grupo de Investigación en Sanidad Acuícola, Inocuidad y
Salud Ambiental, Escuela de Acuicultura y Pesquería, Facultad de Ciencias Veterinarias,
Teresa Pérez Nieto Marine Research Centre (CIM-UVIGO), University of Vigo, Vigo,
Pontevedra, Spain
Santiago Pascual Ecology and Biodiversity Department, Institute of Marine Research,
Kerry Perkins Sea Life Brighton—Merlin Entertainments, Brighton, UK
Jane L. Polglase Institute of Life and Earth Sciences, School of Energy, Geoscience,
Infrastructure and Society, Heriot Watt University, Edinburgh, Scotland, UK
María Prado-Álvarez Aquatic Molecular Pathobiology Group, Institute of Marine Research,
Yaosen Qian Ganyu Institute of Fishery Science, Lianyungang, China
Jing Ren Key Laboratory of Mariculture, Ministry of Education, Institute of Evolution and
Marine Biodiversity, Ocean University of China, Qingdao, China
Katina Roumbedakis Association for Cephalopod Research (CephRes), Naples, Italy
Carlos S. Ruiz ANFACO-CECOPESCA, Ctra. Colexio Universitario, Vigo, Pontevedra,
Spain
Ana María Santana-Piñeros Grupo de Investigación en Sanidad Acuícola, Inocuidad y
Salud Ambiental, Escuela de Acuicultura y Pesquería, Facultad de Ciencias Veterinarias,
Dhikra Souidenne National Museum of Natural History of Paris, Biologie des Organismes et
Ecosystèmes Aquatiques (BOREA), Research Team: Reproduction and Development, Evo-
lution Adaptation, Regulation CNRS 7208, Sorbonne Université, UCN, IRD 207, Paris,
France
Antonio V. Sykes Centro de Ciências Do Mar, Universidade Do Algarve|CCMAR, Faro,
Portugal
Juan M. Vieites ANFACO-CECOPESCA, Ctra. Colexio Universitario, Vigo, Pontevedra,
Spain
Qingqi Zhang Ganyu Jiaxin Fishery Technical Development Co., Ltd., Lianyungang, China
Contributors xv
Xiaodong Zheng Key Laboratory of Mariculture, Ministry of Education, Institute of Evo-

lution and Marine Biodiversity, Ocean University of China, Qingdao, China
Letizia Zullo Centre for Synaptic Neuroscience and Technology, Fondazione Istituto Italiano
Di Tecnologia, Genoa, Italy
Introduction
1
Camino Gestal, Santiago Pascual, Ángel Guerra, Graziano Fiorito,
and Juan M. Vieites
Abstract
Cephalopods are valuable seafood for human consumption, and some of them are good
candidates for aquaculture. In addition, they have evolved many characteristic features that
make them interesting models for research. The recent inclusion of cephalopods in the Directive
2010/EU regulates the use of animals for scientific purposes and obliges cephalopod researchers
to promote the best health and welfare practices during aquarium maintenance or aquaculture
procedures. The identification of diseases of cephalopods, and the pathogens that cause them, is
consequently of major interest to improve cephalopod welfare and husbandry. This work has
been designed as a short, easy to follow ‘handbook,’ with the aim of presenting fundamental
aspects of the anatomical and histological structures as well as the identification of different
pathogens, the resulting histopathology, and the diagnosis of diseases in cephalopods. We hope
it will provide a useful contribution that will also encourage marine pathologists, parasitologists,
veterinarians and those involved in fishery sanitary assessment, aquarium maintenance, and
aquaculture practice to increase our knowledge about the pathology of cephalopods further.

Keywords
Cephalopods Pathology Parasites Infectious diseases Fisheries Aquaculture
Seafood
Knowledge of pathologies of cephalopod mollusks in the

C. Gestal (&) wild is very limited. The information available is mainly
Aquatic Molecular Pathobiology Group, Institute of Marine based on postmortem examination of animals after capture,
Research, Spanish National Research Council (CSIC), 36208 which limits the identification of the etiological agent
Vigo, Pontevedra, Spain responsible for the disease. Most recently, pathologies of
e-mail: [email protected]
cephalopods have also been identified in laboratory and
S. Pascual Á. Guerra small-scale culture conditions; it is predicted that the
Ecology and Biodiversity Department, Institute of Marine
Research, Spanish National Research Council (CSIC), 36208 increasing interest in industrial cephalopod aquaculture will
Vigo, Pontevedra, Spain increase the risks of their occurrence (Sykes and Gestal 2014).
e-mail: [email protected] Identifying pathogens and the resulting diseases, and the
Á. Guerra potential risks to animals’ health due to mechanical damage
e-mail: [email protected] or injuries from capture or in the laboratory are considered
G. Fiorito some of the main requisites for improving welfare and
Association for Cephalopod Research (CephRes), Naples, Italy husbandry for these animals, as required in ‘assessment of
e-mail: graziano.fi[email protected] health and welfare’ of the Directive 2010/63/EU.
J. M. Vieites Cephalopods (i.e., nautilus, cuttlefish, squid, and octopus)
ANFACO-CECOPESCA. Ctra. Colexio Universitario, 16, 36310 are members of the phylum mollusca. The taxon currently
e-mail: [email protected] numbers about 800 species, representing a large diversity of
© The Author(s) 2019 1

C. Gestal et al. (eds.), Handbook of Pathogens and Diseases in Cephalopods,
https://doi.org/10.1007/978-3-030-11330-8_1
2 C. Gestal et al.
forms and adaptations. These are exclusively marine inver- in wild cephalopods, while the collection, transfer, aquarium
tebrates distributed in all areas of the world, from the intertidal maintenance and weakening of animals under stress may
areas to deep sea. facilitate and increase the development of the diseases
The interest in cephalopods has increased considerably (Hanlon and Forsythe 1990; Hochberg 1990). Wild cephalo-
over the last few decades, mainly because they (i) represent a pods are also intermediate, paratenic, or definitive hosts of a
very important target for fisheries with high market value; range of parasites with different life cycle strategies. They
(ii) constitute an important resource of seafood for human occupy an ecological niche that makes them vulnerable to
consumption, with a high protein and polyunsaturated fatty infection by specific groups of parasites, which are transmitted
acid content; (iii) are characterized by features of their to the definitive host, namely fish, marine mammals, or birds.
biology and physiology which are novel in design and An association between relative species diversity of parasites
evolutionary adaptation (Albertin et al. 2015; Shigeno et al. and cephalopod life cycle characteristics has been observed in
2018); (iv) are the sole invertebrates included in the list of Atlantic waters, suggesting that the ecological niche of a
regulated species by the Directive 2010/63/EU (Fiorito et al. cephalopod species is more important in determining its risk of
2015; Di Cristina et al. 2015). parasitic infection than its phylogeny (González et al. 2003).
Coleoid cephalopods have been used for millennia as Despite the increased interest in cephalopods as seafood
seafood by humans across the world and across different and the recommendations of Food Safety Authorities on
food cultures (Mouritsen and Styrbæk 2018). Cephalopod parasite risk in fishery products, currently only fragmentary
landings reached about 4 million tons in 2016 (FAO 2017), information on pathogens and diseases in cephalopods
although a fall of approximately one-quarter from that total exists. This information has been mainly gathered from
was reported for 2017 (G. Pierce, pers. communication). The opportunistic sampling plans within commercial fisheries or
continuously increasing demand from the market, the market surveys, and it is small in comparison with infor-
decline in fishing overall, and the search for a more sus- mation available for other commercially important taxa
tainable food resource have all contributed to promote a (Pascual and Guerra 2003; González and Pascual 2018). At
great interest in cephalopod aquaculture over the last decade, the present, there is no available information on the risk that
with an important, associated research effort in the field cephalopod parasites pose to human consumers. In order to
(Iglesias et al. 2014). carry out good Regulatory Science, (which is described as
Considered classically as ‘marine guinea pigs’ (Grimpe the scientific and technical foundations upon which regula-
1928), cephalopods have been studied for more than one tions are based) knowing what risks cephalopod disease pose
century for the uniqueness of their biology (Grimpe 1928; to consumers will be a key point. Future research should be
Packard 1972; Marini et al. 2017). They have evolved many addressed to this, together with building the knowledge base
characteristic features that make them ‘organisms of interest’ overall, which is also a critical point in this research area.
for the study of the evolution of neural and behavioral Although human consumption of cephalopods worldwide is
complexity. Despite their typical molluscan design and body much lower than that of fish, potential risk should be man-
plan, cephalopods possess a highly differentiated aged appropriately. As an example, González and Pascual
multi-lobular brain, a camera eye resembling that of verte- (2018) pointed out that ‘risk management should configure
brates, a ‘closed’ circulatory system, a sophisticated set of and consistently implement policies to ensure that scientific
sensory organs and fast jet-propelled locomotion. Cephalo- evidence is translated into action, while also considering
pods, and squid in particular, are also the animals that aspects such as the key general principles established in EU
donated to neuroscience the giant axon, the classic prepa- food law (necessity, proportionality, minimum effect on
ration that allowed the discovery of how neuronal action competence, and guarantee of level playing field) that
potentials and nerve propagation worked, together with the guarantee and protect the functioning of markets.’ The use of
ionic mechanism of action potentials. certified biobanking in fish (González et al. 2018) can aid the
The identification and management of diseases are some establishment of a similar network for sampling and col-
of the major hurdles in the development of the aquaculture lection of traceable cephalopod parasites.
industry. The accurate identification of the different organs at Knowledge of the most important pathogenic agents
histological level and the knowledge and management of identified in cephalopods has been reviewed in volume III
infectious and non-infectious diseases that may affect cul- (1990) of the seminal serial work ‘Diseases of Marine
tured species are a priority for both the aquarium mainte- Animals,’ edited by Otto Kinne. A general overview of each
nance and aquaculture of cephalopods. group of pathogens, together with a compilation of infor-
A range of diseases has been described in cephalopods, mation on microorganisms and parasite species identified per
caused by a wide variety of pathogens, belonging to many cephalopod host species, is included in the original work
phyla, including fungi, viruses, bacteria, and protozoan and (Hochberg 1990; Hanlon and Forsythe 1990). In more recent
metazoan parasites. Bacterial infections have been identified years, a review by Castellanos-Martínez and Gestal (2013),
1 Introduction 3
and some additional papers on specific pathogens or para- We hope this will provide a useful contribution that will
sites added additional data on the knowledge of cephalopod also encourage marine pathologists, parasitologists, veteri-
parasitology and diseases. narians and those involved in fishery sanitary assessment,
However, to the best of our knowledge, no guide to aquarium maintenance, and aquaculture practice, to increase
histological identification has yet been published; this book our knowledge regarding the pathology of cephalopods
aims to contribute to fill this gap. It originates as one of the further.
outcomes of the activities of the COST Action FA1301,
Cephs In Action, which established an interdisciplinary
network for improvement of cephalopod welfare and hus-
bandry in research, aquaculture, and fisheries. References
The first part of the book offers tools that advise one on
how to make an accurate pathological analysis. Several Albertin CB, Simakov O, Mitros T, Wang ZY, Pungor JR,
Edsinger-Gonzales E, Brenner S, Ragsdale CW, Rokhsar DS
chapters provide a review of sampling methodology (in-
(2015) The octopus genome and the evolution of cephalopod neural
cluding necropsy and postmortem examination), organ and morphological novelties. Nature 524:220–224
anatomy, as well as a detailed description of the histology of Castellanos-Martínez S, Gestal C (2013) Pathogens and immune
larval stages and adults for three species of cephalopods response of cephalopods. J Exp Mar Bio Ecol 447:14–22
Di Cristina G, Andrews P, Ponte G, Galligioni V, Fiorito G (2015) The
(Sepia officinalis, Loligo vulgaris, and Octopus vulgaris).
impact of Directive 2010/63/EU on cephalopod research. Invert
We consider these species as valuable ‘morphotype’ models Neurosci 15:8
of the taxonomic groups Sepioidea, Myopsida, and Octo- EU (2010) Directive 2010/63/EU of the European Parliament and of the
poda, which include most of the species with highest culture Council of 22 September 2010 on the Protection of Animals used
for Scientific Purposes. Official J Euro Union 33–79
potential (Iglesias et al. 2014).
FAO (2017) Yearbook of fisheries and aquaculture statistics. FAO
Additionally, knowledge of organ architecture and tissue annuaire, November, 2017, Rome
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analyze pathological conditions. The histological identifica- welfare of cephalopods in research—a consensus based on an
initiative by CephRes, FELASA and the Boyd Group. Lab Animal
tion of organs of the selected species of cephalopods is
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makes a cephalopod a suitable host for parasites? The case of
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Shigeno S, Andrews PL, Ponte G, Fiorito G (2018) Cephalopod brains: Springer, Netherlands, pp 97–112
An overview of current knowledge to facilitate comparison with
vertebrates. Front Physiol 9:952
Open Access This chapter is licensed under the terms of the Creative The images or other third party material in this chapter are included in
Commons Attribution 4.0 International License (http://creative the chapter’s Creative Commons licence, unless indicated otherwise in a
commons.org/licenses/by/4.0/), which permits use, sharing, adaptation, credit line to the material. If material is not included in the chapter’s
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made.
Importance of Cephalopod Health
and Welfare for the Commercial Sector 2
Juan M. Vieites, Carlos S. Ruiz, Felicidad Fernández,
and Roberto C. Alonso
Abstract
We witness the expansion of cephalopod fisheries and their growing importance in the
world’s fisheries production. Despite this, only 4 of the 28 taxonomic families are
commercially exploited. The rational exploitation of resources could provide large quantities
of high-quality cephalopods and would only require further development in harvesting
techniques. The intrinsic nutritional value of the cephalopods and the progress of extraction
and processing technologies would allow for an expansion of the range of products attractive
to consumers, including current non-commercial species. This atlas presents a review of
general pathology in octopus, cuttlefish, and squid from different regions of the world. This
topic is closely linked to food safety concerns, and it can also be considered a tool for
assessing the state of populations. This review provides a resource for teaching and guidance
in universities, research centers, public and private laboratories, processing and transforma-
tion companies, as well as for administrations in their legislative processes.

Keywords
Cephalopods Pathology guidance Seafood Commercial sector
The 2010 FAO review of cephalopods of the world (Jereb and between 1 and more than 2 million tons, which represents 50%
Roper 2010), considers the existence of 28 families, although of the total catch of cephalopods worldwide. The impressive
the most commercially available species are focused on the increase in squid production over the past 30 years is mainly
families Sepiidae, Loliginidae, Ommastrephidae, and due to the discovery and subsequent exploitation of resources
Octopoteuthidae. The number of cephalopod species covered in the southwestern Atlantic, mainly Illex argentinus, as well
by commercial fishing has continued to grow significantly as an increase in the production of other target species, mainly
since 1984, as a result of the increasing market demand and the Dosidicus gigas in the East Pacific and Todarodes pacificus in
expansion of fishing activities in new fishing grounds and the Northwest Pacific.
deeper waters. Species of the Ommastrephidae family are the Regarding the evolution of the catches of all cephalopods,
most important commercial fishery among cephalopods. Doubleday et al’s (2016) data show a general upward trend in
According to FAO (2016), during the decade from 1997 to the period 1955–2012. Within this general trend, we highlight
2007, the annual world catch of Ommastrephidae varied that, after reaching the maximum level of 4.3 million tons in
2007, the increase in total cephalopod catch slowed for some
J. M. Vieites (&) C. S. Ruiz F. Fernández R. C. Alonso
ANFACO-CECOPESCA, Ctra. Colexio Universitario, 16, 36310 years. However, in 2012, catches surpassed again and, in
Vigo, Pontevedra, Spain 2014, they surpassed 4.5 million tons, according to the 2016
e-mail: [email protected] FAO report. In successive reports (FAO 2018), a dramatic
C. S. Ruiz drop in the cephalopod catch was recorded in 2016, although
e-mail: [email protected] there seem to be signs of recovery in 2017. Catches of octo-
F. Fernández puses (family Octopoteuthidae) have been shown, in global
e-mail: [email protected] figures to be more stable than those of squids. Since 2008, both
R. C. Alonso catches of cuttlefish and octopuses have remained relatively

https://doi.org/10.1007/978-3-030-11330-8_2
6 J. M. Vieites et al.
stable between 300 and 350,000 tons, respectively, although technological development allows to add value to cephalo-
this represents a decrease in the case of cuttlefish and an pod products, making them attractive to broader layers of
increase in octopuses, as compared to previous years. consumers. In one of the last discussion forums
From the 2018 FAO report on the state of fisheries and (MAFE 2012) it became clear that, currently, consumers
aquaculture, we can mention that, after five years of continu- consider food as the guarantor of their future health. The
ous growth, which began in 2010, catches of cephalopods high-protein content, the abundance of essential amino acids,
stabilized in 2015, but fell in 2016, when the three main spe- and the low-fat content of cephalopods make them an ideal
cies of squids recorded a combined loss of 1.2 million tons. food to be part of healthy and balanced diets. The data
The potential for the fishery of all species of the exploitable collected in nutritional tables such as those prepared by
Ommastrephidae family is estimated between 6 and 9 million ANFACO-CECOPESCA (2018) and the USDA (2018)
tons. A large number of squids of this family, which lacks show that the squid form a homogeneous group with
ammonium, are considered little exploited. These include high-protein content, in which essential amino acids abound
Sthenoteuthis pteropus, Ommastrephes bartramii, Martialia (with slight differences in level with respect to octopuses)
hyadesi, Todarodes sagittatus, Sthenoteuthis oualaniensis, plus a low-fat content, giving a healthy profile. The signif-
Nototodarus philippinensis, Dosidicus gigas, and the cir- icant numbers of authorized declarations of health proper-
cumpolar and subantarctic species Todarodes filippovae. ties, to which the products derived from cephalopods can be
The assessment of the availability of commercial and accepted under the provisions of the EU (2012), support the
other less exploited species faces the difficulties derived nutritional quality of cephalopods.
from the short life of the cephalopods and their adaptive From the reports on the state of world fisheries, it should be
strategies to ensure survival against stressful environmental noted that in the last 40 years the percentage of cephalopods
conditions, including those caused by intensive fishing. production to world production increased from 2 to around
Therefore, both stock assessments and predicting important 5%. The manufacturing industry requires constancy in pur-
fluctuations in catches and landings are difficult (Pierce and chases. The important fluctuations experienced by the main
Guerra 1994; Robert et al. 2010). Barring uncertainties in the species of cephalopods of commercial interest raise practical
assessment of fishing potential, the exploitation of these issues in the control of conformity of the specifications of
species could provide large quantities of high-quality commercial quality of the raw materials since they condition
cephalopods and would require only further development the specifications of the final products. This entails the exe-
in harvesting techniques. cution of a series of controls based on self-control, on the
On the consumption forecast of products derived from maximum content of certain contaminants and microbiolog-
cephalopods, the aforementioned FAO 2018 and 2016 reports ical criteria, and on the organoleptic or sensorial characteris-
show that Spain, Italy, and Japan are the main consumers and tics that determine the acceptability of the raw material.
importers of these species. Thailand, Spain, China, Argentina, With this panoramic view it presents, our Atlas covers
and Peru were the largest exporters of squid and cuttlefish, aspects directly linked to food safety and quality. The Atlas
while Morocco, Mauritania, and China were the main presents an overall review of the general pathology in
exporters of octopus. Vietnam is expanding its cephalopod octopus, cuttlefish, and squid from other regions of the
markets, including squid, in Southeast Asia. Other Asian world. Regarding morphological aspects, it describes macro
countries such as the Republic of Korea and India are also and microscopic lesions and their consequences for organ-
important suppliers. In South America, there is an increasing isms, both in the wild and from mariculture. In addition,
interest in the Humboldt Squid (Dosidicus gigas), which is there is a chapter on tissue and organ regeneration and others
being exported from Peru to more than 50 countries, and on viruses and parasites.
efforts are being made to develop new products. In 2013, the Given the expected future projection of cephalopods in the
main markets, especially Japan and the European Union, world diet, the Atlas is considered a reference publication for
remained strong, despite the difficult economic situations and teaching and guidance in universities, research centers, public
the high prices of these species. In the 2014–2015 period, the and private laboratories, processing and transformation
largest increases in the markets were of octopus, rather than of companies, as well as for use by administrations in their leg-
squid and cuttlefish. However, the reduction in catches islative processes.
resulted in a shortage of supply in 2016 and 2017.
On consumer preferences according to the FAO 2016
report, the use of squid for human consumption is extensive 2.1 Concluding Remarks
and diverse. There is an increasingly wide range of raw,
refrigerated, frozen, dry, canned, and prepared products. The chapter justifies the importance of cephalopod health
Despite the aversion of cultural origin of the inhabitants of and welfare for the commercial sector and the usefulness of
northern countries to the consumption of cephalopods, the handbook under the panoramic view presented.
2 Importance of Cephalopod Health and Welfare for the Commercial … 7
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92-5-105383-9
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nology of Anfaco-Cecopesca: “Innovation and Biotechnology for a istry of Agriculture, Food and Environment (Spain), Spain
more competitive Marine and Food sector” 10.9.2018 (Available in Pierce GJ, Guerra A (1994) Stock assessment methods used for
http://www.anfaco.es/es/index.php) cephalopod fisheries. Fis Res 21:255–285
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Steer M, Leporati SC, Lourenço S, Quetglas A, Sauer W, Gillan- modelling of the De Lury depletion model: strengths and limitations
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(10):406–407. https://doi.org/10.1016/j.cub.2016.04.002 ICES J Mar Sci 67:1272–1290
EU (2012) Commission Regulation No 432/2012 of 16 May 2012 USDA (2018) United States Department of Agriculture Agricultural
establishing a list of permitted health claims made on foods, other Research Service National Nutrient Database for Standard Refer-
than those referring to the reduction of disease risk and to children’s ence. Tables available in three web pages: https://ndb.nal.usda.gov/
development and health (Official Journal of the European Union L ndb/foods/show/4641?fgcd=Finfish+and+Shellfish
136/1, https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ: +Products&manu=&lfacet=&format=&count=&max=35&offset=
L:2012:136:0001:0040:en:PDF 140&sort=&qlookup; https://ndb.nal.usda.gov/ndb/foods/show/
FAO (2016) The state of world fisheries and aquaculture 2016. 4644?fgcd=Finfish+and+Shellfish+Products&manu=&lfacet=
Contribution to food security and nutrition for all, Rome, p 224 &format=&count=&max=35&offset=140&sort=&qlookup; https://
FAO (2018) The state of world fisheries and aquaculture. Fulfilling the ndb.nal.usda.gov/ndb/foods/show/4653?fgcd=Finfish+and
objectives of sustainable development, Rome, http://www.fao.org/3/ +Shellfish+Products&manu=&lfacet=&format=&count=&max=
i9540en/I9540EN.pdf 35&offset=140&sort=&qlookup
Jereb P, Roper CFE (2010) Cephalopods of the world: an annotated and
illustrated catalogue of cephalopod species known to date. FAO
Commons Attribution 4.0 International License (http:// the chapter’s Creative Commons licence, unless indicated otherwise in a
creativecommons.org/licenses/by/4.0/), which permits use, sharing, credit line to the material. If material is not included in the chapter’s
adaptation, distribution and reproduction in any medium or format, as Creative Commons licence and your intended use is not permitted by
long as you give appropriate credit to the original author(s) and the statutory regulation or exceeds the permitted use, you will need to obtain
source, provide a link to the Creative Commons licence and indicate if permission directly from the copyright holder.
changes were made.
Part I
Functional Anatomy and Histology
Functional Anatomy: Macroscopic Anatomy
and Post-mortem Examination 3
Ángel Guerra
Abstract
Understanding the relationship between form and function of living beings is an intimidating
challenge. The recognition and interpretation of physiological and pathological processes
require a previous knowledge of regular morphology and anatomy of the external and internal
structures and organs of any living creature. Cephalopods span an awesome range of shapes
and scales, and the variations between species are crucial for correct interpretations. This
chapter covers the gross morphological and anatomical main characteristics of different
cephalopod species, as well as necropsy protocols and methods of euthanasia. This
knowledge is decisive to a suitable understanding of the modifications caused by injury,
infection, or disease, especially for those people who are not familiar with these remarkable
marine molluscs.

Keywords
Cephalopods Gross morphology Functional anatomy Euthanasia Necropsy
Palaeontologists have identified three distinct fossil clades

3.1 Classification
that are entirely extinct. All members of these clades were
squid-like, but had straight external shells. They flourished
The Cephalopoda is an ancient class of the Phylum Mollusca
in Palaeozoic oceans between the Ordovician (488 mya) and
dating from the Upper Cambrian Period (around 500 million
Triassic periods (200 mya). The shells of some of these
years ago; mya). Cephalopods constitute one of the most
species reached nearly 10 m in length. The most well known
complex groups of invertebrates and the most evolved of
of these fossil records are the nautiloids, ammonoids, and
molluscs. This group has been among the dominant large
belemnites. Some of the shelled ammonites that were the
predators in the ocean at various times in geological history.
dominant elements of the marine fauna during the Mesozoic
Its evolution is related directly to the development of
were of 3 m in diameter. Increase in brain size and com-
low-pressure buoyancy mechanisms. They have acquired the
plexity, development of effective sense organs, and changes
ability to regulate buoyancy, followed by reduction and
in the skin concurred to the development of sophisticated
internalization of the shell and the development of the
behaviours. These traits make cephalopods the most active
mantle musculature. Some 17,000 fossil species are known,
and intriguing of the molluscs (Nixon and Young 2003).
most of them provided with an outer calcareous shell, whose
There are two major divisions within present-day
abundance and distribution have experience important fluc-
cephalopods: the Nautiloidea with six species of the pearly
tuations throughout the different geologic eras. Clearly, the
nautilus (Fig. 3.1), which are the only living cephalopods
lineages of extinct taxa were prolific and diverse.
with outer shells, and the Coleoidea, which is represented by
Á. Guerra (&) about 800 species, containing the cuttlefishes and bobtail
Ecology and Biodiversity Department, Institute of Marine squids (Fig. 3.2), long-fin and short-fin squids (Fig. 3.3),
Research, Spanish National Research Council (CSIC), 36208 vampire squids, Dumbo octopuses and octopods. This last
Vigo, Pontevedra, Spain group includes Argonauta species whose females produce an

https://doi.org/10.1007/978-3-030-11330-8_3
12 Á. Guerra
Class: Cephalopoda Cuvier, 1797

Subclass: Nautiloidea Agassiz, 1847
Family: Nautilidae Blainville, 1825 (Pearly or chambered nautilus)
Subclass: Coleoidea Bather, 1888
Superorder: Decapodiformes Leach, 1817
Order: Spirulida Haeckel, 1896
Fam: Spirulidae Owen, 1836
Order: Sepioidea Naef, 1916
Suborder: Sepiida Keferstein, 1866
Fam: Sepiidae Keferstein, 1866 (Cuttlefishes)
Suborder: Sepiolida Naef, 1916
Fam: Sepiadariidae Fischer, 1882
Fam: Sepiolidae Leach, 1817 (Bobtail squids)
Order: Myopsida Naef, 1916
Fam: Australiteuthidae Lu, 2005
Fam: Loliginidae Lesueur, 1821 (Long-fin squids)
Order: Oegopsida Orbigny, 1845
Fam: Architeuthidae Pfeffer, 1900 (Giant squid)
Fam: Brachioteuthidae Pfeffer, 1908
Fam: Batoteuthidae Young and Roper, 1968
Fam: Chiroteuthidae Gray, 1849
Fam: Joubiniteuthidae Naef, 1922
Fam: Magnapinnidae Vecchione and Young, 1998
Fam: Mastigoteuthidae Verrill, 1881
Fam: Promachoteuthidae Naef, 1912
Fam: Cranchiidae Prosch, 1847
Fam: Cycloteuthidae Naef, 1923
Fam: Ancistrocheiridae Pfeffer, 1912
Fam: Enoploteuthidae Pfeffer, 1900
Fam: Lycoteuthidae Pfeffer, 1908
Fam: Pyroteuthidae Pfeffer, 1912
Fam: Gonatidae Hoyle 1886
Fam: Histioteuthidae Verrill, 1881
Fam: Psychroteuthidae Thiele, 1920
Fam: Lepidoteuthidae Naef, 1912
Fam: Octopoteuthidae Berry, 1912
Fam: Pholidoteuthidae Voss, 1956
Fam: Neoteuthidae Naef, 1921
Fam: Ommastrephidae Steenstrup, 1857 (Short-fin squids)
Fam: Onychoteuthidae Gray, 1847
Fam: Thysanoteuthidae Keferstein, 1866
Superorder: Octopodiformes Berthold and Engeser, 1987
Order: Vampyromorpha Robson, 1929
Fam: Vampyroteuthidae Thiele, in Chun, 1915 (Vampire squids)
Order: Octopoda Leach, 1818

Suborder: Cirrata Grimpe, 1916 (Dumbo octopuses or Cirroctopods)
Fam: Cirroteuthidae Keferstein, 1866
Fam: Stauroteuthidae Grimpe, 1916
Fam: Opisthoteuthidae Verrill, 1896
Suborder: Incirrata Grimpe, 1916
Fam: Alloposidae Verrill, 1881
Fam: Argonautidae Cantraine, 1841
Fam: Ocythoidae Gray, 1849
Fam: Tremoctopodidae Tryon, 1879
Fam: Eledonidae Grimpe, 1921
Fam: Octopodidae Orbigny, 1839 (Octopuses)
Fam: Enteroctopodidae Strugnell et al., 2013
Fam: Amphitretidae Hoyle, 1886
Fam: Bathypolypodidae Robson, 1932
3 Functional Anatomy: Macroscopic Anatomy and Post-Mortem … 13
Fam: Megaleledonidae Taki, 1961 groups. The aphorism “live fast, die young” describes quite
Order uncertain well the life history strategy of these coleoid cephalopods,
Superfamily: Bathyteuthoidea nov. which have epipelagic and benthic habitats. Nevertheless, as
Fam: Bathyteuthidae Pfeffer, 1900
Fam: Chtenopterygidae Grimpe, 1922
indicated by O’Dor (In Darmaillacq et al. 2014), «that phrase
Fam: Idiosepiidae Fischer, 1882 “live fast, die young” perhaps should be expanded to “live
fast and smart, to leave offspring fewer enemies». Specifi-
cally, the available evidence of species of neritic cephalo-
pods is that they complete their life cycle in one or two years
and in some small species even in a shorter time. Beside this
short lifespan, other characteristics shared by all members of
this category are a similar type of predation, which places
them at the upper trophic levels of the ecosystem. Like most
cephalopods, neritic species have a single ovarian cycle.
However, many of them have long spawning periods, in
which the larger peak of new hatchlings is synchronized
with environmental conditions providing them with suitable
oceanographic factors and appropriate prey, which ensures
high survival rates, but, conversely, noteworthy mass mor-
tality. Consequently, cephalopod populations are highly
unsteadied responsive to change in physical, chemical, and
biological environment. Fecundity of the species within this
category is very variable, from a few hundred eggs in cut-
tlefish, to several hundreds of thousands in octopus.
Fig. 3.1 Nautiluses or chambered nautiluses are the sole living
Cephalopods do not have a true larva, because they lack of a
cephalopods with an external shell distinct metamorphosis. Hatchlings of a number of species
are planktonic and have a distinctively mode of life from
older conspecifics (paralarvae). The neritic species share
external calcareous structure (Fig. 3.4), which is not a true
some degree of dependency on the seabed: some of them lay
shell but a brood chamber.
and eat at the bottom (demersal species, e.g. Loliginidae),
Currently, the most widely accepted classification is the
but others, such as cuttlefish and octopuses, are truly benthic
proposed by Young et al. (2018):
species.
The “oceanic and deep-sea category” of species encom-
passes the taxonomically diverse families of epi-, meso-, and
3.2 Ecology: General Aspects bathypelagic, as well as bathybenthic cephalopods. The
ecological knowledge about this group is still scarce.
As indicated by Boyle and Rodhouse (2005): «Any ecolog- Although they share many basic characteristics, their life-
ical approach always tries to take account of the interacting styles are more different between these taxa than with the
factors of evolution, genetics, physiology, and behaviour of neritic forms.
the organisms as well as their relationships with the Like all division into categories, the one we present here
stochastics parameters of the environment. Consequently, is artificial and there are some species of difficult location,
these studies involve multidisciplinary approaches, which are even within the same family. Thus, for example, in the
not always easy to obtain for marine animals». This difficulty Ommastrephidae, there are several species of different gen-
also affects cephalopods which occupy a great variety of era (Ommastrephes, Sthenoteuthis, Martialia, Dosidicus)
habitats in all of the world’s oceans (Fig. 3.5) and have a large that are truly ocean dwellers. Conversely, species of the
variety of life history strategies. Another difficulty with these genus Illex, Todadores, Todaropsis, and Nototodarus,
studies is the disparity of knowledge currently held about although they typically have an offshore distribution, are
cephalopod species, which, on the other hand, increases day frequently present on the continental shelves. There are
by day. For these reasons, we are going to provide here some many other examples of this or similar scenarios, and Jereb
generalities concerning the great ecological categories in and Roper (2005, 2010) and Jereb et al. (2016) catalogues
which it is possible to classify living cephalopods. provide account for all families.
The broad category of “coastal and shelf species” (neritic In any case, cephalopods are important to the ecosystem
species) covers the relatively well-known cephalopod as both predator and prey as well as reservoirs of parasites.
14 Á. Guerra
Fig. 3.2 Two species representatives of the order Sepioidea. Photographs by J. Hernández-Urcera and J. L. González
SSFs; however, IF are supported by a few species, mainly

3.3 Fisheries and Aquaculture belonging to the families Loliginidae, Ommastrephidae, and
Octopodidae (see Arkhipkin et al. 2015 for review).
Cephalopod catches worldwide account for around 4 million Historically, the consumption of cephalopod products has
tons per year in 2016 (about 4% of total marine products). been highest in the countries of Asia (Japan, Thailand,
Although in recent times the total world catch from marine Taiwan, and China). Among European countries, Spain,
and freshwater fish stocks appears to have peaked and may Portugal, Italy, and Greece are the traditionally high con-
be declining, the catch of cephalopods has continued to sumers of cephalopods. In the rest of the globe, per capita
increase as fishers concentrate efforts away from more tra- cephalopod consumption is low.
ditional finfish resources. Cephalopods fisheries can be The life cycle characteristics of cephalopods mean that
divided between small-scale fisheries (SSFs) and industrial their fisheries are intrinsically difficult to assess and manage.
fisheries (IF). The SSFs are of great importance in terms of The level of exploitation of some stocks exploited in IF is
job opportunities, and they contribute significantly to the quite high, and some of them are actually overexploited.
economy of many coastal communities. Methods of capture Increasing of scientific knowledge for assessment and
in SSFs are very diverse (pots, traps, lures, etc.), and the management purposes is needed.
catches are mainly consumed in fresh. IF methods of capture The increasing demand of cephalopods and some of
are mainly jigging and trawling, and the catches are mainly biological traits (high growth rates and short life spans)
commercialized frozen. Numerous species are caught in make cephalopods ideal candidates for commercial
Fig. 3.3 Two species representatives of the order Myopsida (genus Loligo, family Loliginidae) and one of the order Oegopsida (genus Illex,
family Ommastrephidae). Courtesy of A. Escánez and J.L. González
aquaculture since they have the potential to rapidly reach small-scale culture of some species has become scientifically
market size. As pointed out by Louise Allcock, former important in the latter half of the twentieth century. Never-
President of the Cephalopod International Council (CIAC), theless, the industrial culture of cephalopods is still in an
in the preface of the book “Cephalopod Culture” by Iglesias incipient state. There are, however, some advances; one of
et al. (2014), «this is a pioneering text, which draws together them is successfully culture octopuses with large hatchlings
a vast array of knowledge on cephalopod culture and pro- using a completely artificial diet. As a result of the numerous
vides the foundations for further advances in this significant studies carried out since 1990 to present days, many culture
field». Moreover, some species are used as model organisms protocols had been optimized. At present, only 19 species of
in neurobiology, robotics, restocking, pharmaceutical cephalopods are being cultivated worldwide: three Nautilus
exploitation of antibacterial anticancer activities reported and Allonautilus species, four cuttlefish, two sepiolids, three
from the ink sac, the use of modified cuttlebone in tissue loliginid squids, and seven octopods. The main bottlenecks
engineering, the many and varied used of cuttlefish oil, and in cephalopod culture were identified to be nutrition and
to study the cephalopod immune system. In consequence, physiology.
16 Á. Guerra
Fig. 3.4 Dumbo octopus (Cirroteuthis sp) is representative of the order Octopoda, suborder Cirrata; Japetella diaphana is a pelagic Octopoda;
Argonatuta argo and Octopus vulgaris are representatives of the order Octopoda, suborder Incirrata. Courtesy of J.L. González
distance between the antero-dorsal margin of the mantle and

3.4 Morphology and Anatomy of the Adult the posterior apex of the mantle (Fig. 3.6). In the
Octopodiformes, ML is measured from the back of the body
Modern cephalopods (subclass Coleoidea) have bilateral to an imaginary line that would connect the centre of both
symmetry, and the body is divided into two defined parts: eyes (Fig. 3.7). Sometimes, other measures such as the total
the cephalopodium or anterior part and the visceropalium or length (TL), which is the distance between the longest arm,
posterior part. The cephalopodium includes the head, the or the extended tentacle, and the back of the animal’s
appendages that surround the mouth and the funnel; the mantle, are used. Mantle length of adult cephalopods varies
visceropalium comprises the mantle, the cavity of the mantle between 6 mm in the genus Idiosepius to around 2 m in the
and its organs, as well as the shell and the fins, the latter if giant squid Architeuthis dux.
present (Mangold 1989).
3.4.1.2 External Form
The head is usually separated from the mantle by the nuchal
3.4.1 External Morphology constriction. It carries the oral appendices and the eyes,
which are usually spherical and of similar size, although in
3.4.1.1 Size Histioteuthis the left eye is much larger than the right. In the
The basic measure of cephalopods is the dorsal length of the occipital region, the head may be completely fused with the
mantle (ML), but also ventral mantle length (VML) can be mantle or attached to it by a nuchal cartilage. A cartilaginous
used. In Sepioidea, Myopsida, and Oegopsida, ML is the capsule contributes to the shape and volume of the head.
Fig. 3.5 Distribution with habitat and depth of selected genera characteristics of different marine zones
This capsule is like a skull that surrounds and protects the The arms of Sepioidea, Myopsida, and Oegopsida are
brain. On both sides of the head, near the neck, the olfactory attached to the outer lip by a buccal membrane that has six,
organs are located. seven, or eight folds. These folds are attached to the dorsal
Decapodiformes cephalopods have ten muscular appen- and ventral margins of the arms through the buccal con-
dages of two types, differing according to their length: eight nectives. The arrangement of such bonds has taxonomic
arms (shorter) and two tentacles (longer). In contrast, value and is also expressed by a formula. Thus, a DDVV
Octopodiformes have only eight arms, lacking tentacles, formula indicates that the buccal connectives are attached to
although the arms are usually longer in proportion to body the dorsal margin of the first two pairs of arms and to the
size than Decapodiformes. Each pair of arms is generally ventral of the following two pairs. Vampyroteuthis and all
different in size from the other pairs. In Vampyroteuthis, two Octopodiformes lack buccal connectives.
of its ten arms have been transformed into long and thin The cross section of the arms of Sepioidea, Myopsida,
filaments that are retracted in a pair of bags placed between and Oegopsida is generally triangular. The inner surface
the dorsal and dorso-lateral arms. The species of Nautilus (oral) is flattened, while the outer (aboral) surface is angular.
have 63–94 arms or short tentacles. The arms are often The suckers are arranged on the oral surface, usually in two
numbered from the dorsal pair to the ventral (which is well rows, although there may be more. Suckers are provided
defined because it is the side of the animal where the funnel with denticulate or smooth chitinous rings. Arm suckers
opens). This gives rise to the “brachial formula” (Figs. 3.6 have been converted into hooks in some oceanic species of
and 3.7). A brachial formula such as 4.13.2 indicates that the Oegopsida (e.g. Taningia danae). Along the lateral angle of
fourth pair of arms (or ventral) to the right of the animal’s the oral surface of the arms, there are web-like integument
body is longer than the second (or dorso-lateral), and this protective membranes, generally supported by muscular rods
one longer than the first (or dorsal), which is longer than the called trabeculae. These protective membranes are well
third. The numbers of the brachial formula can be Arabic or developed in some species. Thus, for example, in Histio-
Roman (Figs. 3.6 and 3.7). This formula has taxonomic teuthis species join two pairs or more arms, while in some
value, especially the Octopodiformes (Jereb and Roper Ommastrephidae (e.g. Ommastrephes bartramii), the ventral
2005, 2010 and Jereb et al. 2016 catalogues provide illus- protective membranes of arm III are very wide and in adult
trated glossaries of technical terms and measurements). females expand into a large, triangular, membranous lobe.
18 Á. Guerra
Fig. 3.6 Nomenclature of diverse parts of the body of Loliginidae and points between which the body measurements should be taken (Drawings
from Guerra 1992)
The so-called swimming keels are flattened and muscular stalks, protective membranes, trabeculae, and a section of the
expansions located on the aboral side of some arms to render arm. The last occurs in the distal tip of the Cirrata and
them more hydrodynamic. Incirrata Octopodiformes. In these cases, along the hecto-
Vampyroteuthis and Octopodiformes cross section of the cotilized arm, there is a spermatophore groove, which end
arms is, generally, circular. All arm suckers of Octopodi- open into a structure formed by the ligula and the calamus
formes lack of chitinous rings. In certain cases, as in the (Fig. 3.7b). The ligula is a spatulate or spoon-shaped, ter-
males of some species of Octopoda Incirrata, there are minal structure of the hectocotylus, which usually contains a
several modified suckers, generally larger than the others, series of transverse ridges and grooves on the oral surface.
which play a role in courtship. Both Vampyromorpha and The calamus is a conical papilla or projection of the base of
Octopoda Cirrata have elongate, fleshy, finger-like papillae the ligula at the distal terminus of the sperm groove, distal to
(cirri) along the lateral edges of the oral surface of the arms, the last sucker.
which length is variable. Cirri are mechanoreceptors. The tentacles are two long appendages in Decapodi-
A membranous sheet of greater or lesser extent can be pre- formes, used for prey capture and capable of considerable
sent between the arms of many Octopodiformes (Fig. 3.7a); extension and contraction. The tentacles can be retracted into
this web gives an umbrella-like appearance when the arms open depression or pockets located in the antero-ventral
are spread out. surface of the head between the bases of the ventro-lateral
One (or more) arm in male cephalopods is modified to be (3) and ventral pair of arms (4) in all Sepioidea species, but
used transferring spermatophores to the female. This not in Myopsid and Oegopsid squids. A tentacle is com-
arm (s) is called hectocotilized arm and the modified portion posed by a peduncle, the carpus or fixing apparatus, and the
hectocotylus. Modifications may involve suckers, sucker tentacular club, which is an expansion of the distal part
Fig. 3.7 Main measurements and terms in Octopoda Incirrata. a Body; b hectocotilized arm. (Drawings from Guerra 1992)
(Fig. 1.6). The central or “hand” portion of the club may The muscles that support the lateral attachment of the funnel
have suckers and/or hooks. The distal, terminal section of to the head, called funnel-adductor muscle, are generally
the club, often characterized by suckers of reduced size is the well developed. In many groups of cephalopods, within the
dactylus. funnel, there is a semilunar muscular flap in the dorsal sur-
The ventral, subconical tube through which the water is face near the distal opening of the funnel—the funnel valve
expelled from the mantle cavity during locomotion and —and also a glandular structure—the funnel organ -, which
respiration, and that also serve to expelled ink, reproductive adopts different forms; the configuration of the funnel organ
and waste products, is the funnel. In Sepioidea, Myopsida, has taxonomic importance, especially in Cranchiidae and
and Oegopsida, the funnel is located within a depression in Octopodidae.
the posterior-ventral surface of the head called funnel The lower lateral margins of the funnel may be fused to
groove. However, in Octopodiformes, it is embedded in the the mantle (e.g. Cranchiidae) or be connected to it by a
tissues of the head, leaving only the apical region free. The cartilage: the funnel-locking cartilage. The cartilage portion
funnel groove of Oegopsid squids has a series of structures found in the funnel of Sepioidea, Myopsida, and Oegopsida
with taxonomic value. Thus, in some genera (e.g. Todor- presents a varied morphology, and it is also a character of
odes, Illex), there are transverse, membranous folds of skin taxonomic importance. There is also a funnel-locking carti-
that form a pocket in the anterior end of the funnel groove, lage in the Argonautoidea superfamily, but the rest of the
called foveola. Other genera, like Ommastrephes, have also Octopodiformes lack this structure. The cartilaginous ridge,
small shallow pockets lateral to foveola in the funnel groove. knob, or swelling on each side of the ventro-lateral, internal
20 Á. Guerra
surface of mantle that locks into the funnel component is 3.4.1.3 Integumental System: The Skin
called mantle-locking cartilage. Both structures funnel and and Elements Contributing to Colour
mantle-locking cartilages form the locking apparatus which and Body Patterns
is essential for locomotion. The epidermis contains three main kinds of cells: epithelial
The mantle of cephalopods is basically a muscular sac. columnar cells, gland cells, and sensory cells. Immediately
The dorsal part of the mantle cavity is small, while the bellow the epidermis, there is a layer (dermis) that possesses
ventral part is larger and lodges the viscera. Cephalopod a series of sacs with pigment—the chromatophores, which
muscles are arranged in three dimensions in closely packed are typically only red, yellow, or brown and determine
blocks, which allow rapid and abrupt contractions. The colour changes in camouflage (Fig. 3.8). Other colours are
contraction and relaxation of the different types of muscle attainable by using a second layer of structures in the
fibres of the mantle allow the expulsion and entry of water cephalopod skin called iridophores and leucophores, which
into the mantle cavity. The best-known peripheral nervous are located in the dermis. Iridophores are stacks of very thin
system is that of the giant axons, which in three steps from cells that are capable of reflecting light back at different
the magnocellular lobe and the paleovisceral lobe of the wavelengths and possibly different polarities. Cuttlefish and
brain innervate the mantle musculature. These axons have a octopuses possess an additional type of reflector cells called
diameter between 0.5 and 1 mm (thousand times greater leucophores. They are cells that scatter full spectrum light so
than the axons of mammals). Its reaction potential is so high that they appear white. By combining reflection from the
that the nerve transmission runs at high speed, allowing the iridophores and leucophores with the correct patterning of
extremely rapid, complete, and instantaneous reaction of the chromatophores, the cephalopod can create a very con-
pallial musculature to give rise to the incomparable hydro- vincing copy of the surrounding conditions. The rest of the
dynamic invention of the jet propulsion. dermis consists of an outer tunic with collagen fibres, the
The mantle musculature in some Oegopsida (e.g. musculature, a layer with nerve fibres and blood vessels,
Cranchiidae) and pelagic octopuses (e.g. Japetella diaphana, and, finally, an inner tunic. In Sepioidea and Incirrata
Fig. 3.4) has been reduced and has a high water content. Octopodiformes, there is a complex musculature that chan-
Many of these species have gelatinous consistency, medu- ges their skin from smooth and flat to rugose and
soid aspect, and the walls of the mantle are translucent so three-dimensional. The organs responsible for this physical
that they allow the internal organs to be seen. change are the skin papillae. Skin texture is an important
The members of the family Lepidoteuthidae have a dis- contribution to body pattering. The photophores, which are
tinct dermal cushions present on the mantle. These dermal bioluminescence cells, are also located in the skin and in
cushions, which are thickening of the skin with abundant different regions of the body, for instance, around the eyes,
vacuoles and connective tissue, are relatively large, dia- in the ventral part of the mantle, arms, tentacles, and even
mond, or hexagonal-shaped structures that cover the whole inside the mantle cavity over the ink sac.
circumference of the mantle and in overlapping arrangement.
Most cephalopods have a pair of fins of varying shapes
and sizes. They are located in the back of the mantle in the 3.4.2 Functional Anatomy
Myopsida and Oegopsida (Figs. 3.3 and 3.6), in its middle
zone in the Octopoda Cirrata (Fig. 3.4) or in its lateral 3.4.2.1 The Shell
borders in Sepioidea (Fig. 3.2). Octopoda Incirata has no At present, the only representatives of this class of the
fins. This pair of muscular flaps is used for locomotion, phylum Mollusca with external shell are Nautilus and
steering and stabilization. Allonautilus. The shell of these cephalopods is divided into
The mantle of many Myopsida and Oegopsida has a chambers bounded by transverse septa, the latter occupying
posterior narrow extension or tail, in which length may be the animal. Through the septa, there is a tissue cord tube-like
very long (Fig. 3.3). The end of the fins and the beginning of form or siphuncle, which intervenes in the control of the
the tail often overlap. This posterior extension of the body is buoyancy of the animal, regulating the relative volume of
often very long in paralarval stages. Some species (e.g. gases and liquids present in the chambers of the shell.
Alloteuthis subulata or A. africana) show a lengthening of The rest of the cephalopods present an internal shell, as it
the tail as the males mature sexually and constitute a sec- occurs in cuttlefish, squids, or it is totally vestigial, or it does
ondary sexual character. not exist as such (Incirrata). Spirula has a flattened spiral
Fig. 3.8 Diagram showing the elements that contribute to colour body patterns and their arrangement in the skin
internal shell that does not intervene in the protection of the to maintain the rigidity of the body in longitudinal sense
animal, although it contributes to maintaining the body during the convulsive contraction phase of jet swimming,
shape and acts as a hydrostatic skeleton. The cuttlebone of but it has lost its protective function. A number of pelagic
the cuttlefishes (Sepia spp) is an intricate structure composed from achieve neutral buoyancy by a reduction of the protein
of a dorsal shield and ventrally placed chamber complex. It content of their tissues and the accumulation of a
is composed of calcium carbonate in its aragonite polymorph low-density solution of ammonium chloride either the coe-
mixed with a small amount of organic matter, a complex of lomic space (e.g. Cranchiidae) or in the vacuoles within the
b-chitin and protein. In ventral plan view, the chamber musculature and connective tissue (e.g. Architeuthis dux).
complex consists of the posterior siphuncular zone, which is Cirrate octopuses possess a well-developed internal shell
characterized by a series of striae corresponding each to the that supports their muscular swimming fins. This is in con-
posterior end of one chamber, and the septum of the trast to the more familiar, finless, incirrate octopuses, in
last-formed chamber. In dorso-ventral vertical section, each which the shell remnant is either present as a pair of stylets
chamber is composed of a complex arrangement of hori- or absent altogether.
zontal septa and membranes and vertical pillars and mem-
branes, intervening in the control of the buoyancy of the 3.4.2.2 Respiratory and Circulatory Systems
animal by regulating the amount of gas and liquid present in Respiratory exchange with the environment occurs through
such chambers. well-vascularized gills suspended in the mantle cavity.
In the Myopsida and Oegopsida squids, the shell, also Nautilus and Allonautilus species have two pairs of gills, but
called gladius or pen, is reduced to a chitinous sheet with a in all Coleoidea there is only a single pair (Fig. 3.9). Due to
central axis and two lateral expansions; the pen contributes the particular orientation of the gills within the mantle
22 Á. Guerra
Fig. 3.9 Respiratory and circulatory system of Octopus vulgaris (partial); a: auricle; aae: abdominal aorta exit; agv: afferent gill vessel; bh:
branchial or gill heart; da: dorsal aorta; agv; efferent gill vessel; g: gill or branchia; gl: gill lamellae; sh: systemic heart; v: ventricle
cavity, water flows between the lamellae of each gill in the the systemic heart to supply the body with blood. Blood
opposite direction of the flow of blood through the tissue. drains to the branchial hearts by the major veins, known as
This originates a countercurrent system that maximizes the the anterior and lateral vena cava or vena cava cephalica.
exchange of gas. The vessels within the gills are known as afferent bran-
Unlike all other molluscs, cephalopods have a closed chial vessels, which drain back to the main ventricle of the
circulatory system. This means that blood flows through a systemic heart. Blood is then pumped from the systemic
series of vessels to return to the heart, rather than bathing heart to the body via the main cephalic artery.
organs in the blood fluid as in open circulatory systems. Oxygenate cephalopod blood is a blue colour due to the
The core of the cephalopod circulatory system is a series presence of copper-containing respiratory pigment haemo-
of three beating hearts. This trio of hearts connects to a high cyanin in solution in the blood, because cephalopods lack of
pressure system of veins, arteries, and capillaries—unique erythrocytes. The oxygen carrying capacity of the haemo-
among all molluscs. Two of the hearts are branchial hearts cyanin is less efficient that the vertebrate haemoglobin.
(Fig. 3.9), which pump blood through the gills for respira- Cephalopods exhibit the highest rates of aerobic metabolism
tion and gas exchange. The third heart is a systemic heart among the marine invertebrates. However, it is very variable,
(Fig. 3.9), receiving the blood that drains from the gills and depending of some environmental factors such as water
pumping that oxygenated blood to the body system. Each of temperature, but also on the performance of their haemo-
the three hearts is innervated by a variety of nerves, though it cyanins (Pörtner et al. 1994). Accordingly with oxygen
appears that the cardiac ganglion—a cluster of nerves—acts affinity, cephalopods can be divided into three groups:
as the controlling pacemaker of the hearts. The two branchial octopuses and some sluggish squids, with relatively low
hearts beat simultaneously, followed by the contraction of oxygen capacity; fast-swimming squids; and Sepia species.
Fig. 3.10 Sepia officinalis

digestive system; an: anus; asg:
anterior salivary glands; be: beak
or mandibles; bm: buccal mass;
cae: caecum; dg: digestive gland
(bilobulate); dgda: digestive
gland duct appendages; int:
intestine; insd: Ink sac duct; oe:
oesophagus; st: stomach
3.4.2.3 Digestive System these glands is poorly known but thought to be primarily in
The digestive system of the cephalopods (Figs. 3.10, 3.11 mucous production. The radula, which is a chitinous and
and 3.12) opens in the mouth. Located at the base of the ribbon-like band, is placed on the floor of the oral cavity; it
arms and tentacles (circumoral appendages), the mouth is the contributes to scraping the food in order to fragment it into
opening of the buccal mass, which contains the beaks, smaller pieces. The radula can be of various types and its
radula, various glands, and the pharynx. Surrounding the structure varies depending on the group. Many Octopus
mouth, the inner and outer lips possess numerous ridges or species are able drilling shells of crustaceans and shells of
papillae. The beaks are two chitinous mandibles bound in other mollusks. These drilling activities are carried out by a
powerful muscles. The dorsal beak is referred as “upper” salivary papilla that lies just below the radula. The papilla is
beak, and it inserts within the “lower” beak to tear tissue muscular, and its anterior face is covered with very small
with a scissors-like cutting action. A pair of glands of the teeth. It is now possible to say that the salivary papilla can
digestive system is associated with the buccal mass, the function as an accessory radula. Some cephalopod species
sublingual and the anterior salivary glands. The function of (e.g. Spirula spirula) lack of radula. Some cephalopods,
24 Á. Guerra
Fig. 3.11 Loligo vulgaris digestive system; a female cut from the int: intestine; ins: ink sac; insd: Ink sac duct; oe: oesophagus; psg:
ventral side of the mantle: b diagram showing the main parts of the posterior salivary glands; psgd: posterior salivary gland duct; st:
digestive system; an: anus; anf: anal flaps; bm: buccal mass; cae: stomach
caecum; dg: digestive gland; dgda: digestive gland duct appendages;
such as Sepia, Octopus, inject neurotoxins into their prey in grind-up food with the aid of digestive enzymes. The
order to immobilize them and facilitate their ingestion; these stomach may be greatly expandable in size and serve as a
are secreted by the posterior salivary glands. The blue-ringed storage area, in species lacking a crop, until food can be fully
octopus of the Pacific, Hapalochlaena maculosa, produces processed. The caecum is a major organ of this system that is
toxins that can be deadly to man. a primary site of absorption. It joins the stomach “upstream”
The portion of the digestive tract between the buccal mass and the intestine “downstream”. Present in some Decapod-
and the stomach is the oesophagus. The lumen of the iformes, the caecal sac is a thin-walled posterior portion of
oesophagus is narrow and slightly dilatable, which is the caecum that lacks the internal, ciliated leaflets charac-
because it passes through the brain and cranial cartilage. teristic of the anterior portion of the caecum (Fig. 3.11b).
This is why cephalopods may chop their prey into small The digestive enzymes enter the caecum in the ducts from
pieces with their beaks and then force the pieces down the the digestive gland (Fig. 3.12), which is the primary organ in
throat with the radula. Often, a portion of the oesophagus is cephalopods that secretes digestive enzymes. The ducts
enlarged to form a crop. This expansion or diverticulum of leading from the digestive gland have outpockets, which are
the oesophagus serves for storing food. It is present in covered with glandular epithelium, and they are called
Nautilus and most Octopodiformes. When there is no crop digestive gland duct appendages. Digestive gland is also
(Fig. 3.12), the oesophagus opens in the stomach. The important in absorption, excretion, and detoxification of
stomach is a cavity generally lined with cuticular ridges to heavy metal accumulations.
Fig. 3.12 Octopus vulgaris

digestive system; an: anus; asg:
anterior salivary glands; bm:
buccal mass; cae: caecum; cr:
crop; dg: digestive gland; dgd:
digestive gland duct; int:
intestine; ins: ink sac; oe:
oesophagus; psg: posterior
salivary glands; st: stomach
After the caecum begins the intestine, which opens into squirted; its proximity to the base of the funnel means that
the anus, situated in the anterior ventral part of the mantle the ink can be distributed by ejected water as the cephalopod
cavity, near the funnel. As a whole, the cephalopod digestive uses its jet propulsion. The ejected cloud of melanin forms a
tract has a U-shape. A pair of muscular palps that arise at the lump approximately the size and shape of the cephalopod,
sides of the anus in most Coleoidea are called anal flaps fixing the predator’s attention while the cephalopod itself
(Fig. 3.11). makes a hasty escape.
A notable anatomical feature of the digestive tract of All cephalopods actively catch and eat live prey, and a
cephalopods is the ink sac. With the exception of nocturnal very range of prey item has been recorded. The diet is
and very deep-water cephalopods, all Coleoidea which dwell probably determined as much by prey availability as
in light conditions have an ink sac. The ink sac is a muscular predator preference. Hunting is essentially visual; however,
bag which originated as an extension of the hind gut chemical cues also probably have a role. Feeding strategies
(Fig. 3.11b). It lies beneath the gut and opens into the anus, are very different. Once trapped, generally using tentacles or
into which its contents—almost pure melanin—can be arms, the prey is drawn in towards the mouth which is
26 Á. Guerra
generally paralysed by the saliva of the posterior salivary

glands and bitten into the beaks. As above commented,
bite-sized of flesh are shallowed. Nevertheless, hard pieces
of their prey are also found in the stomach contents, which
can be used for preliminary prey’s identification. However,
to identify prey in cephalopod stomach contents the most
accurate is to use molecular techniques. It is well known that
cephalopods have high requirements for protein and rela-
tively low requirements for high-quality lipids: the cepha-
lopod diet must consist of over 60% protein and 4% lipids.
After ingestion, the already fragmented meal enters in
digestive tract. The characteristic fast growth rate of
cephalopods (3–10% body weight d−1) sets high require-
ments for digestion and assimilation. The digestion is a
complex process. It starts externally at the prey, where
salivary enzymes are injected after perforation by the beaks
or salivary papilla. Although the exact biochemical mecha- Fig. 3.13 Schematic representation of the excretory (renal) complex
and associated circulatory system in Octopus. Arrows show the
nisms in different species are not fully known, it is consid- direction of the blood flux; bh: brachial heart; bha: brachial heart
ered that pre-digestion is probably limited to loosening appendages; orsm: opening of renal sac to mantle cavity; ps:
muscle attachments. Partially digested food is then ingested pericardical sac; ra: renal appendages; rpc: renopericardial sac; rs:
and enters the crop, in octopus, or goes to the stomach, in renal sac; sh: systemic heart; vc: vena cava. Modified from Martin AW
and Harrison FM. Excretion. In Wilbur and Yonge, C.M. (eds.)
cuttlefish and squid, where digestive enzymes from the Physiology of Mollusca, Vol. II. Academic Press, New York, 1966
digestive gland initiate digestion. Enzyme-bound soluble
nutrients pass from the crop to the stomach in octopus or
directly to the stomach in cuttlefish and squid, where fibrillar to pump the secreted waste into the sacs, to be released into
proteins and other macromolecules are degraded until a the mantle cavity through a pore (the renal papilla). The
semi-liquid mass of partly digested food (chyme) is formed. main extra-renal organs involved in ammonia excretion are
The chyme is then separated by the caecum to be transported the gills. In this case, the waste is directly excreted from tits
to the digestive gland or to form faecal pellets. Once in the epithelium to the seawater. The rate of release is lowest in
digestive gland, nutrients are dissolved and absorbed by the shelled cephalopods Nautilus and Sepia as a result of
pinocytosis in the digestive gland cells, where intracellular their using nitrogen to fill their shells with gas to increase
digestion occurs. This process can take from 4 to 8 h, buoyancy. Other cephalopods use ammonium in a similar
depending on the size of the meal, animal, and temperature. way, storing the ions (as ammonium chloride) to reduce their
The high rate of consumption leads to interesting specula- overall density and increase buoyancy. A remarkable feature
tions about the fuelling by cannibalism that is relatively of the renal system of cephalopods is its infestation by
frequent in many cephalopods, especially in the long dicyemid mesozoans, which relationship with the host is
migrations undertaken by many shoaling squid species. apparently symbiotic.
3.4.2.4 Excretory System 3.4.2.5 Reproductive System and Reproduction

Because protein is a major constituent of the cephalopod Sexes in cephalopods are separate. The reproductive system
diet, large amounts of ammonia (NH4+) are produced as of the females consists of an ovary, which leads to one or
waste. This waste is excreted in solution by several routes. two oviducts, the oviductal gland (single or paired), and the
Excretion from the blood system takes place in a Decapodiformes of the nidamental and accessory nidamental
well-differentiated renal system surrounding the venous glands (Figs. 3.14 and 3.15) The ovary is located at the back
return to the systemic heart. Filtered nitrogenous waste of the mantle cavity, and in it the oocytes are formed. The
(primary urine) is produced by ultrafiltration from the blood function of nidamental glands is to produce the outer coat for
in the pericardial cavity of the branchial hearts, each of eggs. The accessory gland has many of the structural fea-
which is connected by a narrow canal to the brachial heart tures of a secretory organ. The basic structural unit is a
appendages (Fig. 3.13). The canal delivers the excreta to a tubule composed of a single layer of epithelial cells con-
bladder-like renal sac and also resorbs excess water from the taining ordered arrays of rough endoplasmic reticulum and a
filtrate. Several outgrowths of the lateral vena cava (renal lumenal surface covered with microvilli, cilia, and structural
appendages) project into the renal sac, continuously inflating specialization presumed to be involved in secretion. The
and deflating as the branchial hearts beat. This action helps lumen of each tubule is filled with a dense population of
Fig. 3.14 Sepia officinalis

female reproductive system; an:
anus; ang: accessory nidamental
glands; bh: branchial heart; fun:
funnel; flc: funnel-locking
cartilage apparatus; g: gill; ins:
ink sac; int: intestine; ng:
nidamental glands; oe:
oesophagus
bacteria. During sexual maturation of the squid, the acces- of some females Decapodiformes or as pockets of the
sory gland changes in colour from white to mottled red oviducal glands seminal or spermatheca in Octopus species
(Fig. 3.15a). The accessory gland of the sexually mature (Fig. 3.16b), a lateral split located in the anterior ventral part
squid has a mixture of red, white, and yellow tubules; in of the mantle, within the mantle, etc. The extruded, explo-
each case, the colour of the tubule is due to the bacterial ded, evaginated spermatophore/s often in form of round bulb
population occupying the tubule. Since the red colour of the is called spermatangium (pl. spermatangia).
gland is due to the pigmentation of the bacteria, the bacteria In males, the spermatozoa produced by the testis are packed
must be responsive to the sexual state of the host, possible and surrounded by membranes, forming the spermatophore
through a change in the nature of the material secreted into (Figs. 3.17b and 3.18b). Therefore, a spermatophore is a
the tubule lumen. tubular structure manufactured by male for packaging sperm,
Semen can be stored in different parts of the female’s capable of holding millions of spermatozoa. A spermatophore
body: a sperm receptacle located on the buccal mass, a is composed by the sperm cord, the cement body, and the
pouch under the eye, specialized structures found in the skin ejaculatory apparatus. It is transferred and attached to the
28 Á. Guerra
Fig. 3.15 Loligo vulgaris female reproductive system; a general view; intestine; ins: ink sac; g: gill; int: intestine; ng: nidamental glands; oe:
b diagram showing main parts; an: anus; ang: accessory nidamental oesophagus; ov: ovary; ovd: oviduct (single in Ommastrephidae, and
glands; bh: branchial heart; dg: digestive gland; f: fin; fun: funnel; in: other Oegopsida are in pair)
female after fertilization begins, and it forms the sper- Sexual maturation is under the control of hormone(s)
matangium after the spermatophoric reaction occurs and the released from the small bodies called optic glands located on
spermatophore has everted. After the spermatozoa are formed, the optical tract, which connecting optic lobes to the brain. At
they pass to the spermatophoric organ through the vas defer- the onset of sexual maturity, there is a rapid gonad growth,
ens. The spermatophoric organ is composed by distinct yolk formation in the ova, and ripening of nidamental and
structures different in Decapodiformes (Figs. 3.17 and 3.18) accessory glands. In most of the coastal and epipelagic spe-
than in Octopodiformes (Fig. 3.18). In Octopus species, it is cies, reproduction is seasonal and afterwards both males and
formed by the seminal vesicle and the prostate, which are the females die shortly after spawning or after a variable time
structures engaged in forming the spermatophore sheaths. taking care of the eggs during the embryonic development
Ripe spermatophores are stored in the spermatophoric sac or (e.g. Octopus vulgaris). The causes of the senescence and
Needham’s sac. This sac opens into de mantle cavity or universal mortality that become after reproductive events are
directly into the water through the terminal organ, which by not still understood, although they seem to be related with
some authors incorrectly denominate penis. Although the physical changes in the optic gland and its secretions. Nev-
terminal organ of some Oegopsida (e.g. Architethis dux) can ertheless, variations of this pattern of monocyclic reproduc-
be extremely long (up to 80% of mantle length), its functioning tion and short lifespan are found or suspected in deep-water
is not that of a true penis. This because spermatophore is benthic octopuses and a range of other species.
transferred by the male generally using a modified arm called a Cephalopod mating usually includes a courtship that
hectocotylus. In some species of Octopus, the terminal organ often involves elaborate colour and body pattern changes.
widens into a diverticulum (Fig. 3.19b). Most females then lay large yolky eggs in clusters on the
Fig. 3.16 Octopus vulgaris female reproductive system; a general dg: digestive gland; g: gill; gl: gill lamellae; ov: ovary; ovd: oviducts;
view; b diagram showing main parts and a section of the oviducal ogl: oviducal gland
glands; c main parts; bh: branchial heart; bv: blood vessels; fun: funnel;
ocean floor or on any other hard substrate. However, other wider range of possibilities than vision. The studies of a few
(e.g. Ommastrephidae and Thysanoteuthidae) spawn neu- nearshore species have revealed complex behaviour and
trally buoyant egg masses that retain a specific location in remarkable capacity for learning. The kind of information
the water column by floating at the interface between water collected by these sense organs, the nature of its effectors or
layers of slightly different densities. Eggs develop by motor apparatus, and the organization of the brain are the
dividing unequally instead of in the spiral pattern of other main factors on which its behaviour depends (Hanlon and
molluscs. It is thought this is a derived mode of develop- Messenger 1996). In the previous sections, the main effec-
ment. After a period of development within the egg, juve- tors or body organs that carry out, or “effect”, the responses
niles hatch out directly without the swimming larval stage an animal makes to a stimulus (e.g. arms, tentacles, suckers,
common to many other molluscs. fins, chromatophores organs, reflecting cells, photophores,
and ink sac) were show. In this section, information about
3.4.2.6 Sensory Systems the sense organs is given. The following one will deal with
Cephalopods (excluding Nautilus) are predatory, agile, the organization of the brain and the peripheral nervous
swift-moving and, except few exceptions, highly visual system, which in these organisms is very important.
animals that can see well under highly varying light condi- Table 3.1 summarizes main information on the sense organs
tions. Nevertheless, cephalopod sensory systems cover a of these peculiar marine molluscs.
30 Á. Guerra
Fig. 3.17 Sepia officinalis male reproductive system; a general view; oe: oesophagus; sp: spermatophores; spgl: spermatophoric gland; spss:
b main parts; an: anus; anf: anal flaps; bh: branchial heart; fun: funnel; spermatophoric sac or Needham’s sac; te: testis; to: terminal organ
flc: funnel-locking cartilage apparatus; g: gill; ins: ink sac; int: intestine; (“penis”)
3.4.2.7 Nervous System (Octopus vulgaris) brain, which is the well-known central
The cephalopod nervous system is divided into a central and nervous system among cephalopods.
a peripheral part. The central part includes the brain proper The peripheral nervous system contains twice as many
and the optic lobes; the large peripheral part includes the nerve cells (350 million) as the central nervous system. All
nervous system of the body and of the arms. peripheral ganglia are lower motor centres. The largest part
The brain is protected by a cartilaginous skull or cephalic of the peripheral nervous system is the five brachial nerve
cartilage. It is arranged around the oesophagus. There is a cords; each has one large axial cord, four much smaller
supra-oesophageal and a sub-oesophageal part, and they are peripheral intramuscular cords, and as many brachial ganglia
laterally connected by a part that may be considered as as the arm has suckers; in addition, each sucker has its own
peri-oesophageal. Each of these parts is further subdivided small sucker ganglion. Most of the neurons in the brachial
into a varying number of lobes. In octopods and decapods, and sucker ganglia are (lower) motor neurons that drive the
25 major lobes have been described; however, some are muscles of the suckers, but they also analyse chemo- and
further subdivided, giving a total number of 37 and 38 lobes, mechanosensory as well as propiosensory inputs.
respectively (Fig. 3.20). It is beyond the aim of this section The most well known of the peripheral ganglia are the
to describe the structures and possible functions of all the 38 stellate ganglia on the inside of the mantle. These ganglia are
brain lobes. For and excellent and comprehensive descrip- lower motor centres for the movement of the mantle. They
tions and reviews, see Nixon and Young (2003). Table 3.2 house the giant synapse and give rise to the stellate nerves.
shows the principal information about the common octopus The giant fibre system is present in Decapodiformes only
Fig. 3.18 Loligo vulgaris male reproductive system; a general view; spermatophoric complex; spo: spermatophoric organ or gland; spss:
b main parts; an: anus; anf: anal flaps; bh: branchial heart; f: fin; fun: spermatophoric sac or Needham’s sac; te: testis; to: terminal organ
funnel; g: gill; ins: ink sac; int: intestine; sp: spermatophores; spc:
and is composed of a chain of three giant nerve cells on each

side. The first-order giant cell lies in the ventral magnocel- 3.5 Post-mortem Examination
lular lobe; its axon runs to the pallio-visceral lobe where it and Recognition of Tissues
crosses to the contralateral side, forming a fusion (Loligo) or Abnormalities
a synapse (Sepia) at the chiasma with its contralateral peer.
From there, the second-order giant cell runs to the stellate 3.5.1 Necropsy and Post-mortem Examination:
ganglion. There, it connects via the giant synapse to the Preliminary Remarks
third-order giant cell (s), which drives the mantle muscula-
ture. The main function of the giant fibre system is fast Although there are some documents on how to necropsy
escape jetting. some species of cephalopods, to date there was no attempt to
Other peripheral ganglia are: the inferior buccal and produce a general guide. The information used to perform
subradular ganglia, which control the buccal and the radula this section has three origins: (i) exiting partial documents
movements; the large gastric ganglion, which innervates the (e.g. dissection technique to Sepia officinalis by A.V Sykes
crop, stomach, caecum, and intestines; the fusiform, cardiac 2016); (ii) information of the excellent atlas on Salmonid
and auricular ganglia, which innervate the vena cava and the diseases by Bruno et al. (2013), and (iii) our own experience.
gill and systemic hearts; and the branchial (or gill) ganglia, Post-mortem examination or necropsy is the procedure of
which innervate the muscles of the gill lamellae. examining a body with the objective of assessing the lesions
32 Á. Guerra
Fig. 3.19 Octopus vulgaris male reproductive system; a general view; spermatophores; spc: spermatophoric complex; spgl: spermatophoric
b main parts; an: anus; bh: branchial heart; bv: blood vessels; d: gland; spsg: spermatophoric sac gland; spss: spermatophoric or
diverticulum of the terminal organ; fun: funnel; g: gill; sp: Needham’s sac; te: testis; to: terminal organ
present and the cause of death. This is achieved through a When it comes to examining a group of individuals
systematic approach and observation of external and internal belonging to a more or less numerous population or group,
structures, organs or tissues, assisted by the collection of records of the number (or the best possible estimation) of
samples for further analysis. affected individuals within the population should be ascer-
Any cephalopod sample can have two origins: wild or tained. This data, along with a detailed history of daily and
cultivated. As the industrial culture of these organisms is total mortality, taking into account the size, sex, maturity
underdeveloped, it is quite unlikely that the sample comes stage, age (when possible), and origin of the stock, will
from a farm. However, the sample could come from indi- allow establishing: (i) the morbidity rate and (ii) the pattern
viduals kept in confinement in the laboratories. In order to of the spread of the disease or abnormality observed.
undertake an appropriate cephalopod health assessment Normally, this preliminary diagnosis made from wild
when the specimen is alive in their habitat, it is very useful to animals or kept in aquaria is not definitive. This emphasizes
have data of the main parameters of the water mass (locality, the need of the necropsy. The description provided in this
water temperature, pH, salinity, and chemical and physical chapter describes the procedures of necropsy with particular
conditions) in which the animal has lived, as well as some reference to obtaining adequate samples of the most com-
characteristics of its habitat. Under farming or rearing con- mon tissues collected for histological examination. This is
ditions, information on changes in the standard cephalopod on the understanding that during necropsy, other samples
behaviour should be noted. Moreover, management practices will also be taken, e.g. for microbiological analysis, as well
as well as diet and feeding response also become of partic- as blood or tissue samples for immunological or molecular
ular relevance. studies, analyses of heavy metals concentrations, or
Table 3.1 Cephalopod sensory organs (modified from Nixon and Young 2003)
Eyes Larger posterior chamber, lens, iris, retina, Rabdomeric type; rabdomera instead Excellent visual ability an
choroid, sclera, and argentea. rod and cone cells in the retina. acuity.
Extraocular eye muscles. Generally a single visual pigment: Polarization sensitivity
Similarity to vertebrate eyes rhodopsin. Most species blind to
colours
Photosensitive Head and mantle Compare sunlight with the one emitted Downwelling
vesicles by their own photophores themselves Camouflage
Statocysts Paired organs in the skull. Direction of gravity and linear Balance and equilibrium.
System 1: Macula–statolith–statoconia acceleration. Infrasound.
System 2: Cresta–cupula Countershading reflexes. Countershading
Rotational acceleration
“Lateral lines” Mechanoreceptors or epidermis lines in Detection of the impact of waves of Detection in the dark
different regions of the body pressure of the surrounding water
Suckers Decapodiformes: Cuttlefishes and squids, Chemoreceptors. Taste, smell, touch, pressure,
with corneal rings Mechanoreceptors. and position of the own body
Octopodiformes: octopuses; no corneal rings Propioreceptors and limbs in space
Olfactory In Coleoidea: a couple of small holes, one Chemoreceptors Smell
organs each side of the head under the eyes and near
the edge of the mantle.
Epithelial structures (rinophores)
Nociceptors In the skin, muscles, and viscera They detect changes at chemical, Pain
thermal, and mechanical level,
associated with cellular damage
statocysts to study of the damaging effects of the impacts of 3.5.3 Euthanasia

artificial sound sources. However, these procedures will not
be covered or discussed in detail in this chapter. The inclusion of “all live cephalopods” in Directive
2010/63/EU that regulates the use of animals for scientific
purposes (European Parliament and Council of the European
3.5.2 Sample Size Union 2010) entails the identification of humane methods
for killing, which is a particular challenge for neuroscience,
The number of specimens sampled for a health assessment as pointed out by the comprehensive paper of Fiorito et al.
will vary according to the objectives of the study. For (2014).
example, certification of freedom of a notifiable disease The first point that should be taking into account is that a
generally follows the guidelines from the Office International general anaesthesia is required for performing surgical pro-
des Epizooties (OIE). cedures followed by recovery. Over the last century, a
To obtain a 95–98% probability of detecting at least one diverse range of substances has been used to induce general
infected cephalopod in a clinically healthy population, this anaesthesia in cephalopods (see Andrews et al. 2013 for a
translates to a minimum of 30 individuals. Conversely, for review). Following Andrews et al. (2013), criteria for
disease investigations, five to ten individuals showing assessment of general anaesthesia in cephalopods include:
abnormal behaviour or the characteristic signs of the con- (1) depression of ventilation and in some cases cessation,
dition will be adequate for necropsy. Despite these specifi- probably accompanied by reduced cardiac activity; (2) de-
cations, many times one will have to work with a single or crease in chromatophore tone (indicative of reduced drive to
few individuals. or from the sub-oesophageal chromatophore lobes); (3) re-
Cuttlefishes, squids, or octopuses removed alive for duced arm activity, tone, and sucker adhesion (particularly
examination should be placed into a smaller container where octopus); (4) loss of normal posture and righting reflex;
further observations can be made before any procedure or (5) reduced or absent response to a noxious stimulus.
the removal of tissues or body fluids. The animal should be The Directive requires that if, for a justified reason (e.g. at
sacrificed (euthanized) by a humane method. the end of project, to obtain tissue for an in vitro study,
34 Á. Guerra
Fig. 3.20 Drawing of reconstruction of the Octopus vulgaris central lobe; lbup: posterior buccal lobe; lbus: superior buccal lobe; lbv:
nervous system (brain) drawn from serial sections; see from the right vertical lobe; lmag: magnocellular lobe; lpv: pallio-visceral lobe; nbr:
side (Young 1971); chpl: chromatophoric lobe; coinb: interbrachial brachial nerve; nlab: labial nerves; nops: superior posterior ophthalmic
commissure; frim: median inferior frontal lobe; frsm: median superior nerves; nol: olfactory nerve; nsp: posterior salivary nerve; nvis: visceral
frontal lobe; gbu: buccal ganglia; gopi: inferior optic ganglia; gsp: nerve; nfun: funnel nerve; pel: lateral pedal lobe; trop: optic tract
posterior salivary ganglia; lbra: brachial lobe; lbum: median buccal
because a humane end point is reached), it is necessary to ending with a final concentration of at least 3.5% in the
kill an animal, it must be done “with the minimum of pain, chamber used for humane killing), possibly enhanced by
suffering, and distress” (Article 6). The methods to euthanize using a chilled solution or with the clove oil active con-
a specimen can be summarized in two main categories: stituent eugenol, followed by immediate mechanical
(1) mechanical methods (cutting between the eyes to destroy destruction of the brain (Andrews et al. 2013).
the brain and/or decapitation); (2) chemical methods—(a) It must be emphasized that the above are initial proposals,
cooling: cool water containing 2% ethanol; (b) magnesium and it is likely that methods will need to be refined, possibly
chloride MgCl2 alone or in combination with eugenol; with the identification of species-specific protocols.
(c) chloroform; and (d) carbon dioxide. All current tech- Annex IV of the Directive includes methods for confir-
niques use immersion in sea water containing the anaesthetic mation of death, and these are discussed in relation to
agent. Time of exposition will depend on the species and cephalopods in the paper of Andrews et al. It should also be
size of the cephalopod. noted that the requirement for humane killing also applies to
Based upon current evidence, the following method is hatchlings. In this last case, killing by direct immersion in
proposed for humanely killing cephalopods such as Sepia fixative would not now be considered acceptable in the EU,
officinalis, Dorytheuthis pealei, Octopus vulgaris, and Ele- although it might be possible to obtain permission to use this
done cirrhosa: at least 15 min immersion in MgCl2 (with a as a method if it could be justified to the National Citizens
rising concentration [optimal rates remain to be determined], Alliance (NCA).
Table 3.2 Number of cells and a summary of main functions of the principal lobes of the Octopus vulgaris brain (modified from Nixon and
Young 2003)
Masses and lobes No of cells Main functions
Analysers of the visual input and regulate visual behaviour
They initiate the programs by which the higher motor centres
Optics lobes 128 940 000 (the basal lobes) produce the various motor activities of the
animal
Centres for simple visual discrimination learning and also involved
in the expression of the various chromatic components
Sub -oesophageal mass Motor centres involves in most action of the animal
Palliovisceral 108 000 Control of muscular activity of the mantle and viscera
Chromatophore 526 000 Management of the chromatophoric’s muscles
Vasomotor 1 307 000 Control of the musculature of blood vessels
Brachial 341 000 Actions of the arms and tentacles
Pedal 241 000 Idem
Supra -oesophageal mass
Vertical 25 066 000 Lobes linked for the management of exploratory actions, learning
Subvertical 810 000 and short and long term memories
Frontal Superior 1 854 000
Inferior 1 085 000 In addition, they are closely related to the functional system
Subfrontal 5 308 000 related to the chemotactile stimuli coming from the suckers
Subpedunculate 144 000 and the mouth, as well as visual stimuli coming from the eyes
Basal
anterior 380 000 Control of posture and movements of head and eyes
dorsal 1 796 000 Control of defence and avoidance strategies
lateral 127 000 Control of the chromatophores and the muscles in the skin
median 245 000 Control of swimming and respiration
Buccal superior 150 000 Motor control of feeding
Peduncle 142 000 Coordinate motor activity, colour changes and ink-injection
Olfactory 136 000 Control of muscles of olfactory organs
Peri-oesophageal mass 581 000 This lobe is particularly involved in defence and fast escape
Magnocellular lobe reactions
The giant fibre system originates there
3.5.4 Necropsy Procedure reference to the relative position of the abnormality or the
sample taken is an essential part of the report. When the
3.5.4.1 External Examination exam of the dorsal region is finished, the same should be
The first operation to be performed is to try to eliminate the done with the ventral one, turning the specimen over.
water that has remained in the specimen’s paleal cavity. The A thorough check of the entire body external surface (which
most convenient is to use an absorbent paper. The specimen should be done under a dissecting microscope a when nec-
should be placed in a tray that prevents further contamina- essary) is anatomical anomalies, such as the integrity of the
tion and which allows performing the work. Above the tray, skin and fins, changes of normal pigmentation for the spe-
and below the animal, an absorbent paper to remove excess cies, loss of arms and/or tentacles, bifurcation of the arms,
water should be placed. It is ideally maintained the specimen anomalies in the copulatory arms, regeneration of arms
at cool temperatures throughout the necropsy, which can be and/or suckers, erosion, ulcers, grossly visible parasites,
achieved by placing ice below the tray. evidence of cuttlebone deformity (Sepia) or muscle atrophy,
The most appropriate is to completely extend (arms and but, above all, tumorous-like conditions in the arms, head,
tentacles included) the specimen in the tray and begin the eyes, and/or the mantle. Keep in mind that in cephalopods it
inspection by its dorsal face, the opposite of where the will not be possible to denote haemorrhages, since their
funnel is located. The mantle, head, and arms should be blood is bluish white. The mouth and the oral cavity should
carefully examined. Any deviation from normality for the be examined recording the presence of any possible, vesi-
species throughout the process of the post-mortem exami- cles, parasites, or abnormality associated with these struc-
nation should be recorded in a notebook. The provision of a tures. Samples of skin and mucous can be taken for
36 Á. Guerra
immediate analysis. Carefully remove a small piece of encysted parasites. As experience increases, all the pro-
approximately 1 cm3 and place immediately in fixative such cedures can be performed using scissors and scalpel.
as 10% buffered formal saline. A gross examination of the The protocol that we recommend for the examination of
eyes should include reference to corneal opacity, cataract, or the internal organs starts by carefully removal each one of
exophthalmia which, although not necessarily pathog- them. After removal, each system should be placed in a Petri
nomonic, can indicate a minor infection or a sign of a more dish, which might be advisable to keep in a refrigerator (4 °C)
serious condition. To remove the eye carefully dissect the for further examination. The most convenient order for the
skin around the orbit using small curved scissors or a scalpel organs removal is as follows: 1°) the reproductive system
until sufficient tissue is available to grip with forceps. Pull (ovary, oviducts, and oviductal glands if there were, in the
the eye forwards in order to expose the associated muscles case of females; testicle, spermatophoric complex, and ter-
and then cut free the entire eye ball, but it is not advisable to minal organ (penis) in males); 2°) respiratory and main parts
try to extract the optic lobes or the ocular nerves, which of the circulatory system (gills, branchial hearts, systemic
should be done when working with the brain. Eye fixation heart, trying to conserve their connections through the asso-
can be made using Carnoy’s solution instead of formalin. ciate blood vessels, as well as the most important vessels
However, when the main interest is parasitological exami- leaving the systemic heart, at least in part of their length). The
nation, this will usually require fixation in 70% alcohol. excretory system is closely linked to these other two systems
For the purpose of histological examination, specimens of and is extracted together with them. Again, place these sys-
this small size can be preserved whole, but making a cut in tems in a Petri dish and keep it in a refrigerator (4 °C) for
the ventral region that allows the fixative to penetrate within further examination; 3°) the digestive system (oesophagus,
the mantle cavity and guarantee proper fixation. For larger posterior salivary glands, crop if present, stomach, caecum,
specimens, fresh samples of tissues (mainly skin ones) or digestive gland duct appendages, intestine and anus). If food
body fluids can also be taken for initial in situ analysis, but remains are found in any part of the digestive system, it is
for histological examination tissues need to be dissected convenient to remove them. The digestive gland and the ink
using scalpel, scissors, and forceps. If the animal has a rigid sac are part of this system. As they break with relative ease,
internal shell (cuttlebone or gladius), it is not convenient to what we recommend to avoid its rupture is to keep them
remove it, because that preserves the rigidity of the body and together with the other parts of the system, and, once in the
the internal organs in their natural position. Petri dish or in an appropriate tray, remove them carefully
avoiding its rupture; 4°) the buccal mass (lips, two mandibles
3.5.4.2 Internal Examination or beak, anterior salivary glands, buccal muscles, and radula)
To access the internal organs the mantle or paleal cavity is is relatively easy to extract using scissors and scalpel. Gen-
opened. There are several ways to approach dissection, erally, when the buccal bulb is removed, fragments of the
however, the choice must prevent or reduce the likelihood of oesophagus remain. After these manipulations, the cuttlebone
the process introducing artefacts, damaging tissues, com- or the gladius can be removed.
pressing, cutting, moving, or displacing organs as well as Complete removal of the viscera is practical for easier
avoiding the risk of contamination. Place the animal with the assessment under a dissecting microscope, e.g. for para-
ventral side facing towards you; gently press the both lateral sitological analysis, however inappropriate, if aseptic
sides of the mantle to open a cavity; then, use the scissors microbiological samples are required. For tissue sampling
with the blunted side facing the inside of the mantle to and depending of necessities, whole organs may be fixed
perform a cut along the mid-ventral line of the mantle that from small individuals (e.g. the entire gastrointestinal tract);
reaches its end the cut can be also performed laterally (as conversely, from larger specimens portions of *1 cm3 of
shown in Fig. 14 to Sepia officinalis and in Fig. 16 to each organ should be removed and fixed. Bouin’s fixative
Octopus vulgaris; then, make two lateral incisions at the end has been recommended for this tissue as gonads in an
point of the first incision, one towards the right and another advanced stage of development can be hard to cut after
towards the left, to expose the internal organs. If during this routine formalin fixation. The digestive may vary in colour
operation a long muscle that holds the mantle internally (it depending of the type of diet, as well as the health status of
occurs in the octopuses) is found, it should be cut carefully the specimen. A sample should be collected using a sharp
and lengthwise, preventing damage of any internal organ. scalpel rather than scissors. An examination of the gas-
Exposure of the entire body cavity is required for assessment trointestinal tract, caecum, and associated tissues can be
and allows access to organs for sampling. Once the internal carried out once relevant microbiological sampling has been
organs are in sight, a preliminary and general inspection performed.
should be carried out. Notes on the general appearance of the Finally, the cranium needs to be opened in order to expose
cavity may include references to the extent of tissue growth and examine the brain. One of the most appropriate ways to
or colour changes, swelling, ascites, adhesions, and absence access the brain is: (1) fix the specimen on a dish, dorsal side up;
(2) remove the skin of the head using a sharp scalpel; (3) re- which is also used for communication with predators and
move the eyes, and the paired large optic lobes are located conspecifics. In addition, the muscular systemic heart, the
laterally and the white bodies over each optic lobe; (4) with a branchial hearts, and contractile blood vessels contributed to
horizontal cut, remove the dorsal part of the cranium cartilage a strong blood circulation within a close circulatory system.
until the central part of the brain, a collection of very soft nerve The particularities set out here, which are not the only ones,
tissue that actually surrounds the oesophagus, is visible. This require special knowledge of the morphology and gross
mass corresponds to the supra and sub-oesophageal regions of anatomy of these organisms, when studies on their
the brain; (5) carefully remove the whole brain. The brain pathologies are focused. On the other hand, this type of
deterioration is rapid, and it is, therefore, important to fixing it. knowledge is crucial when considering an adequate protocol
The two most common ways offixing the brain are freezing and for external and internal examination. Also its sacrifice, in
the use of fixative solutions. Each method has its advantages; the case that it would be necessary, must be considered
(6) when all sub-oesophageal brain tissue is totally removed, attending to their particular characteristics.
the statocysts become visible. They are two cavities located at Cephalopods also play an important role in the trophic
the posterior-ventral region of the cranium cartilage. Avoid web of marine ecosystems. The importance of cephalopods
damaging the thin cartilage between the brain and the statocyst as hosts for parasites, which may travel up the food chain to
cavities. When the anterior, lateral, and posterior outlines of the top predators, such as marine mammals and man, has been
statocysts are visible, a block of cartilage containing the two emphasized elsewhere.
statocysts can be cut out of the head. The statocysts can be also Finally, the growing importance of cephalopods in dif-
approaches from the ventral, which is the most appropriate way ferent research’s fields, aquaculture, and human consump-
when operating with anesthetized animals. The tissue should fix tion requires a top priority and wider knowledge of their
for light microscopy or scanning electron microscopy. Sta- health. This knowledge includes detailed information about
toliths are two white opaque structures located within the sta- the cause, mechanisms of development (pathogenesis),
tocysts, which are using for ageing. It should be preserved dried morphological and anatomical alterations, and their clinical
or in ethanol 70%. manifestations.
Maintaining an organized and systematic approach to the
necropsy is an important aspect of the procedure, and careful
observations made during this examination will provide References
valuable information not only immediately, but conse-
quently during the interpretation of the histological sections. Andrews PLR, Darmaillacq A-N, Dennison N, Gleadall IG, Hawkins P,
All tissues samples must be clearly identified with a Messenger JB, Osorio D, Smith VJ, Smith JM (2013) The
reference code when sent for processing to ensure that there identification and management of pain, suffering and distress in
is no risk of incorrect reporting. cephalopods, including anaesthesia, analgesia and humane killing.
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Arkhipkin AI, Rodhouse PGK, Pierce GJ et al (2015) World Squid
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2018
made.
Functional Histology: The Tissues
of Common Coleoid Cephalopods 4
Ramón Anadón
Abstract
The knowledge of the organization of normal tissues and the changes occurring during
physiological or pathological processes is basic to interpret the relationship between structure
and function. There are numerous microscopic studies focused on different cephalopod organs
that are based on the use of high-resolution methods as the transmission electron microscopy.
However, there is no comprehensive basic histological guide to the different tissues in
common species of cephalopods. To fill this gap, we present a careful description of the
normal histological organization of cephalopods. Through 35 plates including 225
photomicrographs and the accompanying text descriptions, this chapter covers the body
systems of three common species of European coleoid cephalopods, the cuttlefish (Sepia
officinalis), the squid (Loligo vulgaris) and the octopus (Octopus vulgaris). The histology of
the three species is presented in parallel, emphasizing those significant between-species
differences. Sections used for study and photomicrographs were mostly stained with
hematoxylin–eosin (H&E), a standard light microscopy method widely accessible for most
laboratories of histology and pathology. Some sections of octopus were stained the Masson’s
trichrome or the periodic acid–Schiff (PAS) methods.

Keywords
Cephalopods Tissues Comparative histology Light microscopy Atlas
resolution methods such as the transmission electron

4.1 Introduction microscopy, which have been comprehensively presented in
a chapter on cephalopods by Budelmann et al. (1997). For
The microscopic anatomy or histology is based on exami-
ultrastructural data, interested readers are directed to this
nation of stained thin sections of the different structures of
chapter, which also includes an exhaustive list of references.
the body. The knowledge of the organization of normal
However, there is no comprehensive histological guide to
tissues and the changes occurring during physiological or
the different tissues in common species of cephalopods using
pathological processes is fundamental to interpret the rela-
standard light microscopy methods available in most labo-
tionship between structure and function. Most textbooks of
ratories of pathology. This chapter covers the “normal”
histology are dedicated to human or mammalian tissues, and
histological organization of three representative coastal
the scant books existing on comparative histology generally
species of Coleoidea. The species studied here are two
do not mention cephalopod tissues at all. There are numer-
representatives of decapodiformes, the cuttlefish (Sepia
ous detailed microscopic studies focused on different
officinalis; Sepiida) and the squid (Loligo vulgaris; Teuthida)
cephalopod organs that are based in the use of high-
and a representative of the octopodiformes, the common
R. Anadón (&) octopus (Octopus vulgaris).
Department of Functional Biology, University of Santiago de The anatomy of the cephalopods has been studied for
Compostela, Campus Vida, Santiago de Compostela, Spain centuries (Swammerdam 1737; Cuvier 1817; Owen 1855;

https://doi.org/10.1007/978-3-030-11330-8_4
40 R. Anadón
Isgrove 1909; Williams 1909; Meyer 1913; Chun 1914). In pallial cavity. The skin of the cephalopods consists of a
this chapter, the microscopic anatomy of main systems of the transparent epidermis formed of columnar epithelial cells
three species is summarily presented in parallel, emphasizing and interspersed mucous cells, and a dermal layer of varying
those between-species differences in tissue organization thickness formed of connective tissue that includes a number
when significant. Photomicrographs were taken of histo- of chromatophores, iridophores and reflecting cells. Light
logical sections of four octopus (two adult males and two microscopy of histological sections shows little detail of the
females, one immature), two squid (a male and an immature epithelial cells, but ultrastructural studies reveal complex
female) and three cuttlefish (two adult males and a female). interdigitation of lateral cell surfaces as well as the presence
Previous to histological methods, live specimens were of an apical microvillous layer (see Lee et al. 2014). A thick
deeply anaesthetized by immersion in seawater containing basal lamina anchors the epidermis to the dermal tissue. The
1% MgCl2 and 1% ethanol (10–15 min) and then euthanized epidermis of the pallial cavity mostly shows flattened epi-
in seawater with 3.5% MgCl2 followed by immediate dermal cells and is deeply modified in some regions of the
mechanical destruction of the brain. After dissection of the funnel (see below) where it may form extensive thickened
various organs and structures, fixation was done in Davidson glandular surfaces.
fluid (formalin, alcohol and acetic acid in tap water). Tissue The dermis mostly consists of a connective tissue formed
blocks were embedded in paraffin wax and sectioned on a of fibroblasts and networks of collagen fibers included in
rotary microtome. Dewaxed sections were routinely stained varied amounts of ground substance that exhibit in many
with hematoxylin–eosin (H&E). Some sections of octopus places conspicuous chromatophore organs and also other
were additionally stained using the Masson’s trichrome or types of pigment cells below. The skin of octopus and cut-
the periodic acid–Schiff (PAS)-staining methods. tlefish, mainly dorsally, contains a number of muscle fasci-
cles that allow rapid changes in the animal appearance of
skin papillae and tubercles caused by erector muscles or its
4.2 Skin (Fig. 4.1) disappearance by contraction of depressor muscles. Collagen
fibrils are abundant in the skin and muscles of cephalopods,
The skin of cephalopods has evolved to separate the internal where they form fiber bundles with various orientations.
medium from the seawater but also to provide a quick These fibers are eosinophilic in H&E stains but are better
changing way adapted for intra-specific communication, distinguished from muscle fibers with the Masson’s tri-
camouflage, or prey attraction (Osorio 2014). The skin chrome stain, showing a similar affinity for colorants as
covers the outer surface of the body, showing highly dif- vertebrate collagen fibers. The thickness and appearance of
ferent regional specializations, as in the suckers, and is the dermis vary along the animal surface. In octopus and
continuous with the inner surfaces of the siphon and the cuttlefish, the dermis is thick and endowed of various
Fig. 4.1 Sections of the skin of (a) (b)

octopus arm (a, b) and squid
mantle (c, d). In a, note the
abundant collagen stained in
green and the iridophores in pink.
In c, goblet cells and secreted
mucus appear red-stained. In d,
note muscle fibers inserted at both
poles of the organ. Chr,
chromatophoric organs; Ep,
epidermis; Ir, iridophores
(reflecting cells). a Masson’s
trichrome. b, c H&E stain. Scale
bars a, d 100 µm; b 200 µm;
c 50 µm
(c) (d)
4 Functional Histology: The Tissues of Common Coleoid Cephalopods 41
bundles of muscle fibers whose contraction transforms the leucophores and reflecting cells with varied structural orga-
skin from smooth to papillae or tubercles (papillary erector nizations of reflective structures (see Budelmann et al.
muscles) or the smoothening of the surface (papillary 1997). Recent studies show that reflecting platelets contain
depressor muscles). This, together with fast changes in condensed proteins coded by the reflectin gene family that is
pigmentation in an instant, allows animals to adopting specific to cephalopods (Crookes et al. 2004; DeMartini
flexibly very different appearances for camouflage or com- et al. 2015). The regular arrangement of collagen fibers in
munication (Hanlon 2007). The dermis of the inner pallial the dermis may also contribute to the skin appearance and
surface is thin and mostly lacks pigment cells. reflectivity.
In many species of pelagic cephalopods, but not in the
octopus, squid and cuttlefish species illustrated here, the skin
4.3 Chromatophores may bear a number of photophores, specialized organs that
emit light.
The chromatophore organs are complex pigment-containing
structures that consist of a large elastic sac (sacculus) filled
of pigment that is contained inside of a chromatophoric cell 4.4 Cartilaginous Tissues (Fig. 4.2)
and a crown of radial muscle fibers that join this cell
(Fig. 4.1d). Specialized histological methods and/or trans- Cephalopods use cartilaginous tissues in a few locations of
mission electron microscopy reveal that radial muscle fibers the body (Cole and Hall 2009). Chondroid tissues form a part
are richly innervated by nerve fibers accompanied of glial of buccal bolsters contributing to mechanical properties of
cells, as well as the unsheathing cells around the chro- the buccal mass. In squid, buccal bolsters show an onion-like
matophore cell. In live animals, chromatophore organs organization of connective cells around a pulpous-like con-
exhibit changing size and colors that vary from black-brown nective center. The cerebral ganglia are surrounded by car-
in melanophores to red or yellow in other chromatophores. tilage in some respects similar to that of vertebrates, although
The contraction of radial muscle fibers extends the pigment chondrocytes exhibit thin-branched processes. Isogenic
sacculus and its relaxation concentrated pigment showing a groups of chondrocytes are frequently observed. A dense
small surface. In histological sections of anaesthetized ani- perichondrium consisting of thin fibroblasts included in an
mals, they appear contracted as black organs. The contrac- abundant matrix of collagen fibers surrounds some surfaces
tion and/or relaxation of different types of chromatophoric of cranial cartilages. In the eye, the equatorial and subequa-
organs combined with the reflection of light by other types torial scleral regions exhibit a cartilaginous skeleton that
of pigment cells located below allow the extremely rapid reminds the scleral bones or cartilages found in many ver-
changes of the coloration patterns in the skin of cephalopods tebrate eyes. In squid and cuttlefish, the scleral cartilage
(Packard 1995). A number of videos of cephalopods show- shows a single or a double layer of radial columnar chon-
ing fast changes of skin pigmentation patterns are freely drocytes embedded in homogeneous cartilaginous matrix. In
available in the Web. octopus eyes, the equatorial sclera exhibits a thick cartilage-
In the skin, eyes and other tissues of cephalopods, there like tissue with dense accumulation of very thin and long
are different types of reflecting cells. In H&E-stained his- radial cells that are well-stained with the Masson’s method.
tological sections, the reflecting cells appear to contain These cells are embedded in scarce matrix, and the inner and
yellowish platelets or granules that are mostly unstained outer tissue surfaces are flanked by thin muscle layers (scleral
(Fig. 4.1b), but granules appear brightly stained in red with muscles). Connective tissues in the base of the octopus cra-
Masson’s trichrome method. The internal organization of nium often may show atypical appearances with very thick
reflecting cells has been thoroughly studied with transmis- collagen fibers embedded in an abundant homogeneous
sion electron microscopy. These ultrastructural studies unstained matrix containing fibroblasts and lacking blood
reveal several types of pigment cells named as iridophores, vessels, which make them difficult to classify.
42 R. Anadón
Fig. 4.2 Sections of cartilage and related skeletal tissues. a, b Chon- chondrocytes. f A typical equatorial scleral cartilage-like tissue of the
droid tissue of a buccal bolster of squid showing an onion-like octopus eye showing dense accumulation of thin and long radial cells.
organization of connective cells around a pulpous-like connective The tissue is flanked by thin muscle layers (scleral muscles; outlined
center. c Section of the cranial cartilage of squid. d Detail of processes arrows). i, j Sections through connective tissue in the base of the
of chondrocytes. e Section of the cranial cartilage of octopus showing octopus cranium showing thick collagen fibers in a homogeneous
branched chondrocytes. f Dense perichondrium of a cranial cartilage. matrix. a–d, g–i, H&E stain; e, f, h, j Masson’s trichrome. Scale bars a,
e Equatorial scleral cartilage of the squid with a single layer of f, h, i, j 100 µm; b, g 25 µm; d, e 50 µm; c 200 µm
4.5 The Shell Contractile activity of the mantle provides the water flux for
respiration and the water jerks for rapid escape and swim-
Some cephalopods have a shell that is located within a dorsal ming of the animal, whereas fin contractions mediate “fly-
sac of the mantle. Two types of shell are found in coleoids, ing.” The mantle consists of a complex tubule-like lamina of
calcified and chitinous. In cuttlefish, the shell (cuttlebone) is muscle tissue covered of connective tunics and skin in its
calcareous (aragonite) with a dense dorsal region and a outer and inner surface. In octopus, the mantle muscle
chambered ventral part with pillars and lamellae. A siphuncle mainly consists of inner and outer layers of longitudinal
complex consisting of specialized epithelium below the shell muscle fibers sandwiching a thick central region of circular
allows the regulated filling of shell chambers with gas, con- (transverse) muscle fibers. Thin layers of radial muscle
tributing to the regulation of the cuttlefish buoyancy. For fibers separate bundles of fibers in both the circular and
further anatomical details of the cuttlebone, see Budelmann longitudinal muscles. In the squid mantle, the muscular
et al. (1997) and Checa et al. (2015). In squid, the shell is a region mainly consists of thick bands of circular muscle
chitinous pen (gladius), a feather-shaped plate, included in the fibers alternating with thin circular bands of radial muscle
dorsal epithelial sac and secreted by the shell gland. The squid fibers. These layers are covered by the inner and outer
pen is thickened in the midline forming a rachis. In octopus, connective tunics, and oblique lattices of connective fibers
the shell relicts surrounded by mantle muscles are appreciable are intercalated with muscle fibers. These connective fibers
only at the insertion of retractor muscles of the funnel. fold or expand with mantle contractions to accommodate
strain gradients (Kurth et al. 2014). The organization of the
cuttlefish mantle is similar to that of squid.
4.6 Muscular Organs
Muscular tissues form a major part of the body of cephalo- 4.7.1 Fins
pods, which are highly specialized active marine predators.
The musculature of cephalopods, as in other mollusks, mostly Squid and cuttlefish have muscular fins attached to the lat-
consists of muscle fibers with contractile material organized in eral side of the mantle, which is lacking in octopus.
oblique band or oblique striation, which is hardly visible in Undulations of these fins, together with mantle contractions
normal histological sections as those shown here. These fibers and water jets, contribute actively to the different phases of
are very thin and long cylindrical cells with the contractile swimming. The lateral fins extend along most the mantle in
material forming an external tubular region around a central cuttlefish but only in the apical region in squid. They are
cytoplasmic core that can be mitochondria-poor (“white” attached over the mantle musculature to a region originated
muscle fibers) or mitochondria-rich (“red” muscle cells) during development from the shell sac. The central muscular
(Mommsen et al. 1981). Fibers bear a single elongated region of the fin is covered of skin similar to that covering
nucleus. Ultrastructural and functional descriptions of the the outer mantle surface. The fin musculature forms two
organization of these fibers including the oblique organization opposite bands separated by a connective layer. In each
of contractile filaments, dense bodies and sarcoplasmic band, there are bundles of muscle fibers with longitudinal
reticulum can be found in Budelmann et al. (1997) and (deep region), transverse (superficial) and dorso-ventral
Rosenbluth et al. (2010). These fibers form the basis of the orientation arranged in a regular pattern. An intramuscular
body muscles, which are most often observed in bands or array of crossed connective fibers probably provides support
bundles of parallel fibers with different orientations (see during gentle fin movements (Johnsen and Kier 1993).
below). Based on their biomechanical features, these mus-
cular masses form muscular hydrostats, muscular organs
which lack typical systems of skeletal support (Kier and Smith 4.7.2 The Funnel, the Closure Apparatus
1985). In addition to the muscle fibers with oblique striation and Funnel Organ (Fig. 4.4)
that form most of the cephalopod muscles, thin cross-striated
muscle fibers with myofilaments aligned in register have been The siphon or funnel is a foot-derived muscular organ that
reported in transverse muscles of tentacles of cuttlefish and projects from the margin of the mantle between the ventral
squid and in intrinsic eye muscles (Kier 1985). mantle corners into a conical tube, the siphon that leads
from the mantle cavity to the exterior. The extended funnel
ventral region allows the free entrance of water in the pallial
4.7 The Mantle and Its Muscle Layers cavity between the it and the ventral mantle during pallial
(Fig. 4.3) dilatation, and make the function of a valve (siphonal
valves) during mantle contraction, forcing the flow of water
The mantle forms the muscle walls of the large pallial cavity through the siphon (water jerks). The muscles of the squid
enclosing the gills and the visceral mass. In squid and cut- funnel valves are formed of thick inner and outer bands of
tlefish, lateral extensions joined to the mantle form the fins. longitudinal fibers and circular (transverse) fibers,
44 R. Anadón
Fig. 4.3 Muscle fibers of the

mantle of octopus (a–c) and squid
(d–f). a Panoramic view of a
transversal section showing bun-
dles of longitudinal fibers in the
inner and outer side of the thick
central region with circular muscle
fibers. b Detail of the central
region showing thin layers of
radial fibers separating thick bun-
dles of circular fibers. Note also
the net of collagen fibers (green
stained) among muscle bundles.
c Section through the outer bun-
dles of longitudinal fibers showing
the net of collagen tissue and thin
bands of radial muscle separating
circular bundles. d Outer region of
the squid mantle musculature
showing circular and radial mus-
cles. e Detail of muscle fibers
running in parallel. f Inner region
of the squid mantle musculature
showing bundles of radial, circular
and longitudinal fibers and fascias
of dense connective tissue. cT,
connective tissue; C, circular mus-
cle; iL, inner longitudinal muscle;
oL, outer longitudinal muscle; R,
radial muscle. In all figures except-
ing E, the outer surface is at the
right. a, d–f H&E stain. b, c Mas-
son’s trichrome. Scale bars
a 500 µm; d, f 100 µm; b, c,
e 50 µm
respectively, with thin bands of radial fibers interspersed. contributes to minimize water turbulences and facilitate the
The muscles are covered of connective tunics that are con- dynamic of the ink clouds ejected in the water during escape
tinuous with the perichondrium of the funnel cartilage. In behavior (Derby 2014).
squid and cuttlefish, this funnel cartilage is covered of a thin
skin and forms a socket for the complementary bouton-like
cartilaginous protrusion of the mantle. Thus, the funnel 4.7.3 The Arms and Tentacles (Figs. 4.5 and 4.6)
articular cartilage is part of the paired closure apparatus
joining the siphonal valves and mantle. Cephalopods bear a characteristic crown of highly mobile
The epidermis of the inner surface of the funnel of squid elongated fleshy appendages (eight arms and, in squid and
(and other cephalopods) forms an extensive and very thick cuttlefish, two tentacles). These appendages are arranged
intraepithelial gland. It consists of a superficial layer of small around the mouth and are essential for feeding. These
epithelial cells of cuboid cylindrical appearance that covers a cephalopod appendages, together with the siphon, are
large mass of tall mucous glandular cells that show nuclei in derived from the embryonic foot. The arms are thicker
basal regions and enlarged secretory regions in upper basally and taper toward the tip. Transverse sections of the
regions. The basal cytoplasm of glandular cells is associated arm shaft show a large cylindrical mass of muscle fibers and
with the fibro-vascular tunic of the epithelial gland. It is connective tissue around an axis formed of a ganglionic
thought that the secretion of this gland adds to ink-jets and nerve cord (see below). The large central circular or ovoidal
Fig. 4.4 a Section of the squid

funnel showing the thick muscle
layers and the funnel organ. b,
c Photomicrographs of the funnel
organ showing details of the thick
glandular epithelium. Note the
large mucous cells basally located
and the small non-glandular cells
apically. d Section of the funnel
passing through the funnel
cartilage forming part of the
closure organ. H&E stain. Scale
bars a 500 µm; b 100 µm;
c 50 µm
muscle mass of octopus consists of a number of transverse extension and contraction and are used for prey capture. The
muscular fibers organized in bundles crossed in various shaft shows the axial nerve cord with thick nerve fibers (see
directions oriented more or less radially and extending below) surrounded by a large mass of crossed bands of
peripherally as muscle trabeculae intercalated with bundles transverse muscle fibers, whereas bands of longitudinal
of compact longitudinal muscle fibers arranged in four sec- muscles in the periphery are small and separated by trans-
tors. This central region is surrounded by thinner bands of verse muscle bands. Thin circular and helical layers of
oblique (helical) muscle fibers and longitudinal muscle fibers muscle fibers surround the large central muscular mass.
surrounded by a layer of circular muscle fibers. Trabeculae Interestingly, the transverse and circular muscle fibers of the
of connective tissue accompany bundles of muscles. For tentacular shaft are thin and cross-striated. Unlike
further details of the octopus arm organization, see Kier and oblique-striated fibers, ultrastructural observations reveal
Stella (2007). Along the inner side surface (facing toward that the contractile material is organized in short sarcomeres
the mouth), they bear numerous suckers on thin peduncles with thick myosin myofilaments of only about 1 µm long,
(decapods) or sessile suckers (two alternate rows in octopus). but this is inappreciable with the conventional light micro-
Muscular bands join these stalked of sessile suckers to the scopy methods used here. The functional properties of these
shaft musculature. The shaft is also connected to protective cross-striated fibers differ dramatically from those of
membranes, lateral flaps flanking the sucker region and oblique-striated fibers and exhibit contraction curves about
swimming keels by muscle bands. These lateral membranes ten times faster than the other arm muscles. For additional
contribute to the hydrodynamic properties of squid facili- information on the functional organization of muscles of
tating arm cohesion and forward or backward swimming and cephalopod arms and tentacles, see Kier and Curtin (2002)
steering. During squid swimming, the arms are pressed and Kier (2016).
together and enclose the tentacles. The fourth ventral pair of The suckers are muscular suction cups on arms and ten-
arms of cuttlefish bears prominent lateral flaps. tacles used in feeding and other functions. The arms and
The pair of tentacles found in squid and cuttlefish are suckers grow continuously, and new suckers are added to the
modified 4th pair appendages inserted in a pocket between growing apex. The size of suckers decreases toward the arm
the arm pairs III and IV (the fifth ventral arm of decapods is tip. In octopus, the suckers are symmetrical protrusions and
named as arm IV). Tentacles consist of a long, highly pro- consist of a muscular cup-shaped acetabulum joined to a
tractible shaft ending in a dilation or club armed with suckers flattened distal ring, the infundibulum, which is the surface
and a terminal region. They are able of considerable fast that attaches to the substrate. Suckers are attached to the arm
46 R. Anadón
Fig. 4.5 Transverse sections of a squid arm (a), tentacle (b) and just below the epidermis with cuboid cells. f Detail of the central region
tentacle club (c) showing their different appearances. In a and c, of the acetabulum showing the insertion of pedicular muscles in the
suckers (S) are visible. Note in C the long pedicle joining the suckers to fascia below a thickened epithelium. N, nerves; B, blood vessel.
the club and lateral protective membranes. C, nerve cord; Lm, g Detail of the border between the acetabulum (A) and infundibulum
longitudinal muscle; Tm, transverse muscle. d Detail of the base of the (I) showing the secretory epithelium producing the ring teeth (star) at
acetabulum (A) separated by a groove from the infundibulum (I) of a left, and the sharp transition between skin zones (outlined arrow). Stars
club sucker. e Detail of the acetabulum outer surface showing the in a, c, d, g indicate the ring teeth. H&E stain. Scale bars a, b 1.2 mm;
insertion of radial muscle fibers (R) in the fascia (outlined arrow) that is c 1 mm; d 200 µm; e 50 µm; f, g, 100 µm
Fig. 4.6 a Transverse sections of an octopus arm showing the core infundibulum showing the transition of the papillae to the normal skin
muscles around the nerve cord (C) and the non-stalked base of a sucker with chromatophores. Note the thick dermis traversed by thin muscle
(S). The sucker is sectioned marginally showing the thick muscle walls fiber bundles which are in contrast with thick infundibular muscle wall.
of the acetabulum (A) and infundibulum (I). b Lateral wall of the e Detail of infundibular epithelial papillae covered of a cuticular layer
acetabulum showing the complex fascia between the epithelium and formed of two different types of lamellae. The inset shows a detail of
muscles. Only a very thin cuticle covers the acetabular epithelium, the cuticle showing the relation between apical borders of epithelial
which shows occasional papillae. c Section of the infundibulum cells and the inner lamella with small pegs. Superficial lamellae appear
showing the papillae of the epithelium (outlined arrow). Muscle bands to be seeded out easily (note detached lamellae in c and d). A, H&E
with different orientation (radial, meridional and circular) are also stain. b–e, Masson’s trichrome. Scale bars a 1.2 mm; b 100 µm; c,
appreciable. The acetabulum is at the left low corner. d Border of the d 200 µm; e 50 µm
or tentacle club via a stalk. The attachment is a short fibers are also visible. After Masson’s trichrome staining, a
cylindrical basal region in octopus (sessile suckers), but a network of oblique collagen trabeculae can be observed in
conical and elongated pedicle in squid and cuttlefish suckers this muscle wall extending between the inner and outer
(stalked). connective membranes and accompanying radial fibers. At
Histological sections of octopus suckers show the the junction with the infundibulum, there are sphincters with
acetabulum and infundibulum formed of the thick-walled numerous circular muscle fibers. In the thick muscle wall of
intrinsic sucker musculature, which is separated from other the infundibulum, there are radial, meridional and circular
external tissues by a continuous connective fascia. In the muscle fibers.
acetabulum (sucker chamber), most muscle fibers extend In the acetabulum, a poorly specialized cuboidal epithe-
radially between the outer and inner connective membranes lium bearing scarce small papillae covers the inner connec-
or fasciae, but bundles of meridional (oriented toward the tive layer. Instead, the inner surface of the infundibulum is
communication with the infundibulum) and circular muscle covered by a papillary epidermis consisting of tall epithelial
48 R. Anadón
cells arranged in conical papillae or denticles. The epithelial cuboidal or flattened epithelium directly attached to the
surface is covered of a thick cuticular layer formed of thin connective fascia surrounding sucker muscles, without any
alternate laminae of small pegs and amorphous material, loose connective tissue separating it from the sucker mus-
which exhibit different staining affinities with the Masson’s cles, which is unlike the complex skin and muscle bundles of
trichrome stain. Pegs and amorphous material are secreted the basis of suckers found in octopus. The suckers of dec-
by tall epithelial cells, and the presence of alternate layers apod arms and tentacles are generally asymmetric. In the
suggests a cyclical renewal of the adhesive surface. At the tentacular club, numerous and simple small suckers lacking
rim around the papillary surface of the infundibulum, over distinction between the acetabulum and infundibulum are
the end of the thick muscular wall, the papillae and pegs found adjacent to the large suckers.
disappear and the surface of the epidermis is only covered of
amorphous cuticular material. This cuticle disappears at the
transition with the normal epidermis. The suckers are highly 4.8 The Digestive System (Figs. 4.7, 4.8, 4.9,
specialized for anchoring to a number of substrates and 4.10, 4.11, 4.12, 4.13 and 4.14)
preys. Combined actions of the acetabular and infundibular
muscles together with the properties of the infundibular Cephalopods are highly evolved carnivorous mollusks that
surface provide the basis for the adhesion. For details on the pursue and capture prey as crustaceans, fish or other
structure and adhesion mechanisms of octopus suckers, see cephalopods using arms and tentacles. Their digestive sys-
Tramacere et al. (2013, 2014). Below the acetabulum, a tem (gut) is highly developed and consists of the alimentary
musculo-connective region connects the arm and sucker. canal formed of specialized compartments united by tubular
Numerous muscle bundles of the acetabulo-brachial extrin- parts. In the coleoids studied in this atlas, the alimentary
sic musculature allow the directional extension and retrac- canal starts in a mouth opening in the large buccal mass. It
tion of the sucker. A detailed description of the extrinsic bears two mandibles or beaks and a radula moved by a
acetabulo-brachial muscles of octopus suckers along the complex muscular system and distal parts of salivary glands.
arm, arm muscles, and interbrachial muscles is found in The mouth is surrounded by lips (buccal funnel) and the arm
Guérin (1908). apparatus. The arms capture and present prey to feed.
In squid and cuttlefish arms and tentacles, the stalked A feature of cephalopods is that the esophagus runs through
suckers exhibit striking differences with those of octopus. the brain, so the prey must be reduced to fragments by the
Vertical sections of the sucker show a pedicular muscle that buccal mass before swallowing. Below the buccal mass, the
enters through the central roof of the acetabulum and inserts esophagus allows food to pass to a stomach that is united to
in a thick connective fascia just below the epithelium of the a large digestive gland and a caecum, and a thick short
roof, which is unlike the octopus acetabulum (Guérin 1908). intestine continues the system to the anus. Specialized sali-
The thickened muscle walls of the acetabulum surround this vary glands accompany the esophagus, their conducts
pedicular insertion, being separated by connective tissue. coursing to the buccal mass. Anatomical differences in the
The junction between the infundibulum and acetabulum is digestive system are noted among the three species illus-
wide, unlike in octopus suckers. Moreover, the epithelium of trated here (see Boucaud-Camou and Boucher-Rodoni
the inner surface of the infundibulum secretes a thick hard 1983). For instance, in octopus a crop separates the esoph-
annular material known as sucker ring teeth, which lacks in agus into proximal and distal parts, and in cuttlefish, the
octopus suckers. The ring teeth perform grappling functions digestive gland is paired.
during prey capture. Recent studies reveal that the charac-
teristic and mechanically robust ring teeth are formed of a
new family of proteins named suckerins, and lack chitin. 4.8.1 The Buccal Mass (Buccal Bulb, Pharynx)
Suckerins self-assemble into a unique type of supramolec-
ular protein network mechanically reinforced by b-sheets The buccal mass is a complex structure surrounded by the
and are embedded in an amorphous matrix (Guerette et al. buccal and peristomial membranes or lips that comprises a
2014; Ding et al. 2014; Hiew et al. 2016). In the outer rim of pair of strong, chitinous beaks moved by large muscles, a
the infundibulum, there is a toothed cuticular epithelium of radular apparatus associated with the buccal cavity, the
tall cells covered of cuticular material that is probably related intrinsic mandibular and radular musculature and nerves,
with sensory neurons. It is followed of a region of folded and salivary glands and papilla. For a detailed description of the
thickened epidermis with mucous glandular cells. The complex anatomy of the buccal mass of squid, including
external skin of the sucker is mostly formed of a smooth muscles, jaws, radula and glands, the reader is directed to
Fig. 4.7 MA1. Sections of the

buccal mass of cuttlefish and
octopus. a Section through the
base of a beak wing showing the
thick epithelium of beccublasts
and the growth of the beak
thickness away from this initial
region. b Section of a portion of
the invaginated radular sac of
octopus showing the lamina with
radular teeth. c Detail of a radular
tooth united to the lamina.
d Section showing the tall
odontoblasts sinthesizing radular
structures. e Section of the buccal
mass of an octopus passing
through the salivary papilla and
the large submandibular salivary
gland (star). The arrow points to
the salivary duct and the outlined
arrow to the subradular ganglion.
f Detail of the mucous secretory
epithelium of the submandibular
gland. a, e, f H&E stain; b–
d Masson’s trichrome. Scale bars
a 100 µm; b 0.5 mm; c 200 µm;
d, f 50 µm; e 1.2 mm
Williams (1909). The functional organization of beak mus- histological sections, adult beaks appear frequently broken
cles of octopus was studied by Uyeno and Kier (2005). In or separated from the epithelium. The beaks are formed by
the following, we only describe the histology of some most and lye on a specialized beak-forming epithelium consisting
characteristic structures including beaks, radula and salivary of tall epithelial cells that secrete chitin and proteins, often
glands. called beccublasts (Dilly and Nixon 1976). The beccublasts
are very tall at the basis of the beak and decrease progres-
sively in height toward apical regions, whereas the beak
4.8.2 Beaks plate is thin near the basal border and increases considerably
its thickness toward the rostrum. The jaws grow constantly
The mandibles, beaks, or jaws resemble a parrot’s beak by additions to their margins and to their unexposed sur-
inverted. The upper mandible is longer, straighter, and more faces. Away from the alar border, the sections of the beak
compressed than the lower which is strongly convex and plate (at least in cuttlefish) show two different regions, the
closes outside the upper jaw (Williams 1909). Each mand- distal one that is continuous with that early appearing at the
ible is formed of two wide and transparent soft chitinous basis and an inner one over beccublasts that shows a dif-
basal plates or wings, which extend apically in a progres- ferent staining with general histological methods. A recent
sively much harder tip or rostrum that forms the cutting biochemical study in octopus has shown that the hydration,
edge, where they are generally brownish-black. In hardening and protein composition of the beak changes from
50 R. Anadón
Fig. 4.8 D1. Sections of the

anterior salivary gland of octopus
(a–c) and the posterior salivary
gland of cuttlefish (d–f).
a Panoramic view showing the
branched secretory tubules. b and
c Secretory portions mainly
consist of mucous gland cells that
stain pale blue (b) or pink (c) with
the Masson’s trichrome and the
PAS method, respectively.
d Panoramic view of the cuttlefish
glandular tissue showing its
branched tubular organization.
e Detail of the mucous cells of
glandular tubules. f Section of a
glandular duct showing the tall
epithelium and the thick
musculo-connective wall. a, d–
f H&E; b Masson’s trichrome;
c PAS method. Scale bars
a 1 mm; b, c, e 50 µm; f 100 µm;
d 500 µm
the base to the apex. In soft basal parts, there are proteins cells with apical microvilli, the odontoblasts. These cells are
belonging to the chitin-binding protein family joined to organized in two layers, the outer cells producing the radular
chitin, whereas in harder apical parts appear proteins of the membrane and the bases of the teeth, and the inner ones
highly modular histidine-rich protein family (Tan et al. producing the cusps. The membrane extends laterally in the
2015). Mandibular muscles are inserted on the basement hyaline shield covering the cavity in which the retracted
connective lamina of the beccublast epithelium (Dilly and radula is lodged. The odontoblasts also secrete the hyaline
Nixon 1976). shield. A subradular connective lamina is intercalated
between odontoblasts and muscles. Histological sections of
octopus show some details of the radula as the composite
4.8.3 The Radular Apparatus origin of teeth, the tall cuticle-secreting odontoblasts and the
connective tissue of the subradular membrane. A pair of
The radular apparatus is an eversible buccal structure com- large protractor muscles (bolster rods), together with the
prised of the radula and various associated structures. The retractor muscles, moves the radula away of and toward the
cephalopod radula consists of a long chitinous membrane sac allowing to rake food into the pharynx. The radular
with regularly attached teeth arranged in transverse rows, muscles are innervated by the inferior buccal ganglion
which is better appreciated in dissected and macerated (Young 1971). For further details of the anatomy of the
radulas (Messenger and Young 1999). It is lodged in a radular complex of squid and octopus, the reader is directed
radular canal that emerges from the radular sac located in the to the studies of Williams (1909) and Messenger and Young
median line. Radular teeth are formed by a set of elongate (1999).
Fig. 4.9 D2. Sections of the

posterior salivary (venom) gland
of octopus. a Panoramic view
showing the loose organization of
branched glandular tubules and
striated ducts. b Section showing
the transition of the striated duct
to the glandular tubule (outlined
arrow). Numerous small blood
vessels are also observed (thin
arrows). c Glandular region
showing cells with pale secretion
granules and cells with brightly
stained granules. Masson’s
trichrome. d The PAS method
stains in purple some glandular
cells. e Transverse section of a
striated duct showing the
characteristic “cistern cells” (thin
arrows). f, g Longitudinal
sections of a striated duct (f) and
one side of a larger duct showing
cistern cells (thin arrows) and
nerve fibers into the epithelium
(outlined arrows). In e–g, stars
indicate the duct lumen. a,
b H&E; c, e–g Masson’s
trichrome; d PAS method. Scale
bars a 1 mm; b, c, e–g 50 µm;
d 100 µm
4.8.4 The Salivary Glands (Figs. 4.8 and 4.9) subapical location. In the basal part of glandular tubules,
goblet cells are replaced by shorter glandular cells filled of
Three types of salivary glands secrete different substances in eosinophilic granules in the supranuclear cytoplasm. Sup-
the buccal mass region contributing to the first phases of porting cells are also found among glandular cells. In cut-
feeding including killing preys. These are the submandibular tlefish, the submandibular gland also exhibits goblet cells
gland and the paired anterior and posterior salivary glands. and granular eosinophilic cells, together with supporting
The submandibular gland consists of a series of invagi- cells, but goblet cells do not form a massive layer.
nated glandular tubules separated by connective-vascular
septa that form a rather compact mass around a central canal
lined by the same type of epithelium. In section of octopus 4.8.5 Anterior Salivary Gland
glands stained with H&E, the glandular tubules show dif-
ferentiated apical and basal (terminal) regions. The apical In octopus, the paired anterior salivary glands are located
part consists of a columnar epithelium formed of large just behind the buccal mass. These glands produce abundant
mucous (goblet) glandular cells with basal nuclei and cyto- mucus. In H&E-stained histological sections, the gland
plasm mostly filled of pale granules and, among goblet cells, appears as a compact mass formed of numerous epithelio-
slender supporting epithelial cells with the nucleus in glandular tubules with a thin connective-vascular layer
52 R. Anadón
Fig. 4.10 D3. Transverse

sections of esophagus and
crop. a Section of the squid
esophagus showing the thick
muscle layers surrounding the
folded mucosa. b, c Details of the
muscular wall and the low
prismatic epithelium showing a
thick cuticle-like layer.
d Esophagus of octopus stained
with the Masson’s trichrome.
Note the abundant connective
tissue and scarcity of muscle
fibers in the submucosa and
mucosal folds more complex than
in squid. e Detail of the mucosa of
octopus esophagus showing the
tall cylindrical epithelium and the
thin cuticle. f Section of the crop
of octopus showing the long and
complex folds of mucosa. a–c,
e–f H&E stain. Scale bars A,
0.5 mm; b, d 200 µm; c,
e 50 µm; f 1.2 mm
surrounding them. Two types of tubules can be distinguished other animals (Ruder et al. 2013). In cuttlefish, the posterior
attending to the appearance of glandular cells. Proximal salivary gland is a massive branched tubular gland with
(toward the ends) parts of tubules show columnar cells with abundant dense connective-vascular tissue among the glan-
basal rounded nuclei surrounded by basophilic cytoplasm dular tubules. Unlike octopus, these glandular tubules show
and apical cytoplasm filled of small basophilic granules. a homogeneous appearance along the gland. The epithelial
Distal tubules are coarser and show a higher glandular wall consists of tall columnar glandular cells with basophilic
epithelium with numerous condensed small nuclei basally basal cytoplasm including the nucleus and a wide apical
and a wide cytoplasm mass filled of eosinophilic granules in region filled of small pale granules, possibly mucous.
which lateral limits between cells are hardly appreciable Among these cells, there are also thin supporting cells with
under light microscopy. The thickest intraglandular ducts condensed nuclei located subapically. The main duct of the
show the same type of glandular epithelium. posterior salivary gland of cuttlefish is lined by a short
columnar non-glandular epithelium with abundant cell
nuclei located at middle heights. This duct is surrounded by
4.8.6 Posterior Salivary Glands (Poisonous abundant circular muscle fibers.
Gland, Venom Gland) In octopus, the posterior salivary gland consists of bran-
ched tubules embedded in a translucent matrix, with two
The posterior salivary glands of coleoids lie dorsal to the types of tubules, secretory (A) and striated (B), which are
mantle cavity behind the cranium. These glands secrete continuous. The spaces among tubules are filled of loose
mixtures of proteins and other substances that are toxic for connective-vascular tissue. The distal A tubules are clearly
Fig. 4.11 Transverse sections of stomach. a–c Sections of squid muscle layers in the thick collagenous fascia joining both sides (d and
stomach showing the muscular wall, the pleated mucosa and the thick e). Note also in d the thick submucosa and the small epithelial folds. f–
cuticle. In c, note long microvillous appendages in the surface of h Details of the differential staining showing the multilayered cuticle.
epithelial cells. d–h Sections through one of the paired central pads of a–c and f H&E staining, d, e, g Masson’s trichrome, h PAS method.
the octopus stomach showing the insertion of the much thickened Scale bars a 1 mm; b 500 µm; c, g, h 50 µm; d 0.7 mm; f 100 µm
54 R. Anadón
Fig. 4.12 Sections of the

caecum of octopus (a–d) and
cuttlefish (e, f). a Panoramic view
of a transverse section showing
the spiral organization of primary
and secondary caecal leafs around
the columella (outlined arrow).
Note the counterclockwise
diminution of the size of leafs and
the alternate arrangement of long
and short lamellae. b Detail of
lamellae of the caecum showing
the lateral leaflets mostly
consisting of high ciliated
epithelium. c Section passing
through the apex of a primary
lamella showing the mucous
tubular glands and the tall
epithelium. d Detail of the base of
a leaf showing muscle fibers
entering in the axis. e Section of
the caecum of cuttlefish showing
leafs with leaflets. f Detail of the
tall ciliated epithelium covering
the leaflets. a, b and e, f H&E; c,
d Masson’s trichrome. Scale bars
a, e 500 µm; b 200 µm; c, d,
f 100 µm
glandular and show at least two types of glandular cells whose striations are better observed in transverse sections of these
cytoplasm appears either bluish (basophilic) or orange tubules. Ultrastructural studies in octopus reveal numerous
(strongly eosinophilic) with the H&E-staining method. basal infoldings of these cells containing mitochondria and
Distal A tubules mostly consist of eosinophilic columnar canaliculi, as well as an apical brush border, which reminds
gland cells that are short or very short in the thin terminal the striated ducts of mammalian salivary glands. Cistern cells
regions, whereas more proximally the epithelium consists of are rather conspicuous in light microscopy sections, because
mixed basophilic and eosinophilic cells. The glandular of their spherical shape, striated cytoplasm and hollow apical
epithelium is continuous with the striated tubules, with a region. These cells are surrounded apically by flattened sup-
sharp transition between them. In the striated tubules, three porting (lining) cells with nuclei located apically. This glan-
types of cell were reported using electron microscopy, striated dular portion is thought to be involved in active ion transport
cells, cistern cells and lumen-lining cells (Matus 1971). The and excretion. In tangential sections to the two types of
striated cells show a wide basal eosinophilic cytoplasm of tubules, it can be appreciated the presence of a layer of flat-
striated appearance with nuclei inside. The cytoplasmic tened thin muscle fibers surrounding the epithelial tubules,

digestive gland of squid (a, b) and
octopus (c–g). a panoramic view
showing branched glandular
tubules open to a large conduct.
b Section showing the complex
appearance of the digestive gland
cells, which appear to form
extensions of cytoplasm toward
the central region. c Panoramic
view showing the two sectors of
the octopus digestive gland, main
and accessory (limit indicated by
arrows). In the low right corner,
the accessory gland tubules are
open to the branched duct of the
gland (asterisks). d–e Portions of
the main digestive gland stained
with Masson’s trichrome showing
the complex appearance of gland
cell cytoplasm, with granules of
various appearances. Note also
the vasculo-connective cords
among acinar units. f Section of
the accessory digestive gland
showing the branched system of
tubules converging on larger
ducts. g Detail of the secretory
units of the accessory digestive
gland showing the complex
star-shaped aspect of the inner
cavity and the homogeneous
appearance of gland cell
cytoplasm. Note the
vasculo-connective cords. a–
c and f H&E; d, e, g Masson’s
trichrome. Scale bars a 0.8 mm;
b, e, g 50 µm; c 1.2 mm;
d 100 µm; f 300 µm
which reminds the myoepithelial cells in mammalian glands. that is accompanied by large vessels. In cuttlefish and squid,
The gland receives rich nerve supply from the superior buccal the esophagus shows a pleated mucosa and submucosa
lobe whose integrity is necessary to poison a crab after surrounded by muscular layers. The epithelium consists of
catching it (Young 1965). Nerve bundles enter the wall of columnar cells covered of a thick cuticle layer. The mucosa
proximal striated tubules and can be observed coursing along lays over a thick submucosa and three muscle layers with
the basal region of the epithelium (Matus 1971). different fiber orientations. The inner muscular layer is thin
and consists of longitudinally oriented muscle fibers,
whereas the outer layer is thicker and consists of circular
4.8.7 Esophagus and Crop (Fig. 4.10) muscle. In octopus, the folds of the wall of esophagus are
long and thick, and its epithelium is much thicker than in
The pharynx is continuous with the esophagus that, at the squid and cuttlefish, showing taller and thinner cells.
level of the cranium, is surrounded by the brain masses, and The octopus esophagus only exhibits a thin cuticular layer.
56 R. Anadón
Fig. 4.14 Transverse sections of

the octopus (a–d) and squid
intestine (e–f). a Panoramic view
showing the folds of the mucosa
including the two pairs of
typhlosoles flanking the bilateral
plane of symmetry (outlined
arrow). b Detail of a fold of the
mucosa showing the rich
vasculo-connective submucosa.
c Detail showing the layers of
circular and longitudinal muscle
fibers between the submucosa and
the outer connective layers. d The
PAS method stains numerous
mucous goblet cells in the
intestine mucosa. e Section of the
squid intestine. Note the lack of
typhlosolis. f Detail of a fold of
the mucosa. Note the scarce
submucosa. a, e, f H&E; b,
c Masson’s trichrome; d PAS.
Scale bars a 0.8 mm; b, d,
f 50 µm; c 100 µm; e 500 µm
The submucosa is thick and the three muscular layers are covered by a very thick chitinous cuticular layer and the
looser arranged than in the other two species. submucosa. Unlike the esophagus, the epithelium of the
In octopus, but not in cuttlefish and squid, a dilated crop squid and cuttlefish stomach shows a tortuous profile and
follows the postcranial esophagus. The crop histology is consists of a single type of cell. The short columnar
similar to that of the octopus esophagus, but the folds of the epithelial cells have their nuclei at intermediate levels and
inner wall are longer and often branched. It is covered of a apical and basal cytoplasm with different appearances. The
similar thick epithelium with thin cuticle, without glandular basal cytoplasm has a striated appearance, which is typical
cells, but the submucosa and muscle layers are thicker than of transporting epithelia. The apical cytoplasm is homoge-
in the esophagus. Thus, the octopus crop forms a very neous and is covered of a conspicuous microvillous-like
expandable part of the esophagus. Peristaltic movements of layer (brush border) and the thick cuticle. These cells secrete
the crop have been reported “in vivo” with ultrasound a chitinous cuticle that is PAS positive and consists of a
sonographic scannings. number of thin lamellae parallel to the epithelium surface.
The submucosa contains both connective and muscular tis-
sues and is continuous with the inner layer of the muscular
4.8.8 Stomach (Fig. 4.11) tunica, which consists of longitudinal muscle fibers. The
thicker outer layer of the muscle tunica consists of circular
The void stomach of squid and cuttlefish shows a deeply muscle fibers. The outer surface is covered of thin and ple-
folded inner surface, whereas the outer surface is rather ated epithelium that lays over connective tissue. The orga-
smooth. The folds of the stomach wall consist of a mucosa nization of the different layers allows the dilation of the
stomach during feeding and suggests that the chitinous epithelium covers the crests and valleys of leaflets, and also
cuticle is highly flexible. the region of the columella (typhlosole) that lacks leafs.
In octopus, the stomach is a very muscular grinding A large number of goblet cells are scattered in the ciliary
organ, reminding one of the gizzards of a bird (Isgrove epithelium mainly in secondary crests. Similar goblet cells
1909). Its central region consists of paired thick dorsal and are observed in the folds of epithelium lining the columella.
ventral pads formed from outer to inner of a thick muscle The folds of the columella and leafs of the caecum have a
layer and a columnar epithelium covered of a very thick central axis with highly vascularized connective tissue (or
cuticle. The lateral walls of this central region are thinner chorion). Along the elongated apical border of primary folds,
and lack muscle layers but exhibit thick collagenous dense there are rows of simple acinar mucous glands called caecal
connective layers to which the lateral wall muscles are glands that open directly into the lumen. These caecal glands
inserted. Transitional regions join this central part of the are formed by secreting cells with a basal flattened nucleus
stomach with the esophagus and the vestibule. and a light vacuolated cytoplasm. In the columella of the
octopus caecum, there is abundant loose connective tissue,
as well as dense connective tissue surrounding the
4.8.9 Vestibule hepato-pancreatic ducts.
In squid, the caecum is less coiled than in octopus
The vestibule is a dilatable small chamber that forms the link although the appearance of leafs and leaflets is similar. As in
between the stomach, spiral caecum and intestine. The octopus, the thick folded epithelium near the columella
mucosa of the vestibule of cuttlefish consists of tall columnar (typhlosole) contains numerous goblet cells. In cuttlefish,
cells with cilia in the apical surface and long glandular caecal leafs are poorly organized by comparison with those
(goblet) cells interspersed with columnar cells. In octopus, of octopus and squid, and have a thick connective axis. In
the vestibule epithelium is cuticularized toward the stomach cases of heavy infestation by coccidia (Aggregata), caecal
and the cuticle disappears suddenly next to the caecum. folds are thickened and partially disorganized.
4.8.10 The Caecum (Cecum, Accessory Stomach, 4.8.11 The Digestive Gland and Accessory
Spiral Stomach, Caecal Sac) (Fig. 4.12) Digestive Gland (Fig. 4.13)
The caecum has been considered the main absorptive organ. The digestive gland (hepatopancreas, midgut gland) is the
It is a thin-walled large sac spirally coiled around a col- largest gland of cephalopods. The digestive enzymes are
umella that is connected with the stomach, the digestive mainly supplied by the digestive gland in alternating phases
gland and the intestine. The partially digested food passes of synthesis and release. It consists of a highly branched
from the stomach to the caecum and is filtered in it, dis- system of glandular tubules covered of a thick epithelium of
carding gross particles that pass directly to the intestine and uniform appearance. Basal conducts have a prominent
allowing food to enter the digestive glands through the lumen, but most tubules show a reduced lumen. The
hepato-pancreatic ducts. The caecum has a complex internal epithelium mainly consists of large columnar cells with basal
structure that consists of numerous leafs and leaflets covered nuclei, an apical region with a brush border and central
of ciliated epithelium extending from the outer surface and regions with numerous large rounded pale granules (lipid
converging toward the columella without reaching it. In droplets, lysosomes) and protein spherules (“boules”). The
octopus, the outer border of the caecum forms three types of tubules are separated by thin vasculo-connective septa con-
leafs, long (primary), intermediate (secondary) and short. taining some muscle fibers. Electron microscopic studies
Primary leafs alternate with secondary ones, and both types suggest the presence of two main types of cell, basal (re-
of leaf are flanked on both sides by very short unfolded placement) cells and chief (columnar) cells (Budelmann
lamellae allowing a regular organization. The primary and et al. 1997), but this is hardly distinguishable with light
secondary leafs exhibit short lateral leaflets coursing longi- microscopy. Chief cells are polymorphic and may exhibit
tudinally along leafs and giving them a comb-like appear- different appearances during the digestive cycle, with
ance in cross section. The axis of leafs and leaflets is thin and secreting and absorptive periods (see Costa et al. 2014). In
continuous with the tissue of the border of the sac. The some phases of digestion, proteins are secreted as “boules”
leaflets exhibit a clear alternate arrangement in squid and from the apical cytoplasm of the cells in an apocrine way or
cuttlefish, but not in octopus where is irregular and leaflets even the whole cell passes to the lumen. In these phases,
often are opposite. In the octopus caecum, too, there is a basal cells with abundant basophilic material can be easily
clear gradient in the thickness of leafs/leaflets, largely observed. Mitotic cells are found often in the epithelium, to
diminishing toward the tip of the spiral. A thick ciliary replace the tall cells that are probably short-lived.
58 R. Anadón
The histological appearance of the digestive gland varies submucosa, extending along the axis of epithelial folds, only
very considerably from sample to sample, even in individ- occasionally branched, and surrounding the muscular layers.
uals sacrificed at the same time after a meal, which has lead The organization of the muscular wall of the intestine
to conflicting reports on its function. comprises an internal layer of longitudinal muscle and an
Associated with the main digestive gland, the octopus has external layer of circular muscle, better observed in octopus
a different part of the gland that is known as the accessory because of its large intestine. In octopus and cuttlefish, too,
digestive gland (pancreatic appendages). This posterior numerous blood vessels are observed in connective regions.
portion of the digestive gland is located around the digestive The end part of the intestine that opens to the anal pore is
gland (hepato-pancreatic) duct to which opens through wide similar in structure to other intestinal parts.
ducts and shows an appearance very different from the main
glandular region. The main gland duct branches in a number
of wide secondary and tertiary ducts ending in a network of 4.8.13 Ink Sac Complex
closely packed tubules or alveoli with striking angled
luminal profiles. This appearance is produced by the large The ink sac of cephalopods is the most characteristic gland of
variations in the height of the pleated epithelium of adjacent these animals. The ink sac originates from an evagination of
alveoli, which are separated by a thin vasculo-connective the intestinal primordium. The ink sac complex consists of the
lamina. Cell limits are not resolved by light microscopy, and ink gland, a large ink sac or reservoir, and the short ink duct
cell nuclei appear irregularly distributed in the eosinophilic (Girod 1881; Williams 1909). The glandular epithelium con-
cytoplasm. The apical cytoplasm shows numerous pale sists of immature proliferating cells in the inner portion,
round structures that may correspond to lipidic droplets. whereas the outer glandular portion consists of a series of
Ultrastructural observations in octopus reveal cells with a connected chambers lined by a cubical glandular epithelium.
large number of longitudinal folds of the plasma membrane, The gland cells are very active in melanogenesis (Palumbo
which explains the difficulty for seeing cell limits with light 2003). Submicrometric small melanin granules are secreted by
microscopy. Electron microscopy of these cells also reveals gland cells and accumulate as ink in the ink sac, being ejected
numerous mitochondria, small vacuoles and large lipid (ink-jet) on demand. The wall of the squid ink sac is formed of
droplets in cytoplasm lamellae (see Budelmann et al. 1997). an outer sheet of connective tissue, a middle sheet of circular
These cells are in contact with the chyme, from which they and longitudinal muscle fibers and an inner sheet of pavement
absorb different nutrients. In the limit with the main part of epithelium (Williams 1909). The reservoir has an additional
the digestive gland, tubules of the two glands entering in layer situated between the epithelium and the muscular layer.
contact are separated by a thin connective-vascular layer. In This layer is about as thick as the other layers combined and is
cuttlefish and squid, pancreatic appendages are not enclosed formed of iridiocytes which reflect light and give the ink sac its
in the digestive gland or surrounded by a common capsule, silvery appearance (Williams 1909). The ink sac opens into
but tubules arising from the hepato-pancreatic duct are in the anal chamber, being separated by two sphincters, and the
contact with the epithelium of the renal sac. chamber opens into the mantle cavity through the anus (Girod
1881; Williams 1909). The released ink is a complex mixture
receiving also contributions of the funnel organ (Derby 2014).
4.8.12 Intestine (Fig. 4.14)
The intestine shows longitudinal folds protruding into the 4.9 Blood and Circulatory System
lumen. In octopus, but not in squid or cuttlefish, the presence (Figs. 4.15, 4.16, 4.17 and 4.18)
of large paired folds (typhlosoles) in opposite (dorsal and
ventral) regions delineates a bilateral symmetry plane in the Cephalopods have a closed circulatory system with arteries
intestine. The typhlosolis epithelium has tall ciliated cells and veins joined by a capillary bed. Two branchial hearts
with nuclei in basal or intermediate levels, and mucous cells pump blood to the gills (branchial circulation) and one
intercalated among them that secrete neutral and acid systemic heart to the remainder body (systemic circulation),
mucopolysaccharides (proteoglycans). In other intestinal in a double circulation way that reminds the double circu-
folds, the epithelium mainly consists of numerous gland lation (pulmonary and systemic) of land vertebrates. The
cells that secrete neutral and acid mucopolysaccharides blood is a viscous fluid that mainly consists of plasma with a
(proteoglycans) and non-ciliated epithelial cells. Toward the high content in hemocyanin, a copper-containing respiratory
anus, the thickness of the epithelium diminishes, but its protein, and also contains hemocytes (amebocytes). Hemo-
features are similar. This epithelium is presumably involved cyanin and hemocytes are produced in the branchial glands
in absorption. The connective tissue is abundant in the and the white body, respectively.
Fig. 4.15 Blood-forming organs and blood. a Branchial gland of e Section of an octopus vessel showing coagulated plasma containing
cuttlefish showing a dense appearance. b Detail of the branchial gland some hemocytes. f Photomicrograph of live octopus hemocytes
showing cell cords that synthesize hemocyanin. Spaces among cords showing large cells and a small hemocyte. g, h Photomicrographs of
are continuous with blood vessels. c Lobe of the white body of monolayers of fixed hemocytes. a–c, e H&E stain; d Masson’s
cuttlefish. d Detail of the white body of an octopus showing the large trichrome; f Nomarski’s differential interference contrast (DIC); g,
number of blood cells (hemocytes) included in a loose h staining with Hemacolor® (see Castellano-Martínez et al. 2014).
connective-vascular meshwork. Note the vasculo-connective trabecula. Scale bars a 1 mm; b, d 50 µm; c 1.2 mm; e 25 µm; f–h 10 µm
60 R. Anadón

systemic heart of octopus (a–c).
a Section of the heart showing the
very thick myocardium with
crossed orientations of fibers and
the lumenal spaces at right.
b Detail of the superficial layers
of myocardium showing in
cross-orientation superficial
myocardial fiber layers. Note the
epicardium (at the left) and the
dense connective meshwork
surrounding muscle fibers.
c Longitudinal view of
myocardial fibers and
accompanying connective
meshwork. d and f panoramic
views of systemic hearts of squid
(d) and cuttlefish (f) showing the
very thick muscle layers. e Detail
of the squid myocardium.
a–c Masson’s trichrome;
d–f H&E stain. Scale bars
a 0.8 mm 500 µm; b 100 µm;
c, e 50 µm; d, f 1.2 mm
4.9.1 Branchial Gland is also responsible for the eosinophilic staining of the content
of blood vessels with the H&E stain. Capillaries of the
In Coleoids, a pair of branchial glands accompanies the branchial gland join to efferent vessels that are brightly
branchial ligaments through the whole length of the gills. red-stained with H&E.
Each gland is contained in a capsule and is profusely vas-
cularized. Under light microscopy, the branchial gland pre-
sents a dense appearance with numerous cords of basophilic 4.9.2 White Body (Hematopoietic Organ)
cells interspersed with blood-filled capillaries. Studies using
electron microscopy reveal a dense cytoplasm in these cells There is general agreement that blood hemocytes of coleoids
with numerous cisterns of rough endoplasmic reticulum. The are mainly produced in the white bodies. White bodies are
secretory cells of branchial glands synthesize subunits of dense masses of small cells enclosed in sinuses that are
hemocyanin, a copper-containing protein that forms organized in lobes located around the optic nerves between
high-molecular-weight complexes in circulating blood of the optic lobe and the eye orbit. In light microscopic sec-
cephalopods and other mollusks. This cephalopod “he- tions, the white body appears as a very dense mass of small
matopoietic” glands provide the plasma with an cells mostly consisting of a round nucleus. A close inspec-
oxygen-transport protein, the most abundant protein of the tion reveals islands of cells with lighter nuclei (differentiat-
blood (5–10% of blood volume), being responsible for the ing hemocytoblasts) among areas of smaller cells with
blue appearance of oxygenated blood. This abundant protein denser nuclei (mature hemocytes). Numerous mitotic cells
Fig. 4.17 Sections of the branchial heart of octopus (a–d) and squid showing cells with large spherical cytoplasmatic inclusions. e Section of
(e–g). a Section showing the large mass of cells that appears to fill most the branchial heart (H) of a squid at the junction with the branchial
of the heart cavity, surrounded by thin muscle and connective layers arteria (A). Br, gill. f Detail of the junction showing the transition
(outlined arrow). b Detail of the heart wall showing from outer to inner between the thick myocardium (My) to the arterial tunica media (Tm).
the thick coelomic epithelium, the connective (Ct) and muscular g Detail of the cardiac muscle. a, d–g H&E stain; b–c, Masson’s
(M) layers and the inner cell mass. c Detail of a musculo-connective trichrome. Scale bars a 0.8 mm; b, f 200 µm; c 100 µm; d, g 50 µm;
trabecula crossing the inner cell mass. d Detail of the inner cell mass e 1.2 mm
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Fig. 4.18 Transverse sections of

blood vessels of octopus (a–d, f) and
squid (e). a Section of the octopus
aorta showing the thick muscle wall
(tunica media) surrounded by abun-
dant collagen connective tissue (tu-
nica adventitia). b Detail of the
muscle layer showing muscle fibers
surrounded of a dense network of
thin connective fibers. c Section of a
medium-sized arteria showing the
thin tunica intima of endothelial
cells on the elastic lamina stained
pink with the PAS method (outlined
arrow). The thin meshwork around
muscle fibers is also PAS positive.
d Section of a small arteria showing
the elastic lamina separating the
intima and the thin tunica media.
e Section of an efferent branchial
vessel (vena) of squid. f Collapsed
large vena of octopus surrounded by
abundant of collagen fibers but
scarce muscle fibers. The outlined
arrow points to the collapsed lumen.
a, b, f Masson’s trichrome; c, d PAS
method; e H&E. Scale bars
a 500 µm; b 16 µm; c, d, f 50 µm;
e 100 µm
are observed in the white body, indicating it is a highly cephalopods are referred to as leucocytes, amebocytes, or
proliferating organ. As indicated above, respiratory pigments hemocytes. These are small ovoid cells are able to migrate to
are located in the blood plasma, and no cells similar to tissues. In histological sections, they are observed in scarce
vertebrate red blood cells are present in cephalopods. numbers inside blood vessels. These cells are better
demonstrated in fresh hemolymph or in hemolymph smears
stained with Romanovsky-type stains (Wright, Giemsa,
4.9.3 Blood May-Grünwall, Hemacolor, etc.), similar to blood cells of
vertebrates. Flow cytometry, phagocytosis assays and
The blood of coleoid cephalopods is a transparent liquid, enzymatic histochemistry, together with electron micro-
turning blue in color when oxygenated. It is about as viscous scopy, have been employed to study cephalopod hemocytes.
as the vertebrate plasma, and its osmolarity is similar to that The major hemocytes identified in octopus are described as
of the seawater although proportions of Na+ and K+ differ granulocytes and are phagocytic cells (Castellanos-Martínez
from seawater (lower and higher, respectively). It mostly et al. 2014). Small hemocytes are also present. Other authors
contains plasma bearing the respiratory pigment, hemo- have recognized haemoblast-like cells, hyalinocytes and
cyanin, which is mainly synthesized by the branchial glands, granulocytes, hyalinocytes being the most abundant (Tron-
whereas blood is poor in cells. The efficiency of cephalopod cone et al. 2015). A single type of granulocyte population
blood as an oxygen carrier compares poorly with that the with variable internal complexity is described in the blood of
blood of vertebrates. The major circulating blood cells of cuttlefish (Le Pabic et al. 2014).
4.9.4 Systemic and Branchial Hearts system, including the distribution of main arteries and veins,
(Figs. 4.16 and 4.17) has been described in detail many times and the interested
reader is referred to Williams (1909), Isgrove (1909) and
The systemic heart consists of a large thick-walled ventricle Wells (1978). Here, only the histology of arteries and veins
joined to the branchial efferent vessels through a pair of is described. Arteries are easily recognizable in sections by
auricles. Two large arteries, the cephalic and posterior aorta, the organization of walls. Three layers or tunicae are dis-
arise from the ventricle. A lacunar system extends in the tinguishable in the walls of large blood vessels of cephalo-
walls from its inner surface to the periphery. The myo- pods, a thin intima, a thick media and a thin adventitia,
cardium consists of a large mass of cardiomyocytes with analogous to that of large vertebrate vessels. The tunica
abundant sarcoplasm that are united among them by inter- intima consists of small endothelial cells attached to a thick
calary discs in a way analogous to the vertebrate car- basal lamina (inner lamina elastica) that in histological
diomyocytes, as shown in electron microscopic studies sections has a pleated appearance. The conspicuous tunica
(Schipp and Schäfer 1969). Blood capillaries enter through media of large arteries contain numerous circular and lon-
the myocardium. The myocardium of the systemic and gitudinal muscle fibers included in an abundant connective
branchial hearts is innervated by nerves from ganglia of the matrix. The thickness of the media diminishes in smaller
visceral system. Among the neurotransmitters released by arteries. Small arteries usually appear contracted in sections.
cardiac nerve fibers are catecholamines or cardioactive Veins resemble arteries but generally have thin walls lacking
peptides of the FMRFamide family. The heart is enclosed in an inner lamina elastica and showing often dilated lumen
a pericardial cavity that is closely related to the renal system filled of blood (Figs. 4.18e–f and 4.22f). Contractile veins as
(see below). branchial efferents and large veins show a thin tunica media.
In coleoid cephalopods, a pair of branchial hearts that lies The cephalopod organs are richly supplied of a number of
in the gill ligament pumps blood to the gills via the afferent small vessels forming series of different diameters, which is
branchial vessel to generate part of the circulatory pressure. easily appreciable in sections of nervous tissue (Figs. 4.24b
These hearts are elongated and appear to be filled partially and 4.25e). The smallest (capillaries) are surrounded only by
by a large mass of cells among which numerous blood pericytes enclosing the thin, often discontinuous endothe-
branching spaces and interstices extend, recognizable by lium (see Budelmann et al. 1997). The largest vessels are
their eosinophilic staining. Most abundant cells are large often accompanied of nerves and its walls receive abundant
polygonal or round cells that are grouped in parenchyma- innervation.
like masses. These cells show a round pale nucleus and
abundant cytoplasm including dark, often very large round
lysosomal inclusions in the cytoplasm. These branchial heart 4.10 The Respiratory System (Fig. 4.19)
cells are responsible of the glandular consistency of the heart
and appear involved in catabolic processes eliminating The two feathery gills of dibranchiate cephalopods protrude
hemocyanin and blood debris (Beuerlein and Schipp 1998; within the mantle cavity allowing the pass of the active
Beuerlein et al. 1998). In addition, blood cells enter among respiratory water flow created by mantle movements through
these cells and may attach them. No well-organized vessels their folds (Wells and Wells 1982). The branchial organi-
are observed into this cell mass. The walls of branchial zation is similar in cuttlefish and squid. The gill is sustained
hearts consist of layers of cardiomyocytes similar in struc- by the gill ligament that contains a large afferent vessel
ture to these found in the systemic heart. At the output side, (branchial artery), muscles and a ganglionic nerve cord that
the transition from the heart to the branchial artery is sharp innervates the gill. From this axis, numerous secondary
and the wall of cardiomyocytes is substituted by the thick branchial lamellae or primary filaments extend into the
musculo-connective layer of the arterial wall. cavity and appear as finely meandering (tertiary lamellae)
increasing in this way the exchange surface of the gill. Each
secondary lamella receives by its proximal side collaterals of
4.9.5 Blood Vessels (Fig. 4.18) the afferent vessel that extends between the two flattened
layers of epithelium (respiratory epithelium) that cover the
The vascular system of coleoids is double, closed and almost lamella, and some perpendicular cells (pillar cells) join these
symmetrical. The anatomy of the cephalopod circulatory epithelia. At the distal end of lamellae, blood spaces join to
64 R. Anadón
Fig. 4.19 Panoramic views and

details of sections of gills of squid
(a, b) and cuttlefish (c, d) and
octopus (e, f). Note the similar
appearance of gill primary and
secondary lamellae of squid and
cuttlefish, and the muscular axis
of the gill (star in a). The
branchial gland is also
appreciable in a and c. e,
f Panoramic view and detail of
gill lamellae of octopus. Note the
different branching pattern (e) and
the thickness of the gill
epithelium (f), by comparison
with decapods. a–e H&E stain;
f Masson’s trichrome. Scale bars
a 1 mm; c, e 500 µm; b 50 µm;
d, f 100 µm
coarse vessels that, together, form the efferent branchial appendages, though the gills, pancreatic appendages and
vessel. In octopus, the gills differ dramatically from those of other structures also contribute to excretion and homeostasis
cuttlefish and squid, because the secondary lamellae are of the internal milieu. The renal and pericardial appendages
branched and tertiary lamellae have the appearance of small are gland-like structures protruding in the renal and peri-
trees rather than laminae. The vascularization also differs cardial coelomic sacs, respectively. In octopus, the renal
from that of the other two species (see Young and Vecchione appendages consist of highly branched structures protruding
2002). In octopus, too, the respiratory epithelium is thicker in the renal sac. They are formed of continuous sheets
than in these species. A thinner epithelium appears to favor covered of two layers of cuboid epithelial cells separated by
the gaseous interchange in most active species. The thin blood sinuses derived of the vena cava system. The epithelial
branchial epithelium also allows direct elimination of sub- cells show a central rounded nucleus and abundant cyto-
stances from blood, such as ammonium and other wastes. plasm. Ultrastructural studies reveal the complex organiza-
tion of these cells, with a microvillous apical border and a
lobulated basal region directly contacting blood (see
4.11 The Excretory System (Fig. 4.20) Budelmann et al. 1997), which are inappreciable in con-
ventional light microscopy sections. In octopus, the renal sac
The coleoids have a complex excretory system consisting of is a unique habitat for parasites and the coelomic surface of
different organs with various functions. The proper excretory the renal appendages appears generally colonized by
organs are the renal and pericardial (branchial heart) numerous dicyemids.
Fig. 4.20 a Panoramic view of

the renal appendages of octopus.
b Section showing the renal
appendages associated with the
pericardial surface. c Detail of the
octorpus renal epithelium
showing the luminal surface of
the epithelium facing faintly
stained parasites (dicyemids).
d Renal appendages of cuttlefish.
e Detail of the renal epithelia
showing two different papillar
appearances, lower facing the
renal sac filled of dicyemids
(bottom) and taller in the opposite
surface. f Detail or the complex
pericardial epithelium shown in
b. a, d, e H&E stain; b,
f Masson’s trichrome. Scale bars
a 0.8 mm; b 500 µm; c, e,
f 50 µm; d 200 µm
is covered with connective tissues, and in the equatorial

4.12 The Visual System (Figs. 4.21 and 4.22) region of the eye, there is a lamina of cartilage that shows a
different organization in octopus and cuttlefish/squids
The visual system of coleoid cephalopods is highly devel- (Fig. 4.2). A connective fascia is also present in the poste-
oped. It consists of a pair of big camera-type eyes connected rior pole of the eye except at the exit of the optic nerve
with paired optic lobes (ganglia) through the optic nerve bundles. Several extra-ocular muscles (both rectus and
(Hensen 1865; Young 1971). The parts of the cephalopod oblique muscles) formed of muscle fiber fascicles allow eye
eye are strikingly similar to those of vertebrate eyes, movements with a surprising similarity with those found in
although they differ in its origin and organization, being vertebrate eyes, though its number and organization largely
considered a notable example of convergent evolution. The vary among cephalopod species, unlike in vertebrates.
inner surface of the eye consists of a neural retina that The retina (Fig. 4.21) is a bilayered structure in the three
continues anteriorly with a simple pigment epithelium species illustrated here, with long photoreceptor cells of a
extending to the lens-producing “ciliary papillae.” More single type, with perikarya located in the deep (inner) layer
anteriorly, this epithelium is continuous with that of the iris. and photoreceptor processes extending in the superficial
The iris lines a pupil circular (squid), horizontal (octopus), or (outer) layer. Note that this convention is different than that
irregular in shape (cuttlefish), and is outwardly followed by a used for vertebrate eyes. In the outer layer, photoreceptor
transparent cornea. External eyelids are found in many processes bear two opposite rhabdomeres (formed of long,
cephalopod species as the octopus and cuttlefish. The retina densely packed microvilli perpendicular to the axis of the
66 R. Anadón
Fig. 4.21 Vertical sections through the retina of an octopus (a, b) and segments. The thin arrow in d points to a defined layer of basal
a squid (c, d), showing the layers of distal photoreceptive segments (“epithelial”) cells below the plexiform layer in the squid retina. e and
bearing the rhabdomeres and pigment granules (Ds), the layer of f Detail of the basal membrane (outlined arrows) separating distal and
proximal photoreceptive segments (Ps) and nuclei (N) of photorecep- proximal segments in octopus and squid, respectively. Note capillaries
tors, and the basal plexiform layer with axonal processes of photore- in the basal lamina (thin arrows), and nuclei of supporting cells (sc) that
ceptors and efferent fibers. Note that the distribution of pigment are obscured by the accumulation of pigment granules. On, optic nerve
granules shown here is typical of light-adapted retinas. Outlined arrows (in c). a, b, f Masson’s trichrome; c, d, f H&E stain. Scale bars a,
in b and d point to the basal lamina separating proximal and distal c 200 µm; b, d 100 µm; e, f 50 µm
process bearing the visual pigment or rhodopsin), contacting to that of vertebrate retinas (inverted retina). Inner processes
laterally with rhabdomeres of adjacent photoreceptors. Using of photoreceptor cells give rise to collaterals forming part of
light microscopy, rhabdomeres appear as a fuzzy layer, but an inner plexiform layer before entering in the optic fiber
these structures can be resolved using transmission electron bundles. Note that to observe these collaterals and their
microscopy (Young 1971). This latter technique allows contacts with efferent axons from the optic lobes, it is nec-
differentiating numerous filamentous mitochondria in the essary the use of Golgi methods or transmission electron
axis of the process, a well as pigment granules that cyclically microscopy.
migrate along the photoreceptor segment depending on The numerous optic nerves (bundles) of cephalopods do
light/darkness conditions. In the outer layer, there are also not form a single nerve as in vertebrates, but exit the eye
supporting (glial) cells with somata located basally and a orbit at its posterior pole and course toward the first optic
long apical process extending till and forming part of the center, the optic lobe, in such a way that bundles arising
outer limiting membrane. Numerous blood capillaries are from the dorsal retina, course toward the ventral optic lobe,
observed between the inner and outer retinal layers. The those ventral ones toward the dorsal region of the lobe and
orientation of photoreceptor segments facing toward the the central ones toward the central region of the lobe, i.e.,
light entering through the pupil (direct retina) is the opposite inverting their relative positions along the dorso-ventral and
Fig. 4.22 Sections through anterior structures of cephalopod eyes. section of a papilla showing lens cells with dense cytoplasm forming
a Section of the octopus eye showing the transition between the retina long, thin eosinophilic processes (outlined arrow, star) that form the
and the ciliary region (“ora serrata”-like; outlined arrow). Star lens. e Section showing the irideal angle of a squid eye, with a thick
indicates the equatorial scleral cartilage. b Section of octopus eye iris (outlined arrow) and ciliary papillae (star) below. f Proximal
showing the ciliary papillae (star) and the base of the iris (outlined region of the iris of an octopus showing inner and outer pigment
arrow). c Transverse section of the lens-forming ciliary papillae, the epithelia and a stroma with abundant collagen, small muscular
upper and lower halves giving rise to the outer and inner hemilenses, bundles and blood vessels. g, h The granule-filled iridophores of
respectively. The homogeneous pale material that surrounds papillae octopus iris appear colorless in H&E stains but pink with the
(stars) consists of innumerable cell processes of papillary cells. Note Masson’s trichrome. Scale bars a, b, e 500 µm; c 200 µm;
also abundant blood vessels in the axes of papillae. d Tangential d, f–h 50 µm
rostro-caudal axes (see below). This cross of bundles was nucleus in basal location, large amounts of black pigment in
named optic chasm by Cajal (1917). The bundles consist of the middle and an eosinophilic apical cytoplasm. This
numerous thin axons accompanied of glial cells and blood epithelial layer is continuous with that of supporting cells of
capillaries. the retina. The abrupt change from the neural retina to the
The ciliary proximal part of the retina is a simple ciliary epithelium reminds the ora serrata of vertebrates.
epithelium formed of cuboidal–prismatic cells with the More distally, this epithelial layer transforms toward the lens
68 R. Anadón
forming a numerous series of radial folds covered of char- pairs of ganglia: pallial, pedal, visceral and buccal. In
acteristic epithelial cells and a vasculo-connective axis (cil- coleoid cephalopods, the number of ganglia has increased by
iary papillae and ciliary ring). The shape and size of the growth, condensation and subdivision of ancestral ganglia,
papillary epithelial cells vary from small cells proximally to forming a large mass (“brain”) around the esophagus com-
tall cell with large nucleus and highly basophilic cytoplasm posed of numerous ganglia and lobes. This brain is partially
sometimes with melanic granules that give rise to a thin encased by cartilage (cranial cartilage) and dense connective
process that extends in parallel with those of neighbor cells tissue. Here, only a brief description of selected ganglia,
toward the lens. The accumulation of these long and thin nerve cords and nerves stained with general methods will be
cytoplasmic processes (crystalline processes) gives rise to provided. For further histological details, readers are directed
the layers of the spherical lens, which is formed of two to specialized monographs as that of Young (1971) on the
unequal halves, a small external and a large internal sepa- nervous system of octopus.
rated by a septum. Two series of papillary folds are In octopus, the different brain lobes (Fig. 4.23) exhibit a
responsible of these portions. A muscular ring joins the cortex of cell perikarya that surrounds an extensive neuropil
scleral cartilage to the ciliary ring, its contraction allowing and tract region. Large differences are noted among lobes in
eye accommodation by displacing the lens perpendicularly the number and size of perikarya, which is easily appreciable
to the eye axis (Schaeffel et al. 1999). in sagittal sections of the brain. In some lobes, cords of cells
Externally to the ciliary papillae, the pigment epithelium enter the central neuropil, forming networks. The location,
continues and covers the inner surface of the iris. In octopus, cell size and histological organization allow distinguishing
cuttlefish and squid, the iris is much thickened toward the lobes, although wide intermediate neuropil areas make dif-
pupil and contains a number of large iridophores filled of ficult to trace boundaries between lobes. In the suprae-
reflecting platelets (reflecting tissue, silver layer) in a central sophageal mass of the brain, the vertical lobes are most
location. These platelets are unstained in H&E sections but easily recognizable centers because the large amounts of
appear brightly red-stained with the Masson’s trichrome small granule cells forming columns in the cortex and its
method. Iridocyte platelets contain condensed proteins of the superficial dorsal location. Rostral to it in superficial loca-
reflectin family (Crookes et al. 2004; Andouche et al. 2013; tions, the superior and inferior frontal lobes and the sub-
DeMartini et al. 2015). frontal lobe exhibit different cortical organization. Just
For the organization of statocysts, olfactory organs and rostral to the inferior frontal and subfrontal lobes, the
other sensory structures, readers are referred to Young superior buccal lobe exhibits a characteristic cortex with
(1971), Wells (1978), Budelmann et al. (1997) and Polese large neurons. Coarse labial nerves extend from this lobe
et al. (2015). rostrally. By comparison with these lobes, deeper regions of
the supraesophageal mass appear as poorly delimited
regions, including the subvertical lobe and several regions in
4.13 The Nervous System (Figs. 4.23, 4.24, the basal lobe system.
4.25 and 4.26) The optic lobes (Fig. 4.24) are easily recognizable in the
three species by their lateral location with regard to the brain
4.13.1 Brain and relation with the eye, as ganglia that receive fibers of the
optic nerves. The optic lobe is covered by a layered super-
The nervous system of coleoid cephalopods (squid, cuttlefish ficial region (cortex) that by analogy with deep layers of the
and octopus) is very complex compared to other inverte- vertebrate retina is named retina profunda, and a central
brates, its level of complexity being comparable to those of region that shows a non-laminar organization with islands of
fishes despite their very different designs. The cephalopod neurons intermingled with extensive neuropil areas. The
nervous system supports a number of complex behavioral cortex is three-layered, with two dense bands of small neu-
traits with multiple dedicated systems for memory, learning rons (granules) flanking an elaborated middle layer or
and control of body movements, dynamic camouflage, plexiform layer. Optic nerve fibers end in this plexiform
reproductive behavior and visceral function (Young 1971, layer contacting with neurons of the granular layers. In both
1995; Wells 1978; Huffard 2013). The ganglionic nervous granular layers, some larger neurons can be distinguished
system of cephalopods has evolved from a more generalized among small cells, indicating a complex neuronal organi-
mollusk organization as that observed in present-day gas- zation. Small axonal bundles cross these layers, giving them
tropods. This nervous system is composed of cerebral gan- a columnar appearance. Below the inner granular layer,
glia joined by connectives and commissures with different groups of neurons become arranged in radial columns
Fig. 4.23 a Photomicrograph of a parasagittal section of suprae- central neuropil with glomerular appearance. d Detail of the inferior
sophagic cerebral mass of octopus showing the different appearance of frontal lobe showing its glomerular neuropil. e Subfrontal lobe showing
the main lobes. Rostral is at the left. Ba, Basal lobe; Bs, Superior buccal the small-celled cortex and a central region with numerous fiber
lobe; cb, cerebro-brachial connective; Fi, inferior frontal lobe; Fs, bundles. f Detail of the buccal superior lobe showing large neurons in
superior frontal lobe; Sf, Subfrontal lobe; V, vertical lobe. b Detail of the cortex. g Detail of the magnocellular chromatophoric lobe of the
the vertical lobe showing the cortex with small-celled columns palliovisceral complex showing big and small neuronal perikarya in the
extending cell cords to the central neuropil or medulla. c Detail of cortex. The connective layer covering the lobe is seen at right. a–f H&E
the frontal superior lobe showing the thick small-celled cortex and the stain; f Masson’s trichrome. Scale bars a 1 mm; b–g 200 µm
separated by abundant neuropil. In more central regions of gland that consists of endocrine cells and is innervated by a
the lobe, the cords appear more heterogeneous forming a tract bearing fibers from peptidergic neurons of the posterior
network and exhibiting large neurons. The optic lobes are dorsal basal ganglion. These peptidergic neurons express the
among the most complex centers of the cephalopod central neuropeptides FMRFamide and/or GnRH, which appears to
nervous system. The medial region of the optic lobes lacks inhibit secretion (Di Cosmo and Di Cristo 1998). The
the three-layered cortex and is joined with the basal complex secretion of gonadotropic mitogenic factor by this gland
of the supraesophagic mass via conspicuous optic tracts varies throughout the sexual cycle and is involved in the
coursing in the optic peduncle, which includes small control of gametogenesis, being regulated by the
peduncle and olfactory lobes. In coleoids, the olfactory lobes axo-glandular innervation.
receive sensory information of a peripheral chemosensory Most lobes of the subesophageal mass exhibit a simple
structure located in the skin behind the eye. Closely asso- histological organization with neuronal perikarya segregated
ciated with the optic peduncle, there is a conspicuous optic to the cortex and a massive central region occupied by
70 R. Anadón
Fig. 4.24 Section of the cerebral mass of a squid showing the optic the thick outer neuropil (oneu). Note also the entrance of optic nerve
lobe (OL) and the optic tracts (OT) connecting with other lobes. Note (ON) bundles. c Detail of a cell cord of the medulla (M) with large and
the cortical laminas (CTX) embracing the core or medulla (M) of the small neurons surrounded by neuropil. d–f Sections of the octopus
lobe. Note also the esophagus (E) surrounded by cerebral masses and optic lobe, showing similar organization as in squid, with small
the white body (WB). PL, peduncular lobe. b Detail of the cortex of the differences. Same abbreviations as used for squid. H&E stain. Scale
squid optic lobe showing outer (OG) and inner (IG) granular layers and bars a 1 mm; d 500 µm; b, c, e, f 200 µm
Fig. 4.25 Transverse section of

the nerve cord of club (a, b) and
tentacle (c, d) of a squid. a,
b Panoramic view and detail
showing the mantle of neuron
perikarya that surround the
central neuropil (neu). Glial and
vascular processes are the better
stained structures in the neuropil.
Note the cerebral–tentacular tract
(btt). c, d In the squid tentacle
cord, the cerebro-tentacular tract
is prominent exhibiting numerous
coarse axons. With regard to the
club, the cord neuropil is more
scarce and the perikarya
(N) small. e Section of octopus
arm nerve cord showing the
peripheral mantle of perikarya
and the central neuropil.
Masson’s trichrome stains in
green the vascular net entering the
cord and the connective
surrounding the cord. Scale bars
a, c 200 µm; b, d, e 100 µm
neuropil with glial cells and capillaries. The size of neurons dedicated to chromatophore control (chromatophoric lobes),
in the cortex varies from small to large or very large in the as well as lobes dedicated to skin, vascular and body
different ganglia. Some tracts of thickened fibers can be movements. The histological appearance of the chro-
observed passing through the neuropil. These conspicuous matophoric lobes of octopus is unusual, with a very thick
tracts interconnect the different ganglia. Different nerves cortical layer with very numerous large neurons and a small
connect these ganglia with other structures. The sube- core of cells and neuropil.
sophageal mass of cephalopods has evolved by fusion and
specialization of several ganglia that are present in other
mollusks. The anterior subesophageal mass of octopus gives 4.13.2 Ganglionic Nerve Cords of Arms
rise to the eight thick arm nerves (ten in squid and cuttlefish), and Tentacles (Fig. 4.25)
to large tracts joining the mass with the supraesophageal
lobes (buccal, frontal) and to a large dorsal commissure. The The control of the complex muscular system and coordi-
arm nerves course toward the arms and to some distance of nation of arms and suckers, and the integration of sensory
the brain acquire ganglionic cells and form the axial nerve information, is provided by the brachial nerves and by
cord that extends all along the center of the arms. The middle additional arm ganglionic structures (Young 1971). The
subesophageal mass receives the statocyst nerves. The center of the arms and tentacles contains a ganglionic nerve
subesophageal mass includes several conspicuous lobes cord that extends along the entire length and is continuous
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Fig. 4.26 Sections of nerves of

octopus (a–c) and squid (d–f). a,
b Transverse sections of nerves
passing through the cranial
skeleton showing the close
arrangement and large diameter of
non-myelinated fibers. Star,
cartilage. c, Tangential section of
the large-celled stellate ganglion
(G) of octopus showing the exit
of a stellar nerve (outlined arrow).
d, e Mantle nerves of squid
showing thin axons (d) and a
giant axon (e), respectively. Thin
arrows point to the profile of the
giant axon. f, Branchial nerve
cord with ganglion cells (outlined
arrow). a, c–f H&E stain,
b Masson’s trichrome. Scale bars
a, b, d–f 100 µm; c 200 µm
with the brachial nerve. The cord consists of a cortical oral surface. The nerve cord organization is similar in cut-
region with perikarya mainly located at its lateral surfaces, a tlefish tentacles.
central region of neuropil and bundles of longitudinal fibers
(cerebro-brachial tracts) on the oral and aboral sides. The
cord innervates the complex musculature of the arms and 4.13.3 Nerves, the Stellate Ganglion
suckers through numerous nerves, and also receives sensory and the Giant Fiber System (Fig. 4.26)
fibers. The histological organization varies between arms
and tentacles. In octopus arms, which bear two alternate Numerous nerves innervate the different parts of the
lines of suckers, an alternate concentration of perikarya that cephalopod body. These consist of fibers of different diam-
innervate the suckers is noted along the cord (ganglionic eters distinguishable from other tissues (muscle, connective)
cord). In squid tentacles, the shaft lacks suckers. In the by its characteristic appearance. Some nerves consist of thick
nerve cord, almost a half of the oral side is occupied by fibers, others of thin fibers and some of a mixture of thick
thick fibers of the cerebro-brachial tract; those of the aboral and thin fibers. In the mantle of cuttlefish and squid, nerves
side are smaller and scarcer. The lateral bands of neurons containing a single giant nerve fiber are also recognizable.
and central neuropil are located between tracts. Numerous The stellate ganglion is one of the larger peripheral ganglia
glial cells are also observed. The cord is surrounded by of the coleoid nervous system. By its superficial location in
connective tissue and accompanied of large blood vessels. the mantle, it is an accessible ganglion for experimentation.
In the tentacular club, the nerve cord is displaced toward the In octopus, it consists of a peripheral cortex with numerous
large perikarya and a core neuropil that receives fibers from third-order giant axons in the stellate ganglion. Stellate
the palliovisceral lobe of the subesophageal mass through a nerves control respiratory movements of the mantle, jetting
coarse pallial nerve. The ganglion connects with the mantle and the expansion of the mantle which causes water to be
through about 20 radial stellar nerves. This ganglion provides inhaled (Packard and Trueman 1974).
the motor fibers that innervate the mantle musculature. In
cuttlefish and squid, the processes of some perikarya of this
ganglion fuse to form the third-order giant fibers that inner- 4.14 Reproductive System (Figs. 4.27, 4.28,
vate the mantle in these species. Most motor fibers arising in 4.29, 4.30, 4.31, 4.32, 4.33, 4.34
this ganglion, however, are not gigantic. In cuttlefish and and 4.35)
squid, too, a pair of first-order gigantic neurons receiving
statocyst fibers is situated in the magnocellular lobe of the 4.14.1 Females
subesophagic mass. These cells mediate the fast escape reflex
in a way analogous to that of Mauthner neurons in many The female reproductive system of coleoid cephalopods
fishes. These first-order giant neurons contact second-order consists of an ovary and one (squid and cuttlefish) or two
ganglion neurons located in the palliovisceral lobes that (octopus) oviducts with associated oviducal glands, which
directly innervate some fast retractor muscles or contact the opens in the gonopore to the pallial cavity (Wells 1978).
Fig. 4.27 Sections of ovaries of immature (a–c) and mature (d, e) female forming elongated crests. Octopus follicles are elongated and
octopus. a Section of showing numerous oogonia and early oocytes pear-shaped, so that in sections of the long stalks show small diameter
attached to an ovarian cord. b, c Detail of an oogonia and oocytes in (arrows; most follicles are sectioned transversely). The outlined arrow
early folliculogenesis. Note in b a big nucleolus, and in b and c the points to an obliquely sectioned follicular stalk. e Detail of a
meiotic chromosomes. d Section of a vitellogenic ovary showing vitellogenic follicle. a–c Masson’s trichrome; d, e H&E stain. Scale
abundant yolk inside oocytes and the follicular epithelium infolds bars a 200 µm; b, c 50 µm; d 800 µm; e 100 µm
74 R. Anadón
Fig. 4.28 Sections of ovaries of squid (a–d) and cuttlefish (e–g). deep infolds of the follicular epithelium accompanied of
a Panoramic view of an immature squid ovary showing some vasculo-connective tissue (arrowheads). Note frequent mitotic figures
previtellogenic follicles (arrows) and a number of oocytes in early in the follicular epithelium. e Section of a mature cuttlefish ovary
folliculogenesis. b Detail of a small oogonia (outlined arrow) and showing large vitellogenic follicles (stars) and smaller follicles in
oocytes in early stages of folliculogenesis with a pole of follicular cells previtellogenic stages (arrows). Note the yellow-stained yolk inside the
(arrowheads). Note that oocytes are in meiotic prophase (zygotene– cytoplasm of large oocytes. f Previtellogenic follicle of cuttlefish
pachytene) judging from the nuclear appearance. c Detail of previtel- showing the reticular appearance of follicular infolds. e Detail of the
logenic follicles (thin arrows) and oocytes in folliculogenesis (arrow- follicular infolds of a large vitellogenic follicle. H&E stain. Scale bars
heads) of the same ovary. d Wall of a previtellogenic follicle showing a 0.8 mm; b, d, g 50 µm; c, f 200 µm; e 1.2 mm
Spermatophore receptacles or spermathecae of octopus are 4.14.1.1 The Ovary of Octopus (Fig. 4.27)
part of the oviducal gland, whereas receptacles of cuttlefish The ovary has the appearance of a racemose gland with
and squid females are located in the head. In decapods, a pair numerous ovarian follicles surrounded by the gonadial coe-
of large nidamental glands and accessory nidamental glands lom. A comparison of the ovaria of immature and mature
opens directly in the pallial cavity near the gonopore. These octopus illustrates the huge changes occurring in ovarian
accessory glands lack in octopus. Oviducal and nidamental follicles (Di Cosmo et al. 2001). In sections of an octopus
glands form the egg case that is essential in the reproductive immature ovary sac, a large number of small developing
strategy of cephalopods (Boletzky 1986). follicles can be appreciated. These follicles are pear-shaped,
Fig. 4.29 a, b Longitudinal tangential section of the oviducal gland of e Section passing more centrally showing secretory ducts of the distal
a mature female octopus shows the organization in sectors consisting of portion (arrows) and sections of three spermathecae (outlined arrows).
glandular units of two types, proximal and distal with respect to the f Spermathecae with sperm released from spermatophores waiting for
ovary. Distal units stain brightly red and the proximal units pale pink. egg fertilization. g Detail of the spermatheca tall epithelium with
Note the sharp limit between the units extending in each gland sector attached sperm heads and long sperm tails directed to the lumen
(arrowed) (b). Star, lumen of mucous region. c Detail of the distal (bundles of tails are appreciable in f). h Tangential section of the
glandular epithelium showing gland cells filled of red-stained secretory oviducal gland and oviduct (ov) of an immature female octopus
granules. Note the basal location of cell nuclei. Star, lumen. d Detail of showing sectors of branching tubules that are broadly separated by
proximal secretory units filled of pale mucous granules. Note pale connective stroma. Star, lumen. H&E stain. Scale bars a, e 1.2 mm;
nuclei of cells lining the lumen (arrows) and dense nuclei of gland cells. b 400 µm; f 500 µm; c, d, g 50 µm; h 1 mm
76 R. Anadón
Fig. 4.30 a Section of the nidamental gland of a mature female apical supporting cells (arrow). The outlined arrows in c, d point to the
cuttlefish showing leafs of glandular tissue around a central region thin connective layers separating glandular bands excepting apically.
showing the gland duct. b Detail of the inner region of leafs showing e Section of the nidamental gland duct showing the tall ciliated
two different glandular regions. c Detail of the basal region of the leaf epithelium. Stars in a–e indicate the glandular and duct lumen. f–h,
showing closely apposed parallel bands of tall glandular cells with Panoramic view and details of the accessory nidamental gland of a
cytoplasm filled of red-stained granules and a dense basal nucleus. Note mature female cuttlefish showing numerous glandular tubules filled of
apical non-glandular cells (arrow). d Detail of the apical region of the secretion. H&E stain. Scale bars a, f 500 µm; b, h 200 µm; c–e 50 µm;
leaf showing parallel bands of tall mucous cells with basal nuclei and g 100 µm
Fig. 4.31 a Section of the testis of an immature squid showing the stages of maturation. d Layer of spermatogonia (arrow) in the tubule
compact organization of testicular cords. b Detail of adjacent testicular border. b Cluster of spermatocytes in leptotene (prophase I of meiosis).
cords (tubules) showing a zonal organization with spermatogonia in the f Cluster of spermatocytes I in zygotene. f Cluster of spermatocytes in
outer layers, followed of cysts of spermatocytes and spermatids in pachytene (bottom) and early spermatids (top). h Cluster of spermato-
maturation. c Section of the wall of a testicular tubule of a mature octopus cytes in metaphase I. i Spermatocytes in telophase I (bottom), and
showing cysts of germ cells in different maturation stages, including spermatids with elongating nuclei (top). H&E stain. Scale bars a 1 mm;
mature spermatozoa. d–i Details of spermatogenic cells in different b 40 µm; c 50 µm; i 16 µm, this bar also applies to d–h
with a long stalk continuous by its proximal end with the entering the stalk and a thicker distal region. The oocyte is
septal ingrowths of the ovary wall and protrude in the ovarian covered by an inner follicular epithelium surrounded by a
cavity. The octopus oocytes are flask-shaped with a thin part flattened ovarian epithelium with thin blood vessels that enter
78 R. Anadón
in the stalk between these layers. The nucleus of immature considerably from early vitellogenic stages. Atretic follicles
oocytes is rounded and exhibits a large nucleolus and a can also be observed in the ovary.
granular appearance. In mature octopus, the ovary contains
very large yolk-filled oocytes, which are covered by a thick 4.14.1.3 The Oviduct
follicular epithelium that shows some longitudinal infoldings. The oviduct of female cuttlefish is a duct with folded inner
The number of follicular cells covering each oocyte is enor- surface that consists of a high ciliated epithelium that lies on
mous. These follicular cells synthesize among others the main a thick connective-vascular tissue. The thin and tall epithelial
protein of yolk, vitellogenin and other substances as lipids cells show basal nuclei. Muscular layers surround externally
that form this egg reserve material. With high magnification, a the connective tissue.
number of yolk platelets are appreciable in the otherwise
homogeneous yolky region. Nuclei are very infrequently 4.14.1.4 The Oviducal Gland and Seminal
observed in sections of these big oocytes. Receptacle of Female Octopus
(Fig. 4.29)
4.14.1.2 The Ovary of Squid and Cuttlefish The oviducal gland of mature female octopus is a compact
(Fig. 4.28) ovoid-shaped gland that exhibits two different sectors, an
The pictured sections of squid ovary were from an immature outer sector consisting of highly eosinophilic gland cells and
female. In this ovary, a large number of immature, pre- an inner one with light gland cells, with a clear limit between.
vitellogenic oocytes of several sizes, are observed. Smaller These sectors produce cement (a mucoprotein) and
oocytes only present very flattened follicular cells and may mucopolysaccharides that act as a polymerizer of the cement,
appear to contact other oocytes, whereas in larger oocytes a to form an egg string for its fixation to appropriate substrates.
thicker follicular epithelium was clearly appreciable. Only a The gland appears organized in compact lobes separated by
few oocytes show infolds of their surface accompanied of a thin connective septa that extend through both sectors, and it
pleated sheet of follicular cells. In larger follicles, numerous is covered with a thin connective-muscular capsule. The
mitotic figures are appreciable in the follicular epithelium. lobes consist of branched glandular epithelial tubules with
The nucleus of oocytes shows chromatin with granular thick walls and small lumen that are separated by very thin
appearance but not a conspicuous nucleolus. septa, giving the gland a quite compact appearance. The
Sections of ovaries of mature female cuttlefish show tubules branch from the central region of the lobe, where the
ovarian follicles in different stages of development, the most lumen of the tubules is larger. The tubules of the eosinophilic
advanced being in vitellogenic stages. The presence of fol- sector consist of two types of cell, glandular and supporting,
licles in different stages of development indicates that cut- the glandular cells showing the abundant cytoplasm filled of
tlefish females may breed at different times. Small round secretory granules that are highly eosinophilic,
previtellogenic oocytes are surrounded by a layer of follic- whereas the small and condensed nucleus is located basally
ular cells whose height increases with development. In more close to the septa. The supporting cells have larger nuclei
advanced vitellogenic follicles, the follicular epithelium located apically and basal processes coursing among glan-
becomes pleated accommodating in the infolds of the oocyte dular cells, but they are masked by the large amount of
surface, having a meandering appearance. These infolds are secretory granules. The non-eosinophilic sector is also
anastomosed forming a complex network. The follicular formed of closely apposed thick branched epithelial tubules
cells are short and are mostly occupied by a round con- with small lumen. In this sector, the supporting cells show
densed nucleus. In some sections, the large and round oocyte clearly their apical nuclei and the basal processes because the
nucleus can be appreciated with uncondensed chromatin light glandular cells do not mask them. The light gland cells
showing an appearance similar to that of lampbrush chro- exhibit condensed nuclei in the basal region. No continuity
mosomes (uncondensed bivalents), and the cytoplasm between eosinophilic and light tubules was observed in the
becomes slightly basophilic. In large vitellogenic follicles, sections examined. In the central region of the eosinophilic
the follicular cells are tall and intensely basophilic, with a lobes, some short tubules showed regions without gland cells
granular nucleus that occupies the middle of the cell. The covered by ciliated epithelial cells, forming a transition
entire follicle is surrounded by connective tissue (follicular toward the gland ducts that are covered by a tall ciliated
theca), and connective tissue and blood vessels enter in the epithelium with undulated surface. In sections showing the
axis of infoldings of the follicular epithelium. This is unlike ducts, they appear filled of a mass of eosinophilic granules
octopus, where the covers of follicles are inconspicuous. The including condensed nuclei, suggesting that secretion of this
oocyte is filled of eosinophilic yolk that by places appears gland sector is of holocrine type.
formed of rounded platelets. The follicle diameter increased
The seminal receptacle of female octopus consists of the toward the centro-apical region. This organization produces
spermathecae, which are closely associated with the oviducal a characteristic pattern of parallel bands when sections are
gland. They are elongated sacs of smooth surface covered of tangential to the lamina surface. The elongated glandular
a columnar epithelium with nuclei located basally. In mated units may be anastomosed at some points. The covering
females, the epithelium is associated with large numbers of cells are small cells with nuclei located mostly apically in
sperm cells attached through their head, whereas the long the epithelium and long basal processes coursing mostly
sperm tails fill the sac cavity accompanied of numerous among gland cells toward the basis of the epithelium or to
eosinophilic globules. Sperm waits there until oviposition. the lines between adjacent secretory bands. These cells are
Unlike in mature females, the oviducal glands of immature ciliated. Toward the tip of the lamina, the epithelium
octopus show a very different appearance, with wide ducts of becomes progressively thinner, most glandular cells disap-
pleated and branched walls, forming numerous alveolar pear and the epithelium changes to a thin highly ciliated
structures. These ducts exhibit a radial arrangement around the epithelium similar to that covering the gland duct. Areas of
oviduct, and each duct and its lobules are separated from those ciliated epithelium can also be observed toward the base of
adjacent by abundant connective tissues. The ducts are cov- laminas. The outer surface of the gland is formed of
ered of a tall ciliated epithelium, and the walls do not show the connective-vascular layers.
appearance of glandular epithelium. The accessory gland of cuttlefish is a large glandular
structure filled of numerous wide tubular and alveolar
4.14.1.5 Nidamental Glands (Fig. 4.30) structures surrounded with layers of connective and vascular
The nidamental gland of cuttlefish is a large gland of tissue. Tubular parts of small diameter are covered with a
massive appearance formed of a number of laminas simple cuboidal epithelium and have reduced lumen,
extending from the periphery toward the center. The lamina whereas the dilated tubules and alveoli are covered by a
is formed of two closely apposed thick layers of glandular– flattened epithelium. Abundant amorphous secretory mate-
epithelial tissue separated by a thin vasculo-connective rial can be appreciated inside of alveoli and tubules. Among
axial region with an unusual appearance. The glandular tubules and alveoli run thin sheets of connective-vascular
cells are organized in parallel bands partially separated by tissue with numerous blood vessels.
processes of ciliated epithelial cells. In these bands, the In immature female squid, the elongated nidamental gland
gland cell nuclei are displaced toward the limit of the axial shows a large number of closely parallel laminas formed of
sheet or to the limit between adjacent bands, whereas the two apposed cuboid epithelia with a thin connective-vascular
abundant cytoplasm with secretory granules is located lamina between them. At the border of laminas, each layer of
(a) (b) (c)
Fig. 4.32 Sections of the ependyme (coiled male duct) of cuttlefish (a, b) and octopus (c). Note large amounts of spermatozoa and in b, the thick
epithelium covering the trabeculas that are characteristic of cuttlefish and squid. H&E stain. Scale bars a 1 mm; b 50 µm; c 500 µm
80 R. Anadón
Fig. 4.33 a Section of the

spermatophoric gland of a mature
male octopus showing different
parts, including a convoluted
glandular region (1; Section 1), a
thick-walled secretory part (3;
Section 3) and a part of the
proximal vas deferens with a
sperm rope inside (outlined
arrow) abutting Section 1.
b Detail of the ciliated epithelium
of the vas deferens. Star, lumen.
c Detail of the convoluted
glandular epithelium in Section 1.
d, e Detail of glandular epithelia
of Section 3 covering the convex
wall (d) and the thicker outer
region (e). Note the different
appearance of basal regions, with
solid invaginations in d and
smooth in e. The apical epithelial
cells are ciliated, as better shown
in e. Stars, duct lumen.
f Section of the thick glandular
epithelium of the spermatophoric
gland of a cuttlefish, with an
appearance similar to that
octopus. The outlined arrow
points to superficial epithelial
(supporting) cells. g Detail of
basal columns of gland cells that
form most of the epithelium.
H&E stain. Scale bars a 1 mm; d,
e 50 µm; b, c, f 100 µm; g 30 µm
epithelium is continuous with that of the adjacent lamina. The The Testis (Fig. 4.31)
epithelium shows frequent apical mitosis and lacks any In immature males of cuttlefish, the testis is massive and
glandular appearance at this immature stage. formed of closely apposed testicular cords or tubules. The
cords show a large number of germinal cells in different
4.14.1.6 Male Reproductive Organs (Figs. 4.31, phases of maturation. In a mature male octopus, the massive
4.32, 4.33, 4.34 and 4.35) testis consists of a number of thick-walled testicular tubules
The male reproductive system of coleoid cephalopods con- with maturing spermatozoa inside the lumen. The tubules are
sists of a single testis and the gonoduct, which comprises the separated by a thin connective-vascular space. The walls of
proximal deferent canal (male duct, spermiduct), blind-end tubules show a sequence of stages of spermatogenesis from
seminal vesicles (spermatophoric glands) and associated gonial stages outside to spermiogenesis near the lumen.
accessory gland, the distal deferent canal and a dilatation Cysts of cells are easily recognized because they show the
that, in mature animals, contains spermatophores, the same appearance. The outermost cell layer is formed of
Needham’s sac. It opens into the pallial cavity through the spermatogonia attached to a connective membrane that at
gonopore (Mann et al. 1970; Wells 1978). some places look like an epithelium. Mitosis is occasionally
Fig. 4.34 a Transverse section

of the accessory spermatophoric
gland (spermatophoric gland
system II, prostrate) of a mature
male octopus showing numerous
glandular tubules abutting the
central duct. b Detail of the
junction of tubules with the duct
(star). c Section of tubules
showing the secretory cells with
nuclei in the outer region and
supporting cells close to the
lumen (arrow). d Section of the
accessory spermatophoric gland
of cuttlefish showing the
glandular tissue. H&E stain. Scale
bars a 800 µm; b 100 µm; c,
d 50 µm
observed in this layer. In other areas, groups of secondary The spermatozoa leaving the octopus testis pass into the
spermatogonia with fine chromatin are prophasic cells. Pri- proximal vas deferens (Mann et al. 1970). In mature octopus,
mary spermatocytes are numerous occupying next locations. this highly convoluted duct consists of a ciliated epithelium
These cells are characterized by their large size (they are the surrounded by connective tissue. The duct is dilated by the
largest cells in the testis) and characteristic chromatin inside presence of large masses (rope) of spermatozoa and has a
the nucleus that reveals that cells of each group are in the rather smooth inner surface with opposite indentations.
same phase of the long first meiotic prophase (leptotene, Numerous blood vessels enter the axis of the tubular complex.
zygotene, pachytene and diplotene). Some cysts also appear
in metaphase I showing paired chromosomes with a lozenge Spermatophoric Gland I (Seminal Vesicle) (Fig. 4.33)
shape. This is the longest phase of the first meiotic division, This complex part of the spermiduct follows the proximal
and the anaphase I and telophase I are rarely observed. Most vas deferens containing a beaded rope of spermatozoa in
cells over the region of primary spermatocytes are small Fig. 4.33a. It is a convoluted structure in which three sectors
spermatids with small round nuclei, because spermatocytes can be distinguished from proximal to distal (Mann et al.
II and second meiotic division are short-lived stages. Cysts 1970). A proximal region is characterized by the labyrinth
of spermatids changing its nuclear shape (elongating) in formed by its finely pleated walls. Its inner walls consist of a
various stages of transformation to spermatozoa can also be high ciliated epithelium showing two layers of nuclei, those
observed close to the lumen. Clumps of spermatozoa with of ciliated cells in the apical region, and those of the
long filamentous nucleus and tangles of long sperm flagella numerous glandular cells in the basal region of the epithe-
occupy the lumen of the tube. lium. The epithelium is finely pleated and a thin basal
vasculo-connective layer separates its different folds. The
Proximal Vas Deferens and Ependymus (Fig. 4.32) third sector shows a C-shaped lumen because a side of the
In male cuttlefish, the ependymus appears as a tortuous tube wall protrudes toward it. In this third region, the ciliated
dilated tube filled of spermatozoa, which in sections appears glandular epithelium is very thick and shows a striking
to consist of a pale cubic or flattened epithelium over a thin appearance. Differences are noted between the thicker
vasculo-connective lamina. In cuttlefish, the tube exhibits epithelium covering the outer, concave lumen surface and
sparse meandering infolds of the epithelium, which is unlike the thinner convex inner surface. In the outer lumen surface,
the octopus. The large amounts of spermatozoa preclude a a large number of nuclei are distributed through most of the
clear observation of the apical surface of these epithelial cells. epithelium thickness. Several rows of dense spindle-shaped
82 R. Anadón
Fig. 4.35 a Transverse section showing a part of the octopus Need- spermatozoa. g Sections of spermatophores passing through the transi-
ham’s (spermatophoric) sac with spermatophores among extensive wall tion between the rope and the ejaculatory apparatus (outlined arrow) and
folds. b Detail of the simple thick epithelium covering the branched folds through different levels of this apparatus (thin arrows). Note the complex
and the connective-vascular axes. c, d Sections of the “female oriented” spermatophore organization. h Transverse section of the terminal organ
ejection apparatus of octopus spermatophores showing the secretion (penis) of a squid showing a spermatophore (outlined arrow) lodged in
spiral in d. Note the layered organization of the ejaculatory apparatus. one of the longitudinal grooves of the wall. Note skin papillae (thin
e Section of cuttlefish elongated spermatophores in Needham’s sac arrow). i Detail of the covering epithelium and the longitudinal and
showing different regions. f Section passing trout the cuttlefish circular muscle layers. a, c, e–i H&E stain; b, d Masson’s trichrome.
spermatophore “male-oriented” pole containing the coiled rope of Scale bars a 500 µm; b I, 50 µm; c, d, f, g, 100 µm; e, h 200 µm
nuclei are located apically, corresponding to nuclei of cili- of glandular cells in the convex surface of this sector is
ated epithelial cells and apical mucous cells. Other glandular clearly lobular, with basal lobes extending in the loose
cells with larger and less-condensed nuclei are distributed connective submucosa. Deep glandular cells show an eosi-
along most of the thickness. Deep glandular cells appear nophilic cytoplasm but do not show secretory granules. In
grouped forming vertical columns corresponding with the tangential sections through the apical part of the epithelium,
lobes of the basal border of the epithelium. The distribution a mosaic of necks of glandular cells of two types is
appreciable. In some parts of the thick epithelium, there are non-glandular regions, the tall glandular cells disappear,
large cells with large nuclei often showing a conspicuous whereas covering cells form then all the epithelium. The
nucleolus. These cells probably correspond to intraepithelial glandular zone lies over a rich vasculo-connective layer. The
sensory neurons. In the region containing these cells, small folds of the inner wall of the sac in the glandular zone
intraepithelial nerve bundles are also seen coursing hori- consist of long primary and short secondary folds sometimes
zontally near the base of the epithelium. Below the epithe- bifurcated, whereas the folds are short and simple in
lium, there is a layer of loose connective tissue that links the non-glandular regions. The folded region surrounds sper-
major turns of the glandular conduct. matophores that in sections appear as long rods or circles
In mature male cuttlefish, the thick epithelium of the depending on the plane of the section. Surrounding the sac,
spermatophoric sector 3 appears quite similar to that of there is a loose connective wall containing numerous blood
octopus. The lobed appearance of the basal glandular region vessels, but apparently lacking muscular tissue.
is more pronounced than in octopus, and thin connective
laminas ascend among lobes till the subapical region. Spermatophores
Spermatophores of octopus and cuttlefish are very long
Spermatophoric Gland II (Accessory Gland, Prostrate) slender structures bearing large packets of spermatozoa that
(Fig. 4.34) consist of two moieties, one that bears the rope of sperma-
This is a massive gland that in octopus consists of a highly tozoa at one end and the other, the ejaculatory apparatus, in
branched system of thick-walled convoluted glandular the other. The spermatophores of cuttlefish were first
tubules that radiate from a central canal. The gland is sur- described in the seventieth century by Swammerdam, who
rounded by a thin connective capsule, whereas thin laminas represented five different parts including the long anterior
of vasculo-connective tissue extend from it inside the gland flagellum, the coiled ejaculatory apparatus, a vitreous por-
and branch separating glandular tubules. Blood vessels are tion, a white substance and a posterior “perlucida”
numerous. The tubules consist of two types of cell. Most of part. Since spermatophores are very large structures (about
the thickness of the epithelium is formed of glandular cells 2–3 cm long in octopus), histological sections provide
with basal nuclei. Other cells are ciliated and show apical details of parts but make difficult to understand the whole
nuclei located near the central lumen. The ciliated nature of organization. Here, only some details of cuttlefish sper-
these cells is better observed in sagittal sections of tubules, matophores will be presented. In sections through the pos-
since in transverse sections the lumen is narrow or very terior region, the coiled rope of spermatozoa shows a thin
narrow. At the junction of proximal tubules with the gland laminar appearance with thin laminas of eosinophilic
duct, the glandular epithelium diminishes its thickness and material separating turns. The heads of spermatozoa are
transforms in a ciliated cuboidal epithelium. oriented obliquely toward the walls and the mass of tails and
In mature male cuttlefish, the massive accessory gland including materials toward the center and anteriorly. This
consists of tubules with glandular and ciliated epithelial spermatangium is surrounded by a thick slightly basophilic
cells that are similar to those described in octopus. inner tunica and a thin eosinophilic outer tunica. These
tunics extend till the tip of the flagellum surrounding the
Distal Vas Deferens (Transition to the Needham’s Sac) other spermatophore structures. The anterior limit of the
In octopus, this part of the gonoduct consists of a tube with spermatangium is a flask-like structure called the cement
the lumen invaded by long epithelial laminas accompanied body of complex shape that after a constriction continues in
of a vasculo-connective axis. Secondary laminas are a coiled layered structure that stains differentially. It disap-
numerous along the longest primary laminas. The epithe- pears in the flagellum. For further data on spermatophores
lium is formed of cuboidal cells with numerous apical cilia, and the spermatophore reaction, see Mann et al. (1970),
showing a homogeneous appearance. Marian and Domaneschi (2012) and Marian (2012).
Needham’s (Spermatophoric) Sac, Spermatophores and Terminal Organ or Penis

Penis (Fig. 4.35) From the spermatophoric sac, octopus spermatophores enter
The Needham’s pouch or sac of mature male octopus is a singly in the terminal spermatophoric duct consisting of the
wide sac of pleated inner walls covered in most parts of a diverticulum and terminal organ or “penis” (Mann et al.,
thick glandular epithelium that contains the mature sper- 1970). In sections, the duct appears covered with a thick
matophores waiting for breeding. The epithelium consists of epithelium that consists of ciliated and mucous glandular
tall ciliated cells with round nuclei. Nuclei of these cells are cells. In the lumen, the single spermatophore is appreciable.
dense and locate at the basal pole of glandular cells, whereas The epithelium shows small undulations and invaginations.
a granular secretion occupies most of the apical cytoplasm. The epithelial duct is surrounded by connective tissue
At the transition of this glandular epithelium to containing a large number of blood vessels.
84 R. Anadón
4.15 Concluding Remarks Crookes WJ, Ding LL, Huang QL, Kimbell JR, Horwitz J,
McFall-Ngai MJ (2004) Reflectins: the unusual proteins of squid
reflective tissues. Science 303(5655):235–238
The chapter presents a basic histological and functional Cuvier G (1817) Mémoires pour servir á l’histoire et a l’anatomie des
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DeMartini DG, Izumi M, Weaver AT, Pandolfi E, Morse DE (2015)
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Structures, organization, and function of reflectin proteins in
the histological organization of their various functional dynamically tunable reflective cells. J Biol Chem 290:15238–
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uncover them the strange organization of tissues and sys- Derby CC (2014) Cephalopod ink: production, chemistry, functions
and applications. Mar Drugs 12:2700–2730. https://doi.org/10.
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which show both some surprising convergences and most Di Cosmo A, Di Cristo C (1998) Neuropeptidergic control of the optic
often large divergences with the body organization of ver- gland of Octopus vulgaris: FMRF-amide and GnRH immunoreac-
tebrates, including man. tivity. J Comp Neurol 398:1–12
Di Cosmo A, Di Cristo C, Paolucci M (2001) Sex steroid hormone
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Acknowledgements We thank José Manuel Antonio Durán of the female of Octopus vulgaris throughout the annual cycle.
(IIM-CSIC) for his technical assistance in necropsies and tissue pro- J Exp Zool 289:33–47
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changes were made.
Tissues of Paralarvae and Juvenile
Cephalopods 5
Raquel Fernández-Gago, Pilar Molist, and Ramón Anadón
Abstract
Cephalopods have a different development to other molluscs and hatch as modified miniature
adults called larvae, juveniles or, in some octopuses, paralarvae. The terminology used to
describe young cephalopods is varied. In Octopus vulgaris and other members of Octopoda and
Teuthida (squids), hatchlings are called paralarvae. They are planktonic stages that swim actively
and prey on live planktonic organisms while undergoing morphological changes mainly due to
the fast growth of the arms relative to the mantle. Sepia officinalis and other members of the
Sepioidea are called hatchlings until they are a week old. All of the specimens studied in this
chapter are newly hatched. For a better understanding of the correct terminology, we will refer to
the cuttlefish and squid specimens as juveniles and to the O. vulgaris specimens as paralarvae. In
this chapter, we provide a detailed view of the anatomy of premature hatchlings of the squid
(Loligo vulgaris) and the cuttlefish (S. officinalis) and of paralarvae of O. vulgaris. We organize
this into sections of the functional “systems” (e.g. respiratory system, excretory system).

Keywords
Histology Anatomy Squid Cuttlefish Octopus Normal structure
animals while undergoing strong morphological changes

5.1 Introduction mainly due to the fast growth of the arms relative to the mantle
(Iglesias et al. 2007). Hanlon and Messenger (1988) named
Cephalopods have a different development to other molluscs
the early Sepia officinalis stages hatchlings until they are a
and hatch as modified miniature adults called larvae, juveniles
week old. All of the specimens studied in the following
or, in some octopuses, paralarvae (Young and Harman 1988).
chapter are newly hatched. For a better understanding of the
The terminology used to describe young cephalopods is var-
correct terminology, we will refer to the cuttlefish and squid
ied. In Octopus vulgaris, the hatchlings are called paralarvae.
specimens as juveniles and to the O. vulgaris specimens as
They are squid-like, swim actively and prey on live planktonic
paralarvae. In this chapter, a detailed view of the anatomy of
premature hatchlings of squid (Loligo vulgaris) and cuttlefish
R. Fernández-Gago (&) (S. officinalis) and of paralarvae of octopus (O. vulgaris) is
Department of Ecology and Animal Biology, University of Vigo, presented by “systems” (e.g. respiratory system, excretory
system), following a functional approach.
P. Molist
Department of Functional Biology and Health Sciences, 5.2 Skin and the Body Wall (Fig. 5.1)
University of Vigo, Lagoas-Marcosende, Vigo, Spain
e-mail: [email protected] The skin consists of epidermis and dermis. The epidermis of
R. Anadón hatchling octopodiformes, O. vulgaris, and decapodiformes,
Department of Functional Biology, University of Santiago de L. vulgaris and S. officinalis, show differences in morphology.
Compostela, Campus Vida, Santiago de Compostela, Spain
e-mail: [email protected] In octopus paralarvae, the epidermis of the head and arms is

https://doi.org/10.1007/978-3-030-11330-8_5
88 R. Fernández-Gago et al.
(a) (b) (c)
(d) (e)
(f) (g)
Fig. 5.1 Longitudinal section of the skin of octopus mantle (a). dermis; Ep, epidermis; gc, goblet cell; Kt, Kölliker organ tufts; lc,
Kölliker organ detail (b, c). Transversal section of the skin of squid lateral cell; Mu, musculature; Ir, iridophores (reflecting cells); and Sc,
mantle (d). Longitudinal section of the skin of cuttlefish mantle (e). secretory cell. a–b, d–g H&E stain and c Richardson blue. Scale bars:
Sections of the skin of squid arm (f). Detail of epidermal line a, f, g 20 µm; b–c 10 µm; d 30 µm; and e 50 µm
epithelium (g). Cha, chaetoblast; Chr, chromatophoric organs; Ed,
5 Tissues of Paralarvae and Juvenile Cephalopods 89
formed by a simple columnar epithelium (Fig. 5.1a). This survival out of the chorion, and Villanueva (1995) suggested
epithelium is lower than in the mantle. Bi-stratified regions of that light reflection by tufts of Kölliker’s organs could help in
this epithelium can be seen in some areas. Among the the camouflage of the planktonic paralarvae.
epithelial columnar cells, there are two types of secretory cells In early juvenile squid and cuttlefish, the epidermis
(Fig. 5.1a) and a characteristic cell of hatchling cephalopods, (Fig. 5.1d, e) is generally composed of large goblet cells
the Kölliker organs (Fig. 5.1b, c). Boletzky (1973) and with an ovoid basal nucleus, but in the arms and surrounding
Brocco et al. (1974) electron microscopy studies show that eyes, this epithelium is something modified. In the internal
these organs are mainly composed of the epidermis and a region of the arms, the epidermis presents gland cells with
dermal muscular layer. The epidermis consists of numerous eosinophilic secretory granules (Fig. 5.1f). In some regions
follicles with specialized epidermal cells (Fig. 5.1c) produc- of the post-embryonic specimens, it is possible to see the
ing an extracellular fascicle of cannular rodlets (Kölliker’s lines of epidermal cells that have a rectilinear arrangement
tufts; Fig. 3.1b, c) (Boletzky 1973). The follicle is centred in a (Sundermann 1983). These lines are seen in the arms’
specialized basal cell called cystoblast or chaetoblast (Brocco external region epithelium and around the eyes. These epi-
et al. 1974) (Fig. 5.1b, c) that is surrounded by several fol- dermal lines consist of ciliated and accessory cells
licular cells or wall cells (Boletzky 1973) (Fig. 5.1c). The (Fig. 5.1g). Accessory cells are slender and elongated with
chaetoblast has a large nucleus and apical microvilli that dense cytoplasm and a microvillous apical border. This type
penetrate the base of each rodlet (Brocco et al. 1974). The of cell has a small and dark nucleus. The ciliated cells are
lateral follicular cells (Fig. 5.1c) are similar to chaetoblasts very voluminous and have a central nucleus.
except for the lack of microvilli. The muscular apparatus The dermis is a narrow layer below the epidermis, formed
consists of a group of striated muscle fibres allowing evagi- by a network of connective tissue with collagen fibres,
nation and spreading of the bundles. fibroblasts and various amounts of extracellular matrix. In
These organs are widely distributed over the surface of this layer, there are small chromatophores with the same
paralarvae until 30–35 days post-hatching (Boletzky 1973), appearance as in adults. The presence of iridophores, leu-
although they are more abundant in the mantle than in the cophores, photophores and reflecting cells was not observed
head, syphon and arms (Brocco et al. 1974). Kölliker’s organs in octopus and squid hatchling specimens. However, in the
disappear after the first few months of post-embryonic life, so cuttlefish dermis, there are abundant chromatophores and
it is assumed they have a function associated with planktonic iridophores or reflecting cells (Fig. 5.1e).
life. There have been several theories regarding its function:
Naef (1923) proposed that these structures help passive
5.3 Musculature (Fig. 5.2)
planktonic transport in the water column and are a basis for
the formation of more complex structures in juveniles and
The muscular mantle wall in octopus consists of three sets of
adults, Portmann (1933), Boletzky (1973) and Mangold et al.
muscular layers orientated in longitudinal, circular and radial
(1971) hypothesized that these organs help the embryo
directions (Fig. 5.2a). In decapods (Fig. 5.2b, c), the
(a) (b) (c)
Fig. 5.2 Arrangement of muscle fibres in the muscular layer of the layer; Lm, longitudinal muscular layer; Rm, radial muscular layer and
mantle. Transversal sections of octopus (a). Longitudinal sections of radial muscle fibres. a–c Masson’s trichome stain. Scale bars: a 10 µm;
squid (b) and horizontal section of cuttlefish (c) mc, circular muscular b 20 µm; and c 50 µm
situation is strikingly different, with two layers in circular ultrastructure in hatchling L. vulgaris has been described by
and radial orientations (Budelmann et al. 1997). As similarly Hopkins and Boletzky (1994). These authors identified five
observed in adults, muscle fibres are arranged in different kinds of cells: basal cells at the margin in the dorsal and
directions although this arrangement is less significant in ventral regions low cells of two distinct morphologies in the
hatchling cephalopods. Specifically, the longitudinal and dorsal zone and tall cells of two distinct morphologies in the
radial musculature is less developed than in adults (Fig. 5.2). ventral zone. In adults, Bizikov (1987) observed three shell
layers: the inner layer or hypostracum, the ostracum or
medium shell layer and the periostracum or outer shell layer.
5.4 Shell (Fig. 5.3) In larvae, the shell is formed by an eosinophilic homogenous
material without any layer.
The internal cephalopod shells can be classified into chiti-
nous pen or gladius and calcified (cuttlebone).
5.4.2 Calcified Shell (S. officinalis)
5.4.1 Chitinous Pen (L. vulgaris) The sepia cuttlebone is a flat, elongated shell with an oval
form (Budelmann et al. 1997). Its dorsal surface is heavily
The squid exoskeleton is a chitinous pen constituted mostly of calcified, while the ventral surface has a smooth anterior part
b-chitin (Hunt and El Sherief 1990; Yun et al. 2013), which is and a striped posterior part (siphuncular zone). Each
in the dorsal part of the mantle. The loliginid pens are broad siphuncular stripe is the posterior edge of an interseptal
and are almost as long as the mantle (Budelmann et al. 1997). space or shell chamber (Budelmann et al. 1997).
The squid pens contain a central axis or rachis, lateral The cuttlebone develops early in embryogenesis (Fioroni
expansions (vane) and the terminal conus. The shell sac in L. 1990). In hatchling cuttlefish, eight to nine complete shell
vulgaris consists of an invaginated monolayer of ectodermal chambers are present (Boletzky 1983). The septa are more
cells with two distinct zones: primary or ventral and secondary spaced than in adults. The lamellar structure provides an
or dorsal zones (Hopkins and Boletzky 1994; Fig. 5.3a). The indicator of growth because it lays down at fairly regular
main function of this structure is maintaining rigidity about the intervals (Boletzky and Wiedmann 1978).
longitudinal axis of the animal during swimming.
In larval squid, the shell sac extends along the dorsal
midline of the mantle. This sac is a membranous envelope 5.5 The Digestive System (Figs. 5.4, 5.5, 5.6,
that surrounds the gladius, whose epithelium presents dorsal 5.7, 5.8 and 5.9)
and ventral differentiation (Fig. 5.3a, b). In the shell sac, the
dorsal epithelium is characterized by flat cells with an The digestive system of larval and paralarval cephalopods is
elongated nucleus, while the ventral epithelium consists of composed of the alimentary canal and digestive glands
cubic cells with a dark cytoplasm (Fig. 5.3b). The shell sac (anterior and posterior salivary glands, and digestive gland).
(a) (b)
Fig. 5.3 Transversal section of the L. vulgaris pen (a). Longitudinal section of the L. vulgaris pen (b). Ed, dermis; Ep, epidermis; G, gladius; and
Mu, muscular layer. a–b H&E stain. Scale bars: a 20 µm and b 10 µm
(a)
(b)
(c) (d)
Fig. 5.4 Longitudinal section of O. vulgaris lateral lobe (a). Horizontal basophilic cell; Ec, eosinophilic cell; Ll, lateral lobe; and Sc, secretory
section of S. officinalis lateral lobe (b). Detail of O. vulgaris submandibu- cell. a, c, d H&E stain and b Masson’s trichome stain. Scale bars: a,
lar gland (c). Detail of L. vulgaris submandibular gland (d). B, beak; Bc, c 10 µm and b, d 20 µm
(a) (b)
(c) (d) (e)
Fig. 5.5 Transversal section of L. vulgaris oesophagus (a). Transversal gland; eW, external wall; Sto, stomach; and ve, vestibule. a–d H&E stain
section of O. vulgaris crop (b). Transversal section of O. vulgaris and e Masson’s trichome stain. Scale bars: a, d 10 µm; b 20 µm;
stomach and vestibule (c). Detail of O. vulgaris stomach epithelium (d). and c 100 µm
Detail of S. officinalis stomach epithelium (e). Cu, cuticle; Dg, digestive
Different regions are distinguishable in the canal: buccal Between the ascending and the descending branches of the
mass, oesophagus, stomach, caecum and intestine. Func- U, the digestive gland and the posterior salivary glands are
tionally, the digestive system is a complex system that plays visible. All regions of the digestive system are already
an important role in the absorption of essential nutrients formed one day post-hatching. Although the histology of the
(Fernández-Gago et al. 2017). digestive tube varies along its length, the following layers
The digestive system of octopus paralarvae and of squid can be observed from inside out: the mucosa, the submu-
and cuttlefish larvae is a U-shaped tube, as in the adults and cosa, the muscular layer and the serosa.
juveniles of the species. The descending U-branch is in Similarly to adults, the buccal mass is formed by different
dorsal position and consists of the buccal bulb and structures that contribute to the feeding process, namely:
oesophagus. A crop is also found in octopus (Fernández-- mandibles, radula, salivary papilla, submandibular gland and
Gago et al. 2017). The stomach and the caecum are located the secretory ducts of the posterior and anterior salivary
in the curve of the U-shaped tube. The ascending U-branch, glands. The overall anatomy of the different parts of the buccal
which is ventral, is formed by the intestine and the anus. bulb is very similar to adults, although with some histological
(a) (b) (c)
Fig. 5.6 Transversal sections of O. vulgaris caecum (a). Detail of L. goblet cell. a–b H&E stain and c Masson’s trichome stain. Scale bars:
vulgaris caecum epithelium (b). Detail of S. officinalis caecum a, c 25 µm and b 10 µm
epithelium (c). Cg, caecal gland; Ci, cilia; eW, external wall; and gc,
differences. The buccal lateral lobes, which form the food in cuttlefish, the stomach epithelium is formed by cylindrical
passage, are underdeveloped in larvae and paralarvae. cells (Fig. 5.5e). The three species have the apical region of
The food passage is lined by a simple cubic epithelium the cells covered by a thin non-stratified cuticle.
with cubical cells instead of the typical prismatic cells of The caecum of cuttlefish and octopus hatchlings, as in
adults (Fig. 5.4a, b). The glandular epithelia associated with adults, shows a histological differentiation between the
the posterior part of the lateral lobes and jaws are absent at external and internal walls. The external wall contains only
these stages. The submandibular gland consists of a simple primary folds of the mucosa that consist of a pseudostratified
glandular epithelium formed by two types of secretory cells: epithelium, whereas in the internal wall the unfolded mucosa
granular and mucous. In octopus paralarvae, the zonal is formed by a simple epithelium (Fig. 5.6a). The caecum of
arrangement of these two types of cell is characteristic. The juvenile squid presents the differentiated regions of the cil-
granular cells are located in the middle of the epithelium, iated organ and the caecal sac. The ciliated organ has the
while mucous cells are in the lateral regions of the gland same differentiation between the internal and external walls
(Fig. 5.4c). However, in squid, both types of cell appear (Fig. 5.6b), but these are missing in the caecal sac (Bidder
mixed (Fig. 5.4d). 1950). No secretory cells have been found in the ciliated
The oesophagus is a muscular tube formed by a mucosa organ of the squid, unlike in the adults. The only gland cells
with folds or villi of different sizes. Unlike in adults, it is present in the caecum of paralarvae are located at the
lined by a low simple epithelium with a thin cuticle. entrance or exit of this organ to the vestibule (Fig. 5.6a),
Moreover, in the hatchling squid, the oesophagus does not lacking secretory cells in the internal and external walls, as
have folds in its mucosa (Fig. 5.5a). The crop of octopus well as the caecal glands typical of adults. However, in
paralarvae is a tubular structure with a distended lumen. In hatchling cuttlefish, the caecum has secretory cells and the
the crop, the mucosa forms small villi or longitudinal folds caecal glands in the apical region of the folds (Fig. 5.6c).
that differ from the adult in the fact that they are not bran- The octopus and squid intestines are a tubular organ that
ched (Fig. 5.5b). presents structural differences at the level of the mucosa. In
In hatchling cephalopods, the stomach is a sac-like organ the ventral region of the mucosa, there are two broad lon-
with a simple folded mucosal epithelium (Fig. 5.5c). In gitudinal folds that resemble the adult typhlosoles (Fig. 5.7a,
octopus and squid, the stomach epithelium is formed by b), but they are comparatively less developed. These folds
cubic cells with a large and central nucleus (Fig. 5.5d), but present a thick ciliated pseudostratified epithelium
(a) (b)
(c)
Fig. 5.7 Transversal section of the intestine of Octopus vulgaris paralarvae (a). Transversal section of the intestine of squid (b). Detail of the
typhlosole epithelium (c). ci, cilia and Ty, typhlosole. a–c H&E stain. Scale bars: a 10 µm; b 20 µm; and c 5 µm
(Fig. 5.7c). The two folds are only present in the region of secretion. The epithelium lining this gland shows a greater
the intestine close to the vestibule, whereas, in the remainder proportion of eosinophilic gland cells than the other two
tube, there are no folds or villi in the mucosa, which consists types (Fig. 5.8b). Due to the absence of secretory cells, the
of a simple squamous epithelium. In the dorsal region, a anterior salivary gland in squid does not yet have a secretory
thickening of this wall was observed (Fig. 5.7a, b). No function, which may be due to its premature state
secretory cells are present in the intestinal mucosa. (Fig. 5.8a).
The posterior salivary glands (Fig. 5.8c, d) of the hatch-
lings have a morphological structure similar to those in
5.5.1 Annex Glands adults. The secretory units of these glands show two types of
gland cells: (i) cells with granular eosinophilic secretion and
Unlike the adults, the anterior salivary glands of the squid (ii) cells with mucous basophilic secretion. However, in the
and octopus hatchlings are simple and have no ramifications case of O. vulgaris paralarvae the yellow granular cells,
(Fig. 5.8a, b). These glands show, in octopus, two types of typical in adults, are not present.
glandular cells with granular secretion, eosinophils and The single digestive gland of newly hatched octopus
basophils as well as a type of gland cell with mucous paralarvae has tubules with a simpler structure than those in
(a) (b)
(c) (d)
Fig. 5.8 Transversal section of the anterior salivary glands of a squid salivary gland; Bc, basophilic cell; Cr, crop; Dg, digestive gland; E,
larva (a). Detail of the anterior salivary gland of an octopus paralarva (b). oesophagus; Ec, eosinophilic cell; Iy, internal yolk; and Psg, posterior
Transversal section of octopus showing the posterior salivary glands (c). salivary gland. a–d H&E stain. Scale bars: a 50 µm; b 10 µm; c 100 µm;
Detail of the posterior salivary gland of a squid larva (d). Asg, anterior and d 20 µm
adults (Fig. 5.9a). These tubules are characterized as having (Fig. 5.9c). However, this absence seems indicate that they
a lumen and a poorly defined cellular organization, showing are premature hatchlings (early juvenile) than a distinctive
only a basement membrane and two types of cell: immature feature of this stage of development. The digestive gland of
digestive cells and storage cells (Fig. 5.9b). The latter cells cuttlefish has a similar structure to that in octopus
show an appearance similar to mature digestive cells of (Fig. 5.9d). The internal yolk of squids and octopuses is
adults, but its cytoplasm appears expanded due to abundant composed of a cluster of yolk platelets of different sizes
yolk plaques and inclusions of different sizes. Lemaire et al. (Fig. 5.9a, c) with the sizes decreasing from the centre to the
(1977) described storage cells and cells that have boules in periphery. This internal yolk is separated from the digestive
the basal region and a microvilli border in the apical region. gland by a thin membrane. While the paralarvae internal
In squid, the two digestive glands appear at both sides of the yolk is formed by a single elongated mass running parallel to
internal yolk. They are tubular and lack yolk plaques the digestive gland, the juvenile squid internal yolk consists
(a) (b)
(c) (d)
Fig. 5.9 Transversal section of the digestive gland of paralarval crop; Da, digestive appendages; dg, digestive gland; lc, basal cell or
octopus (a). Detail of octopus main digestive gland and digestive gland immature cell; ly, internal yolk; and Rc, reserve cell. a–d H&E stain.
appendages (b). Longitudinal section of L. vulgaris digestive gland (c). Scale bars: a 100 µm; b 50 µm; c 20 µm; and d 25 µm
Longitudinal section of the digestive gland of larval cuttlefish (d). Cr,
of anterior and posterior masses located at the back of the basophilic cells with a large nucleus near the base of the
mantle. cell. In hatchling cephalopods, the branchial glands are
present (Fig. 5.10a). Similarly to adults, these are dense
structures formed by basophilic cells with a central round
5.6 Circulatory System (Fig. 5.10) large nucleus (Fig. 5.10b). However, the well-developed
cord structure present in adults is not seen in hatch-
5.6.1 Branchial Glands lings. These glands have a similar structure in squid and
octopus.
In adults, the branchial glands are a dense paired structure.
These glands extend the whole length of the gills. The
branchial gland is contained in a capsule consisting of a 5.6.2 White Body (Hematopoietic Organ)
columnar cell epithelium (Dilly and Messenger 1972). It
presents a uniform appearance with numerous acini groups The hematopoietic organ or white body is a multilobular
of cells. These acini are formed by some characteristic gland behind the eyeball in cephalopod adults. In octopods
(a)
(b) (c)
Fig. 5.10 Transversal section of L. vulgaris circulatory and respira- branchial heart; Ebv, efferent blood vessel; Gl, gill ligament; Hs,
tory systems (a). Detail of L. vulgaris branchial gland (b). Transversal systemic heart; Iy, internal yolk; Pl, primary lamella; and Wb, white
section of the L. vulgaris head showing the eye and the associated body. a–c H&E stain. Scale bars: a–c 50 µm and b 100 µm
white body (c). Abv, afferent blood vessel; Bg, branchial gland; Bh,
and decapods, this gland is well vascularized and has rela- two auricles, branchial hearts, and a single ventricle, systemic
tively few collagen fibres (Johnson 1987). Similarly to heart. The vessel system is formed of arteries and veins which
adults, the white body of the hatchling cephalopods is are linked by a capillary network and a peripheral sinus and
located between the eye and the optic lobe and is made up of lacunae where a gas exchange occurs.
a tight group of small blood cells (Fig. 5.10c). The systemic heart is a powerful muscle with a lumen
which is divided by a muscular heart septum into two
equally large chambers (Kling and Schipp 1987). The main
5.6.3 Systemic and Branchial Hearts moving force of blood circulation is produced by the heart
and is supported by other contractile organs (Kling and
The cephalopods have a completely closed circulatory sys- Schipp 1987). Therefore, the function of this is to be the
tem which is well developed with an extended artery and vein central motor of the high-pressure circulatory system. The
net. The system is like the circulatory system of vertebrates branchial hearts are deep red to yellow-brown colour with a
(Schipp 1987). Among the Cephalopoda, the Coleoidea have much branched narrow lumen (Schipp and Schäfer 1969).
This organ also has other different functions: to assist (Budelmann et al. 1997). In O. vulgaris, each gill has a
filtration for the formation of primary urine, to store central cavity, with the primary lamellae on each side. The
important substances, to aid in the excretion of catabolites primary lamella is folded along its long axis so that a series
and in the immune defence mechanism (Fielder and Schipp of secondary lamellae are formed, alternating on the two
1987). sides of the primary ones (Wells and Wells 1982). The
The wall of the ventricle and the auricles has three main secondary lamellae are branched and form tertiary lamellae.
layers: epicardium, myocardium and endocardium. The epi- The main afferent vessel runs along the dorsal margin of the
cardium is formed by flat extended epithelial cells with gill and down the inner surface of each primary lamella.
microvilli. The myocardium has a different structure in the From here, it branches along the crest of the secondary
systemic and branchial hearts (Budelmann et al. 1997). It is a lamella. Cuttlefish gills are less complex. These are divided
spongy layer containing muscle fibres and polygonal cells into a series of compartments by membranous partitions.
rich in the cytoplasm and containing an ovoid nucleus. This One such runs vertically along the length of the gill, dividing
layer is more developed in the systemic heart and is com- it into two. The afferent vessel from the branchial heart runs
posed of many muscle layers. However, in the branchial along the groove formed at the bottom of the gill. The
heart, this layer is only formed by one or two layers of corresponding efferent vessel is external (Thompsett 1939).
densely innervated muscle fibres (Budelmann et al. 1997). As The secondary and tertiary lamellae are crossed by the
it is observed in adults, the myocardium is formed by car- arteries and veins of the second and third orders. Between
diomyocytes similar to the vertebrate cardiomyocytes the veins and arteries of the third order, the blood runs in
(Schipp and Schäfer 1969). The third layer is an incomplete “lacunae”. This is the region of gas exchange (Schipp et al.
endothelium towards the lumen, but sometimes it is not 1979; Wells and Wells 1982).
developed at all (Fielder and Schipp 1987). This layer is In the hatchlings of octopus and squid, the gills are
formed by a continuous basal lamina with flattened scarcely branched, in contrast to the deeply branched gills of
endothelial cells and myocytes (Kling and Schipp 1987; the cuttlefish (Fig. 5.11). Paralarvae and juveniles only have
Budelmann et al. 1997). a primary lamella (Fig. 5.11a), which in its distal portion
The post-hatching cephalopod ventricles and auricles ends in a bulbous protrusion, where a blood sinus is found
have a large lumen. These organs have poorly developed (Fig. 5.10b). Similarly to adults, it is possible to observe the
walls. Paralarvae and juveniles have a strong musculature in gill gland as well as the afferent vessels from the branchial
the systemic heart similar to adults. The branchial heart in heart (Fig. 5.10b). The epithelium of the lamellae varies
hatchling cephalopods has a large lumen, unlike in adults. In from cubic to squamous in different gill areas of octopus,
the organ wall, it is possible to see the typical cells of this while this epithelium is squamous in squid (Fig. 5.11a, c). In
organ with a dense black vacuole (Fig. 5.10a). juvenile cuttlefish, the gill shows differences in height and
inner structure of the concave and convex epithelial surfaces
of secondary folds (Fig. 5.11b). The height of the epithelium
5.7 The Respiratory System (Figs. 5.10 of the concave side is nearly twice that of the outer side
and 5.11) epithelium and appears more vacuolated (Schipp et al.
1979). The inner epithelium (concave side) is formed by tall
The dibranchiate cephalopods (coleoids) have a pair of gills cells exhibiting some vesicles and a large vacuole
protruding from an expandable mantle cavity where the flow (Fig. 5.11d). The outer (convex) epithelium is a simple
of respiratory water passes through their folds. This water squamous epithelium with a well-developed microvillous
flow is produced by the powerful contractions of a highly apical border (Budelmann et al. 1997).
elastic mantle muscle system (Budelmann et al. 1997).
A pair of branchial hearts generate part of the circulatory
pressure and pump the blood via the afferent branchial vessel 5.8 The Excretory System (Fig. 5.12)
(branchial artery) that lies in the gill ligament, to the gill
lamellae where it is oxygenated. It is passed to the system- In cephalopod excretion, the organs involved are the renal
atic heart by active pulsations of the efferent branchial vein complex, with the renal and digestive appendages, and the
(Schipp et al. 1979). The cephalopod gills serve for respi- branchial heart complex, with their appendages or pericar-
ratory and excretory functions (Schipp et al. 1979). dial glands and the brachial heart (Furuya et al. 2004). The
The gills are composed by lamellae, which are organized digestive gland and the white body also collaborate in this
symmetrically in Sepia and asymmetrically in octopods function (Boucher-Rodoni and Mangold 1988).
(a) (b)
(c) (d)
Fig. 5.11 Longitudinal section of the Loligo vulgaris gill (a). a, c H&E stain and b, d Masson’s trichrome stain. Scale bars: a 50 µm;
Horizontal section of S. officinalis gill (b). Detail of the L. vulgaris b 100 µm; and c–d 25 µm
gill epithelium (c). Detail of the S. officinalis gill epithelium (d).
5.8.1 Renal Appendages dense lysosomes with high acid phosphatase activity
(Budelmann et al. 1997). Below the epithelium lies a blood
In adult octopods, the renal appendages are protrusions in the “lacuna” with an incomplete layer of endothelial cells (only a
coelom cavity with grooves and folds that cover the vena few fibroblasts and collagenous fibres), but also an extensive
cava and its branches. These appendages consist of a network of fine oblique striated muscle cells is observed. This
single-layered columnar epithelium with apical microvilli. network of muscle fibres is responsible for the rhythmic
The renal appendages of decapods have the same structure contractions of the renal appendages, which support the flow
that has been observed in octopus, with deep grooves and of the haemolymph and urine (Budelmann et al. 1997).
folds (Furuya et al. 2004) coated by a columnar epithelium Furuya et al. (2004) established that the renal appendages
with microvilli. However, these cells are characterized as the differ between juveniles and adults of different species, the
presence of numerous mitochondria in the apical region and latter always exhibiting a more complex surface of renal
in the area of the basal labyrinth, as well as by many large appendages than in juveniles. In octopus paralarvae, the
(a)
(b) (c)
Fig. 5.12 Transversal section of renal appendages of an O. vulgaris view (c). Bh, branchial heart and Ra, renal appendages. H&E stain.
paralarva protruding in the renal coelom (a). Detail of the epithelium of Scale bars: a–b 25 µm and c 10 µm
renal appendages (b). L. vulgaris digestive appendages, transversal
renal appendage consists of a single-layered columnar inner pancreatic epithelium) lie in parallel, separated by a
epithelium covering the vena cava and its branches capillary system and a small amount of connective tissue
(Fig. 5.12a, b). In comparison with the adult, it is a simple (Budelmann et al. 1997). The juvenile pancreatic appen-
saccular structure without groove and folds. The renal dages are less folded and show a simple cubic epithelium
appendages of squid show the same organizational structure (Fig. 5.12c).
as in octopus paralarvae, but these are more developed than
in adult octopus.
5.8.3 Branchial Heart Appendages or Pericardial
5.8.2 Digestive Appendages Glands
In squid, the digestive appendages consist of two different The cephalopod pericardial gland is a structure connected
epithelia. The outer epithelium consists of a single-layered with the branchial heart that projects into the coelom. In
columnar epithelium with microvilli. It is connected to the cuttlefish, these appendages are conical and have grooves
terminal epithelia of the renal appendages. In the centre, and folds. Apparently, these structures are not developed in
two pancreatic epithelial sheets (the outer renal and the post-hatching stages (Derby 2014).
(a) (b)
(c)
Fig. 5.13 Transversal section of O. vulgaris showing the ink sac (a). Di, ink sac duct; Ig, ink gland; iy, internal yolk; and Ri, ink sac or
Longitudinal section of the L. vulgaris ink sac (b). L. vulgaris funnel reservoir. a–c H&E stain. Scale bars: a 25 µm and b–c 50 µm
organ, transversal view (c). Gc, goblet cell; Gz, basal germinal zone;
5.8.4 Ink Gland Complex (Fig. 5.13) 5.8.5 Ink Sac
The Coleoidea species have ink sacs and produce ink, but This gland is exocrine and produces the ink. The epithelium
some coleoids have lost their ink sac. For example, this is of the gland complex inner wall is formed by a columnar
true in the case of the octopod species in the Cirrata suborder glandular epithelium, whereas the outer wall is formed by
(Budelmann et al. 1997). The ink gland complex consists of iridophores (Mangold et al. 1989). The ink gland is divided
three parts: the ink gland, the ink reservoir and the ink sac by lamellae that are composed of muscle and collagenous
duct (Budelmann et al. 1997). The ink is produced by the fibres. The gland centre has a tubuloalveolar structure with a
secretions of two glands, the ink gland (in the ink sac) and cubical epithelium (Budelmann et al. 1997). In hatchling
the funnel gland (Derby 2014). The combined secretion of cephalopods, it is possible to see the ink gland complex and
these two glands, produced in different quantities, leads to its structures (gland sac and duct). The gland presents a
ink different forms (Derby 2014). cubic epithelium, while the ink sac wall is formed by a
squamous epithelium (Fig. 5.13a, b).
5.8.6 Funnel Organ This organ is present in hatchling squid, cuttlefish and
octopus. In juvenile squids (Fig. 5.13c), this organ has the
The funnel organ is the second gland contributing to the ink same morphology as observed by Voss (1963) for Sepioidea.
secretion (Derby 2014). This is a mucous gland on the This gland has a glandular epithelium composed of colum-
funnel inner surface (Budelmann et al. 1997). In adult nar goblet cells with clear cytoplasm and a basal nucleus.
squids, this gland is formed by columnar goblet cells with a
dark cytoplasm and a nucleus in the basal region (Laurie
1888). This organ has morphological differences between 5.9 Central Nervous System or Brain
different species. This organ is usually W-shaped in octo- (Fig. 5.14)
puses, but in Sepioidea it consists of a dorsal inverted
V-shaped and paired lateral pads (Voss 1963). Cephalopods have the most complex brain of all inverte-
brates (Young 1977, 1979). The central nervous system can
(a) (b)
(c) (d) (e)
Fig. 5.14 Horizontal section of the S. officinalis brain at the level of ineu, inner neuropil; oc, optic commissure; OG, outer granular; OL,
the optic commissure (a). Horizontal section of the O. vulgaris brain optic lobes; oneu, outer neuropil; p, pedal lobes; PL, peduncular lobe;
(b). Detail of S. officinalis optic lobes (c). Detail of L. vulgaris optic st, statocyst; Sv, subvertical lobe; and V, vertical lobe. a, c–e Masson’s
lobes (d). Detail of the outer layer of the S. officinalis pedal lobe (e). trichrome stain and b H&E stain. Scale bars: a 300 µm; b 100 µm; and
Ba, basal lobe; ctx, cortical laminas; E, oesophagus; IG, inner granular; c–d 20 µm
morphologically be divided into sub- and supra-oesophageal species (Yamazaki et al. 2002). The tactile memory centres
areas, partitioned by the oesophagus. On each side of the grow faster than the swimming centres in benthic species.
peri-oesophageal lobes are the large optic lobes which However, the optic lobes increase with growth in pelagic
comprise an outer cortex and a central medulla. The brain species (Yamazaki et al. 2002). Basically, the eyes and
areas are divided into many lobes (Young 1977, 1979); in nervous system of a variety of hatchling cephalopod species
the Coleoidea, brain has been characterized by twenty-five follow the same pattern; they vary interspecifically in size,
major lobes (Young 1971). The central mass lobes have a proportion and complexity (Wild et al. 2015).
pattern of central neuropil (nerve fibres) and a surrounding The optic lobes of hatchling squid, cuttlefish and octopus
cortex (cell bodies). The brain lobe proportions change are similar in relative size and tissue organization to those of
between hatchling, juveniles and adult stages of life (Frösch adults (Fig. 5.14a, b). The outer cortex is formed by three
1971). These changes have been correlated with morpho- layers: two of them contain small neurons that are densely
logical developments and changes in behaviour and habitat packed (outer and inner granular layers), and in the middle,
(Messenger 1973; Nixon and Mangold 1996; Shigeno et al. there is a layer of optic nerve fibres (outer neuropil or
2001). The modes of brain development vary between plexiform layer) (Fig. 5.14c). Below the inner granular
(a) (b) (b)
(c) (d)
Fig. 5.15 Horizontal section of O. vulgaris connection (*) between digestive gland; N, neurons; neu, neuropil; OL, optic lobes; p, pedal
the pedal lobe and stellate ganglion (a). Detail of O. vulgaris stellate lobes; psg, posterior salivary gland; Rad, radula sac; and Sg, stellate
ganglion (b). Horizontal section of S. officinalis buccal ganglion (c). ganglion. a H&E stain and b–d Masson’s trichrome stain. Scale bars:
Detail of S. officinalis buccal ganglion (d). Bl, buccal lobe; dg, a, c 100 µm; b 20 µm; and d 50 µm
layer, the neurons are arranged in columns separated by commissure lies tightly adjacent to the dorsal optic com-
neuropil which are more abundant in the medulla missure (Wild et al. 2015).
(Fig. 5.14a, b). However, in the hatchlings of L. vulgaris a The cerebral ganglia in hatchling cephalopods are less
thin second plexiform layer (inner) is seen just below the developed, lacking the complex organization observed in the
inner granular layer (Fig. 5.14d) (Wild et al. 2015). From adults. Nevertheless, the general architecture of the brain
each optical lobe, an optic tract crosses the brain forming the formed by suboesophageal and supra-oesophageal lobes has
optic commissure (Fig. 5.14a). The optic commissure is well been observed (Fig. 5.14a, b). Moreover, ganglia which are
developed and can be divided into dorsal (Fig. 5.14a) and connected with the cerebral ganglia via connectives and
ventral optic commissures (Wild et al. 2015). The peduncle commissures are also observed along the body.
(a) (b)
(c) (d)
Fig. 5.16 Longitudinal section of the O. vulgaris head showing an photoreceptor segments bearing rhabdomeres and pigment granules;
eye (a). Longitudinal section of the O. vulgaris optic nerves and optic El, eye lens; I, iris; IG, inner granular cells; OG, outer granular cells;
lobe (b). Detail of O. vulgaris ciliary body and iris (c). Detail of the O. oneu, outer neuropil; and R, retina. a–e H&E stain. Scale bars:
vulgaris retina (d). Bs, basal region of distal segments of the visual cell a 100 µm; b 25 µm; c 50 µm; d 10 µm
filled with pigment granules; Cm, ciliary body; Ds, distal region of
Concerning the suboesophageal mass, the hatchlings In addition to the review of Young (1971), more recently
show a developed pedal lobe (Fig. 5.14a, b). A brachial lobe Wild et al. (2015) published a specialized monograph on the
entailing short brachiopedal connectives is described in nervous system of six species of hatchling cephalopod using
hatchling cuttlefish (Wild et al. 2015). The histology and 3D modelling.
supra-oesophageal mass is more complex, and it is composed
laterally by the basal and peduncular lobes and anteriorly by
the vertical and subvertical lobes (Fig. 5.14a, b). Moreover, 5.10 Sensory System (Figs. 5.16 and 5.17)
in the most anterior part posteriorly to the buccal mass, a
buccal lobe can also be observed (Fig. 5.14a). The arrange- Cephalopods have numerous sensory organs (Budelmann
ment of these lobes is simpler than those in adults. The 1994; Budelmann and Tu 1997) such as large and
vertical lobe, in adults, has a very characteristic shape with well-developed eyes, a paired statocyst, a system similar to
five lobules. Most of the lobes in the hatchlings are formed by the fish lateral line, extraocular photoreceptor organs. Some
a central extended neuropil surrounding by densely packed octopuses are also sensitive to chemical and tactile stimuli.
groups of neurons. The size of the neurons varies from small
to large (Fig. 5.14e). Group of fibres forming nerves connect
these lobes with small ganglia located along the body. In the 5.10.1 The Visual System (Fig. 5.16)
hatchling cuttlefish and octopus, it is possible to see the
neuropil of the stellate ganglia receiving fibres from the pedal All cephalopods possess eyes although there are differences
lobe through a coarse pallial nerve (Fig. 5.15a, b). Moreover, in their morphology. The eyes are mostly spherical, but
the muscular organ (bolsters) of the buccal mass receive sometimes oval, telescopic or stalked (Wentworth and Muntz
innervation from the buccal lobe (5.15c, d). The connection 1989; Young 1970, 1991). The Coleoidea eyes contain all the
of the buccal lobe in adults has been described by Young major components of a complex vertebrate eyes (Budelmann
(1971), “the superior buccal lobes send nerves directly to the et al. 1997), namely: cornea (except those of Todarodes, Illex
posterior salivary glands and, via inferior buccal ganglia and Ommastrephes), iris, retina, pupil (which can be circular,
(which are connected to the superior buccal lobes by inter- horizontal or irregular in shape) and rigid lens that are sus-
buccal connectives) to the anterior salivary glands, the pended by ciliary muscles which allow for some near vision
muscles of the jaws and the radula”. (Messenger 1981, 1991; Hartline and Lange 1984).
(a) (b) (c)
Fig. 5.17 Longitudinal section of the O. vulgaris retina (a). Longi- pigment granules and Ds, distal photoreceptive segments bearing the
tudinal section of L. vulgaris retina (b). Longitudinal section of the S. rhabdomeres and pigment granules. a–c H&E stain. Scale bars:
officinalis retina (c). Bs, basal segment of the visual cell filled with a 10 µm and b–c 20 µm
(a) (b)
(c)
Fig. 5.18 Horizontal section of an O. vulgaris paralarva (a). Close view of statocysts (b). Detail of sensory areas of statocyst. Br, brain; Ey, eyes;
Hc, hair cells; OL, optic lobes; Sp, supporting cells; St, statocyst. a–c H&E stain. Scale bars: a 100 µm; b 50 µm; and c 25 µm
The cephalopod iris is formed of five layers: external differences in the thickness or development of some of its
epithelium, chromatophore layer, iridophore layer, muscle layers are observed (Fig. 5.17). The plexiform layer is more
and collagen fibre layer and pigment epithelium (Froesch developed in the first few days post-hatching. In Loligo, the
1973). The cephalopod retina is less developed than those in pigmentary granules in the apical rhabdomere region are
vertebrates. It is formed by receptor cells (primary sensory scarce (Fig. 5.17b).
cells) and efferent fibres. The visual information from the
retina runs to large optic lobes through the optic nerve
(Budelmann 1994). The behaviour response of embryos to 5.10.2 The Statocysts (Fig. 5.18)
visual stimulus and their ability to learn visual features of
potential prey indicate that the visual system, including the These structures are situated in the cranial cartilage and
optic lobes, is functional before hatching (Darmaillacq et al. show a variety of forms in cephalopods (Budelmann et al.
2014). 1997). In octopus, this is a spherical sac (Fig. 5.18a, b) with
In the hatchling octopus, squid and cuttlefish, the eyes are a balance system and an angular acceleration system divided
completely developed and have the same structure than in into nine segments (Young 1960). However, in decapods,
adults (Fig. 5.16). In some structures such as the retina, this structure is irregularly shaped and has three gravity
receptor systems and an angular acceleration system (Ste- circulatory system, a low level of development has been
phens and Young 1982). The equilibrium receptor organs found in the branchial hearts and also in the systemic heart.
provide information regarding body position. Different ani- In hatchling cephalopods, the branchial hearts present a large
mals have different requirements for this organ depending on lumen that is absent in adults. Likewise, systemic a poorly
lifestyles and habitats. In general, fast-moving species orient developed wall is observed in the systemic heart of all the
themselves with respect to the direction of gravity (and other studied species. In paralarvae and juvenile, the systemic
linear accelerations), as well as respond to rotatory body heart does not have the strong musculature observed in the
movements (angular accelerations) (Neumeister and Budel- systemic heart of adults.
mann 1997). Consequently, the equilibrium receptor organs In some instances, the degree of development in hatchling
include two different receptor systems: a gravity receptor cephalopods is similar to that in adults. This happens, for
system for the detection of position relative to the direction example, in defensive systems, the ink complex, the struc-
of gravity and an angular acceleration receptor system for tures that help in the prey capture and the visual system. In
the detection of rotatory movements (Neumeister and hatchling octopus, squid and cuttlefish, the eyes are com-
Budelmann 1997). pletely developed. Only in the retina, differences in the
Both receptor systems are formed by two types of cells, thickness or development of some of its layers are observed.
hair cells which are voluminous and have a large nucleus Finally, there are tissues that change throughout the
and microvillous supporting cells (Fig. 5.18c). The gravity development to adapt to different habitats. This occurs in
receptor system comprises the macula epithelium and the structures such as the L. vulgaris and S. officinalis epidermis
accessory statolith. The hair cells of the macula are in dif- where a large number of globet cells are observed differ from
ferent arrangements: concentric and half circles or in 360° or the characteristic epithelium of adults. Another example is
180° radial fan-like. The other receptor system, an angular the characteristic Kölliker cells that appear only in the epi-
acceleration system, is formed by the crista epithelium with dermis of octopus paralarvae, disappearing after the first
the cupula. This structure forms ridges that run inside the month of post-embryonic life.
statocyst in the three dimensions of space. The study of all tissues and systems in premature
The crista segments are composed of primary sensory hatchling of squid (L. vulgaris) and cuttlefish (S. officinalis)
hair cells and two types of secondary hair cells (Budelmann and of paralarvae of octopus (O. vulgaris) allows us to
1994). Along each crista segment, the hair cells are arranged conclude that although most systems are present, the
in two or four regular rows. Just like in the macula, between majority of them show a lower degree of development than
the hair cells, there are supporting cells. in adults. Some systems, mainly those related to sensory
systems, present a similar development, and others change
their morphological characteristics.
5.11 Concluding Remarks
Acknowledgements We would like to thank Antonio Sykes
The histological anatomy study of premature hatchlings of (CCMAR–CIMAR) who generously donated the paraffin blocks from
which the sections of S. officinalis were prepared.
squid (L. vulgaris) and cuttlefish (S. officinalis) and of par-
alarvae of octopus (O. vulgaris) shows that most systems
and their characteristic structures are present. However,
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Part II
Pathogens and Related Diseases
Cephalopod Diseases Caused by Fungi
and Labyrinthulomycetes 6
Jane L. Polglase
Abstract
This chapter describes infections of cephalopods by both the Labyrinthulomycetes (formerly
fungi, now protists) and organisms still classified as fungi, with information on how to
diagnose them. Both types of infection are rare, but those by Labyrinthulomycetes in captive
cephalopod populations can increase with time and may last for three or more years, raising
concerns about such ubiquitous organisms for cephalopod culture.

Keywords
Cephalopod diseases Infection Fungi Labyrinthulomycetes Labyrinthulomycota
Thraustochytrids Aplanochytrids Labyrinthula
commonly molluscs) and the labyrinthulids (not considered

6.1 Introduction here but also opportunist pathogens of marine grasses and in
land grasses in saline soils). The common factor that allows
As well as considering the rare cephalopod infections by
the Labyrinthulomycetes to become pathogenic is stress in
organisms currently recognised as being within the fungal
the host.
kingdom, this chapter covers diseases caused by organisms
known most commonly as Labyrinthulomycetes. As their
name suggests, the Labyrinthulomycetes were originally
6.2 Thraustochytrid/Aplanochytrid
thought to be fungi. However, these single-celled organisms,
Infections in Cephalopods
with unique “ectoplasmic nets”, are now classified as pro-
tists. Bennet et al (2017) provide the most comprehensive,
In molluscs, thraustochytrids proper and aplanochytrids
recent description of the group, while Moss (1986,
(collectively referred to here as thraustochytrids) are found
Chap. 11) provides excellent images of diagnostic features
as important pathogens or parasites of bivalves and also of
of these organisms.
nudibranchs (McLean and Porter 1982), octopus and squid.
Labyrinthulomycetes are most commonly found as mar-
Only one thraustochytrid infection has been described to
ine decomposers or remineralisers of complex organic
date in European cephalopods. This was a fatal, progressive,
material, but can be symbionts, parasites or pathogens. There
ulcerative dermal necrosis of lesser or curled octopus, Ele-
are three major groupings within the Labyrinthulomycetes:
done cirrhosa (Polglase 1980, 1981) found on the east coast
the thraustochytrids and aplanochytrids (regularly found as
of Scotland. The disease (originally spread from one indi-
opportunist pathogens or parasites of invertebrates, most
vidual) infected an entire research aquarium, despite strin-
gent efforts to remove it, for over four years, during which a
small number of other infected animals were also brought in
J. L. Polglase (&)
from the wild. A similar condition had also been observed
Institute of Life and Earth Sciences, School of Energy, sporadically in the 1970s/80s in Octopus vulgaris, at the
Geoscience, Infrastructure and Society, Heriot Watt University, Stazione Zoologica, Naples, Italy (J. B. Messenger, pers.
Riccarton, Edinburgh, EH11 1QH, Scotland, UK comm. to Polglase).

https://doi.org/10.1007/978-3-030-11330-8_6
114 J. L. Polglase
Fig. 6.2 Moribund Eledone cirrhosa, showing final stages of

slow-progressing Labyrinthulomycete infection with major dermal
lesions. Arrows indicate lesions; e = eye; f = funnel. Scale bar: 20 mm
Fig. 6.1 Eledone cirrhosa showing symptoms of a rapidly progressing
multi-lesion thraustochytrid infection. Arrows indicate lesions; e = eye;
f = funnel. Scale bar: 12 mm
also seen in the gills and eyes (producing a cataract-like
Key signs were patches of inactive chromatophores, from appearance) and in the lower dermis, but there was no evi-
which the epidermis was then lost (Fig. 6.1). Affected animals dence of a stage of systemic infection. Interestingly, labyr-
constantly rubbed their skin and shook their arms, as if itching. inthulids were also isolated from some of the lesions, but
The lesions deepened, multiplied and spread, either rapidly or they appeared to be secondary to the thraustochytrid. Elec-
more slowly in individuals with fewer lesions, revealing the tron microscopy confirmed the diagnostic features of a
unpigmented connective tissue and muscle layers below thraustochytrid (see Fig. 6.8), but it was not possible to
(Fig. 6.2). The cause of the death was not clear but could have re-infect from cultures derived from the lesions.
been metabolite imbalance (see Polglase 1980). In the light Many haemocytes of infected animals responded by
microscope, clear zones of tissue destruction were seen in the adopting the elongate “prefibroblast” morphology, seen in
lesions around rounded cells approximately 6 µm in diameter haemocytes moving towards the surface of mechanical
(Figs. 6.3 and 6.4). The cells divided to produce diads, triads, wounds (Polglase et al. 1984). Groups of haemocytes were
tetrads and larger cell clusters, which point to thraustochytrid seen to produce incomplete capsules on the edge of the
involvement (Figs. 6.3 and 6.4). necrotic zones around the protists, but the thraustochytrids
Only one organism was consistently seen in histological were never completely encapsulated and no dermal plug was
and electron microscope sections from all infected animals. ever formed across the lesion.
This was a thraustochytrid tentatively identified from its life The condition appeared to be highly contagious (Polglase
history in culture as Ulkenia amoeboidea, but more recent 1980) and made it extremely difficult to keep the Eledone in
current work indicates that is a novel species and genus, with the aquarium for research purposes.
some metabolic differences from other thraustochytrids. In The only thraustochytrid infection in squid was found in
severe infections, small numbers of thraustochytrids were the Northwest Atlantic. This is the gill disease in captive
6 Cephalopod Diseases Caused by Fungi and Labyrinthulomycetes 115
Fig. 6.3 Mallory’s triple stained light microscope section showing from the basal membrane (bm) and then be lost (asterisk). c = chro-
thraustochytrids in the epidermis of Eledone cirrhosa. Arrows show matophore; e = epidermis; i = iridophore; t = thraustochytrid.
clear zones where the host tissue has been dissolved by the lytic Scale bar: 40 µm
enzymes of the ectoplasmic net causing the cells to separate initially
short-finned squids, Illex illecebrosus, reported by Jones (Fig. 6.7). Ultrastructural and semi-thin sections confirmed
(1981) and Jones and O’Dor (1983). the organism was a thraustochytrid. In semi-thin sections,
In 1977–79, this disease affected squid kept in a facility at diads, triads and tetrads of cells could be seen beneath the
Dalhousie University, Canada, designed to produce the best gill epithelium, with evidence of substantial tissue damage
conditions possible for research on these delicate animals from the activities of the lytic enzymes from the ecto-
(the Aquatron). In 1977–78, only two or three animals died, plasmic net but not close to the cell bodies. In advanced
atypically, before mating, while the others remained healthy. infections, just as in E. cirrhosa, the overlying epithelium
Those that died (Jones and O’Dor 1983) had a large number was disrupted, releasing the thraustochytrids into the
of small (1.5 mm) white nodule-like lesions in their gills mantle cavity.
(Figs. 6.5 and 6.6) and, in the later stages of heavy infec- Although some cells thought to be squid amoebocytes
tions, also on the inside of the mantle; “sufficient numbers were found close to or even partially enclosing the thraus-
for these to be considered a likely cause of death” (Fig. 6.6). tochytrids, there were no instances of complete encirclement
By the end of the 1979 squid “season”, however, the lesions or any degeneration of the thraustochytrids within the
had become the main cause of mortality. They were never lesions. Jones and O’Dor were not able to isolate the
observed in animals in the wild and appeared after the ani- thraustochytrid into pure culture for identification but spec-
mals had been kept for 3 weeks or longer in the Aquatron. ulated that it was a species of Schizochytrium. Lack of pure
Light and electron microscopy again showed that only a cultures also meant that re-infection experiments could not
single organism (spherical cells about 7 µm in diameter) be undertaken, so there is no final proof that this was a
could be identified in the lesions, often in large numbers pathogen.
116 J. L. Polglase
Fig. 6.4 Section of Eledone

cirrhosa skin from which the
epidermis has been lost, allowing
the thraustochytrid (t) to move
down into the dermal layer.
Arrows indicate areas where
tissue lysis is occurring surround
the dividing thraustochytrid;
c = chromatophore,
CL = opportunist ciliate. Scale
bar: 12 µm
Fig. 6.5 Female northern

short-finned squid, Illex
illecebrosus, ventral dissection.
Gills show small white lesions
typical of the thraustochytrid
disease. Scale bar 40 mm
(Courtesy of Jones and O’Dor)
Fig. 6.6 Illex illecebrosus gill with scattered white lesions on filaments. Scale shows mm (Courtesy of Jones and O’Dor)
Fig. 6.7 Thraustochytrids within a lesion in the gill of Illex illecebrosus, note cells dividing to form diads, triads and tetrads. Scale bar 30 µm
(Courtesy of Jones and O’Dor)
118 J. L. Polglase
6.3 Diagnosing a Labyrinthulomycete and secrete the ectoplasmic net system. They are posi-
Infection Using Electron and Light tioned at the point at which the ectoplasmic net leaves the
Microscopy cell body and vary in structure between species. In all
cases, however, ectoplasmic reticulum converges to
Almost all Labyrinthulomycetes have three unique, diag- become a dense array, which may have the appearance of
nostic features (see Bennett et al. (2017) and Moss (1986) a plug at the “top” of the net (Fig. 6.8).
for detailed descriptions). Electron microscopy shows all • An ectoplasmic net. This consists only of unit membrane,
three, but high powered light microscopy generally reveals continuous with that of the sagenogenetosome. It con-
the ectoplasmic net and its effects. tains no organelles, only occasionally a few membranous
structures. In thraustochytrids and aplanochytrids, the net
• Unique, subcellular organelles, called sagenogeneto- takes the form of a root-like system and is known to
somes. These have been compared to spider spinnerets secrete both digestive enzymes and transport nutrients
Fig. 6.8 Transmission electron

micrograph of the thraustochytrid
from Eledone cirrhosa, showing
the sagenogenetosome (s); cell
wall with layers of scales (w); the
ectoplasmic net (e); n = nucleus;
g = golgi; m = mitochondria with
tubular cristae. Scale bar:
1.75 µm
back to the cell body. The net is also known to contain to the next and increased with severity with time. Treatments
actin and myosin (Preston and King 2005), and in apla- for thraustochytrid infections have only been tried with
nochytrids, this allows the cells to move with a crawling bivalves with limited success, with heat shock for QPX
motion, when observed by light microscope. disease in hard clams being the most successful (Dahl et al.
• The cell wall of the Labyrinthulomycetes (Moss 1986) 2011). These observations are an indication that thraus-
consists of a unit membrane over which circular scales, tochytrids could be a major, long-lasting problem in
2–3 nm thick, are laid in layers in varying numbers, cephalopod rearing facilities, if the animals are stressed or
depending on the species and age of the cell. These scales mechanically damaged.
are made either of fucose or galactose, again dependent
on species (Honda et al. 1999).
6.5 Infections Caused by Organisms Still
Other features of Labyrinthulomycetes useful for diag- Classified as Fungi
nosis are the production of biflagellated zoospores, easily
seen in culture. These are not always produced, but where Only five infections of cephalopods by organisms classified
they are, they swim with a “flipping” motion and have an as fungi have been observed to date. The same fungus is
anterior flagellum, which is covered with brush-like masti- responsible for two infections, one in an octopus and one in
gonemes, and a posterior, shorter, “whiplash” flagellum squid. This is the deuteromycete, Cladosporium sphaeros-
(Moss 1986, Chap. 11). Thraustochytrids and aplanochytrids permum. Cladosporium is a ubiquitous, salt-tolerant, gen-
are, however, extremely plastic in the life stages they can erally saprobic fungus, which has a range of beneficial and
present under different nutrient and environmental condi- deleterious habits (Bensch et al. 2012). It was first found as
tions (Fossier Marchan et al. 2018). It is therefore essential an opportunist infection of a mechanical wound in an
to sequence to identify to genus and species level. Mo et al. aquarium kept lesser octopus, E. cirrhosa (Polglase et al.
(2002) provide a comprehensive methodology, with details 1984), (Figs. 6.9 and 6.10), but these authors reported that it
of appropriate primers. could be cultured on agar plates and would re-infect wounds
(Fig. 6.11). Almost complete healing of the original wound
was observed, except where fungal hyphae ran though the
6.4 Implications of Labyrinthulomycete dermal connective tissue out to the exterior. No marked
Infections for Cephalopod Production necrosis or oedema was associated with the hyphae (pics),
but the connective tissue stained less deeply and had an
In the E. cirrhosa infection, one infected individual infected attenuated appearance. Increased numbers of rounded
an entire aquarium, because of its reliance on brick-lined haemocytes were present, but no encapsulation of the
tanks. In both cases, the infection carried over from one year hyphae was observed.
Fig. 6.9 Eledone cirrhosa; skin

wound (w) infected with
Cladosporium sphaerospermum.
Arrows indicate transparent
mycelium. Scale bar: 100 µm
120 J. L. Polglase
Fig. 6.10 Cladosporium

sphaerospermum mycelium
growing from dermis of a wound
in Eledone cirrhosa,
c = chromatophores.
Scale bar: 25 µm
Fig. 6.11 Hyphae (arrowed) of Cladosporium sphaerospermum growing back into Eledone cirrhosa dermis after re-inoculation. Arrows indicate
hyphae. c = chromatophore. Scale bar: 50 µm
Harms et al. (2006) reported a case in which a European

cuttlefish, Sepia officinalis, developed an eruptive skin lesion 6.6 Concluding Remarks
of the dorsal mantle, which biopsy and subsequent culture
revealed contained haemocyte granulomas, again induced by All these infections, both by the Labyrinthulomycetes and
a Cladosporium sp. infection. The lesion was successfully organisms still classified as fungi, have been reported in
treated by excision under anaesthesia, but the animal died captive animals, so stress and/or mechanical damage from
two months later and was found to have a multi-systemic handling are again likely to have been contributory factors,
fungal infection. allowing the infections to establish and develop. There are,
Scimeca (1994) provides a previously unpublished, brief however, still very small numbers of these reports and none
report of a Fusarium infection of the mantle of a nautilus. The to date of true fungal infections in cephalopods from the
fungus was identified using an indirect fluorescent antibody wild raising the question, “What is it about cephalopods that
test for hyphae of the Fusarium group. In this case, the hyphae keeps them free from attack by the fungi commonly found in
extended from the deep ulcer, which was the focus of the the sea, which infect other invertebrates and vertebrates to a
investigation, into the musculature of the mantle. much higher degree?”.
Finally, Scimeca and Oestmann (1995) mention a fungal
infection, presumably therefore by a mycelial organism, in Acknowledgements Jane Polglase thanks Dr. Gwyneth M. Jones,
Seatech Ltd., Halifax, Nova Scotia, Canada, and Dr. Ronald O’Dor,
the hood of Nautilus pompilius. The abstract of this con-
Dalhousie University, Halifax, Nova Scotia, Canada, for kind permis-
ference presentation does not give any further details. sion to reproduce their images.
122 J. L. Polglase
References thraustochytriaceous parasite by host amoebocytes. J Parasitol

68:243–252
Mo C, Douek J, Rinkevich B (2002) Development of a PCR strategy
Bennett RM, Honda D, Beakes GW, Thines M (2017) Labyrinthu- for thraustochytrid identification based on 18S rDNA sequences.
lomycota. In: Archibald J, Simpson A, Slamovits C (eds) Handbook Mar Biol 140:883–889
of the protists. Springer, Cham, Switzerland, pp 507–542 Moss ST (1986) Biology and phylogeny of the labyrinthulales and
Bensch K, Braun U, Graenewald JZ, Crous PW (2012) The genus thraustochytriales. In: Moss ST (ed) The biology of the marine
Cladosporium. Stud Mycol 72(1):1–401 fungi. Cambridge University Press, Cambridge, pp 105–130
Dahl SF, Perrigault M, Liu Q, Collier JL, Barnes DA, Allam D (2011) Polglase JL (1980) A preliminary report on the thraustochytrid(s) and
Effects of temperature on hard clam (Mercenaria mercenaria) labyrinthulid(s) associated with a pathological condition in the
immunity and QPX (Quahog Parasite Unknown) disease develop- lesser octopus, Eledone cirrhosa. Bot Mar 23:699–706
ment: I. Dynamics of QPX disease. J Invert Pathol 106(2):314–321 Polglase JL (1981) Thraustochytrids as potential pathogens of marine
Fossier Marchan L, Lee Chang KJ, Nichols PD, Mitchell WJ, Polglas JL, animals. Abstract published in Bull Br Mycol Soc 16(suppl 1):5
Gutierrez JT (2018) Taxonomy, ecology and biotechnological (Full manuscript now available on Research gate)
applications of thraustochytrids: a review. Biotech Adv 36(1):26–46 Polglase JL, Dix NJ, Bullock AM (1984) Infection of skin wounds in
Harms CA, Lewbart GA, McAlarney R, Christian LS, Geissler KA, the lesser octopus, Eledone cirrhosa, by Cladosporium sphaeros-
Lemons C (2006) Surgical excision of mycotic (Cladosporium sp.) permum. Trans Brit Mycol Soc 82:577–580
granulomas from the mantle of a cuttlefish (Sepia officinalis). J Zoo Preston TM, King CA (2005) Actin-based motility in the net slime
Wildlife Med 37(4):524–530 mould Labyrinthula: evidence for the role of myosin in gliding
Honda D, Yokochi T, Nakahara T, Raghukumar S, Nakagiri A, movement. J Eukaryot Microbiol 52(6):461–475
Schaumann K, Nigashihara T (1999) Molecular phylogeny of Scimeca JM (1994) The cephalopods in invertebrate medicine (2nd
labyrinthulids and thraustochytrids based on the sequencing of the edn). Wiley, pp 112–125
18S ribosomal RNA gene. J Eukaryot Microbiol 46(6):637–647 Scimeca JM, Oestmann D (1995) Selected diseases of captive and
Jones G (1981) Thraustochytrid pathogens. Bull Brit Mycol Soc 16 laboratory-reared cephalopods. Poster presented to a meeting of the
(suppl 1):5–6 International Association for Aquat Ani Med 26:79. Recorded in
Jones G, O’Dor RK (1983) Ultrastructural observations on a thraus- online proceedings at https://www.vin.com/apputil/content/
tochytrid fungus parasitic on the gills of squid (Ilex illecebrosus defaultadv1.aspx?id=3977208&pid=11257&
Lesueur). J Parasitol 69: 903–911
McLean N, Porter D (1982) Yellow spot disease of Tritonea diomedea
Bergh (Mollusca: Gastropoda: Nudibranchia): encapsulation of the
made.
Virus and Virus-like Particles Affecting
Cephalopods 7
María Prado-Álvarez and Pablo García-Fernández
Abstract
This chapter compiles the information available to date regarding virus affecting different
species of cephalopods. A clear evidence of a virus-related disease on cephalopods was not
stablished yet. However, the first description of a virus-like in Octopus vulgaris was observed
in nodular tumors that finally caused the death of the animal. It is noteworthy that not too
much effort has been focused on this area to date. However, the incidence of viruses in
cephalopods might be further investigated since the attention on these species as an
alternative to the aquaculture sector is increasing rapidly over last years and huge efforts are
being made to stablish new cultures.

Keywords
Cephalopod diseases Virus Octopus Cuttlefish Nautilus Squid
morphology observed under transmission electron

7.1 Introduction microphotographs. Further analysis would be necessary to
deepen on the effect caused by virus on these animals, espe-
Virus-like descriptions on cephalopods are rather scarce and cially considering the increasing interest on these animals as
quite spread over time. However, the incidence of viral an alternative product to satisfy the global demand on the
diseases on bivalve and gastropod mollusks has been aquaculture sector. However, advances in this scope become a
extensively studied (Arzul et al. 2017). Since first descrip- big challenge to the scientific community in part due to the
tions in 1971 in Octopus vulgaris and Sepia officinalis, only difficulty to obtain specific cell lines and culture systems.
three more observations were reported in cephalopods in
more species of the genus Octopus and Nautilus and in the
squid Loligo pealei (Table 7.1). 7.2 Iridoviridae
Family classification was initially made based on similarities
to viral mammalian diseases caused by virus (Farley 1978). First observation of virus-like particles affecting a cephalopod
In more recent studies, molecular diagnosis was applied to was made in 1971 in O. vulgaris (Rungger et al. 1971). Nodular
amplify genomic material and search for homology in public tumors on tentacles of 1 mm diameter were observed at early
databases. However, it is important to remark that classifica- stages. However, these tumors can develop and grow up to
tion made on this chapter is based only on the information 5–10 mm and be extended to the ventral surface and also the
available to date which in most cases were made based on siphon. The progress of the infection leads to self-mutilation of
affected areas, and death occurred after 3–5 months in captiv-
M. Prado-Álvarez (&) P. García-Fernández
Aquatic Molecular Pathobiology Group, Institute of Marine ity. Electron micrographs of tumor tissues at early stage showed
Research, Spanish National Research Council (CSIC), 36208 organized virus-like particles that appeared in groups of 300
Vigo, Pontevedra, Spain particles with hexagonal and electrodense internal cores and
120 nm diameter. It was also observed an envelope surround-
P. García-Fernández ing these particles in disaggregated tissues. Mortality was

https://doi.org/10.1007/978-3-030-11330-8_7
124 M. Prado-Álvarez and P. García-Fernández
Table 7.1 Summary of descriptions of viruses and virus-like particles in cephalopods

Year Cephalopod sp. Virus family Reference
1971 Octopus vulgaris Iridoviridae Rungger et al. (1971)
1971 Sepia officinalis Reoviridae Devauchelle and Vago (1971)
1988 Loligo pealei – Pers. Comm. to authors (Hanlon and Forsythe 1990)
1988 Octopus spp. – Pers. Comm. to authors (Hanlon and Forsythe 1990)
2006 Nautilus spp. Iridoviridae Gregory et al. (2006)
2015 O. vulgaris Nodaviridae Fichi et al. (2015)
2018 O. vulgaris Malacoherpesviridae Prado-Álvarez et al. (in preparation)
described to be due to starvation, self-mutilation, or the pre- they could be spherical and infect a wide number of hosts
sumed viral infection associated with these tumors. In 1978, including vertebrates, invertebrates, plants, protists, and
Farley classified this virus particle as Iridoviridae (Farley fungi (Lefkowitz et al. 2018).
1978). The family Iridoviridae comprises icosahedral
double-stranded DNA viruses and mainly affects ectothermic
vertebrates, insects, and crustaceans (Chinchar et al. 2017). 7.4 Nodaviridae
Morphology similar to an Iridovirus was found in renal
tubular epithelial cells of Nautilus spp. (Gregory et al. 2006). Finally, a betanodavirus (family Nodaviridae) was described
Viral particles were enveloped and measured 176 nm in affecting O. vulgaris (Fichi et al. 2015) particularly in white
diameter. Moreover, this was the first characterization that lesions observed in the skin, and also in eye and branchial
included molecular identification and sequencing to confirm heart tissues. Molecular detection by PCR and quantitative
the similarity to an amphibian Iridovirus. PCR confirmed the incidence of betanodavirus. However,
one animal showing similar symptom seemed to be affected
by a nodavirus-like particle. Virions belonging to this family
7.3 Reoviridae are non-enveloped, 30 nm in diameter, and single-stranded
RNA (Artimo et al. 2012).
Devauchelle and Vago (1971) reported a no-pathogenic
incidence of a virus in the European cuttlefish, Sepia offici-
nalis. These non-enveloped particles were observed on 7.5 Unclassified Viruses
epithelial cells on the stomach and were 75 nm in diameter.
Initial studies suggested that these particles could correspond Hanlon and Forsythe revised in 1990 the descriptions of
to the family Reoviridae. Reoviridae are viruses with virus-like particles in cephalopods made to date (Hanlon and
icosahedral symmetry and double-stranded RNA. However, Forsythe 1990). Apart from the two descriptions detailed
Fig. 7.1 In situ hybridization label- (a) (b)

ing of ostreid herpesvirus. Positive
cells are labeled in blue. Gill tissue of
adult O. vulgaris (a). Detailed area at
higher magnification (b). Scale
bar = 10 µm
7 Virus and Virus-like Particles Affecting Cephalopods 125
above, two more annotations were made based on personal References

communications to the authors. One was in the squid Loligo
pealei on which cytoplasmic inclusions were found in the Artimo P, Jonnalagedda M, Arnold K et al (2012) ExPASy: SIB
digestive gland and the other on different species of octopus bioinformatics resource portal. Nucleic Acids Res 40:W597–W603
in New Zealand and the east and west coasts of USA. Arzul I, Corbeil S, Morga B, Renault T (2017) Viruses infecting marine
molluscs. J Invertebr Pathol 147:118–135
More recently, an interspecific study of the incidence of the
Chinchar VG, Hick P, Ince IA et al (2017) ICTV report consortium.
ostreid herpesvirus demonstrated that the virus was widely ICTV virus taxonomy profile: Iridoviridae. J Gen Virol 98:890–891
spread in oyster-culturing areas. This virus belongs to the Davison AJ, Eberle R, Ehlers B et al (2009) The order Herpesvirales.
family Malacoherpesviridae and caused massive mortalities in Arch Virol 154:171–177
Devauchelle G, Vago C (1971) Particles of viral asepct in stomach cells
spat and juvenile oysters (Prado-Álvarez et al. 2016). This
of the cuttle-fish Sepia officinalis L. (Mollusca, Cephalopoda). C R
family comprised of enveloped double-stranded DNA viruses, Acad Sci Hebd Seances Acad Sci D 272(6):894–6
150–200 nm in diameter, and affects to mollusks (Davison et al. Farley C (1978) Viruses and viruslike lesions in marine mollusks. Mar
2009). A collection of samples of O. vulgaris including par- Fish Rev 40:18–20
Fichi G, Cardeti G, Perrucci S et al (2015) Skin lesion-associated
alarvae at different stages and adult animals were analyzed by
pathogens from Octopus vulgaris: first detection of Photobacterium
molecular techniques. Homology searches on positive results swingsii, Lactococcus garvieae and betanodavirus. Dis Aquat
showed the highest similarity to the ostreid herpesvirus-1 µVar Organ 115:147–156
(unpublished data). Moreover, ostreid herpesvirus-specific Gregory CR, Latimer KS, Pennick K, Benson K, Moore T (2006)
Novel Iridovirus in a Nautilus (Nautilus Spp.). J Vet Diagnostic
probes for in situ hybridization were positively labeled on
Investig 18:208–211
epithelial cells of the gills (Fig. 7.1). Further analysis should be Hanlon RT, Forsythe JW (1990) Diseases of Mollusca: Cephalopoda.
carried out to decipher if this virus could be the causative agent Diseases caused by microorganisms. In: Kinne O (ed) Diseases of
of any pathology in these animals. marine animals, vol III. Biologische Anstalt Helgoland, Hamburg,
Germany, pp 23–46
Lefkowitz EJ, Dempsey DM, Hendrickson RC, Orton RJ, Siddell SG,
Smith DB (2018) Virus taxonomy: The database of the International
7.6 Concluding Remarks Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res 46:
D708–D717
Several virus and virus-like particles were described on Prado-Álvarez M, Darmody G, Hutton S, O’Reilly A, Lynch SA,
Culloty SC (2016) Occurrence of OsHV-1 in Crassostrea gigas
cephalopods. However, a clear incidence associated with a Cultured in Ireland during an exceptionally warm summer.
virus-related disease was not well described to date. It Selection of less susceptible oysters. Front Physiol 7:492
deserves to be further investigated as cephalopods could be Rungger D, Rastellt M, Braendle E, Malsberger RG (1971) A viruslike
considered as an alternative to be aquacultured and satisfy particle sssociated with lesions in the muscles of Octopus vulgaris.
J Invertebr Pathol 17:72–89
the global demand.
made.
Bacteria-Affecting Cephalopods
8
Rosa Farto, Gianluca Fichi, Camino Gestal, Santiago Pascual,
and Teresa Pérez Nieto
Abstract
Bacterial pathogens contribute to obtain an unsuccessful production of cephalopods. An
updated overview of the knowledge of these pathogens must be a valuable tool to improve their
aquarium maintenance and aquaculture. The present work provides a description of the main
bacterial pathogens associated with larval stages of cultured Octopus vulgaris, and juvenile and
adults of several cephalopods. Vibrio species, reported with ability to cause vibriosis in
aquaculture, are the main bacteria associated with skin lesions in adults. Different species of
Pseudomonas and Aeromonas, among others, have also been detected. Furthermore,
gram-positive bacteria such as Bacillus have been also described. Among them, V.
alginolyticus, V. carchariae, V. parahaemolyticus, V. splendidus and V. lentus have also been
isolated from sterile organs or fluids of animals and their potential as invaders proved. However,
only V. alginolyticus or V. lentus has the ability to cause lesions, and, in addition, the last one is
proved as the causative agent of death in octopuses. Other organs such as eyes of squids are also
colonized by Vibrio species or Micrococcus sp., and recently Photobacterium swingsii and
Lactococcus garvieae have been reported associated with a retrobulbar lesion in octopus.
Rickettsial-like organisms (RLO) are also detected in the gills of the octopus, having a
detrimental effect on the respiratory gaseous exchange of the animals. Cultures of octopus
paralarvae show a genetically diverse community comparable to those reported previously from
other marine hatcheries. Bacteria included in the Splendidus clade is the dominant group in all
conditions, except in one of them, where V. alginolyticus, V. proteolyticus or Pseudomonas
R. Farto (&) T. P. Nieto

Marine Research Centre (CIM-UVIGO), University of Vigo,
36331 Vigo, Pontevedra, Spain
T. P. Nieto
G. Fichi
Istituto Zooprofilattico Sperimentale delle Regioni Lazio e
Toscana, 56123 Pisa, Italy
e-mail: Gianluca.fi[email protected]
C. Gestal
Aquatic Molecular Pathobiology Group, Institute of Marine
Research, Spanish National Research Council (CSIC), 36208
S. Pascual

https://doi.org/10.1007/978-3-030-11330-8_8
128 R. Farto et al.
fluorescens are the main detected groups. Furthermore, Shewanella or Pseudoalteromonas

undina have also been identified. All this shows that pathogenic bacteria are frequent
microorganisms associated with aquarium maintenance and culture of cephalopods, and special
attention on maintaining a well-balanced community of microorganisms should be applied.

Keywords
Cephalopod diseases Paralarvae Microbial community Pathogenic bacteria
Splendidus clade Rickettsia-like organisms
8.1 Introduction respect to that of reared ones (Roura et al. 2017). Although
aquaculture systems are designed to imitate the natural
Incidence of diseases, several of them caused by bacteria, is environment, the maintenance of adequate conditions during
one of the most important problems which avoid obtaining a all culture production is problematic. Furthermore, diets
successfully production in the aquaculture of cephalopods or provided are less diverse than in natural media. Therefore, it
its suitable maintenance in captive conditions (García-- can produce alterations in microbial community of seawater
Fernández et al. 2016; Sykes and Gestal, 2014). and/or surface (external or internal) of cephalopods. This
Knowledge on bacterial species associated with cephalo- provides an opportunity for the dominance of bacteria,
pods culture and its ecological role would be essential for which show the ability to invade the host, and in the end, to
improving industrial culture production. Seawater have a cause a fatal infection. On the other hand, this is also
variable microscopic population density (Harder 2009), and it facilitated when the surface of the animal is injured.
contains a high microbial diversity depending on In 1990, Hanlon and Forsythe described the main bac-
physico-chemical and geographic factors such as the tem- terial pathogens associated with juvenile and adults. In fact,
perature of the water, latitude and salinity. The ability of they included data from published literature, and their own
bacteria to colonize surfaces (epibiotic bacteria) is very well data collected from analyses resultant of 14 species of dif-
documented, and they comprise well-organized bacterial ferent cephalopods for 13 years. Species of Vibrio, mainly,
communities associated with marine organisms, which are but also members of the genera Pseudomonas, Aeromonas,
influenced by temporal changes in the environment. However, Staphylococcus or Streptococcus, among others, have been
some bacteria are specifically and persistently associated with associated with healthy octopuses or squids in nature. Some
particular marine animals, and they are not found in seawater of these genera have also been associated with the ulcerated
or on other animals (Thakur et al. 2004; Sharp et al. 2007). skin of octopuses and squids from their natural environment
This can explain the species-specific vulnerability to patho- or laboratory-maintained, being this, their route of entrance
gens previously suggested for Octopus bimaculoides and O. in animals, and progressing later until a fatal infection.
maya (Hanlon and Forsythe 1990). These ‘microepibionts’ Furthermore, a possible food-borne was the suggested route
are multi-functional and are involved in obtaining nutrients, of entrance for V. carchariae, which was associated with
acquiring new genetic traits and providing some measure of sudden death of laboratory-maintained octopuses, which
chemical defence against pathogens (Wahl et al. 2012). In showed lack of any external of behavioural symptoms.
these epiphytic communities, developing biofilms is the most Similarly, an unknown route of entrance, not associated with
common, and quorum sensing modulates behaviour of many ulcerated skin, was proposed for cuttlefish which suffered a
biofilm-associated bacteria, affecting symbiotic relationships highly virulent systemic infection. Squids with eye damage
and host interactions (Maximilien et al. 1998; Parsek and associated with bacteria were also reported, but no further
Greenberg 2005). Co-infection is another important factor information on the route of infection was given. However, it
affecting bacterial diseases, in fact in previous studies parasite is unknown if bacteria are the initial cause of infection, with
infection enhanced bacterial invasion in fish (Rodrí- the exception of one study, where V. alginolyticus was
guez-Quiroga et al. 2016). proved as causation of lesions in octopus. Similarly, there is
Differences in culturable bacteria most frequently isolated a lack of information on culturable bacteria associated to
from skin of apparently healthy, wild or laboratory- different stages of cephalopods in reared conditions.
maintained adult squids have been previously found. In In attempting to be comprehensive, we reviewed the
fact, a higher diversity is shown in the former ones (Hanlon knowledge about bacterial disease in the most representative
and Forsythe 1990). Similar data are observed in the gas- European cephalopod species to date, detailing the main
trointestinal microbiome of wild octopus paralarvae with bacterial pathogens associated with juvenile or adults.
8 Bacteria-Affecting Cephalopods 129
Furthermore, data from bacterial associated with larval 8.2.1 Microbial Community Counts
stages of cultured octopuses (unpubl.), recently qualitatively
sampled ‘in situ’ by us were also included. 8.2.1.1 Water Samples
The common signs caused by bacteria on cephalopods, Culturable bacteria was isolated on three different dates cor-
and, when possible, images, are shown. In addition, it is responding to Experiment 1, 2 and 3 from seawater associated
included a brief description of characteristics of bacteria, with different stages of an O. vulgaris culture in Galicia
diagnostic and treatments used against bacteria. (Table 8.1). After 13–15 days of paralarvae culture, samples
were taken on the same day in all stages of each Experiment.
Seawater inside the hatchery was filtered until 1 µm for all
8.2 Potential Pathogenic Bacteria for Larval experiments, but in addition, a high wattage UV treatment was
Development Stages also applied for Artemia (Artemia salina) culture. Moreover,
seawater filtered until 20 µm and treated with low wattage UV
Bacterial septicaemia was occasionally observed in paralar- was analysed for comparative purposes (Experiment 4).
vae (13–15 days old) of O. vulgaris reared in aquaculture Standard conditions of natural photoperiod, seawater tem-
tanks, which could be causative of the dead of the paralarvae perature (19–23 °C) and salinity of 33‰ were maintained.
(Fig. 8.1a, b). The identification of the aetiological agent Larval tanks (1000 L) containing 5 larvae l−1 were reared
was not established by the authors. Seawater quality, and following the protocol described by Iglesias et al. (2004). In this
maintenance and cleaning precautionary measures were study, paralarvae were fed with Artemia nauplius or Artemia
suggested as factors that could facilitate the growth of nauplius complemented with Maja brachydactyla zoeas or
pathogenic bacteria that as a result caused the infection. freeze-dried feed (made from different species of crustaceans).
Previous studies have revealed that uncontrolled factors in Water samples (1 ml) were serially diluted in sterile sea-
the culture have an important impact on the establishment water and spread out (0.1 mL) over plates of Tryptic Soy Agar
and evolution of the microbial community in the culture supplemented up to 1% (w/v) NaCl (TSA-1) and Thiosulphate-
tanks of Artemia (Verschuere et al. 1997). Similar results Citrate-Bile-Sucrose (TCBS) in duplicate. Colonies were
were also found for culture of clams (Kwan and Bolch counted after 4 days of incubation at 22 °C, and the results
2015). It was also shown that an effective balance of expressed in colony-forming units (CFU) per ml. Whereas
microbial populations included in the same community TSA-1 was used for total heterotrophic bacteria counts, TCBS
ensures the final rearing success (Verschuere et al. 1997; was for presumptive Vibrio ones. The last one was used since
Kwan and Bolch 2015; Shi et al. 2017). Vibrio species are a significant component of culturable marine
Recently, it has been proved that the intestinal micro- bacterial populations (Thompson et al. 2004; Montes et al.
biome, highly diverse, of octopus paralarvae reared in cap- 2006; Beaz-Hidalgo et al. 2008; Guisande et al. 2008).
tivity and feed with Artemia is rapidly become less diverse Colonies of each morphology from TSA-1 or TCBS were
and is mainly suggested as a consequence of culture con- picked off and spread on TSA-1 to obtain pure cultures, and
ditions (Roura et al. 2017). However, to date, there is an then inoculated on Tryptic soy broth with 1% (w/v) NaCl
absence of published data available on diversity of bacteria and 15% (v/v) of glycerol for their conservation at −80 °C.
from cephalopod hatcheries. Recent qualitative preliminary
analysis of seawater associated with different stages of O. 8.2.1.2 Microbial Counts
vulgaris hatchery culture has focused to characterize and Total heterotrophic bacteria associated with the octopus
identify the culturable bacteria. cultures are shown in Table 8.1. The results showed that
Fig. 8.1 O. vulgaris paralarvae

reared in aquarium conditions.
a Histological section of the con-
nective tissue of paralarvae infected
by bacteria (arrow). b Detail show-
ing the strong septicemia observed
in the whole paralarvae. H&E stain.
Images a and b by courtesy of Dr.
R. Fernández-Gago. Scale bars a,
b 20 µm
130
Table 8.1 Bacterial counts and species identified in different paralarval stages of O. vulgaris hatchery
Experiment 1 Experiment 2 Experiment 3 Experiment 4
WIH1 WOA ATW ZTW PTW + AZ PTW + DA PTW + DAZ DAWT PTW + A PTW + ACF WIH2
Culturable bacteria counts
Total heterotrophic (TSA-1) (CFU/ml) 7 102 – 1 104 1.3 104 2 104 4 102 1.5 102 1 101 7.5 105 4 106 1.8 104
(± 7 103) (± 5 103) (± 3 102) (± 5 101) (± 2 105) (± 7 103) (± 7 103)
Presumptive Vibrios (TCBS) (CFU/ml) – – 4.8 102 2.5 102 9 102 1.9 102 1 102 1 101 5.8 104 7 105 4.8 103
(± 2.1 102) (± 1.5 102) (± 1 102) (± 1 102) (± 1.5 103) (± 5 104) (± 2 103)
Species identified
Splendidus Clade
V. atlanticus/V. tasmaniensis +(32) +(23) +(1, 2, 3, 8, +(4, 5, 7) +(25, 38)
13)
V. gigantis/V.crassostreae/V.pomeroyi +(34) +(31, 35, 36) +(33) +(6, 14) +(11)
V. gallaecicus +(39)
V.hemicentroti/V.lentus +(10) +(26, 28, 29)
V. alginolyticus +(27)
V. neptunius +(30)
V. proteolyticus +(18, 19) +(21) +(12)
P. fluorescens +(17, 20) +(16)
Pa. undina +(24)
Sh. marinintestina/Sh. sairae/Sh. +(9, 22) +(15)
schelegeliana
In brackets SE for bacteria counts and strain for species identified. WIH1 seawater inside the hatchery filtered until 1 µm; WOA: seawater outside Artemia culture; ATW Artemia tank water; ZTW Zoeae tank
water; PTW + AZ octopus paralarvae tank water mixed with Artemia and zoeae; PTW + DA octopus paralarvae tank water mixed with disinfected Artemia; PTW + DAZ: octopus paralarvae tank water mixed
with disinfected Artemia and zoeae; DAWT disinfected Artemia water tank; PTW + A octopus paralarvae tank water mixed with Artemia; PTW + ACF octopus paralarvae tank water mixed with Artemia and
freeze-dried food; WIH2 seawater inside the hatchery filtered until 20 µm and treated with low wattage UV
R. Farto et al.
there are strong evidences that the food (live or freeze-dried) 0% NaCl or in TCBS and susceptibility to O129 vibriostatic
supplied to the larvae constitutes the main source of the agent (2, 4-diamino-6, 7-di-isopropylpteridine phosphate;
bacteria associated to the cultures of the octopus larvae. 150 lg/disc).
A significant percentage (50–75%) of total heterotrophic These assays indicated that all the strains were
bacteria grew in TCBS, and as it was confirmed in this study, gram-negative motile rods and that they were positive for all
most of them were included in genus Vibrio (Table 8.1). tests except for being able to grow at 0% NaCl and the
Their counts remained uniform in most of the culture con- glucose metabolism. The two last tests allowed discrimi-
ditions studied. The lowest value was detected in the cultures nating three groups of strains; (1) strains showing
treated with disinfectant, whereas the highest was detected in aerobic/anaerobic glucose metabolism, being all of them
that containing Artemia mixed with freeze-dried feed. These unable to grow at 0% NaCl (facultative anaerobes);
results are in agreement with those of scallop larval culture, (2) strains without aerobic/anaerobic glucose metabolism,
where concentrations of Vibrio varied only between 102 and being all of them unable to grow at 0% NaCl; (3) strains
103 cels ml−1 (Nicolas et al. 1996). However, higher counts without oxidative/fermentative glucose metabolism (aer-
from a culture water of Artemia (103–104 cfu ml−1) were obes), but with ability to grow at 0% NaCl.
detected in early times of the culture period (Verschuere All these tests presumptively grouped all facultative
et al. 1997). Variations in Vibrio counts are frequent in anaerobes in genus Vibrio. All of them were able to grow
hatcheries. In fact, an increase of Vibrio counts associated to well on TCBS and allowed discriminating between strains
clam’s hatchery was previously found after the rise of with ability to use sucrose (Table 8.2). Although, this
environmental temperature (Mechri et al. 2012). medium is used for the isolation of Vibrio spp., it is also
Finally, in Experiment 4, the total heterotrophic bacteria possible, but with poor growth, for other genera (Guisande
and the presumptive Vibrio were increased in 2-log or 3-log et al. 2004). In fact, this was the case for our bacteria
units, respectively, with respect at those from water inside identified as P. fluorescens.
the hatchery used for larval culture. Variations in the treat- In order to confirm this presumptive identification, sec-
ment of the seawater (mainly due to filtration) could explain ondary biochemical tests were performed for the anaerobic
these results. facultative group by using the commercial phenothypic test
API 20E (BioMerieux) (Table 8.2). This system is frequently
used for the identification of fish pathogens, since its database
8.2.2 Characterization of Culturable Bacteria includes an important number of them. However, it is well
Associated with Octopus Paralarvae known that several reactions, among them decarboxylases
Culture [arginine dehydrolase (ADH), lysine decarboxylase (LDC),
ornithine decarboxylase (ODC)], must be compared with
To carry out the characterization of bacteria associated with conventional biochemical tests, since if there are differences,
the different culture conditions studied, a total of 38 strains data from the last are preferable as reference (Buller 2004;
corresponding to the colonies that had different morpholo- Popovic et al. 2007). In addition, they were also included in
gies on TSA-1 or TCBS were selected. For their character- this study, and they were determined as described by Montes
ization, primary and secondary identification biochemical et al. (1999). Presence of Vibrio was confirmed by the system,
tests and PCR with specific primers were performed, but the and its database allowed determining that the strains were
final identification and/or assignation to closely related closely related to V. alginolyticus, V. proteolyticus and
species was performed by sequencing of 16S rRNA gene. V. splendidus.
All strains were characterized by phenotypical tests and It is well known that V. splendidus is phylogenically
only 30 by sequencing of 16S rRNA gene. The closely related to other species all of them termed as V.
closest-neighbouring species, which shared a similarity splendidus-related group (Splendidus clade), which is the
value in the 16S rRNA sequences of 98%, were used to most diverse among Vibrionales (Sawabe et al. 2007). API
identify the isolates. Molecular identification was assigned to 20E was unable to discriminate these species since strains
the remaining strains (8), when the strains identified by 16S included in the same group showed variable biochemical
rRNA and the unidentified ones shared the same pheno- profiles (Table 8.2). It is also difficult by using numerical
typical profile. taxonomy or molecular methods (Lago et al. 2009; Revised
by Oden et al. 2016).
8.2.2.1 Phenotypical Characterization
Firstly, the strains were phenotypically characterized by 8.2.2.2 Molecular Characterization
using a set of primary identification tests: gram staining, In a previous study, the combined specificity of V. tas-
motility, oxidase, catalase, oxidative or fermentative glucose maniensis (VTS/VT) and V. splendidus (VTS/VS) primer
metabolism on O/F Basal Medium (O/F), growth ability at sets offered the best coverage (86%) in terms of separating
132 R. Farto et al.
Table 8.2 Characterization and Identification of Vibrio strains associated with of Octopus vulgaris hatchery
Groups identified by sequencing assignmenta
Splendidus clade V. alginolyticus V. neptunius V. proteolyticus
V. atlanticus/ V. gigantis/ V. gallaecicus V. hemicentroti/
V. tasmaniensis V. crassostreae/ V. lentus
V. pomeroyi
Tests to identify strains
Sequence (16S rRNA) 98.7% 99.5% 99.8% >98.5% 99.9% 99.9% 99.9%
identity (%) with the
closest neighbour
Amplification with specific primers
VTS/VT (V. +(10/12) +(4/8) + +(1/4) − − −
tasmaniensis)
VTS/VS (V. splendidus) +(4/12) +(4/8) − − − − −
VN1/VN2 (V. neptunius) − − − − − + −
Biochemical characteristics
TCBS (sucrose +)b +(8/12) +(4/8) − − + + −
TCBS (sucrose −) b
+(4/12) +(4/8) + +(4/4) − − +(3/3)
Arginine dihydrolase + + + + − + +
(ADH)b
Lysine decarboxylase − − − − + − +
(LDC)b
Ornithine decarboxylase − − − − + − −
(ODC)b
ß-galactosidasec +(4/8) +(7/8) − + − − −
Gelatinase c
+(5/8) + − + + + +
Citrate utilizationc −(5/8) − − − − + +
Voges Proskauer c
− − − − + − +
NO2 productionc + + + + + − +
c
Fermentation/Oxidation
Amygdalin + +(5/8) + +(1/2) + + −
Arabinose −(7/8) − − − − − −
Inositol + +(7/8) + + + − +
Melibiose −(6/8) −(7/8) − − − − −
Rhamnose +(5/8) −(5/8) − − + − −
Sorbitol +(7/8) +(6/8) − +(1/2) + − +
Sucrose +(5/8) +(6/8) − − + + +(2/3)
a
Multiple alignment of sequences was created by ClustalW in Genious Editor version 12.0.6 (Biomatters; http://www.geneious.com/). This included 1.138
positions after the removal of ambiguous ones. A phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) version 6
(Tamura et al. 2013). This was performed using the neighbour-joining method and Tamura–Nei distance model, with the calculation of cluster stability by
bootstrap analysis with 1000 replicates. All available species of each genus closest to studied strains were selected
b
Tests performed by using conventional biochemical tests
c
Tests performed by using API 20E. All strains were negative for H2S production, hydrolysis of urea or presence of tryptophane deaminase; and they were
positive for indole production and fermentation/oxidation of D-glucose or D-mannitol (all of them performed by using API 20E). In brackets number of
positive or negative strains of all assayed
several of the species included in the Splendidus clade, and primer sets, respectively (Table 8.2). Negative amplification
from other Vibrio species (Lago et al. 2009). In this study, a was shown by the remaining strains characterized as She-
positive amplification was shown in all strains which were wanella (Sh.) or Pseudomonas; on the contrary, a positive
characterized in the Splendidus clade, except in six (13, 26, amplification with the primer set VTS/VT was shown by
28, 29, 31, 33). In fact, from the positive ones, 84% (16/19) strain 24, which was identified by 16S rRNA gene analyses
and 42% (8/19) of strains reacted with VTS/VT and VTS/VS as Pseudoalteromonas (Pa.) undina. Furthermore, a
V. neptunius primer set was highly specific, showing only from marine biofilters at a recirculation aquaculture system
cross-reaction with V. parahaemolyticus species from 44 (Snoussi et al. 2006; Borges et al. 2008). Other species of
tested species (Lago et al. 2009). Similarly, in this study only Pseudomonas were also associated with oyster larvae (Farto
one strain, which was identified by 16S rRNA gene analyses et al. 2006) and with Artemia culture (Verschuere et al.
as V. neptunius, was positive (Table 8.2). Since these primer 1997). Finally, Shewanella or Pa. undina were also identi-
sets were developed, the number of species included in the fied associated with culture water containing octopus par-
Splendidus clade has increased (revised by Oden et al. alarvae and commercial feed or Artemia of this study. These
2016). In this study, it is shown that they have cross-reaction groups were also previously found from healthy oyster
with them. Nevertheless, these results revealed that the pri- cultures (Farto et al. 2006).
mer sets are a valuable tool for a fast detection of Splendidus It is well known that several of the groups isolated in this
clade, and to separate this clade from other Vibrio species. study may have a role in larval mortality in cultures. In fact,
Additionally, a sequencing of 16S rRNA gene analyses several bacterial included in the Splendidus clade are con-
was performed according to the method described by Gui- sidered pathogenic for bivalve molluscs (oyster, clam, scal-
sande et al. (2008), in order to confirm all these results. The lop, mussel) (Revised by Beaz-Hidalgo et al. 2009; Revised
strains assigned to each phylogenetic group are shown in by Kwan and Bolch 2015; Vanhove et al. 2015; Cheikh et al.
Table 8.1, and the sequence identity (%) with the closest 2016) and also cephalopod molluscs (Farto et al. 2003;
neighbour in Table 8.2. These results allowed to confirm Iehata et al. 2016). Similarly, V. alginolyticus was patho-
that 81% (31 of a total of 38 strains) of strains were facul- genic for carpet shell clam larval and juvenile (Gómez-León
tative anaerobes, and the remaining was aerobic. Among et al. 2005; Mechri et al. 2015), fish (Liu et al. 2004; Zheng
facultative anaerobic bacteria, the dominant genus identified et al. 2017), crustacean (Jayaprakash et al. 2006; Xu et al.
was Vibrio (100%). The highest number (63.2%) of Vibrio 2013), scallop (Riquelme et al. 1996), red abalone
strains was included in Splendidus clade, and they were (Anguiano-Beltrán et al. 1998), associated with infected
assigned to different groups depending on their closest Chilean octopus eggs (Iehata et al. 2016), with ability to
similarity with species (Table 8.2). In addition, one strain cause skin lesions to young octopus (Hanlon and Forsythe
was identified as V. gallaecicus. 1990), and isolated from skin ulcers and/or different organs
The 16S rRNA gene analyses allowed a more complete of cultured cuttlefish (Sangster and Smolowitz 2003). V.
identification since V. gallaecicus, V. neptunius, V. prote- proteolyticus has been previously identified as part of the
olyticus, Pa. undina and P. fluorescens were identified. Vibrio consortium isolated from diseased corals (Cervino
Furthermore, several strains were shown more closely rela- et al. 2008), and were identified as pathogens of Artemia
ted to specific species of Splendidus clade, V. alginolyticus spp. (Verschuere et al. 2000). Another pathogen described
or Shewanella. for Artemia sp. is V. neptunius (Verschuere et al. 2000;
Austin et al. 2005), being also for oyster (Prado et al. 2005;
8.2.3 Pathogenicity of Culturable Bacteria Guisande et al. 2008). There are several strains of Pa. undina
Associated with Octopus Paralarvae described as biological controllers of virus (Maeda et al.
Culture 1997), however, other were pathogens for gilthead sea
bream or scallop (Pujalte et al. 2007; Sandaa et al. 2008).
The Splendidus clade is the most abundant in marine sam- The genus Pseudoalteromonas was also previously found
ples associated with several marine animals, water column, associated with infected Chilean octopus eggs (Uriarte et al.
occurring in bacterioplankton and sediments (revised by 2011; Iehata et al. 2016).
Pérez-Cataluña et al. 2016), and also are part of regular Then, although there is an absence of published data
components of farmed aquatic animal microbiota (Montes available from octopus hatcheries, the culturable species
et al. 2003; Guisande 2004; Baez et al. 2008; Kwan and observed are comparable to those reported previously from
Bolch 2015; Oden et al. 2016). other marine hatcheries. This diversity resembles that of
In this study, these strains were the most abundant in all coastal seawater (Thompson et al. 2005), and several of them
culture conditions analysed, except in the conditions of can cause mortality in hatchery production. Previous studies
Experiment 1, where dominant groups were strains identified have shown that differences in dominance of a specific group
as V. proteolyticus, V. alginolyticus or P. fluorescens. Sim- can be explained by variations in interactions within the total
ilarly, V. alginolyticus was also the predominant species bacterial community due to alterations in environmental
associated with clam or gilthead sea bream hatcheries conditions of cultures. Those changes can encourage domi-
(Snoussi et al. 2006; Mechri et al. 2012); P. fluorescens was nance and diversity of more virulent species (Kwan and
also found associated with the sea bream hatchery, and it Bolch 2015). So, in our study, all cultures of octopus larvae
was one of the autochthonous denitrifying bacterium isolated analysed showed similar levels of mortality, and also a
134 R. Farto et al.
genetically diverse community, but different between showing deep wounds in arms or head mantle of octopus
Experiment 1 and Experiments 2–3. (Hanlon and Forsythe 1990; Farto et al. 2003).
Further studies are needed to understand the equilibrium Previously, until 36 bacteria species had been isolated
among a community of microorganisms, and how changes in from skin ulcers of diseased octopuses or squids, most of
husbandry practices select for more virulent genotypes. them from cultured conditions. Particularly, the majority
corresponded to gram-negative bacteria. From these, the
mainly ones were different species from the genus Vibrio
8.3 Potential Pathogenic Bacteria with ability to cause vibriosis such as V. anguillarum, V.
for Juvenile and Adults alginolyticus, V. parahamolyticus and V. splendidus. Dif-
ferent species of Pseudomonas and Aeromonas, and pre-
Several bacteria have been associated with diseased cepha- sumptive Cytophaga-like strains were also detected.
lopods (octopuses, squids and cuttlefish). Although most of Furthermore, gram-positive bacteria were also detected such
them have been isolated from skin lesions, they were also as Bacillus and Staphylococcus (Hanlon and Forsythe 1990;
from sterile organs or fluids (gill heart, reproductive organs, Farto et al. 2003; Tsai et al. 2012; Fichi et al. 2015).
haemolymph) and others (eye). Particularly, mainly several However, only V. alginolyticus or V. lentus have been
species of Vibrio, such as V. alginolyticus, V. para- confirmed with ability to cause lesions in octopus (Hanlon
haemolyticus, V. splendidus or V. lentus, and recently Pho- and Forsythe 1990; Farto et al. 2003). All these studies have
tobacterium (Ph.) swingsii and L. garvieae have been also shown that is frequent that skin lesions contain different
reported. This fact confirms their potential as invaders, and species of bacteria simultaneously.
also the positive septicaemia described in several cases, A brief description of several bacteria indicated in this
which is associated with advanced stages of infection. The section is shown below.
halting of disease progression and the promotion of healing,
after antibiotic treatment of several animals, also show a role Vibrio alginolyticus and Vibrio parahaemolyticus
of bacteria in diseases of animals (Hanlon and Forsythe
1990; Sherrill et al. 2000; Sangster and Smolowitz 2003). Both V. alginolyticus and V. parahamolyticus are motile
However, most of bacteria were not confirmed as the cause gram-negative bacteria in Vibrionaceae family,
of death of animals, and it is uncertain if they were the initial Gammaproteobacteria class, which have been isolated fre-
cause of infection. In this section, a brief reference to those quently from marine and coastal waters throughout the
bacteria not confirmed as the causative agent of death and/or world. Particularly, in a clam hatchery located in the
the initial cause of infection are included. Mediterranean coast has been shown that V. alginolyticus
strains were the most dominant, and V. parahaemolyticus
strains represent only 2% of all Vibrionaceae isolated
8.3.1 Miscellaneous Bacteria Associated (Mechri et al. 2012). Both have been associated with marine
with Skin Lesions organism’s diseases, and molluscs among them. This is
detailed for V. alginolyticus in Sect. 8.2 (Pathogenicity).
There are several studies showing a variety of bacteria with Furthermore, an exhaustive description of infection stages of
ability to colonize skin lesions in octopus maintained in this species was previously reported for cultured sepoids,
captivity. Crowding and increased contact among cultured suggesting, in addition to skin lesion, other routes of
octopus are proposed as the more probable cause of physical entrance in animals (Sangster and Smolowitz 2003). On the
injury, developing into ulcerations due to colonization of other hand, in diseases affecting small abalone, Spanish
bacteria, which would induce a fatal infection affecting toothcarp and shrimp was isolated V. parahaemoyticus
multiple organs in the same individual, if untreated. Simi- (Alcaide et al. 1999; Liu et al. 2000; Choi et al. 2017).
larly, this would be the process for wild-caught squids Both species were also isolated from skin ulcers of O.
maintained in the laboratory or cuttlefish maintained during joubini, but only V. alginolyticus was proved with ability to
a 12-year period in exhibition aquariums. Furthermore, produce ulcers in this species (1 g) at concentrations of 106
several captured wild octopuses displayed skin lesions, CFU ml−1, after performing an incision on mantle in order to
which became worse under laboratory conditions (Hanlon provide an invasion site for bacteria (Hanlon and Forsythe
and Forsythe 1990; Sherrill et al. 2000; Farto et al. 2003). 1990). Furthermore, both species were also isolated from
The clinical signs most frequently described for octopus skin lesions of cultured O. vulgaris (Fichi et al. 2015). Small
include skin ulcers in the head through which bacteria and larger lesions were detected (Fig. 8.2), showing a white
penetrate, progressing into an advanced infection stage appearance similar to those previously described (Hanlon
Fig. 8.2 White skin lesion on lower concentrations. In fact, there is a well growth up to 8%
head in an adult female of of NaCl and 40% of strains were described with ability to
O. vulgaris cultured in Italy
grow also at 10% NaCl. On the contrary, V. parahamolyticus
exhibits poor growth in media with 6% NaCl and 10% NaCl is
inhibitory (Mechri et al. 2015). Moreover, both are able to
growth between 15 and 42 °C. Although they can live in a
wide range of temperatures their growth is favoured by the
increase of temperatures, and for example, in V. para-
hamolyticus their outbreak of diseases has been associated
with thermal induction (Liu et al. 2000; Huang et al. 2001).
A fast preliminary identification is proposed through the
number of steps described in Sect. 8.2 (Characterization of
culturable bacteria). Other commercial identification systems
(API ZYM, API 20NE, etc.) were also proposed for their
identification. Although several of primers developed have
cross-reaction with both species, specific primers were also
and Forshyte 1990; Farto et al. 2003). Moreover, it was also proposed. Particularly, for V. alginolyticus that correspond-
isolated from gill heart, evidencing their potential as invader. ing to the heat shock protein 40, and for V. parahamoltycus
Similarly, it was shown for V. parahaemolyticus in squids PCR that detects a fragment (including a non-coding region
and cuttlefish being isolated from haemolymph and other and a phosphatidylserine synthetase gene) termed R72H
organs (Hanlon and Forshyte 1990; Sangster and Smolowitz (Buller 2004; Mechri et al. 2015).
2003). In addition, to bacterial species, Aggregata octopiana Sensitivity or resistance at the same antibiotic was shown
oocysts and betanodavirus were also found in the same skin by strains of both species depending on their origin. Both are
lesion (Fichi et al. 2015). Co-infection was previously resistant to a wide range of antibiotics commonly used in
shown as an enhancing factor for bacterial invasion in fish aquaculture (ampicillin, erythromycin, tetracycline, strepto-
(Rodríguez-Quiroga et al. 2016). mycin and chloramphenicol). Flumequine and streptomycin
Additionally, both species can infect humans. Whereas, are promising candidates for therapy against V. alginolyticus
the infections of V. alginolyticus are frequently associated and V. parahamolyticus, respectively (Mechri et al. 2015).
with wound infections, external otitis or cellulitis, acute Alternative promising measures to antibiotics to prevent the
gastroenteritis are caused by V. parahamolyticus. The strains diseases caused by these species such as the use of anti-
of V. parahaemolyticus affecting humans have the ability to biofilm agents (palmitic acid, nanoparticles) were recently
produce a thermostable direct haemolysin (TDH) and/or a proposed (Chari et al. 2017; Santhakumari et al. 2017).
thermostable-related haemolysin (TRH). To date, these Another hopeful treatment are the peptides produced by
toxins have not been produced by most of strains associated Bacillus subtilis which were proved with antimicrobial
with diseases in aquatic animals. On the other hand, there are activity against both species, and in addition, had a protec-
limited studies on ability of human clinical isolates to cause tive effect against V. parahaemolyticus when they were
diseases in marine organisms, but was proved that human incorporated into the diet of shrimps (Cheng et al. 2017).
clinical or environmental strains were infectious for both Water treatment by using photolysis therapy was effective in
humans and abalones (Lee et al. 2003). On the contrary, controlling V. parahamolyticus (Malara et al. 2017).
recently, the lack of effectiveness in causing disease in a
shrimp host was proved for human clinical strains (Choi Vibrio splendidus-related group (Splendidus clade)
et al. 2017). Differences between strains of V. alginolyticus
affecting humans or marine organisms are still unavailable. Splendidus clade, Vibrionaceae family, Gammaproteobacteria
Both species swarm across TSA-1% and completely class, consists of a group of species phylogenically closely
covers the plate in 24 h at 22 °C. Differences in colour of related with V. splendidus. To date, the species are:
colonies are shown in TCBS, and they are yellow and green V. artabrorum, V. atlanticus, V. crassostreae, V. celticus,
for V. alginolyticus and V. parahaemolyticus, respectively. V. cortegadensis, V. cyclitrophicus, V. chagasii, V. for-
Photographs of culture and microscopic appearance of tis, V. gallaecicus, V. gigantis, V. hemicentroti V. kanaloae,
organisms are detailed in texts such as Buller (2004). V. lentus, V. pomeroyi, V. pelagius, V. splendidus,
They are also halophilic. Although most of them show an V. tasmaniensis and V. toranzoniae. Recently, it has been
optimum growth with NaCl concentrations of 2–4% of NaCl, proposed the exclusion of 5 species (V. atlanticus, V. corte-
particularly, V. alginolyticus is more tolerant to higher and gadensis, V. chagasii, V. fortis, V. pelagius), but still they
136 R. Farto et al.
arms, and also to other conspecifics sharing the tank. Death

was not observed in any of animals at least for four months.
Histopathological lesions were characterized by dense
aggregations of bacteria, ulceration, loss of skin epithelium,
and haemocyte infiltration and inflammation of the infected
area (Fig. 8.3c, d).
Splendidus clade includes gram-negative rods, motile and
facultatively anaerobic bacteria with ability to growth on
TCBS, ability to produce acid from a wide variety of sub-
strates, and also with facility to grow by using an extensive
number of substrates as sole carbon source. These lasts
phenotypic features are the mainly basis to differentiate the
species within Splendidus clade, but it is still difficult since
variations in the same phenotypic test are reported among
different strains included in the same species and different
research groups.
A fast preliminary identification is proposed through the
number of steps described in Sect. 8.2 (Characterization of
culturable bacteria). Firstly, a phenotypical characterization
with primary identification tests including the decarboxy-
lases (ADH, LDC, ODC). Secondly, since Splendidus clade,
is the most abundant in marine environment, the use primers
VTS/VT and VTS/VS primer sets are a valuable tool for a
fast detection of Splendidus clade. Finally, the 16S rRNA
gene analyses are also a valuable tool to resolve distinct
genera, but it is insufficient to discriminate closely related
species such as those included in the Splendidus clade. If a
final identification will be necessary, then a multilocus
sequence analysis (MLSA) technique, which is based on the
sequencing of multiple housekeeping genes, should be
applied (Pérez-Cataluña et al. 2016; Oden et al. 2016).
The susceptibility of V. splendidus to several antibiotics
was recorded (chloramphenicol, flumequine, nitrofurantoin,
Fig. 8.3 O. vulgaris from maintained in aquarium conditions. nifurpirinol, oxonic acid and potentiated sulphonamide), and
a Macrophotography of the dorsal mantle showing small black spots their use was successful for the treatment of infection in fish
on the skin. b Detail of small ulcers surrounding the black spots.
c–d. Histological sections of the skin lesions showing ulceration and
(Revised by Austin et al. 2007). Chloramphenicol and
loss of skin epithelium (arrows) (c). Dense aggregations of bacteria, gentamycin were also highly effective in the treatment of
haemocyte infiltration and inflammation of the infected area (arrow squids or cuttlefishes (Hanlon and Forsythe 1990). Recently,
heads) H&E stain. Scale bars c, 500 µm; d, e 200 µm other alternative strategies to control an outbreak have been
effective, such as the use of a combination of different
phages (Li et al. 2016). Treatments to control disease caused
form a clade composed of at least 13 species (Oden et al. by other species of Splendidus clade are still unavailable.
2016).
Most of them have been associated with marine organ-
ism’s diseases, including molluscs (Sect. 8.2). In 8.3.2 Miscellaneous Bacteria Associated
wild-caught O. vulgaris, without apparent damage, we fre- with Eye Damages or Associated Tissues
quently detected small black spots on their dorsal mantle,
and in some occasions small ulcers surrounding the spots Previously, several Vibrio species were proved as invaders
(Fig. 8.3a, b). Bacteria isolated, which were identified by of squid eyes, having hardly affected the cornea. Particularly,
using sequencing of 16S rRNA gene, were closely related to V. anguillarum, V. charchariae or V. harveyi were cultured
V. splendidus/V. atlanticus/V. kanlaloae. However, when from the posterior chamber of squids. Furthermore, Micro-
octopus were kept in the aquarium tanks, the ulcerations coccus sp. was also detected in the vitreous humour, the
were much more evident and spread infection to head and posterior lens surface and the haemolymph of affected
Fig. 8.4 Retrobulbar lesion in an

adult male of O. vulgaris cultured
in Italy: a White fluid material
leaked from the retrobulbar lesion
after the removal of the ocular
bulb. b Retrobulbar-confined
cavity (2–3 cm diameter) excised
from the head
squids. Authors proposed a primarily involving damage to other fish pathogen bacteria, called LG agar, has been
the cornea, which would be previous to the infection developed by Chang et al. (2014). However, some molecular
(Hanlon and Forsythe 1990). Recently, Ph. swingsii and L. techniques, such PCR amplification of the internal tran-
garvieae were isolated from a white fluid material leaked scribed spacer (ITS) region, sequencing of the 16S rRNA
from a retrobulbar lesion after the removal of the ocular bulb gene, multiplex PCR and DNA microarray have been
in an adult male common octopus. Until removing the ocular developed and allow to identify L. garvieae (Chang et al.
bulb, no gross lesion was observed. The lesion appeared as a 2014; Gibello et al. 2016; Meyburgh et al. 2017).
confined cavity of 2–3 cm diameter (Fig. 8.4a, b). The treatment of L. garvieae infection in fish is based on
lincomycin, oxytetracycline and macrolide antibiotics.
Lactococcus garvieae A characteristic of this bacterium is the resistance to clin-
damycin, but resistance to erythromycin, streptomycin,
This is a gram-positive bacterium, which causes diseases in tetracycline, oxytetracycline, florfenicol and some quino-
several aquatic and terrestrial animals (Tsai et al. 2012). In lones have also been reported (Gibello et al. 2016).
fish, it is responsible for hyperacute haemorrhagic septi-
caemia, but it has also been identified in bacterial outbreaks Photobacterium swingsii
in aquatic invertebrates, such as the giant freshwater prawn,
Macrobrchium rosenbergii (Vendrell et al. 2006; Tsai et al. This is a motile gram-negative small coccobacillus in Vib-
2012; Meyburgh et al. 2017). This bacterium has been rionaceae family, Gammaproteobacteria class. It has been
reported in several species of fish, but it has also detected in isolated and characterized for the first time from oysters
other marine animals, such as a bottlenose dolphin, Tursiops during a vibriosis in Mexico and from the haemolymph of
truncatus (Evans et al. 2006) and a sea turtle, Caretta caretta wild spider crabs collected in Canary Islands, Spain, by
(Fichi et al. 2016). Furthermore, it is considered an emerging Gómez-Gil et al. (2011). It grows in TCBS agar, TSA
pathogen related with handling or ingestion of raw fish and supplemented up 2% NaCl (TSA-2), marine agar, and blood
seafood for humans (Gibello et al. 2016). In cephalopods, L. agar in the temperature range of 4–37 °C, and in the salt
garvieae was previously isolated in the muscle of a squid concentration range from 3 to 6% (Gómez-Gil et al. 2011,
collected in a restaurant as the source of infection in a human Fichi et al. 2015). In TCBS, the colonies of Ph. swingsii are
endocarditis in Taiwan, but there was the possibility that the green, small, round with smooth border, 2–3 mm diameter,
squid resulted positive for this bacterium due to a cross while in TSA-2 and marine agar they are white (Gómez-Gil
contamination with other raw fish (Wang et al. 2007). et al. 2011; Fichi et al. 2015). Some differences were
L. garvieae, Streptococcaceae family, Bacilli class, is an observed among the six strains isolated by Gómez-Gil et al.
ovoid coccus, not-motile, which grows from 4 to 45 °C, and (2011), regarding the growth conditions and the biochemical
at 6.5% NaCl (Meyburgh et al. 2017), on several common reactions.
media. On blood agar and Columbia-colistin-nalixic acid The strain isolated from octopus grew from 22 to 37 °C
(CNA) agar, the colonies are small, white, alpha haemolytic and at the 3% salt concentration and it tested positive for
and catalase negative. Several commercial identification oxidase and catalase, and it is possible the identification by
systems, such as API strips, BD Phoenix, the Vitek system API 20 E or API 20NE systems. The identification of Ph.
or MicroScan are able to identify it, but some strains can be swingsii was applied by the sequencing of 16S rRNA gene.
misidentified as L. lactis or Enterococcus spp. (Gibello et al. By the fact that this bacterium has been isolated from oysters
2016). A selective medium to differentiate L. garvieae, from during a vibriosis outbreak and from a retrobulbar lesion in
138 R. Farto et al.
Fig. 8.5 O. vulgaris gills main-

tained in aquarium conditions. His-
tological sections of an adult
showing rickettsiales-like organisms
(arrows) at the base of the gill
epithelium with distension of the
basal membrane and hypertrophy of
the infected cell. H&E stain. Scale
bars a 100 µm; b 50 µm
the common octopus, there are strong evidences of its Light microscopic examinations of paraffin sections or
pathogenic role, but this should be confirmed by an exper- indirect immunofluorescence with specific immune serum are
imental infection. the methods used for their identification. Sequencing of 16S,
The isolated strain resulted susceptible to the vibrio static internal transcribed spacer (ITS) or 23S ribosomal DNA
O129 at 10 and 150 µg, but no other antibiotic test sus- sequencing are also strategies used to characterize these bac-
ceptibility was performed. teria. Moreover, some specific primers set have been devel-
oped to identify Rickettsia in salmonids (Mauel et al. 1996).
Limited studies about treatment of RLO to control disease
8.3.3 Rickettsial-like Organisms (RLO) are published. The supply of medicated feed (oxytetracy-
cline and florfenicol) was effective for the treatment against
RLO are gram-negative bacteria, which have been associated the necrotizing hepatopancreatitis bacterium (NHPB), which
with infection and mass mortalities in several aquatic ani- is a RLO-affecting crustaceans (Gollas-Galván et al. 2014).
mals. Particularly, cases of molluscs bivalves have increased
since the first description in clams was reported (Azevedo
et al. 2006; revised by Romalde and Barja 2010; Ceuta and 8.4 Pathogenic Bacteria for Adults
Boehs 2012; revised by Gollas-Galván et al. 2014). These
organisms have been found mainly within the epithelial cells In this section, a brief reference to those bacteria confirmed
of the mantle, gills digestive gland, connective tissue and as the causative agent of diseases and mortality in octopus
hepatopancreas of several molluscs (revised by are included.
Gollas-Galván et al. 2014).
Similarly, they were detected in the gills of the common
octopus O. vulgaris. They were observed like basophilic 8.4.1 Vibrio lentus
intracytoplasmatic microcolonies of about 102 µm (70–150)
infecting the epithelial cells of the gills (Fig. 8.5a). Invaded This is a gram-negative bacterium, which was firstly
host cells became hyperthophic (Fig. 8.5b) and necrosis described as an environmental species being associated to
were occasionally observed (Gestal et al. 1998). No signif- reared Mediterranean oyster (Macián et al. 2001), and later
icant harm or signs of disease have been observed in the also associated with larvae cultures of scallops, and turbot
hosts, since usually a few cells were affected. However, (Le Roux et al. 2004). Studies to assess the diversity of
conditions of stress, high animal density or crowding system bacteria based on 16S rRNA showed that this species seems
under intensive rearing increased the occurrence of diseases. to be also associated with mussel or adult turbot hatcheries,
Under these conditions, RLO were able to have a detrimental and surface of algae (Montes et al. 2006; Wang et al. 2009;
effect on the respiratory gaseous exchange of the octopus Kwan and Bolch et al. 2015). Moreover, it is one of the
and special attention and controls were needed. intestinal autochthonous bacteria from the intestinal tract of
RLO are obligate intracellular organisms, highly fastidi- the common carp (Chi et al. 2014), and recently it was
ous, non-motile, non-spore-forming and highly pleomorphic. revealed as a protective agent against vibriosis caused by V.
Unfortunately, artificial media to isolate these organisms are harveyi in gnotobiotic sea bass larvae (Schaeck et al. 2016).
still unavailable for most of them, being difficult their iso- On the contrary, this species was also associated with lesions
lation and characterization. To date, only one species was in lobsters (Chistoserdov et al. 2005) or octopus. Particu-
able to grow on agar plates or broth (revised by larly, in octopus, V. lentus was isolated from skin lesions and
Gollas-Galván et al. 2014). the gill heart of diseased individual captured from their
Fig. 8.6 V. lentus on plates,

48 h, 22 °C. a TSA-2. b TCBS
natural marine environment, and it was able to induce both different stages of cultured or aquarium-maintained cepha-
skin lesions and mortality in healthy octopuses (0.5–1 kg) lopods. However, it is still unknown if most of them are the
maintained in the laboratory (Farto et al. 2003). initial cause of infection.
The signs caused by bacteria in octopuses were round To understand the equilibrium among a community of
hard lesions in the arm or head mantle, which can evolve to microorganisms, and how changes in husbandry practices
skin losing and muscle beneath exposition when the time is select for more virulent genotypes are important targets to be
advancing. These symptoms were similar to those of stages 3 addressed in the future.
and 4 of the disease described by Hanlon and Forsythe Prevention techniques are needed to avoid bacterial
(1990) for O. joubini. In addition, the bacteria was able to infections in cephalopods maintained in aquarium systems,
colonize gill heart, and induce lesions and mortality after which include good welfare practices, cleaning of tanks and
72 h of exposition, depending on immunity of each indi- control of water and food quality supplied. However, once
vidual (Farto et al. 2003). the presence of the pathogen is confirmed, treatments to
V. lentus, Vibrionaceae family, Gammaproteobacteria combat the infection are needed. To date, most of treatments
class, is a rod-shaped bacteria, motile, unable to growth at to control diseases caused by bacteria-affecting cephalopods
37 °C or with 7% of NaCl. It shows 0.2–0.4 mm colonies, are based on applying antibiotics. This therapy is becoming
which are, round, transparent, non-pigmented and unable to more restrictive as a consequence of generating multi-drug
swarm after 48 h on TSA-2 at 22 °C (Fig. 8.6a); on TCBS, the resistant pathogens and accumulation of toxic compounds in
colonies are green (Fig. 8.6b). Other phenotypical character- farmed organisms, causing long-term adverse health effect to
istics were previously described in detail (Macián et al. 2001; humans and other animals. Therefore, special attention
Farto et al. 2003). should be focussed on the use of alternative methods in order
This species is closely related to other species, which are all to protect larvae, juvenile and adult cephalopods against
included in the Splendidus clade. Specific primers have not pathogens and improve their survival, and also safeguard
been developed, and the number of steps described in human health and environment.
Sect. 8.2.1 (Splendidus clade) is suggested for their
identification.
Acknowledgements Authors would like to thank Raquel Fernán-
V. lentus showed a wide antimicrobial susceptibility dez-Gago (University of Vigo) who has generously donated images
pattern, having the highest, with amoxicyllin, cefotaxime, (Fig. 8.1a, b) and tissue sections of bacterial-infected O. vulgaris par-
chloramphenicol or piperacillin (Farto et al. 2003). Safer larvae, and María Gómez-Costas (University of Vigo) who has col-
laborated in Sect. 8.2.
strategies, such as probiotics or others, are still unavailable,
for the treatment of infection in octopus.
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Destoumieux-Garzón D (2015) Outer membrane vesicles are
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were made.
Protist (Coccidia) and Related Diseases
9
Sheila Castellanos-Martínez, Camino Gestal, Santiago Pascual,
Ivona Mladineo, and Carlos Azevedo
Abstract
Coccidia of the genus Aggregata is the most widely distributed coccidian in cephalopods.
Damages caused to the hosts include mechanical (tissue injury), biochemical (malfunction of
digestive enzymes), and molecular (affects cellular immune response) effects. However,
coccidiosis is not a fatal disease to the cephalopod host; it severely weakens its innate
immunity making it vulnerable to secondary infections. Therefore, coccidia of the genus
Aggregata are considered the most dangerous parasite for cephalopods affecting wild species
of notable economic importance for fishery and aquaculture activity. The pathology caused by
coccidiosis to the most important European cephalopod species is the subject of the present
chapter.

Keywords
Cephalopods Diseases Histopathology Ultrastructure Coccidian Apicomplexa
9.1 Introduction
S. Castellanos-Martínez (&)
Instituto de Investigaciones Oceanológicas, UABC, Ensenada,
22860, Mexico Cephalopods are hosts for several parasitic organisms.
e-mail: [email protected] Although most of them are known for scientific community,
C. Gestal gaps with respect to their entire biology remain. Coccidian
Aquatic Molecular Pathobiology Department, Institute of Marine protozoans of the phylum Apicomplexa are among the com-
Research, Spanish National Research Council (CSIC), 36208 monest parasites infecting the digestive tract of cephalopods;
however, a limited number of species have been described
worldwide. All of them are included in the genus Aggregata.
S. Pascual
Their life cycle requires an intermediary host, a crustacean,
Ecology and Biodiversity Group, Institute of Marine Research,
Spanish National Research Council (CSIC), 36208 Vigo, and a definitive host, the cephalopod, where the parasite
Pontevedra, Spain develops into the infective stage, the esporozoites, which are
e-mail: [email protected] contained inside the sporocysts that are released to the ocean
I. Mladineo in the feces of the cephalopod host. The apparent simplicity of
Institute of Oceanography and Fisheries, 21000 Split, Croatia coccidia becomes a challenge to clarify very basic aspects that
are not yet clear such as taxonomy, phylogeny, life cycle,
C. Azevedo distribution or effects on their hosts. Two coccidia species,
Laboratory of Cell Biology, Institute of Biomedical Sciences
Aggregata octopiana and Aggregata eberthi, both infecting
(ICBAS/uP), University of Porto, 4050-013 Porto, Portugal
e-mail: [email protected] European hosts are the best know species to date. Coccidiosis

https://doi.org/10.1007/978-3-030-11330-8_9
144 S. Castellanos-Martínez et al.
is highly prevalent in most of the studied cephalopod hosts parasites causative of coccidiosis (Hochberg 1983). The
and reaches severe levels of infection that compromise the intensity of infection can be as high as 82 106
host well-being at functional, biochemical, and molecular sporocyst/digestive tissue of the cephalopod host (Pascual
levels. Traditional techniques of detection are still used until et al. 1996; Gestal 2000). The prevalence of infection varies
advanced protocols are being designed. Detection of coccid- among the cephalopod species. The highest prevalence (98–
iosis is particularly important in species of aquaculture rele- 100%) is recorded in O. vulgaris and S. officinalis (Pascual
vance such as Octopus vulgaris and Sepia officinalis, both et al. 1996). In contrast, the lowest prevalence of infection
caught from the wild for rearing and naturally infected by (3%) has been recorded in the flying squid, Todarodes
Aggregata spp. Hence, the study of coccidiosis in cephalo- sagittatus and all of them in the NE Atlantic (Gestal et al.
pods including characterization, detection, and eradication of 2000). In the wild O. vulgaris from Adriatic and Ionian seas,
the parasite is under research. The mentioned protistan that 100% of prevalence were observed, whereas 98% were
infects cephalopods hosts is far from being well known. recorded in octopuses from Tyrrhenian Sea with no mortalities
However, the up-to-date knowledge regarding biological associated with coccidiosis reported from both localities
aspects of the parasites and histopathological damage caused (Tedesco et al. 2017).
to the cephalopod host is presented in this chapter. Identification of Aggregata spp. relies on the morpho-
logical characterization of sporogonial stages (size, shape,
number of sporozoites per sporocyst and ornamentation
9.2 Etiology and Epidemiology sporocyst) and parasite–host specificity. To date, the best
known species are A. octopiana, parasite of O. vulgaris, and
Coccidiosis is caused by an obligate, intracellular protozoa A. eberthi, which infects S. officinalis. A. octopiana char-
classified in the phylum Apicomplexa, family Aggregatidae. acterizes because of the spiny sporocyst and eight sporo-
To date, 10 species have been described worldwide in octo- zoites inside each. In contrast, smooth sporocysts harboring
pus, squid, and cuttlefish, being Aggregata the single genus three sporozoites characterize A. eberthi (Fig. 9.1) (Dobell
recognized (Table 9.1), and all the species known are 1925; Gestal et al. 1999). The Aggregata species from the
Table 9.1 Aggregata species recorded worldwide in cephalopod hosts. (–) denotes not available
Aggregata sp. Hosts Localities Cyst wall Sporozoites GenBank accession References
number number
octopiana O. vulgaris Mediterranean Spiny/smooth 8 LC186909-LC186925 Tedesco et al. (2017)
NW Atlantic Spiny 8 KC188342 Gestal et al. (1999);
Castellanos-Martínez et al.
(2013)
Adriatic Sea – – DQ096837 Kopečná et al. (2006)
eberthi S. officinalis NE Atlantic Smooth 3 KC188343 Labbé (1895);
Castellanos-Martínez et al.
(2013)
Adriatic Sea Smooth – DQ096838 Kopečná et al. (2006)
bathytherma Vulcanoctopus NE Pacific Smooth 14–17 – Gestal et al. (2010)
hydrothermalis
andresi Martialia SW Atlantic Smooth 3 – Gestal et al. (2005)
hyadesi
patagonica Enteroctopus SW Atlantic Smooth 8 – Sardella et al. (2000)
megalocyatus
valdesensis Octopus SW Atlantic – 4–8 – Sardella et al. (2000)
tehuelchus
sagittata T. sagittatus NE Atlantic Smooth 4–8 – Gestal et al. (2000)
dobelli Enteroctopus NE Pacific Smooth 9–22 – Poynton et al. (1992)
dofleini
millerorum Octopus NE Pacific Smooth 8–10 – Poynton et al. (1992)
bimaculoides
kudoi Sepia elliptica NW Indian Smooth 6–12 – Narasimhamurti (1979)
9 Protist (Coccidia) and Related Diseases 145
Fig. 9.1 Comparison of the num- (a) (b)

ber of sporozoites per sporocyst.
a Histological section of O. vulgaris
cecum showing eight sporozoites of
A. octopiana. b Histological section
of S. officinalis cecum showing
three sporozoites of A. eberthi.
Scale bars a 15 µm; b 20 µm
Adriatic differs morphologically from those in the NE oocysts on the tissue (Figs. 9.3 and 9.4) simply opening
Atlantic, Tyrrhenian, and Ionian seas in the appearance of gently the mantle cavity of the cephalopod (Sykes and
the cyst wall (smooth in the former, spiny in the latter) and Gestal 2014). Extraintestinal infection has been recorded in
some minor variations in the size of oocysts and sporocysts cephalopods reared in floating cages from the NE Atlantic.
(Tedesco et al. 2017). All the species show the same number Either the site of extraintestinal infection, the parasite causes
of sporozoites (being eight), and the characterization of the hypertrophy of invaded cells, hemocytic infiltration and
SSU rRNA gene that complements morphological and activates phagocytosis by hemocytes (Fig. 9.5) (Gestal et al.
morphometric descriptions provides molecular support that 2002a; Pascual et al. 2006). In the Adriatic Sea, Aggregata
distinguish among Aggregata species from NE Atlantic, spp. has been also identified in experimentally reared O.
Adriatic, Ionian, and Tyrrhenian seas (Kopečná et al. 2006; vulgaris (250–500 g) fished from wild (Island of Brač,
Castellanos-Martínez et al. 2013; Tedesco et al. 2017). Adriatic Sea) and held individually in 4 m2 concrete tanks.
Phylogeny indicates three lineages of which Adriatic isolates After 8 months of rearing without expected weight gain,
significantly separated from those of the Ionian and animal ceased the feeding, became excitated and aggressive,
Tyrrhenian seas although being more related to Ionian iso- lacked the capacity of camouflage, and developed epidermal
lates than the rest. These observations all together evidence lesions on the mantle and arms (Fig. 9.5), with visible mil-
the existence of different Aggregata species or subspecies iary protozoan oocysts (Mladineo and Jozić 2005).
complex in octopuses from different seas (Tedesco et al. Extraintestinal gamogonial and sporogonial Aggregata
2017). stages infecting arms and gills triggered notable infiltrated
hemocytes and replacement of the host tissue (Mladineo and
Bočina 2007).
9.3 Pathogenesis
Coccidiosis is a disease that affects the digestive tract of

cephalopods. The Aggregata spp. life cycle is heteroxenous;
asexual merogonial stages develop inside the gut of the
intermediate host, which is a crustacean (Fig. 9.2). The
infective stage called merozoite invades the intestinal
mucosa of the crustaceans and develops intracellularly into
meronts that remains awaiting until the intermediate host is
eaten by the cephalopod. Merogonial stages ingested by the
cephalopod are able to infect the whole digestive tissue tract
of the mollusk and epithelial cells in additional tissues.
Unusual organs of infection such as the connective tissue of
gills, mantle, arms, visceral mass and mesentery of the host
have been recorded when it harbor an intense infection. It is
named extraintestinal coccidiosis, and most of the time, it is Fig. 9.2 Merogonial (mr) stages of A. octopiana infecting the gut of
macroscopically identified because the presence of white Palaemon serratus (Bismark brown stain). Scale bars 100 µm
Fig. 9.3 Macroscopical detection

of A. octopiana in O. vulgaris.
a Gross observation of white
oocysts (arrows) on the muscle of
the siphon and visceral mass.
b Detail of white round and ovoid
oocysts observed infecting the vis-
ceral mesentery. c White round
oocyst (arrows) observed infecting
the serosa on the octopus gut
After merogonial stages are ingested by the cephalopod, the lumen is covered by a wide cuticle that reduces the
the establishment of the infection inside the mollusk takes nutrient intake (Garri and Lauria de Cidre 2013). A similar
place. The infection initiates with gamogony, the formation but thick cuticle covers the crop and esophagus (Garri and
of gametes, which occurs by unequal fission of merozoites Lauria de Cidre 2013) that make nutrients and space avail-
inside the cephalopod digestive tract. Macrogametes (♀) and able for infection, although in low frequency (Gestal et al.
microgametes (♂) are formed. The macrogamete is the most 2002a). Therefore, the target organs of infection are the
conspicuous one due to their round to ovoid in shape, central non-cuticularized cecum, a tubular, spiraled organ and the
nucleus, and large nucleolus that stains darker than the rest gut of the cephalopod (Gestal et al. 2002a). In these tissues,
of the cell when H&E is used (Fig. 9.6). Although the the macrogamete once fertilized by a microgamete produces
infection can be recorded in the whole digestive tract of a zygote that in turn suffers from multiple fission, and gives
the mollusk, no infection is recorded in the stomach because rise to the sporogonial stage where oocysts, sporocysts and
the infective sporozoites are formed (Gestal et al. 2002a)
(Fig. 9.7). The ultrastructural analysis by transmission
electron microscopy (TEM) of the sporoblast formation
shows the presence of multiple nuclei inside each oocyst
(Fig. 9.8), while each sporoblast has a single nucleus and a
thin sporoblast cover (Fig. 9.9). The development of each
sporoblast into sporocyst results in the formation of the
sporozoites inside (the specific number is species specific)
and a thickening of the sporocyst wall (Fig. 9.10). The
infection by macrogametes and initial sporogonial stages
induces a strong hemocytic infiltration, distention and rup-
ture of the tissue infected (Fig. 9.11). The cecum wall is
covered by longitudinal folds lined with cylindrical, simple,
and ciliated epithelium (Garri and Lauria de Cidre 2013) that
can be totally replaced by the parasite (Fig. 9.12). The
infection by coccidia triggers hemocytic defensive activities
Fig. 9.4 Detailed macroscopic observation of A. octopiana oocysts. such as phagocytosis of sporogonial stages, encapsulation of
a White round and ovoid oocysts of A. octopiana. b Purified sporocyst of oocysts and gametes by flattened hemocytes and then con-
A. octopiana observed by light microscopy. c Sporozoites of A. octopiana
free from the sporocyst. Scale bars a 0.4 mm; b 15 µm; c 5 µm nective tissue (Fig. 9.12) in order to isolate the parasite
(Castellanos-Martínez and Gestal 2013). Mature sporocysts
Fig. 9.5 Extraintestinal infection

of A. octopiana. a Epidermal
lesions on the arms of O. vulgaris
showing visible infection by proto-
zoan oocysts (arrow). b Detail of
epidermal lesion showing numerous
white oocysts of coccidia (arrow).
c Connective tissue of O. vulgaris
infected by oocyst. d Connective
tissue of the octopus arm destroyed
by sporogonial stages of A. octopi-
ana and after parasite release. Scale
bars c, d 200 µm
Fig. 9.6 Gamogonial stages infect-

ing O. vulgaris cecum. a Macroga-
metes (arrows) infecting the folds of
the cecum epithelium. b Detail of
macrogamete infecting the fold of
O. vulgaris cecum c Comparative
view of macrogametes (ma) and
microgametes (mi) infecting the
cecum epithelium. d Detail of
microgametes infecting digestive
epithelia and surrounded by hemo-
cytes. Scale bars a 100 µm; b,
d 30 µm; c 60 µm
Fig. 9.7 Sporogonial development of coccidia Aggregata sporozoites enclosed in sporocyst. c Oocyst containing immature
spp. a Zygote after starting multiple division in cecum epithelium of sporocysts (arrowhead) and surrounded by hemocytes. d Oocyst with
O. vulgaris. Note numerous hemocytes (arrowhead) that surround the mature sporocysts of A. octopiana. Note macrogamete (m) close to the
parasites. b Advanced multiple division toward the formation of oocyst and sporozoites (s) inside sporocysts. Scale bars a, b 100 µm;
sporocyst. The nuclei observed in the periphery will give rise to c 200 µm; d 10 µm
are released to the lumen of the cephalopod digestive tract in the host against oxidative stress, is up-regulated in the cecum
order to discharge the parasite to the aquatic medium in the of cephalopod with high parasite load. However, peroxire-
feces of the mollusk and make them available for infecting a doxin is weakly expressed in hemocytes of such hosts,
crustacean intermediate host (Hochberg 1983). The release suggesting that coccidia might suppress the respiratory burst
of the parasite causes detachment of the epithelial cells and and, thus, affect the octopus cellular immune response
rupture of the connective tissue. As a result, the mucosal (Castellanos-Martínez et al. 2014a, b).
folds atrophy and ulcers in the digestive tissue tract of the
host appear (Fig. 9.13). Coccidiosis originates acidification
of the mucosal lumen that affects the stability of the diges- 9.4 Diagnosis
tive enzymes and nutrient absorption. It is exacerbated by
the destruction of the mucosal intestinal epithelium after Signs of infection are not easily visible in the infected cepha-
parasite release that also impedes the nutrient uptake of the lopod hosts. Gross detection of coccidia can be realized by
cephalopod host (Gestal et al. 2002b). Signs of coccidiosis in direct observation of macroscopic white cysts when the host is
cephalopods include malabsorption syndrome, decrease of highly infected (Mayo-Hernández et al. 2013), and small pie-
cephalopod condition, the number of hemocytes, plasmatic ces of digestive epithelia can be observed in feces as a result of
protein, and even the concentration of copper, the respiratory severe infection (Dobell 1925; Hochberg 1990). Fecal oocyst
pigment of hemocyanine in the hemolymph, is affected count is a common method for detecting coccidia, although it is
(Gestal et al. 2002b, 2007). Intense coccidiosis also alters the not yet standardized in cephalopods. Oocyst and sporocyst
cephalopod immune response. Immune genes involved in detection in feces is being under standardization as a diagnostic
pathogen recognition and regulation are up-regulated in the approach in O. vulgaris. Presently, the challenge is focused on
cecum of highly infected hosts indicating active detection of the design of molecular probes for detecting coccidia in the
parasite-derived ligands (Castellanos-Martínez et al. 2014a). cephalopod feces that avoid the need to sacrifice the host, as is
The antioxidant gene peroxiredoxin, responsible to protect currently done, and provide certainty on experiments that uses
Fig. 9.8 Formation of sporoblast showing multiple nuclei inside each

Fig. 9.9 Sporoblast development. Ultrastructural aspects by TEM.
oocyst. Ultrastructural aspects by TEM. HN: host nucleus, Pn: parasite
a Early sporoblast showing a single nucleus and a thin sporoblast cover.
nucleus Scale bar 8 µm
b Late sporoblast showing a thicker cover. Scale bars a, b 30 µm
Fig. 9.10 Phase contrast microscopy photography of A. octopiana

oocyst showing inside the sporocysts containing sporozoites. Note the
sporocyst (sw) and oocyst wall (ow). Scale bar 30 µm
Fig. 9.11 Hemocytic infiltration in the cecum epithelium of O.

vulgaris. a Oocysts surrounded by numerous hemocytes (arrowhead)
accompanied by distention (asterisk) of the tissue. b Pericyst reaction
against oocyst triggered by hemocytes (arrowhead) which formed a
barrier around the cysts, connective tissue (ct), distention (asterisk) of
the tissue. Scale bars a, b 200 µm
Fig. 9.12 Cecum of O. vulgaris infected by A. octopiana and host among sporocyst c Mature oocyst of A. eberthi partially emptied and
immune response. a Longitudinal folds of cecum showing the tissue encapsulated by hemocytes. Note sporocyst (arrowhead) outside the
totally replaced by coccidia at different stages of development; m: oocyst and attacked by hemocytes. d Oocyst encysted by fibrous layer
macrogamete, asterisk: oocyst. b Oocyst of A. eberthi phagocytosed by of connective tissue (ct) as a result of pericyst reaction against the
hemocytes. Note the lack of oocyst border and hemocytes dispersed parasite. Scale bars a 0.4 mm; b 60 µm; c, d 100 µm
of coccidiosis in cephalopods that can be hard to detect.

However, malabsorption syndrome, decrease in the number
of hemocytes, plasmatic protein, and iron in hemolymph, as
well as up-regulation of immune genes, are among the
notable signs of disease and may be accompanied by dis-
charge of small pieces of tissue lumen. The age of cepha-
lopods when they are infected by Aggregata spp. for the first
time has not been determined; however, specimens as small
as 5 mm mantle length have been found infected in the Ria
of Vigo (NW Atlantic). Any risk group based on age or
length has been determined and studied for differences in
severity effects of the infection. However, clear differential
effects have been noted regarding the number of sporocyst
infecting the digestive tract of hosts. The cephalopod rearing
is still depending on wild specimens; therefore, detection of
coccidian species has become an important task to prevent
fattening of severely infected hosts. Preventive strategies of
coccidiosis are difficult to apply in cephalopods, and no
coccidiostats are available to date. Currently, avoiding the
intake of infected food is the best strategy to prevent coc-
cidia infection or reinfection, particularly, to those speci-
mens reared in the wild.
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made.
Protist (Ciliates) and Related Diseases
10
Dhikra Souidenne and Hidetaka Furuya
Abstract
Ciliates are one of the most common protistan parasites in cephalopods. In this chapter, we
have undertaken to describe the biology and diversity of parasitic ciliates in European
cephalopods and give diagnosis elements to identify the known species. We briefly
summarize available data on the ciliates parasitizing the gills and skin of European
cephalodops (Ancistrocomidae) and the endoparasitic forms observed in the digestive tract
and renal appendages (Opalinopsidae). Ancistrocomidae ectoparasites have been observed in
Octopus vulgaris. Opalinosidae family harbours two parasitic genera: Opalinopsis and
Chromidina. Species diversity of these two genera seems to be underestimated in Europe.

Keywords
Parasitic ciliates Opalinopsidae Opalinopsis Chromidina Ancistrocomidae
10.1 Introduction 10.2 Ancistrocomidae (Chatton and Lwoff

1931)
Ciliates are one of the most frequently encountered protistan
parasites in cephalopods. In addition to the endoparasitic Ancistrocomidae ciliates have been described parasitizing
forms observed in the digestive tract, ciliates have been skin and gills of Octopus bimaculoides (Forsythee and
described as ectoparasites parasitizing the gills and skin of Hanlon 1991). In European cephalopods they have been
different cephalopods. identified in Octopus vulgaris parasitizing gills (Fig. 10.1)
with a high prevalence, and in some occasions the skin.
However, no Ancistrocomidae parasites have been observed
in Sepia officinalis.
Free living and attached forms can be observed, measur-
ing 17–25 µm in length and showing oval or pyriform
shaped, with a large centrally located nucleus and a food
vacuole in the distal end of the body. Fresh preparations show
that the ciliation pattern typically surrounds all the body.
D. Souidenne (&)
Muséum National d’Histoire Naturelle de Paris, Biologie Des Histologically submucosal inflammatory infiltrates produc-
Organismes et Ecosystèmes Aquatiques (BOREA), Research ing bronchitis were observed in heavily parasitizied octopus.
Group: Reproduction and Development, Evolution, Adaptation,
Regulation, CNRS 7208, Sorbonne Université, UCN, IRD 207,
43 rue Cuvier, Paris, France
H. Furuya
Department of Biology, Graduate School of Science, Osaka
University, Toyonaka, Osaka 560-0043, Japan

https://doi.org/10.1007/978-3-030-11330-8_10
154 D. Souidenne and H. Furuya
Fig. 10.1 Ancistrocomidae

ciliates parasitizing the gills of
O. vulgaris. a–b General aspect
of gills infected by the ciliates
showing free living and anchored
forms. c–d Detail of the gill
epithelium where anchored
ciliates with pyriform shape and
large centrally located nucleus are
observed. a–d: H&E. Scale bars
a 200 µm; b 200 µm; c100 µm;
d 20 µm (pictures courtesy of
Dr. C. Gestal)
Foettinger (1881) and Dobell (1908) distinguished two

10.3 Opalinopsidae Hartog (1906) genera
(Synonyms: Chromidinida,
Chromidinidae) • parasites of the renal appendages of cephalopods:
Chromidina Gonder (1905)
Although cephalopods and fishes share a wide range of • parasite of the liver and intestine of cephalopods:
parasite groups that can infest both of them, only one family Opalinopsis Foettinger (1881).
of parasites, Opalinopsidae Hartog (1906) (synonyms to
Chromidinida, Chromidinidae), is restricted to cephalopods Main differences between the two genera are summarized
and can never infect fishes. in the table below (Table 10.1):
Opalinopsidae are, after the dicyemids, the most common
parasites in cephalopods. Their classification is mainly based
on their morphology. Gonder (1905) and Dobell (1908) 10.3.1 Opalinopsis, Parasites of the Liver
initially described the Apostomes Opalinopsidae as holotri- of Cephalopods, in Europe
chious protistan parasites of cephalopods.
The macronucleus of Opalinopsidae is a complex, con- Parasites of the genus Opalinopsis are restricted to the
tinuous network distributed in parasite body. Regarding a digestive tract of cephalopods. The only study to avoid
mode of reproduction, there are two ways of interpretation: repetition of Opalinopsis in Europe was reported by Foet-
Foettinger (1881) believed budding is a multiplication mode tinger (1881), who gave detailed description for these par-
in Opalinopsidae, while Dobell (1908) regarded it as a asites. Later, Chatton and Lwoff (1931, 1935) studied
segmentation. In most of Opalinopsidae, developmental Opalinopsis by analogy to Chromidina in order to evaluate
stages are very labile and sensitive to seawater. Their their distinctive criteria. To date, only two species of
reproduction mode and complete life cycle remains to be Opalinopsis were described and named by Foettinger
determined. In addition, molecular data still needed to con- (1881). The following descriptions are bibliographical syn-
firm the monophyly of the Opalinopsidae family. thesis between Foettinger (1881), Dobell (1909), Chatton
10 Protist (Ciliates) and Related Diseases 155
Table 10.1 Characteristic differences of Opalinopsis and Chromidina

Opalinopsis Chromidina
Number of 2 6
morpho-species
described
Common characteristics
Ciliature Holotrichious, helicoidal ciliature, very dense
Nucleus Fragmented nucleus, highly crosslinked, dissociated in uniform masses, spherical or vesiculous
Distinctive characteristics
Shape Ovoid Vermiform
Microhabitat Liver and intestine of cephalopods Renal organs of cephalopods
Host habitat Benthic, mesopelagic cephalopods Pelagic and mesopelagic cephalopods
Mouth No mouth observed Oral blank for tomite stages but no
buccal cavity
Nutrition Diffusion Eat renal cells when attached to the
renal appendages or feed by diffusion
when free in the urine
Host (genus) Alloteuthis, Heteroteuthis, Histioteuthis, Sepia, Sepietta, Sepiola, Octopus Widely in cephalopod genera
Mobility Free in the liver or fixed massively to the epithelium of the hepatic channels Attached their anterior end to the renal
and the intestine by their anterior widened end characterized by epithelium, but detached individuals
distinguishable papillum, kinetie ciliature and infraciliature can swim in the urine.
Kinetie With gaps Without gaps
Vacuole Presence of a contractile vacuole in the posterior end Present only for the tomite stage
Macronucleus Macronucleus organized as a network in the medulla zone Macronucleus organized as a network
throughout the cell
Micronucleus Unique micronucleus with ellipsoidal shape Unique micronucleus streamlined shape
Number of kineties 30 kineties never reaching neither the anterior end nor the posterior end 12–14 kineties
(both ends are bare)
Trichocyst Absent Present
Multiplication Equatorial split Division of the distal region in several
segments. Each segment develops into
the adult stage
Physiology Survives for a long time in sea water Die in the presence of sea water
and Lwoff (1931, 1935), Hochberg (1971, 1982, 1983, direction. The trophotomont is attached to its microhabitat
1990) and Souidenne et al. (2016) descriptions and author’s (liver/intestine) by a rostrum (Hochberg 1971). Cytostome,
observations on the liver of freshly fished cephalopods. rosette or oral cilia are lacked (Foettinger 1881; Gonder
1905; Dobell 1909; Hochberg 1971).
10.3.1.1 Opalinopsis sepiolae (Foettinger 1881) Kineties are oblique and forming a curved radiation, widely
O. sepiolae is a parasite of the liver of Sepiola rondeletti in spaced, starting from the central part of the body and have gaps
the gulf of Naples. Foettinger reported the infection 17% of at some parts (Foettinger 1881; Chatton and Lwoff 1935).
examined hosts and, if present, these ciliates are very dense. A fragmented nucleus is observed in a few live speci-
Bodies are ovoid, covered with short vibrative cilia, and mens. This type of nucleus is dissociated in small fragments,
have a pointed or round big anterior extremity. The size which can be relinked together in a single nucleus afterward.
ranges 60–120 µm length and 30–62 µm width near the Generally, the nucleus has network shape; small nuclei
anterior end, and 30–44 µm at the posterior end (from the linked in a spread, spherical aspect or in sticks shape.
smallest specimens to the biggest specimens). Mobile Multiplication of O. sepiolae is mainly by transversal
specimens always have their anterior end in their swimming segmentation of the body. The division plane results that
posterior half is shorter that the anterior half. However, 10.3.2.1 Chromidina elegans Foettinger (1881)
Foettinger (1881) observed just once, two individuals con- (Synonym: Benedenia elegans)
jugation marked by the fusion of the two bodies followed a C. elegans have been first described in Naples by Foettinger
traversal division. The survivals in sea water probably can (1881) from the renal appendages of Sepia elegans d’Or-
leave the host and swim in the water to infect a new host. bigny, 1825. Chatton and Lwoff (1935) redescribed this
However, the complete life cycle and the transmission of the species from cuttlefishes from Banyuls-sur-Mer, France. C.
infection mode are still unkown. elegans can also infect Sepia orbignyana Ferussac, 1826,
Illex coindetti Vérany, 1837; Todarodes sagittatus Lamarck,
10.3.1.2 Opalinopsis octopi (Foettinger 1881) 1798 and Octopus salutii Vérany, 1839 in France and Eng-
Foettinger (1881) has observed O. octopi for the first time when land (Hochberg 1971, 1982, 1983). There is no available
he examined Octopus tetracirrhus (Delle Chiaje 1830) and information about the prevalence of this Chromidina species.
later it was found in Octopus macropus by Hochberg (1971). C. elegans is considered to be a typical species of the
O. octopi has been obtained from O. tetracirrhus (Foet- genus Chromidina and this is reason why it was redescribed
tinger 1881; Gonder 1905; Hochberg 1971) in Naples (Italy) by Chatton and Lwoff (1935) and Souidenne et al. (2016).
and Banyuls (France) (Foettinger 1881; Hochberg 1990). The trophotomont is vermiform, that reaches 1.4 mm
Today, there is no solid proof that O. octopi differs from length. It is easily distinguishable from other Chromidina by
O. sepiolae. The only difference is the host species (Foet- its club-like apex and 14 Kineties (Collin 1915; Chatton and
tinger 1881). It needs to be confirmed that Opalinopsis has Lwoff 1935; Souidenne et al. 2016).
host specificity. Occasionally, some trophotomonts of C. elegans grow
rapidly and extend up to 5 mm length and they become
10.3.2 Chromidina in Europe hypertrophonts.
Apostome ciliates, Chromidina Gonder (1905), inhabit in 10.3.2.2 Chromidina coronata

the renal sacs of pelagic cephalopods, while the dicyemids C. coronata was described from O. vulgaris by Foettinger
infect mainly the benthic cephalopods (Furuya et al. 2004). (1881), then, from Eledone cirrhosa by Gonder (1905), and
They are specific to this microhabitat because they feed from from Illex coindetii by Dobell (1909). Foettinger (1881) did
cephalopod tissues and fluids (Hochberg 1971; Souidenne not mention the prevalence or mean intensity, but described
et al. 2016). They have a characteristic nuclear system ‘a the dense condition in the renal appendages when parasites
chromidial system’. Chromidina species were reported in 25 were present. C. coronata is very similar to C. elegans in
cephalopod species. Today, only six species of Chromidina body length, body shape, nuclear aspect. However, C.
have been described (Souidenne et al. 2016). coronate is easily distinguishable from C. elegans and the
other Chromidina species by the claviform apex and the
• Life cycle crown of long cilia surrounding the anterior end.
A hypothetical life cycle was deduced from the different

development stages observed and the existence of a crus- 10.4 Concluding Remarks
tacean intermediate host was suggested by analogy to other
apostomes. The adult stage, unlike the other apostomes, is Chromidina ciliates are host-specific to the pelagic squids
vermiforme and called trophotomont. It can reach 2 mm and octopus. However, they are found occasionally in ben-
length. When the trophotomont is extended posteriorly with thic or epibenthic cephalopods when these hosts have a
only one long bud, the budding process is called monotomy. pelagic development stage: like E. cirrhosa, O. salutii,
Later, this bud will develop into a vermiform adult identical to Scaeurgus unicirrhus … implying that they can encounter
the founder trophotomont. When the trophotomont is exten- Chromidina (typically present in the water column, avoiding
ded posteriorly with a chain of small ciliated buds or tomite, competition with dicyemids present near the seabed and
the budding process is called palintomy. Tomite stage is infecting the benthic cephalopods).
probably in charge of the transmission of the infection from a The monophyly of Chromidina is supported among
host to another (Landers 2010; Souidenne et al. 2016). Oligohymenophorea, Apostomatia, Astomatophorida
(Souidenne et al. 2006). However, molecular information of
• Diversity in Europe Opalinopsis is not available, thus, its phylogenetic position
is unclear. The molecular data are essential to clear the
Only two species of Chromidina have been reported in relationship between Opalinopsis and Chromidina and to
Europe. support the monopholy of the Opalinopsidae family.
10 Protist (Ciliates) and Related Diseases 157
Fig. 10.2 Life cycle of the Chromidina (modified from Furuya et al. 2004; on C. elegans, modified from Foettinger 1881)
Table 10.2 Summary of Opalinopsidae parasites of European cephalopods

Ciliate Microhabitat Host species Locality Author(s)
parasite of of the
cephalopods parasite
O. sepiolae Liver Rossia macrosoma Norway (Atlantic Ocean) Hochberg (1971)
Sepietta oweniana France (Mediterranean)
Sepiola atlantica England (English channel)
Sepiola rondeletii Italy, Monaco, France Foettinger (1881); Gonder (1905); Dobell
(1909); Collin (1915); Chatton and Lwoff
(1935)
O. octopi Liver O. macropus Italy (Mediterranean) Hochberg (1971)
O. tetracirrhus Italy (Mediterranean) Foettinger (1881); Gonder (1905); Hochberg
(1971)
C. elegans Renal S. elegans, S. Italy, France Foettinger (1881); Gonder (1905); Dobell
appendages orbignyana, I. coindetti, (Mediterranean Sea, (1909); Collin (1915); Chatton and Lwoff
T. sagittatus, O. salutii Banyuls-sur-Mer), England (1935); Hochberg (1971); Souidenne et al.
(English Channel) (2016)
C. coronata O. vulgaris, E. cirrhosa, Italy, France Foettinger (1881); Dobell (1909); Chatton and
Sepiola rondeleti, S. (Mediterranean Sea, Lwoff (1935); Hochberg (1971); Souidenne
unicirrhus, Illex Banyuls-sur-Mer), England et al. (2016)
coindetti (English Channel)
To date, only eight species of Opalinopsidae have been Dobell CC (1909) Some observations on the infusoria parasitic in
described, and only four have been reported in Europe. This Cephalopoda. Q il microsc Sci 53:183–199
Foettinger A (1881) Recherches sur quelques Infusoires nouveaux
suggests that the diversity of Opalinopsiadae is parasites des Céphalopodes. Arch Biol 2:345–378
underestimated. Furuya H, Ota M, Kimura R, Tsuneki K (2004) Renal organs of
Their impacts on their host individuals are still unknown. cephalopods: a habitat for dicyemids and chromidinids. J Morphol
Some authors suggest that they may be a symbiont (Hochberg 262(2):629–643
Gonder R (1905) Beiträge zur Kenntnis der Kernverhältnisse bei den in
1990; Furuya et al. 2004; Souidenne et al. 2006). Further Cephalopoden schmarotzenden Infusorien. Arch Protis 5:240–262
studies on these enigmatic ciliates are needed to understand Hartog M (1906) Protozoa. In: Harmer SF, Shipley AE (eds) The
the host–parasite relationship (Fig. 10.2 and Table 10.2). Cambridge natural history, vol 1. pp 1–162
Hochberg FG (1971) Some aspects of the biology of cephalopod
kidney parasites. Ph.D. dissertation, University of California, Santa
Barbara
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Mus Vict, Melbourne 44:109–145
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(Foettingériidés+Opalinopsidés). Preuve de sa valdité. C R Hebd
Landers SC (2010) The fine structure of the tropho-tomont of the
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Chatton E, Lwoff A (1935) Les ciliés apostomes 1. Aperçu historique et
tol 6(4):271–279
général; étude monographique des genres et des espèces. Arch Zool
Souidenne D, Florent I, Dellinger M, Justine JL, Romdhane MS,
Exp Gen. 77:1–453
Furuya H, Grellier P (2016) Diversity of apostome ciliates, Chro-
Collin B (1915) A propos de Chromidina elegans (Foettinger). C R
midina spp. (Oligohymenophorea, Opalinopsidae), parasites of
Hebd Acad Sci 160:406–408
cephalopods of the Mediterranean Sea. Parasite 23:33
Dobell CC (1908) The structure and life-history of Copromonas subtilis
n. g., n. so.: a contribution to our knowledge of the Flagellata.
Quart J Micr Sci 52:75–120
made.
Dicyemids
11
Hidetaka Furuya and Dhikra Souidenne
Abstract
Dicyemids are the most common and characteristic endosymbionts found in the renal sac of
benthic cephalopods. In this chapter, we introduce biology and diversity of dicyemids of
European typical cephalopods, Octopus vulgaris and Sepia officinalis. The diphasic life cycle
of dicyemids consists of vermiform stages formed asexually and an infusoriform stage
developed sexually. Their morphology varies depending on the development stage. Recent
molecular studies suggested that dicyemids belong to lophotrochozoans. In Europe, 16
dicyemid species were described from 17 cephalopod species.

Keywords
Dicyemids Mesozoa Vermiform Infusoriform Renal sac
cephalopod molluscs. The body length of dicyemid species

11.1 Introduction
ranges from 0.1 to 10 mm. Typically, two or three dicyemid
species are found in individuals of each cephalopod species
The renal organs of cephalopods are the renal complex (renal
and most are host specific (Furuya 1999). Currently, about 140
and pancreatic appendages) and the branchial heart complex
dicyemid species have been recorded from cephalopod hosts
(branchial heart and pericardial appendage). Prior to release,
distributed in a variety of geographical localities: the Okhotsk
urine is collected in a renal sac, the fluid-filled coelom of
Sea, Japan Sea, western and eastern North Pacific Ocean,
which is a unique environment providing living space for a
waters around New Zealand, North Indian Ocean, Mediter-
diversity of endosymbionts (Hochberg 1982; Furuya et al.
ranean, western North and eastern Atlantic Ocean, Gulf of
2004b). There are phylogenetically distant parasitic organ-
Mexico, and Antarctic Ocean (Catalano 2013;
isms, trematodes, dicyemids, and chromidinids, in the kidney
Castellanos-Martinez et al. 2016; Furuya 1999; Furuya and
of cephalopods (Nouvel 1945; Hochberg and Short 1983;
Tsuneki 2003; Hochberg 1990; McConnaughey 1951; Nou-
Hochberg 1990; Furuya et al. 2004a). Dicyemids (Phylum
vel 1947; Short 1991). The dicyemids are heavily infecting the
Dicyemida) are the commonest and most characteristic
renal organs in their host cephalopods (Fig. 11.1). However,
endosymbionts that are found in the renal sac of benthic
this is somewhat surprising; no damage has ever been
observed in the tissues. This means that dicyemids are not
H. Furuya (&) pathogens to their cephalopod hosts. There must be interac-
Department of Biology, Graduate School of Science, Osaka tion between dicyemids and their host renal organs; it is
University, Toyonaka, Osaka, 560-0043, Japan possible that dicyemids are beneficial for their hosts.
In this chapter, we introduce biology of dicyemids and
D. Souidenne dicyemid species of European typical cephalopods, O. vulgaris
National Museum of Natural History of Paris, Biologie des
Organismes et Ecosystèmes Aquatiques (BOREA), Research and S. officinalis.
Team: Reproduction and Development, Evolution Adaptation,
Regulation CNRS 7208, Sorbonne Université, UCN, IRD 207,
UA 55 rue Buffon, Paris, France

https://doi.org/10.1007/978-3-030-11330-8_11
160 H. Furuya and D. Souidenne
cylindrical cell called the axial cell, which extends to 100 mm

in length in the largest dicyemid.
Regarding the number of somatic cells, the dicyemid is a
multicellular animal that is composed of the fewest in number
of cells in metazoans except for aberrant myxozoans. This
organization does not correspond to metazoan two-layered
construction of endoderm and ectoderm, and dicyemids have
neither body cavities nor differentiated organs.
Infusoriform embryos are ovoid and have both antero-
posterior axis and dorsoventral axis. Embryos mostly consist
of 37 or 39 cells (Short 1971; Furuya 1999), which are more
differentiated than those of vermiform stages (Matsubara and
Dudley 1976; Furuya et al. 2004b). Internally, there are four
Fig. 11.1 Dicyemids on the renal appendage of Amphioctopus
fangsiao. The dicyemids attach on the surface areas of renal large cells called urn cells, each containing a germinal cell
appendages. Scale bar: 1000 lm. Abbreviations RA, renal appendage; that probably gives rise to the next generation (Fig. 11.2). At
RC, renal coelom the anterior region of embryo, there is a pair of unique cell
called the apical cell (Fig. 11.5), each containing a refringent
body composed of magnesium inositol hexaphosphate
(Lapan 1975a). The external cells are mostly ciliated. Infu-
11.2 Life Cycle soriform embryos swim while spinning the body.
The bodies of vermiform stages might be simplified as a
The diphasic life cycle of dicyemids, with a characteristic reflection of their specialization in their parasitic habitat
asexual phase, likely evolved as an adaptation to parasitism composed of renal tubules (Nouvel 1947). By contrast, infu-
(Fig. 11.2), presumably to enable them to adapt to the soriform embryos seem to represent the true level of organi-
cephalopod renal organs. The life cycle of dicyemids consists zation due to free-swimming organisms (Furuya et al. 1997).
of two phases of different body organizations (Fig. 11.2). The However, the body organization of infusoriform embryos
first phase is the vermiform stages, in which the dicyemid cannot be regarded as achieving the grade of tissue level.
exists as a vermiform embryo formed asexually, and as a final
form, the nematogen, or rhombogen. The second phase is the
infusoriform embryo that develops from a fertilized egg. The 11.4 Relationship with Cephalopods
shift from an asexual mode to a sexual mode of reproduction
may be caused by a high population density in the cephalopod Dicyemids are usually found to be heavily infecting the renal
kidney (Lapan and Morowitz 1975). Vermiform stages are organs in their host cephalopods (Fig. 11.1). No damage has
restricted to the renal sac of cephalopods, whereas the infu- ever been observed in the infected renal tissue, so dicyemids
soriform embryos escape from the host into the sea to search apparently do no harm to their cephalopod hosts. Lapan
for a new host. However, it is not clear how infusoriform (1975b) has even suggested that dicyemids facilitate host
larvae develop into vermiform stages in the new host. excretion of ammonia by contributing to acidification of the
urine. In addition to the normal muscular contraction of the
renal appendages, the ciliary activity of dicyemids present in
11.3 General Morphology the kidneys maintains a constant flow of urine, and as a
result dicyemids assist in removal of urine. Thus, dicyemids
Vermiform stages, vermiform embryos, nematogens, and are symbiotic, rather than parasitic, in their relationship with
rhombogens are similar in shape (Fig. 11.2). The body surface cephalopods.
of dicyemids has numerous cilia and the folded structure, The majority of the dicyemid species studied were found
which is considered to contribute to absorb nutrients more to be host specific (Furuya 1999). Typically, two or more
efficiently from urine (Bresciani and Fenchel 1965; Ridley species of dicyemids are present in each host species or each
1968; Furuya et al. 1997). The number of peripheral cells is host individual. There is a certain relationship between the
species specific and constant. At the anterior region, 4–10 calotte configuration of vermiform stages and the
peripheral cells form the calotte, of which cilia are shorter and co-occurrence pattern in hosts (Fig. 11.6). Vermiform indi-
denser than in more posterior peripheral cells (Fig. 11.2). The viduals live specifically within the renal sac. Their anterior
calotte shape varies, depending on the species, and adapts to region, termed a “calotte”, is critical in adapting to their
attach to the various regions of renal tissues in the host kidneys habitats in the renal sac. They insert the distinct anterior
(Furuya et al. 2003a) (Figs. 11.3 and 11.4). There is a central region into renal tubules or crypts of the renal appendages of
11 Dicyemids 161
Fig. 11.2 Dicyemid life cycle (modified from Furuya 2016). The posterior cells are somewhat specialized as uropolars. The development
dashed line indicates an unknown process involved in the infection of a of hermaphroditic gonads (infusorigens), gametegenesis around the
new cephalopod and development into adult forms. In vermiform gonads and development of two types of embryos all proceed within
stages (nematogen, rhombogen, and vermiform embryo), a large the cytoplasm of the axial cell. Abbreviations AG, agamete; AN, axial
cylindrical axial cell is surrounded by peripheral cells. A calotte is cell nucleus; AX, axial cell; CL, calotte; DP, diapolar cell; DV,
formed by four to ten anterior peripheral cells (propolars and developing vermiform embryo; IN, infusorigen; MP, metapolar cell;
metapolars). The other peripheral cells are diapolars and two of those PP, parapolar cell; PR, propolar cell; T, trunk; UP, uropolar cell
11.5 Systematic Position
Van Beneden (1876) regarded the dicyemids as intermedi-

ate in the body plan between Protozoa and Metazoa, and
thus gave them the name Mesozoa. This phylum included
other several microscopical enigmatic organisms, Tri-
choplax, Haplozoon, Neresheimeria, Salinella, and
orthonectids, which were not assignable to any of phyla.
Most of these organisms were subsequently belonged to the
other phyla (Hyman 1940). Only dicyemids and orthonec-
tids were often united into a single phylum Mesozoa. Later,
Hochberg (1990) and Kozloff (1990) treated them inde-
pendently as a separate phylum, Dicyemida and Orthonec-
tida, in each review. However, they were still treated as the
Mesozoa in the many zoological textbooks, because of their
unclear relationships among animals. Several zoologists
regard the simple organization of dicyemids to be the result
of specialization for parasitism (Nouvel 1947; Stunkard
Fig. 11.3 Light micrographs of stained sections through the renal 1954; Ginetsinskaya 1988). However, Hyman (1956),
organ of A. fangsiao. The niche separation occurs between dicyemid
Lapan and Morowitz (1975), and Ohama et al. (1984)
species inhabiting in the renal appendages. Dicyema akashiense
(arrowhead) lives in the fold of renal appendages, while D. helo- concurred that dicyemids are primitive multicellular
cephalum (arrow) attached on the surface of renal appendages. Scale organisms. Since dicyemids have several protozoan-like
bar: 20 lm. Abbreviations EP, epithelial cell of renal appendage; RC, features, an affinity to the protozoans has been pointed out
renal coelom; V, vein
(Czaker 2006; Noto and Endoh 2004). Current analyses of
molecular sequences have revealed that, rather than truly
the host (Ridley 1968; Furuya et al. 1997) or attach to sur- primitive animals that deserve the name “mesozoan”, they
faces of the renal appendages with a flat anterior region probably belong to the lophotrochozoans (Katayama et al.
(Furuya et al. 2003a; Furuya 2005, 2006) (Fig. 11.3). 1995; Kobayashi et al. 1999; Aruga et al. 2007; Suzuki et al.
Fig. 11.4 European dicyemid

species. a C. polymorpha; b M.
vespa; c D. typus; d Dicyema
moschatum; e Dicyemennea
eledones; f P. truncatum
11 Dicyemids 163
Fig. 11.5 Schematic drawings of vermiform larvae with 22 peripheral enveloping cell; G, germinal cell; L, lateral cell; LC, lateral caudal cell;
cells (a, b), vermiform larvae of C. polymorpha (c, d), vermiform larvae MD, median dorsal cell; MP, metapolar cell; PD, paired dorsal cell; PP,
of M. vespa (e, f), infusoriform larvae with 39 cells (g–i) (modified from parapolar cell; PR, propolar cell; PVL, posteroventral lateral cell; RB,
Furuya and Tsuneki 2003; Furuya 2016). a, f, lateral view; b, c, sagittal refringent body; SC, short cilia; T, trunk; U, urn cell; UC urn cavity; UP,
section; d, ventral view; e, dorsal view. Abbreviations A, apical cell; AG, uropolar cell; VC, ventral caudal cell; VI, ventral internal cell; V1, first
agamete; AX, axial cell; C, couvercle cell; CA, capsule cell; CL, calotte; ventral cell; V2, second ventral cell; V3, third ventral cell
DC, dorsal caudal cell; DI, dorsal internal cell; DP, diapolar cell; E,
2010; Mikhailov et al. 2016; Lu et al. 2017). Despite their known to have a relatively wide host range (Furuya 1999). In
extremely reduced body plan, dicyemids still appear to European dicyemids, for instance, Dicyema macrocephalum
exhibit some degree of cell differentiation (Ogino et al. van Beneden (1876) appears to infect five cephalopod species
2011). In this reason, the name Mesozoa is not suited for belonging to three genera. The other species with a wide host
their phylogenetic place; the Dicyemida, which is the first range have been described mostly without using characters of
name of dicyemids by Khrone (1839), has been used as the the infusoriform embryos, so it will be necessary to examine
phylum name of dicyemids in 1999 (Furuya 1999). the cellular composition of the infusoriform embryos of these
other species to confirm their occurrence in more than one
species.
11.6 Diversity of Dicyemids in Europe O. vulgaris, the common octopus, is the commercially
most important species in the European cephalopods.
Cephalopods are commercially important in European Therefore, this species has been well studied for the parasite.
countries. Many species of cephalopods have been examined Four species of dicyemids, Conocyema polymorpha,
for parasites in European waters. Sixteen species of dicye- Dicyema paradoxum, Dicyema typus, and Dicyemennea
mids were described from 17 species of cephalopods, so far lameerei, have been described from O. vulgaris in the
(Table 11.1). From one to four dicyemid species have been Mediterranean Sea, the English Channel, and the Eastern
recorded in a single species of cephalopods. Most dicyemid North Atlantic Ocean (Table 11.1). Typically, two or three
species are host specific. However, 18 dicyemid species are species of dicyemids are present in each host species or each
host individual, all four species were never simultaneously

present in a single-host individual (Furuya et al. 2003b).
Dicyema and Dicyemennea are common, and the largest
number of species is placed in these genera. Several other
genera are monotypic or contain only a small number of
species. C. polymorpha is the unusual dicyemid, which is
irregular in shape and lacking external cilia (Fig. 11.7). It
has relatively small-sized species with 12 peripheral cells.
The vermiform larvae are cuneiform and have distinct
calottes, which consist of only single tier of four propolar
cells. In this species, four parapolar cells are located in the
posterior part of propolar cells instead of metapolar cells
(Fig. 11.5).
S. officinalis is also commercial species in Europe coun-
tries; it is a common inhabitant of the English Channel and
Mediterranean Sea. Four species of dicyemids are also
recorded from this host cephalopod, namely, Dicyemennea
gracile, Pseudicyema truncatum, Dicyema whitmani, and
Microcyema vespa (Table 11.1). M. vespa is unusual in that
the body forms a syncytium and the calotte is irregular
(Fig. 11.7). In M. vespa, the calotte region can only be
recognized in the larval stage (Fig. 11.5) (Furuya et al.
2001). P. truncatum is the most common species observed
with the highest prevalence, nearly 80%. In contrast, D.
whitmani is a very rare species that has been found in only
one cuttlefish host individual collected off Naples, Italy
(Furuya and Hochberg 1998).
The dicyemid fauna in the coastal areas of the United
States and Japan has been well studied (Furuya 2016), as
well as in European waters. The cephalopod fauna in
European waters is similar to the Japanese waters, not to the
Fig. 11.6 Co-occurrence pattern of calotte shapes usually detected
within host individual. (1) Stylized drawings of the main three types of
United States, because no cuttlefish (Sepia) lives in the
regular calotte configurations and a type of irregular configuration coastal areas of the United States. From the ecological
found in vermiform stages. Type I, conical-shaped, is divided into two viewpoint, O. vulgaris and S. officinalis are comparable to
types (Type Ia and Ib); Type II, cap-shaped; Type III, discoidal; the Japanese species, Octopus sinensis and Sepia esculenta,
Type IV, an irregular configuration. Shaded and striped areas indicate
propolar cells and metapolar cells, respectively. (2) When two species
respectively. O. sinensis has been regarded as the same
of dicyemids are present, two distinct calotte shapes, conical and species as O. vulgaris (Gleadall 2016). S. esculenta is a
discoidal, are usually observed. (3) When three species of dicyemids common cuttlefish in Japanese waters. However, Cono-
are present, three types of calotte configurations are usually observed, cyema and Microcyema species have never been found in
conical (two grades) and discoidal. (4) When more than four species of
dicyemids are present, at least one species is characterized by its rare
these host species and the other Japanese cephalopods. The
irregular shaped calotte that spreads, connects with other individuals, presence of various genera may be characteristic of the
and forms a syncytium dicyemid fauna in Europe.
11 Dicyemids 165
Table 11.1 Dicyemids from cephalopod species in European waters

Cephalopods Dicyemids Locality References
Octopoda
Bathypolypus Dicyemodeca Mediterranean (Spain) Nouvel (1961)
sponsalis delamarei
Eledone D. eledones Eastern North Atlantic Ocean (Sweden, Norway), Wagener (1857); Whitmann (1883);
cirrhosa English Channel (France), Mediterranean (Italy, Hartmann (1906); Nouvel (1947); Dhikra
France) et al. (2016)
D. lameerei English Channel (France) Nouvel (1947)
Eledone D. moschatum Mediterranean (Italy, France) van Beneden (1876); Whitmann (1883);
moschata Hartmann (1906); Nouvel (1947)
D. eledones Mediterranean (Italy, France) Wagener (1857); Whitmann (1883);
Nouvel (1947)
Octopus Dicyema Mediterranean (Italy, France) Whitmann (1883); Nouvel (1947)
defilippi microcephalum
Octopus D. paradoxum Mediterranean (Italy, Monaco, France) van Beneden (1876); Whitmann (1883),
macropus Nouvel (1947)
Octopus Dicyema banyulensis Mediterranean (Italy, France) Furuya and Hochberg (1998)
salutii Dicyema benedeni Mediterranean (Italy, France) Furuya and Hochberg (1998)
D. eledones Mediterranean (Italy, France) Wagener (1857); Whitmann (1883);
Hartmann (1906); Nouvel (1947)
O. vulgaris C. polymorpha Mediterranean (Italy, Monaco, France) Whitmann (1883); Hartmann (1939);
Nouvel (1947)
D. paradoxum English Channel (France), Mediterranean (Italy, von Kolliker (1849); van Beneden (1876);
France) Whitmann (1883); Nouvel (1947)
D. typus English Channel (France, England), Eastern North van Beneden (1876); Nouvel (1947)
Atlantic Ocean (France), Mediterranean (Italy,
Monaco, France)
D. lameerei English Channel (France), Eastern North Atlantic Nouvel (1947)
Ocean (France), Mediterranean (Italy, Monaco,
France)
Sepioidea
Sepia elegans D. macrocephalum Mediterranean (Italy, Monaco, France) van Beneden (1876); Whitmann (1883);
Hartmann (1906); Nouvel (1947)
Dicyema Mediterranean (Italy, Monaco, France) van Beneden (1876); Nouvel (1947)
schulzianum
S. officinalis D. whitmani Mediterranean (Italy, France) Furuya and Hochberg (1998)
D. gracile English Channel (France), Mediterranean (Italy, Wagener (1857); Whitmann (1883);
Monaco, France) Nouvel (1947)
M. vespa English Channel (France), Mediterranean (Italy, van Beneden (1882); Nouvel (1947)
Monaco, France)
P. truncatum English Channel (France), Eastern North Atlantic Whitmann (1883); Nouvel (1947)
Ocean (France), Mediterranean (Italy, France, Spain)
Sepia D. gracile Mediterranean (France, Spain) Wagener (1857); Whitmann (1883);
orbignyana Nouvel (1947)
P. truncatum Mediterranean (Italy, Monaco, France) Whitmann (1883); Nouvel (1947)
Sepiolioidea
Rondeletia Dicyema rondeletia Mediterranean (Italy, Monaco, France) Nouvel (1944)
minor D. schulzianum Mediterranean (Italy, Monaco, France) van Beneden (1876); Whitmann (1883);
Nouvel (1947)
(continued)
Table 11.1 (continued)

Cephalopods Dicyemids Locality References
Rossia P. truncatum Mediterranean (Italy, Monaco, France) Whitmann (1883); Nouvel (1947)
macrosoma
Sepetta D. rondeletiolae Mediterranean (Italy, Monaco, France) Nouvel (1944)
neglecta
Sepietta D. macrocephalum Mediterranean (Italy, Monaco, France) van Beneden (1876); Whitmann (1883);
obscura Nouvel (1947)
Sepietta D. macrocephalum Mediterranean (Italy, Monaco, France) van Beneden (1876); Whitmann (1883);
oweniana Nouvel (1947)
D. rondeletiolae Mediterranean (Italy, Monaco, France) Nouvel (1944)
Sepiola D. moschatum Mediterranean (Monaco, France) Whitmann (1883); Nouvel (1947)
rondeleti
Sepiola D. macrocephalum Mediterranean (Italy, Monaco, France) van Beneden (1876); Whitmann (1883);
steenstrupiana Nouvel (1947)
D. microcephalum Mediterranean (Italy, Monaco, France) Whitmann (1883); Nouvel (1947)
Fig. 11.7 Light micrographs of

C. polymorpha (a, b) and M.
vespa (c). Scale bars represent 20
lm. Abbreviations AX, axial cell;
CL, calotte; IF, infusoriform
embryo; IN, infusorigen; V,
vermiform embryo
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Commons Attribution 4.0 International License (http://creativecom- the chapter's Creative Commons licence, unless indicated otherwise in a
mons.org/licenses/by/4.0/), which permits use, sharing, adaptation, credit line to the material. If material is not included in the chapter's
made.
Metazoa and Related Diseases
12
Santiago Pascual, Elvira Abollo, Ivona Mladineo, and Camino Gestal
Abstract
Cephalopods and their metazoan parasites have coevolved in wild fisheries for many years. In
fact, helminth larvae and parasitic copepods have been recorded in cephalopods worldwide.
This is not surprising considering the important role cephalopods play in the transfer of
energy and contaminants in marine food webs. Nerito-oceanic ommastrephid squids are by far
the most noticeable trophic bridge for helminth parasites in the marine realm, coastal octopus,
and cuttlefish serving as primary host for crustaceans. Although it is highly likely that
parasitic infections occurred, relatively little is known about the pathogenic potential of
metazoan parasites in naturally infected cephalopods. It is stated that heavy parasitic
infections may probably cause host morbidity or poor condition but signs of disease are
singularly rare with very few specimens exhibiting disease conditions. Unfortunately, neither
robust scientific evidence nor available material is available to support this statement. It is
more likely that metazoans may deplete energy stores of infected cephalopods, which are
directed toward tissue repair and the host’s defense mechanisms. Parasitic infection may thus
be considered an environmental stressor and as such a source of uncertainty in the evaluation
of the potential productivity of cephalopod populations.

Keywords
Metazoan parasites Pathogens Trematodes Cestodes Nematodes Crustaceans
Seafood security
S. Pascual (&)
12.1 Introduction
Vigo, Pontevedra, Spain Metazoan parasites comprise a polyphyletic group made up
e-mail: [email protected] of six parasitic taxa: flatworms (Platyhelminthes), tape-
E. Abollo worms (cestodes), trematodes (flukes), roundworms (nema-
Centro Tecnológico del Mar, Fundación CETMAR, 36208 todes), acanthocephalans, and crustaceans. They exhibit
complex life cycles and reproductive strategies, with a
remarkable high diversity and prevalence in marine
I. Mladineo
Institute of Oceanography and Fisheries, 21000 Split, Croatia ecosystems. Ecto- and endoparasitic metazoans including
e-mail: [email protected] monoxenous/heteroxenous and specialist/generalist species
C. Gestal have been largely recorded in the different components
Aquatic Molecular Pathobiology Group, Institute of Marine (zooplankton, fish, large fish, marine mammals, and sea-
Research, Spanish National Research Council (CSIC), 36208 birds) of the trophic cascades characteristically defined in the
Vigo, Pontevedra, Spain marine realm (Rohde 2002).

https://doi.org/10.1007/978-3-030-11330-8_12
170 S. Pascual et al.
Recently, spatially explicit modeling revealed that Euro- provinces than in the Mediterranean basin. Furthermore, the
pean cephalopod distributions match contrasting trophic size/weight/sexual maturity structure of a given cephalopod
pathways (Puerta et al. 2015), and therefore it is expected that grouping is recognized as the key categorical predictor of
cephalopods are common hosts for metazoan parasites. Such parasite epidemiological values determining the intra- and
statement is not surprising considering that cephalopods are interspecific variability of the metazoan fauna of European
key element in the food web, its foraging behavior and diet cephalopod stocks. Generally, no significant differences in the
facilitates endoparasite transmission. Furthermore, cephalo- infection rate of males and females are observed, but as the
pods inhabit in a wide array of biotopes (shallow to deeper size/weight/maturity increase the infection values substantially
waters)/ecosystems (benthic to pelagic), and its varied social raise. Additionally, parasite recruitment may vary depending
structure and behavior capabilities (solitary, scholar) may also on the definitive host distribution (Kuhn et al. 2016) but
enable an ectoparasitic recruitment into major cephalopod especially, at the mesoscale, the recruitment in the mesozoo-
stocks. The above both arguments provide us a broad per- plankton and hyperbenthos are affected by the oceanographic
spective to understand the great availability of ubiquitous regime (Pascual and González 2007; Gregori et al. 2015). The
microhabitats offered by cephalopods to colonization by latter authors gave evidence that at in upwelling systems par-
metazoan parasites. In fact, the reported species composition of asite faunas are impoverished, whereas downwelling relaxing
the metazoan fauna-infecting cephalopods being characterized conditions propitiate optimal conditions to successful. Simi-
by a relatively uniform and limited composition remains far larly, ontogenetic shifts in cephalopod diet from planktonic
from saturated, with empty microniches to be colonized. invertebrates and small fish planktophages on one side to lar-
The general qualitative character of the metazoan fauna of gest fish preys on the other may contribute to age-related
European Atlantic populations of octopus/cuttlefish/squid variations in the helminthofauna (from trematodes to
(coastal-slope species) and short-finned squid (slope-shelf cestodes/nematodes). These shifts may also enhance the
and nerito-oceanic species) is almost the same as that of accumulation process of parasites which largely favors the
Mediterranean populations; only a few differences in species typical skewed binomial distribution of metazoans in cepha-
composition were observed, which clearly reflects that lopod populations.
infection by metazoans (mostly at larval stage) are nonspe- Fragmentary information on the metazoan fauna of
cific. Overall, the community structure of parasitic metazoans European cephalopods based on opportunistic sampling
of European cephalopods is similar among ecologically and plans within commercial fisheries or market surveys has
taxonomically close species. On the basis of 2000 individuals been produced in 20 papers published in the last 30 years
comprising 10 cephalopod species collected at a microgeo- accounting from the latter revision provided by Hochberg
graphic area (Galician waters, NW Spain), González et al. (1990). This knowledge progress made on the biodiversity,
(2003) found some associations between parasite relative pathology, and ecological relationships of metazoan para-
species diversity and cephalopod life cycle characteristics. sites affecting cephalopods reflects a poor coverage for
Results showed that those species with similar risk of species/geographical areas. There can be no doubt of the
becoming infected with a given parasite fauna belong to one lack of sufficient critical mass of European scientists in this
of three ecological groupings (coastal, intermediate, or field, but in a comparative analysis with other commercially
nerito-oceanic). It was suggested that the ecological niche of a important taxa, a historical negligible financial support for
cephalopod species is more important in determining its risk research on cephalopod diseases animal group was noted
of parasitic infection than is phylogeny. (Pascual and Guerra 2003). The rate of knowledge progress
Mostly, the narrow range of metazoan parasites found in on cephalopod diseases becomes, therefore, a vexing,
European cephalopod populations is thus characterized by unbalanced question in fisheries research.
wide host specificity. In the life cycles of the reported par- There is, nevertheless, some current regional and national
asitic helminths, cephalopods may be considered second surveillance plans for zoonotic parasites implicated in human
intermediate or transport/paratenic hosts serving as trophic allergy (following the scientific opinion on parasite risk in
bridges for parasite flow to top predators (final hosts) (Pas- fishery products published by the European Food Safety
cual et al. 1996a; Abollo et al. 1998). Authority; EFSA 2010) which may serve as a promising
Moreover, the composition of the parasitocoenoses of platform for future biodiversity studies on metazoan parasites
European cephalopods seems to remain stable over time. of cephalopods. The use of already certified biobanking tools
Another issue is the marked differences noted in the infection in fish parasite research (González et al. 2018) can also aid to
rate. The demographic infection parameters of a given cepha- establish network opportunities for sampling and collection
lopod species within a particular ecological group may vary of traceable metazoan cephalopod parasites.
among ecoregions and even province of a particular European Against the overall background, the strategy defined in
marine realm. As a rule, infections by metazoan parasites are this chapter is not to make a comprehensive literature review
significantly higher in northern European seas and Lusitanian of host–parasite systems, but illustrate the role of the most
12 Metazoa and Related Diseases 171
prevalent and relevant metazoan parasites as pathogens and France and England (Hochberg 1990). Again, the several
diseases-related agents in European cephalopods. Thereafter, morphs recognized present taxonomic uncertainty. Deroge-
scientific focus for the different taxa which is discussed nes varicus were reported in Sepia officinalis in the coast of
under the new challenge perspectives. Plymouth, England, where more than 80% of the specimens
over 10 cm mantle length appeared to be infected.
Lecithochirium sp. was reported in Octopus vulgaris in the
12.2 Metazoan Parasites as Pathogens North East Atlantic, NW Spain (Fig. 12.1b–d). This hemi-
urid shows a fusiform body, with a sub-terminal oral sucker,
12.2.1 Trematodes a small pharynx, big acetabulum, or ventral sucker, with
conspicuous acetabular cleft, and excretory vesicle with a
Several monogeneans have been described from European characteristic Y-shape.
cephalopods (Hochberg 1990), but some forms were con- With some exceptions, most reports of trematodes in
sidered incertae sedis and the type material (holotype, cephalopods show a low prevalence of infection, acting as
paratype, and syntype) is not largely available. The single paratenic hosts. Metacercaria and adult digeneans infected
extent of evidence came by Llewellyn (1984), who reported the digestive system (especially, the stomach and caecum)
gyrodactylids as epidermal browsers in the mantle cavity and and despite they can form oval cysts with thin, transparent
on the gills of Alloteuthis squids from the North Sea and the envelopes no significant host tissue reaction was noted.
English Channel off France and England. These monoge-
neans are thought to be recruited to the squids through direct
contact of adults at the time of mating. 12.2.2 Cestodes
Cephalopods are also parasitized by digenean trematodes,
at larval stages (metacercaria) or adults, acting as second Larval and post-larval stages of cestodes repeatedly have
intermediate, paratenic, or final host, but never as first been described from European cephalopods (Hochberg
intermediate host (Hochberg 1990). Metacercariae of didy- 1990). The prevalence and intensity of infection are high in
mozoid are the most important group of digeneans which theuthoid squids, showing a wide range of sizes and shapes
infects oceanic squids, and some of them have been in relation to the squid host (Fig. 12.2a, b). Plerocercoid
described in Illex coindetii (Fig. 12.1a) or Todaropsis larvae belonging to the orders Tetraphyllidea and Try-
eblanae in European waters (Hochberg 1990). Other groups panorhynchidea dominate among cestodes (Pascual et al.
of digenean trematodes were reported in 10 species of 1996b), which become sexually mature in the digestive
squids, cuttlefish, and octopus from the French and Italian tracts of elasmobranch fishes, sharks, and rays. The scolex of
coasts (Mediterranean) and from the English Channel off Tetraphyllidean larvae has characteristically four large
Fig. 12.1 Digenean trematodes

from European cephalopods.
Whole specimens (ventral view)
of didymozoid from from Illex
coindetii (a), and adult
Lecitochirium sp. (b, c, c) from
Octopus vulgaris showing oral
and ventral sucker, pharynx,
acetabular cleft, and excretory
vesicle. View at light microscopy
(a, b), and (c, d) scanning
electron microscopy (SEM).
Scale bars: A: 200 µm; B:
200 µm; C. 200 µm; D 100 µm
Fig. 12.2 Larval cestodes tetraphyllidean taken from a variety of sucker in apical (m, n) view of scolex. Characteristically, in some
cephalopod species of Europe. Note the biomass of plerocercoids cases, the scolices are invaginated as seen in scanning electron
(a) and the range of sizes and shapes (b) in relation to the squid host. microscopy (o) and histological section (p). (e–h) Alcoholic Gill’s
Light microscopy of two typical body architecture of tetraphyllideans Haematoxylin. (p) H&E. Scale bars: C: 1 mm; D: 5 mm; E: 100 µm;
(c, d), showing the phenotypic plasticity of their scolices: with F: 100 µm; G: 100 µm; H: 500 µm; I: 200 µm; J: 100 µm; K:
wavy-edged bothridia (e–g), smooth-oval bothridia (i–k), and 100 µm; L: 100 µm; M: 200 µm; N: 500 µm; O: 500 µm; P: 250 µm
scalloped-edged bothridia (l). Tegument (h) and apical and bothridial
leaf-like bothridia. The genus Phyllobothrium is the most Despite the yet unresolved uncertainty of their taxonomic
common and widely dispersed in decapod and octopod affinities, these forms are well recognized by their smaller sizes
cephalopods from European waters (Fig. 12.2c, d). Phyl- and bothridia with a characteristic number of suckers.
lobothriid larvae are divided into three groups by the form of Larval Tetrarhynchidean metacestodes characteristically
their bothridia: with wavy-edged bothridia (Fig. 12.2e, g), with four hook-armed tentacles are well represented by
smooth-oval bothridia (Fig. 12.2i, k), and scalloped-edged Nybelinia plercocerci in European cephalopods. Nybelinia is
bothridia (Fig. 12.2l). An apical sucker is also observed (M, commonly encountered found in a variety of cephalopods in
N). They show one accessory sucker by bothridia and one the NE Atlantic and Mediterranean cephalopods (Pascual
apical sucker. The tegument of the body is covered by et al. 1996a). N. lingualis (Cuvier 1817) localizes in
microvilli or microtrics (Fig. 12.2h). Characteristically, in intestinal mesenteries, on the ovary, on the linings of the
some cases, the scolices are invaginated (Fig. 12.2o, p). coelomic cavity, and on the external covering of the stomach
Plerocercoids are mainly lying free in the lumen or (Fig. 12.3a). The scolex is composed of four sessile both-
attached to the organs of the digestive tract (stomach, cae- ridia showing four tentacles or proboscids armed by small
cum, and rectum) (Fig. 12.2p), although sometimes found helicoidally placed hooks (Fig. 12.3b). They can be
free in the liver, mantle cavity, and even leaving the host. observed evaginated (Fig. 12.3a, b, d), or invaginated into
Plerocercoids move freely between these organs, especially the body (Fig. 12.3c). Despite the intensity of infection can
when changes in temperature and other physico-chemical be significantly high in larger squids and the tentacles are
factors occurred in postmortem condition. This movement of used to anchor the host’s tissues, no noticeable report on the
large forms has been suggested to impair the marketability of pathological effect of these cestodes was found in the liter-
the infected specimens considerably due to the unaesthetic ature, with the exception of specific light infiltration of
appearance of the fish product. hemocytes in the infected area (Fig. 12.3e).
Other Tetraphyllidean larvae have been reported from Most reports of cestodes in European cephalopods simply
European cephalopods within the Scolex spp. species complex. document the presence of cestodes, site of infection, sample
Fig. 12.3 Larval

tetrarhynchidean of Nybelinia
lingualis from a variety of hosts.
Stereomicroscopy (a) and light
microscopy (b) image of the
typical body architecture of
tetrarhynchideans, showing four
sessile bothridia with four
tentacles armed with hooks in
spirals. Detail of the hooks
observed invaginated into the
body (c) or evaginated (d).
Histological section showing
specific light infiltration of
hemocytes surrounding the
infected area (e) H&E stain. Scale
bars: A: 250 µm; B: 250 µm; C:
250 µm; D:250 µm; E: 100 µm
locality, and some demographic infection parameter. Despite identify the anisakids and their distribution in the organs and
singular effort has been concentrated on the ecological rela- tissues of the host (Abollo et al. 2001; Melani et al. 2014;
tionships of cephalopod–cestode systems (e.g., Gaevskaya Serracca et al. 2013; Pico-Duran et al. 2016; Cost et al.
and Nigmatulli 1978; Pascual et al. 1996b) as a whole neither 2016). Larval stages of Anisakis simplex sensu stricto and A.
pathological nor disease symptoms have been mentioned or pegreffii have been identified parasitizing Octopus vulgaris,
figured associated to the cestode infections. Eledone cirrhosa, Sepia officinalis, and other sepiidae spe-
cies, and with higher prevalence have been identified in
loliginids and ommastrephids in European waters (Abollo
12.2.3 Nematodes et al. 1998, 2001). As a rule, anisakids are found covering
the outer and inner membranes of internal organs, especially
In the last years, larval ascaridoid nematodes are by far the the gonads (ovary and testes), nidamental glands and on the
most commonly reported parasitic agent in European wall of stomach. Sometimes they were found on the coe-
cephalopods. Survey of anisakids with zoonotic and human lomic membrane of the mantle wall (Fig. 12.4a, b). In
allergic potential in cephalopod food products has been a cephalopods at fresh postmortem condition, the viable ani-
target of some national surveillance plans. In fact, despite in sakid larvae can be also found actively moving within the
the older literature specific identification for nematode larval mantle cavity. This provokes commercial rejection by con-
types in cephalopods has been suggested as a main taxo- sumers due to the unaesthetic appearance of squid products.
nomic concern, from the Hochberg’s revision (1990) General morphological diagnostic characteristics of larval
molecular methods have been largely applied to specifically stages of Anisakis, such as anterior end showing a boring
Fig. 12.4 Anisakid nematodes

from various cephalopod species
in Europe. Third-larval stages are
easily recognized
macroscopically (a, b) coiled and
encysted in different organs
(arrows). Light microscopy
images of the anterior (c) and
posterior (d) extremities with the
characteristic ventriculus (e) of
Anisakis simplex showing some
morphological structures (striated
cuticle, boring tooth, excretory
pore, lips, esophagus, intestine,
anal gland, anus, and mucron) in
lateral view. Cystidicola
sp. nematode from Octopus
vulgaris (f, g). Anterior (f) and
posterior (g) extremities showing
the characteristic pseudolabia and
mucron, respectively. Scale bars:
C: 50 µm; D: 30 µm; E: 150 µm;
F: 20 µm; G: 20 µm
tooth, mouth, lips with papilla and excretory pore, ventricu- the gills of teuthoids (L. vulgaris, T. eblanae), cuttlefish
lous with specific length and shape, and tail end showing the (Sepia officinalis, S. elegans, S. orbignyana, Rossia caroli,
anus and mucron terminal, are shown in Fig. 12.4c, e. Sepietta oweniana) and octopus (Octopus vulgaris, E.
Some nematodes other than ascaridoids occasionally have moschata, Bathypolypus sponsalis) (Gestal et al. 1999b)
been observed in European cephalopods. As an example, (Fig. 12.6a–e).
spirurida larval nematodes of a cystidicolid were found Heavy infestation by the postembryonic stages of the
encapsulated in the external and internal walls of the crop, and siphonostomid copepod Pennella sp. has also been com-
on the connective tissue sheath surrounding the digestive monly reported in the gills of several commercially impor-
gland and intestine of the common octopus O. vulgaris caught tant cephalopod species from temperate waters of the NE
at NW Spain (Gestal et al. 1999a). Larval of Cystidicolidae Atlantic (Pascual et al. 1996a). The spatiotemporal distri-
shows mouth dorsoventrally elongated with a pair of pseu- bution of this mesoparasitic copepod revealed a marked
dolabia with conical protuberances, two lateral cephalic alae. aggregated and seasonal pattern of parasites that fits well
The esophagus shows an anterior short muscular part and with their mating behavior in the gills of cephalopods
posterior part longer and glandular. Post-anal tail short, with (Pascual et al. 2001). Adults of the cyclopoid copepod of the
weakly nodulose truncated mucron (Fig. 12.4f, g). genus Octopicola, Octopicula superbus have been identified
Most recently, some efforts have been made to show the in European octopuses at the English Channel, Mediter-
histopathological effect of nematode infections (Pascual et al. ranean, and Atlantic at NW Spain (Fig. 12.6i–l).
1996b; Gestal et al. 1999a). Early infections consisted of Although in the older literature the cephalopod-copepod
necrotic tissue displaying a light inflammatory reaction fol- relationships have been categorized as highly host specific,
lowed by haemocyte infiltration. In more advanced case of most of the species were considered commensals. They
infection, the response of cephalopod tissues to invading moved on the skin (of head, arm, or mantle), mantle cavity,
nematodes was a typical cell-mediated immune response (Ford and gills, and feed on mucous (Fig. 12.6f–h, m–o), thereby
1992), with parasite encapsulation as an immune strategy to they were considered not true parasites when affecting wild
avoid parasite migration and destruction of host tissues cephalopod populations. In fact, the infected cephalopods did
(Fig. 12.5a–h). Most infected organs displayed evidence of not appear stressed and no damage to the tissues was reported.
mechanical compression and displacement of host tissue ele- However, pennellid larvae deserve a special mention. Cor-
ments at sites close top or in direct contact with parasitic larvae, relation between heavy gill infections and poorer squid con-
with varying degrees of cellular infiltration. The spaces sur- dition at the infrapopulation level has also been demonstrated
rounding worms were usually coated with tissue fragments, cell (Pascual et al. 2005). This work provides strong evidence that
debris, and extensive secretion of mucous. Anisakids were thus mechanical lysing of large areas of functional tissues pro-
postulated as responsible for parasitic castration in those duced by pennellids contributes to the variability in squid
heavily infected mature squids as a consequence of partial growth, being one of the multiple categorical predictors of
destruction and alteration of gonad tissues (Abollo et al. 1998). size-at-age data in several infested cephalopod species com-
mercially exploited in European waters.
12.2.4 Crustaceans
12.3 New Coming Challenges
Few published reports dealt with the crustaceans infecting
European cephalopods. Branchiurans and cymothoid isopods Modern conception of fisheries management under the
have been accidentally found on the skin and in the mantle Common Fishery Policy and the H2020 Research and
cavity of cuttlefish (Sepia and Sepiola species). The majority Innovation Framework is based on two driven pillars: the
of crustacean reports refer to copepods: harpacticoids of ecosystem-based concept and the accommodation of fish
Cholydia intermedia from the mantle cavity and gills of a production systems to the new seafood system challenge.
cirroteuthid cephalopod collected in the Faroe-Shetlands The final goal is to conquer a better understanding of the
Channel; lichomolgids on the gills of Sepia officinalis and natural and anthropogenic impacts on fish resources at the
T. sagittatus from NW Mediterranean (Rosas and Banyuls) ecosystem level to render productive ecosystems and healthy
and the Adriatic (Trieste) (Hochberg 1990; Costanzo et al. seafood products.
1994), and from the gills of Illex coindetii off the Atlantic
coast of the Iberian Peninsula (Lopez-González and Pascual
1996); females and males of a cyclopoid copepod parasite in 12.3.1 Seafood Security
Octopus vulgaris from Banyuls (Hochberg 1990).
Larval stages of Lernaeoceridae assigned to Pennella Parasitic diseases have been largely recognized as a bottleneck
varians have been largely recorded at the Mediterranean on that hampers fish production systems (Shinn et al. 2014).
Fig. 12.5 Histopathology caused

by larval nematodes in various
cephalopod species in Europe.
Histological sections showing
different aspects of the
encapsulation process of nematode
larvae in squids (a, b, c, d) and
octopuses (e–h). (a) Early infection
showing an encapsulated larva of
Anisakis simplex with the Y-shape
lateral chords, and hemocytic
infiltration. (b) Histological section
showing heavy hemocyte
infiltration in response to the
infection by larval nematodes
(arrowheads). (c) Later anisakid
infection showing extensive
secretion of mucous in the spaces
surrounding the worms.
(d) Histological section of
connective-muscular tissue of the
testis of squid showing mechanical
compression and displacement of
host tissue elements (*, cell debris).
(e–h) Later infection in Octopus
vulgaris showing nematodes larvae
encysted in connective tissue
capsule with various degrees of
hemocyte infiltration.
(g) Cystidicolid larvae infecting the
connective tissue of the digestive
gland. (a, b, c, e, f, g) H&E stain.
(d, h) Masson’s trichrome stain.
Scale bars: A: 100 µm; B: 100 µm;
C: 200 µm; D: 250 µm; E:
100 µm; F: 200 µm; G: 20 µm; H:
100 µm
However, as it was summarized in this chapter, apart from estimate the impact of aggregated metazoan infrapopulations
histopathological evidences the role of metazoans as etio- on the condition and productivity of cephalopod populations,
logical agents of pathologies in cephalopod species, stocks, or especially for those species that have fast-growing potential in
individuals still remains unexplored. Of particular interest is to open-caged systems. Similarly, considering that multiple
Fig. 12.6 Various ontogenetic

stages of the most prevalent
copepod (Pennella sp.) from the
gills of many cephalopod species
of Europe. (a–c) Copepodid with
characteristic second antenna (as);
(d) chalimus with polar filament
(fp); (e) free-living adult male.
Histological sections of squid
gills showing pennellids (arrows)
associated to mechanical
displacement of host tissues (f);
secretion of mucous (*) (g);
mating behavior (arrow) of an
adult male and female (h).
Lichomolgids Octopicola
superbus (habitus, lateral) from
the gills of octopuses (i–o).
Free-living adults showing the
specific morphological characters
of the thorax, rostrum, and legs,
with specific distribution of spiny
and seta (*) (i–l). Histological
sections of octopus gills showing
Lichomolgids free living or
anchored to the host tissue (m–o).
(f–h) Masson’s trichrome stain.
(m–o) H&E stain. Scale bars: A:
100 µm; B: 100 µm; C: 100 µm;
D: 100 µm; E: 100 µm; F:
200 µm; G: 200 µm; H: 150 µm;
I: 300 µm; J: 300 µm; K:
100 µm; L: 50 µm; M: 200 µm;
N: 150 µm
infections are common in nature, it would be desirable to have zoonotic potential (i.e., cause anisakidosis in humans)
analyze the synergistic/antagonistic effect of the different but also they affect the marketability of seafood products.
parasitic agents in the well-being of cephalopod populations. Exposure to the anisakidosis pathogens at high rate in the
mantle cavity of European squids should be properly eval-
uated as a causative vector in risk analysis of this emerging
12.3.2 Seafood Safety zoonotic and allergic disease in southern European countries
(EFSA 2010).
Problems in the rational utilization of commercial fisheries To achieve the above challenges, systematic study on
include the management of biohazards along fish production the biodiversity of the metazoan parasitic fauna of Euro-
value chains. The high pathogenicity for man of the anisakid pean cephalopods should clearly be a hot spot. Consid-
larvae and their high prevalence and abundance in various ering the larval stage for most recorded metazoans and in
commercially exploited fish and cephalopod species draws many cases the absence of type specimens, biobanking,
particular attention to this biohazard (EFSA 2010). Parasites and genetic markers are relevant tools to assure the
identification of the etiological agent, the characterization Gaevskaya AV, Nigmatullin CM (1981) Several ecological aspects of
of the hazard and the implementation of critical control the parasitic relationships of the flying squid (Sthenoteuthis
pteropus, Steenstrup, 1855). (In Russian). Biol. Nauki (Mosk.)
points. 1:52–57. (Translation available from F.G. Hochberg)
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changes were made.
Aquarium Maintenance Related Diseases
13
Antonio V. Sykes, Kerry Perkins, Panos Grigoriou,
and Eduardo Almansa
Abstract
This chapter reviews the mechanical (physical) and chemical (water quality) related
pathologies that have been reported since cephalopods are maintained, reared or cultured in
captivity. For the first time, it builds up on the existing knowledge from researchers and
aquarists (which are represented as authors of the chapter) in order to provide the most
updated and inclusive revision on this theme. It is organized in terms of pathologies that are
reported and eventual described for one or more species, which are commonly kept for
research and display purposes, and exemplified with photos when possible. It includes
pathologies of the mantle, arms, eyes, shell; egg infections, malformations of the shell and
eggs; and causes of disease or mortality related with water quality focusing on pH and trace
elements.

Keywords
Cephalopoda Chemical related pathologies Egg infections Malformations
Shell fracture Skin wounds
2012; Ponte et al. 2013; Smith et al. 2013; Fiorito et al. 2014;
13.1 Introduction Di Cristina et al. 2015). Unfortunately, at time of publication
(2010) and until the end of 2017, the Directive omitted
Since 2013, cephalopods used in experimentation are pro- important information regarding the housing and husbandry
tected in the European Union through the application of conditions of this class. This has prompted the cephalopod
Directive 2010/63/EU (EU 2010). The application of this law community to join and write the first set of guidelines for the
generated an impact in research and aquaculture (Sykes et al. use of this class in the laboratory (Fiorito et al. 2015). Under
this framework and that of COST Action CephsInAction
A. V. Sykes (&)
Centro de Ciências Do Mar, Universidade Do Algarve|CCMAR, (FA1301—http://www.cephsinaction.org/), the community
Campus de Gambelas, Faro, Portugal has suggested the definition of mandate minima [minimal
e-mail: [email protected] requirements in Annex III (Care and Accommodation) and
K. Perkins Annex IV (Humane killing)] for cephalopod to be included in
Sea Life Brighton—Merlin Entertainments, Marine Parade, the update of Directive 2010/63/EU. Through a consensus
Brighton, BN2 1TB, UK
approach on published data, the community has established
the species-specific requirements for the care and accom-
P. Grigoriou
modation of cephalopods.
HCMR, Gournes Pediados, P.O. Box 2214 71003 Irakleion, Crete,
Greece The health and welfare of cephalopods are directly related to
e-mail: [email protected] how these animals are housed and treated. ‘Pathologies regis-
E. Almansa tered under captive conditions derive most of the times from
Instituto Español de Oceanografía, Centro Oceanográfico de bad welfare practice’ (Sykes and Gestal 2014). Interestingly,
Canarias, Santa Cruz de Tenerife, Canary Islands, Spain despite this cephalopods have been used as laboratory animals

https://doi.org/10.1007/978-3-030-11330-8_13
182 A. V. Sykes et al.
for more than a century (Dröscher 2016), most of the existing and Forsythe (1990a) characterized four stages of ulceration:
literature regarding pathologies observed in cephalopods in (a) Stage 1—The epidermis is destroyed mostly or completely,
captivity was published at the end of the 1980s and beginning of chromatophores become nonfunctional, with oblong or ellip-
1990s, resulting from the booming interest on culturing indi- tical shape or they disintegrate (originating a slight grey colour
viduals of this class (Sykes et al. 2014b). in the skin). The dermis becomes thicker with collagen, but the
Pathologies in cephalopods are related to the method of layers of muscle cells are disrupted, and the tissue display an
capture, transportation (from the type of container used, to increased number of amoebocytes and blood vascular vessels;
biting or cannibalism, when animals are transported in (b) Stage 2—Total destruction of the epidermis, and total
group), handling (from rough materials used in nets to destruction of the chromatophores with ulcerated zones
slipping from the hand of the operator), housing (seawater increasing in size over the mantle surface. Amoebocytes are
systems not meeting the minimal requirements of seawater present in the superficial layers of the dermis. Bacteria populate
quality, space, illumination, etc.) and husbandry (live prey and embed exposed surface and tissue; (c) Stage 3—Full pen-
may become predators and try to eat the cephalopod) con- etration of the skin and muscle layers (deep wounds). Fast
ditions (Forsythe et al. 1987; Hanlon 1990; Boyle 1991; progression of ulcers and bacteria spread to the ventral part of
Oestmann et al. 1997; Fiorito et al. 2015). Good (positive) or the mantle; Stage 4—Spreading of lesions to the head and arms.
bad (negative) welfare practice in research, maintenance, Wounds in Stages 3 and 4 are fatal. All cephalopods are known
rearing or culture conditions will determine the existence of to be susceptible to the infection of the skin by bacteria (For-
pathologies in cephalopods in any of these. The cause of sythe et al. 1987; Hanlon et al. 1988). The most common sec-
pathologies in cephalopods may be divided in viral, bacte- ondary bacterial infections associated with skin wounds were
rial, fungi, parasite, chemical and mechanical (Sykes and reported by Sherrill et al. (2000), Hanlon et al. (1984), and Ford
Gestal 2014). This chapter focuses on mechanical (physical) et al. (1986) for cuttlefish, octopus and squid species, respec-
and chemical (water quality) related pathologies, which will tively. Nonetheless, cephalopods are known to have the ability
be presented according to the pathologies observed in one or to fully recover from arm injuries or amputation through
more situations identified above for all. regeneration (Feral 1978, 1979, 1988; Rohrbach and Sch-
midtberg 2006; Tressler et al. 2014; Shaw et al. 2016).
13.2 Mechanical (Physical) Damage Related 13.2.1.1 Mantle and Arms

Pathologies Mantle tip damage, resulting from hitting the tank walls, is
known to occur in octopus, squid and cuttlefish (Forsythe
13.2.1 Skin Wounds et al. 1987; Hanlon et al. 1988; Scimeca and Oestmann
1995; Hanley et al. 1998; Scimeca 2011). It is characterized
Apart from being the outer barrier of the muscle tissue beneath, by extensive, deep ulcerative dermatitis and cellulitis
the skin of coleoid cephalopods is used to express behaviour (Fig. 13.1a, b), with exposure of the cuttlebone tip in cut-
through the dynamic patterns of the chromatophores (How et al. tlefish (Fig. 13.1e, f, g). One way of mitigating such damage
2017), communication and camouflage (Hanlon and Messen- is to apply the soft-sided tanks technology (Hanley et al.
ger 1996). The skin also has cells with the ability to sense light 1999). Another way is lowering density or increasing the sex
(Kingston et al. 2015) and, eventually, smell (Campinho et al. ratio of females (Sykes et al. unpublished results). Ford et al.
2017). This integumentary system is composed by (from top to (1986) reported a skin lesion that led to the split of the
bottom): (a) a mono-stratified epidermis consisting of columnar mantle and exposure of the gladius in Lolliguncula brevis.
epithelial cells with a microvillous border (Smith et al. 2011), In octopus and cuttlefish, the mere contact of the suckers
which is intermingled with mucus-secreting cells (Hanlon et al. of one individual to another is sufficient to cause skin
1984); and (b) a deeper dermis (thicker than the previous), damage (Forsythe et al. 1987; Hanlon et al. 1988; Hanley
composed of connecting tissue (with numerous blood vessels et al. 1998). Cuttlefish copulate on a head-to-head position,
and nerves and some amoebocytes), and containing chro- where the arms of both are tangled and males exert some
matophores, iridophores, and leucophores (Andouche and strength to keep the female in the position. Males also fight
Bassaglia 2016). for the opportunity of copula (either with a female or another
Because of the delicate nature of the skin, the handling, smaller male, if the latter is showing signs that may lead to
transportation and housing of cephalopods should be made with sexual misleading; Brown et al. 2012) and this will result in
utmost care to prevent tears or other trauma to the skin tissue. multiple skin wounds (Fig. 13.1d, g). Either cuttlefish or
Any skin wound is an open door for opportunistic secondary octopus can show extended biting wounds (Fig. 13.1c)
infections (particularly bacterial), which can be fatal if resulting from the latter, with eventual internal organ
untreated (Fiorito et al. 2015). Hanlon et al. (1984) and Hanlon exposure [Fig. 6.3 in Sykes and Gestal (2014)].
13 Aquarium Maintenance Related Diseases 183
Fig. 13.1 Mechanical damage of cephalopod skin: a Skin abrasion wall; f Detail image of wound described in E, where the lack of skin
(ulcerative dermatitis of the mantle apex) in the head of juvenile and the appearance of the distal tip of the cuttlebone is now visible;
Octopus vulgaris (probably derived from hitting the tank wall); g Mantle skin wounds in female S. officinalis (arrow on the left shows a
b Generalized skin wound with probable bacterial sepsis in a senescent similar ulcerative dermatitis of the mantle apex as in E and F, while the
O. vulgaris female; c Extended biting marks in the connection of the arrow on the right shows multifocal ulcerative dermatitis provoked by
arms with the head in an O. vulgaris juvenile caused by male fighting; male cuttlefish suckers during the act of copulation); h Skin wound near
d Minor biting marks in the arm of an O. vulgaris juvenile; e Ulcerative the mantle cavity in Nautilus pompilius; i Skin wound in the tissue right
dermatitis of the mantle apex in Sepia officinalis, due to hitting the tank above the eye in N. pompilius
There are reports (Reimschuessel and Stoskopf 1990; 13.2.1.2 Eyes

Budelmann 1998, 2010) regarding autophagy or automuti- Pathologies of the eyes in cephalopods have been reported to
lation syndrome (OAS) to occur in at least four species of occur in octopus, squid and cuttlefish (Forsythe et al. 1987;
octopus (Octopus dolfleini, O. vulgaris, O. bimaculoides and Hanlon 1990; Sykes and Gestal 2014).
O. maya). This syndrome is reported to affect both mantle Forsythe et al. (1987) reported that the eye ball of octo-
and arms. There is no consensus on if it caused by a sub- puses may sometimes swell and rupture, being fatal within 2
stance (released by the octopus) or eventually by a virus or days. According to these same authors, Hanlon et al. (1989b)
bacteria. and Hanlon and Forsythe (1990a), a similar condition may
In squid, skin lesions related to tank wall contact often also occur in squid. However, in the latter the cause is the
result in internal infections (apart from the skin itself), which incrustation of bacterial colonies due to tissue abrasion,
are chronic and develop slowly, requiring weeks or months to which turns the lens and the corneal covering of the eye
be fatal (Forsythe et al. 1987). According to the same authors, opaque and the eye larger than normal. This condition may
the most common of these infections is a localized necrosis of also be found in octopus and cuttlefish (Fig. 13.2a and b,
mantle tissue that surrounds the wound, spreading from the respectively).
mantle apex towards the head and looking like ‘an opaque Idiopathic bulbus protusions of one or two eyes have
dagger-shaped abscess in the lateral mantle tissue’. been registered during reproduction in cuttlefish (Sykes and
In Nautilus, Scimeca (2011) reported skin abrasion, Gestal 2014) and affect both male and female (Fig. 13.2b, c).
associated with a deep infection originated by a fungus, According to Hanley et al. (1998), this condition also affects
which generate pigment loss (Fig. 13.1h, i). There have also juveniles and may eventually lead to the eye pathology
been several anecdotal reports of hood discolouration, with previously described above. There are no reports of alike
fast spreading growth, being these patches often colonised idiopathic bulbus protusions of the eye in nautilus. However,
by other pathogens (Barord 2014). a similar skin abrasion, associated with a deep infection
Fig. 13.2 Mechanical damage

of cephalopod skin 2: a Opaque
lens in O. vulgaris; b Opaque lens
and idiopathic bulbous
protrusions of the eye in S.
officinalis males (on the left and
right individuals, respectively);
c Idiopathic bulbous protrusions
of both eyes in a S. officinalis
female; d Skin wound in the
tissue right above the eye in
Nautilus pompilius
originated by a fungus, which will generate pigment loss has North et al. 2017). The buoyancy and movement in the water
been seen in captive individuals (Fig. 13.2d) and previously column of cuttlefish is attained through the cuttlebone by:
reported by Scimeca (2011). The inexistence of idiopathic (a) varying the volume of gas space in these chambers and,
bulbus protusions in nautilus is most likely due to the therefore, of the overall cuttlebone density by moving liquid
anatomy of the eye, which is more primitive than that of into or out of those via an osmotic process (Denton et al.
other cephalopods. However, excessive mucus production 1961; Denton and Gilpin-Brown 1973) and (b) being highly
around the eyes can suggest a more serious condition or porous (93%) and having a low specific gravity (Checa et al.
infection and further diagnostics would be required to 2015). The gas inside the cuttlebone has traces of CO2, 2–
determine that it is not a potentially fatal mucodegenerative 3% of O2 and the remaining is N2 (Denton and Taylor 1964).
disease (Barord 2014). Despite having a low occurrence (Sherrill et al. 2000), the
fracture of the cuttlebone is one the most common
pathologies reported to occur when holding cuttlefishes in
13.2.2 Shell Wounds/Fracture captivity. We believe that the higher the occurrence, the
lower the welfare the overall rearing conditions, which might
Cuttlefish have an internal shell, commonly known as the be related to available space, tank colour, light conditions or
cuttlebone or sepion, which corresponds to approximately even sex ratios (Sykes and Gestal 2014). It is commonly
9% of the animal volume, acts as a rigid buoyancy device, seen in mature animals due to fighting or banging against the
and is made of a matrix of calcium carbonate (Denton 1961; tank walls (Hanlon and Forsythe 1990b; Hanley et al. 1999).
Denton and Gilpin-Brown 1961, 1973; Birchall and Thomas Cuttlebone pathologies in captivity can be categorized
1983; Jacobs 1996), in its aragonite polymorph with a into fractures that are (a) partial, in the distal (striated
mixture of b-chitin and other protein complexes (Florek siphuncular zone) and proximal tips (Fig. 13.3a and b,
et al. 2009; North et al. 2017). This structure is made of a respectively); (b) full transversal (Fig. 13.3c and e); and
dorsal shield that covers the lamellar matrix below. The (c) full longitudinal (Fig. 13.3i and l). Any of these may or
latter is arranged as over a hundred superposed narrow may not include the rupture of the tissues that separate the
chambers and organic membranes. The chambers have a cuttlebone from the viscera (Fig. 13.3h) and the possible
complex internal arrangement of calcified pillars (made from consequent exposure to seawater (Fig. 13.3f, g and k). When
0.01 mm thick septa supported by intracameral walls) that the latter happens the individual will die in a few days.
allow resisting external pressures greater than 1 MPa There is an additional pathology related to the cuttlebone.
(Hewitt 1975; Birchall and Thomas 1983; Checa et al. 2015; This is characterized by the disruption of the mantle tissue of
Fig. 13.3 Shell fracture in S. officinalis: a fracture at the distal tip of fracture of animal in (g), where part of the digestive system (stomach
the cuttlebone; b fracture at the proximal tip of the cuttlebone; and caecum) has passed between the shell parts; i detail of the broken
c fracture in the middle of the cuttlebone; d live female where the shell and tissue regeneration; j detail of tissue regeneration of shell;
cuttlebone is not present; e fracture in the middle of the cuttlebone with k male exhibiting a shell fracture and exposure of digestive system
increased production of the shell; f and g shell fracture in a female and a organs (stomach, caecum and digestive gland duct appendages) through
male (respectively), where the animal is trying to re-unite the shell by the skin; l detail of shell fracture of animal in (k)
massively producing a solution of calcium carbonate; h detail of shell
the shell sac due to senescence and the natural displacement i and j) (Checa et al. 2015) and up to 100 mL of slightly
of the cuttlebone from its normal site due to its floating milky fluid that accumulates between the dermis and the
capacity (Fig. 13.3d). The animals that show this condition cuttlebone (Hanley et al. 1998).
die a few days later after the loss of the cuttlebone (Sykes, Feeding conditions (malnutrition) will also affect the
unpublished results). proper development of the cuttlebone, generating aberrant
It is not however clear if in these two conditions cause forms in juveniles and adults (Boletzky 1974). Black lines in
of death is directly related to the fracture/loss of the cut- the striated siphuncular zone [Fig. 13.4b; (Keupp 2012)] are
tlebone or to wounds that are generated. Table 6 of Fiorito usually seen in underfed or starving animals.
et al. (2015) resumes the most common bacteria found in Black lines have also been recorded in nautilus and are
wounds of several cephalopod species. None of the bacteria often found with new shell growth in captivity. However,
in that table were reported to be linked to wounds of such they are not considered to be detrimental to the health or
nature. welfare of the nautilus or linked to nutritional deficit (Barord
Cuttlebone fracture also occurs in nature as studied by 2014). Further analysis of nautilus shells under scanning
Boletzky and Overath (1991). The full longitudinal fracture electron microscope (SEM) suggests that the individuals
in S. officinalis was reported to occur during rearing by these kept in aquaria display a shell made of disorderly crystalline
authors, Hanlon and Forsythe (1990a) and Scimeca (2011). structures when compared to those living in the wild (Moini
When exposed to a fracture, the cuttlebone will be ‘re- et al. 2014). It is hypothesised that these differences are due
generated’ by a similar process to the construction described to lower Ca and Mg concentrations in the shell; and the
by Boletzky and Overath (1991) and Čadež et al. (2017) resulting brittleness or shell deformities are due to the lack of
with the secretion of a chitin-protein complex (Figs. 13.3f, g, equilibrium in the biomineralisation process.
Fig. 13.4 Fungus or parasites in

cephalopods eggs: a–c fungi
infestation in Octopus insularis
eggs (photos courtesy of Érica
Vidal); d–f sponges attached to S.
officinalis eggs; g–f fungi
infestation in O. vulgaris eggs
filter prior to the application of the disinfection agent (being

13.3 Egg Infections and Cephalopods the latter either physical or chemical). Fungal activity may
Malformations be prevented in cuttlefish by individualizing the eggs and
using a special tank setup that promotes their gentle move-
One of the major issues when keeping eggs of cephalopods ment and proper oxygenation (Sykes et al. 2006, 2014a).
through embryogenesis is the eventual spread of fungus This setup has been replicated for rearing other cephalopod
(Sykes et al. 2006). However, reports of such occurrence are eggs through embryogenesis under captivity.
very scarce. In cuttlefish, fungal activity may be prevented Despite cuttlefish S. officinalis non-viable eggs
by using the methods described in Sykes et al. (2014a). (Fig. 13.5) were classified and described by Sykes et al.
Fungal infections in cephalopods were reported to be very (2014a), similar reports are lacking for other cephalopods.
limited and are mostly relate to eggs and embryos (Sykes As regards S. officinalis, the most typical non-viable eggs are
and Gestal 2014; Fiorito et al. 2015). They are reviewed in those described as orange (Fig. 13.5a), grey and white
detail in Chap. 4, but, in here, we report the existence of (Fig. 13.5b), and malformations (Fig. 13.5c). It is known
fungus and possible parasites in cephalopod eggs kept in a that non-viable eggs also occur in nature but, until now,
captive environment. Both eggs of Octopus insularis there is not a trend that might explain the high variability of
(Fig. 13.4a–c) and Octopus vulgaris (Fig. 13.4g–f) dis- their occurrence in captivity (Sykes et al. 2013, 2017).
played mycelia covering the chorion at early stages of Overall, malformations are even less reported than
embryonic development in Érica Vidal laboratory (Center existing pathologies in cephalopods. Malformations of the
for Marine Studies, Universidade Federal do Paraná, Brasil) cuttlebone have been reported in literature for S. officinalis
and Eduardo Almansa laboratory (Instituto Español de (Ruggiero 1980; Battiato 1983) and other cuttlefish (Keupp
Oceanografia, Centro Oceanográfico de Canarias, Spain), 2012). A cuttlebone with a full transversal fracture might
respectively. In September 2013, at the Sykes laboratory regenerate (Fig. 13.3j) with an excess of calcified material
(CCMAR, Universidade do Algarve, Portugal), Sepia offic- in the dorsal shield (Fig. 13.6a). While repairing the cuttle-
inalis eggs obtained in captivity aborted their development bone, cuttlefish uses a similar process of lamellar
due to an infestation by siliceous sponges that penetrated the deposition to the normal construction being self-organized
chorion (Fig. 13.4d–f). The causes for such infestations are layer-by-layer as seen with CT-Scan (X-ray computed
surely due to the seawater quality in the systems, which most tomography; Fig. 13.6c and d). The same cuttlebone shows
probably did not have any sterilization or any appropriate signs of possible decay of the last lamella and black lines in
Fig. 13.5 Non-viable eggs of S.

officinalis, according to the
nomenclature given in Table 11.2
of Sykes et al. (2014): a Orange
eggs; b Grey and White eggs;
c Malformation eggs
the striated siphuncular zone (Fig. 13.6b). The use of massive death of animals. Hanlon and Forsythe (1990b)
x-ray-computed tomography will allow a better knowledge reported that at least two authors have verified the existence
on the process of cuttlebone repair (Tiseanu et al. 2005) or of ‘tumours’ in cuttlefish and wild octopus, but the
eventually in nautilus (Hoffmann et al. 2018), even in living description of such is related to something completely dif-
organisms. ferent to what is seen in Fig. 13.7d. Interestingly, malfor-
Other types of malformations exist and are related to the mations (in this case, the split of an arm into 3 new arms)
eye (Fig. 13.7a, b), fin (Fig. 13.7a, c), arms (Fig. 13.7e, f) might develop due to regeneration of an arm after a wound
and mantle to arm tissue (Fig. 13.7g) development in cut- by biting (Fig. 13.7h, i). Arm regeneration is described in
tlefish and octopus. This was previously reported by Hanlon literature for cuttlefish (Tressler et al. 2014) and octopus
and Forsythe (1990b). Neoplasia (Fig. 13.7d) is not com- (Shaw et al. 2016). There are no causes reported for these
mon in cephalopods (Scimeca 2011) but will result in malformations.
Fig. 13.6 Cuttlebone regeneration

in S. officinalis: a and b anterior and
posterior image of cuttlebone that
suffered a transversal shell fracture
in the middle and regenerated with
an increased amount of shell in the
upper part; c and d CT-Scan of the
same cuttlebone from the side and
from above respectively, where the
growth rings are perfectly visible
(images courtesy of René
Hoffmann)
Fig. 13.7 Malformations in

development of specific organs in
cephalopods: a malformation in
development of the S. officinalis
fin and eye in a newly hatched
hatchling; b malformation of the
eye in a S. officinalis hatchling;
c malformation of the fin in a S.
officinalis juvenile; d neoplasia in
the head of a newly hatched S.
officinalis; e and f—
malformations of the arms in O.
vulgaris paralarvae (only 3 arms
and absence of arms,
respectively); g malformation of
the connecting muscle of the
mantle to the arms in a O.
vulgaris juvenile; h and
i development of three arms in
one arm that was subject to biting
in O. vulgaris juveniles
A low seawater pH has an effect on the carriage of oxygen

13.4 Chemical (Water Quality) Damage blood pigments but not in all the species, such as S. officinalis
Related Pathologies juveniles which displays an acid–base regulatory ability
(Gutowska et al. 2008, 2010a). However, as a response, this
Cephalopods are marine animals, and as such, they are species will increase the calcification of its cuttlebone that may
influenced by the seawater chemical quality, at collection or affect its buoyancy function (Gutowska et al. 2010b). On the
after being filtered in open, semi-open and closed systems. other hand, hypercapnia promoted reduced growth, extended
According to Sykes and Gestal (2014), the impact of embryogenesis and smaller cuttlebones (with denser cuttle-
diseases/lesions increases with the physicochemical quality bone laminae) in embryos and hatchlings (Sigwart et al. 2016).
and existing stress (which might be generated due to han- Colmers et al. (1984) reported the absence or malfor-
dling or housing conditions), which might promote a sup- mation of the statolith and the gravity receptor system and
pression of the immune response and proliferation of also the absence of cupulae of the angular acceleration
opportunistic microorganisms. The guidelines for the care receptor systems in seven species of coleoid cephalopods
and welfare of cephalopods in research (Fiorito et al. 2015) (Octopus joubini, O. maya, O. bimaculoides, S. officinalis,
provide a list of water quality criteria for optimum health and Loligo vulgaris, L. pealei, and L. plei). This condition was
welfare of cephalopods in its Appendix 2 and their routine described to affect the control and orientation in cephalopods
monitoring, while Hanlon (1990) and Smith et al. (2011) and was concluded that it was due to the lack of strontium in
described the basic filtering of the seawater systems used for seawater (Hanlon et al. 1989a).
housing cephalopods. Species-specific conditions for every Still concerning rearing in regards to seawater trace ele-
life stage may also be found in Iglesias et al. (2014). ments, despite the copper role in cephalopod haemocyanins
(Decleir et al. 1978; Thonig et al. 2014), Paulij et al. (1990) (Project SEPIABREED PTDC/MAR/120876/2010 and UID/Multi/
verified that copper concentrations of 50–200 ppb Cu2+ 04326/2016), PROMAR (Project SEPIATECH 31.03.05.FEP.002)
and Mar2020 (Project SEPIACUL MAR-02.01.01-FEAMP-0053)
exerted shorter embryogenesis and premature hatching in S. funded this work. EA acknowledges the funding of OCTOMICs
officinalis, while Establier and Pascual (1983) reported that (Project AGL2017-89475-C2-2-R) and OCTOWELF (Project
0.08 ppm of copper will provoke total mortality in freshly AGL2013-49101-C2-1-R) to this chapter. This work benefited from
laid eggs in the same species. According to Smith et al. networking activities carried out under the COST ACTION FA1301,
and is considered a contribution to the COST (European COoperation
(2011) copper is toxic to all cephalopods. There are some on Science and Technology) Action FA1301 ‘A network for
reports (Kuba, personal communication) that an excess of improvement of cephalopod welfare and husbandry in research, aqua-
copper in seawater might generate a similar orientation culture and fisheries’ (http://www.cephsinaction.org/).
problem and death in S. officinalis. This is probably due to
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Commons Attribution 4.0 International License (http://creative the chapter's Creative Commons licence, unless indicated otherwise in a
commons.org/licenses/by/4.0/), which permits use, sharing, adaptation, credit line to the material. If material is not included in the chapter's
made.
Regeneration and Healing
14
Letizia Zullo and Pamela Imperadore
Abstract
Cephalopods are animals endowed with a high regenerative potential. They can regenerate
missing or injured structures such as the cornea, the shell, the arms and tentacles and even
peripheral nerves and brain centers. As much as regeneration and healing cannot be
considered as pathologies sensu stricto, they are always accompanied by conditions such as
inflammation, tissue degeneration and potentially by infection. No treatments are currently
available for any of these cases. Although they often get resolved autonomously, the early
identification and monitoring of post-traumatic events is fundamental in the context of animal
welfare.

Keywords
Tissue damage Traumatism Wound healing Regeneration process
or to a more severe damage to internal structures, such as the

14.1 Introduction
pallial or optic nerves and tracts, causing deafferentation of
targeted organs. That is, alteration of the external appearance
Wild animals often carry on their body signs of pre-
is not always the direct consequence of skin lesions but can
experienced traumatic events. Tissues and structures such as
be the secondary effect of an injury located elsewhere in the
skin, fins and arms are reported to be subject to injury in the
body. This aspect is particularly relevant in the context of
wild as well as in captivity, mainly due to capture and
animal welfare as it allows identifying the primary target to
transport, autotomy, mating and competition (by con-
hit to ensure the animal wellness. In response to body
specifics or predators) but also aggression and cannibalism
damage, most of the cephalopods activate regenerating
(Budelmann 1998; Bush 2006, 2012; Hanlon et al. 1984).
processes in various organs such as cornea, peripheral
In order to discriminate among mild to severe body
nerves, arm and tentacles (Imperadore and Fiorito 2018).
damages, it is important to have a good understanding of the
Regeneration of the arms is particularly important as when
cephalopod physiology. As an example, abnormal body
one or more arms are damaged, the impairment of several
coloration can be due either to a superficial skin damage,
functions, such as swimming, prey manipulation and pos-
which causes chromatophores expansion or skin retraction,
turing can occur (Tressler et al. 2014). This begins with a
characteristic process of skin and tissue wound healing
L. Zullo (&)
Centre for Synaptic Neuroscience and Technology, Fondazione
consistent within and across species (Fossati et al. 2013,
Istituto Italiano Di Tecnologia, Genoa, Italy 2015; Lange 1920; Shaw et al. 2016; Tressler et al. 2014).
e-mail: [email protected] Cephalopod skin has the vital role of protection against
P. Imperadore pathogens. The latter function becomes crucial when keep-
Association for Cephalopod Research (CephRes), Naples, Italy ing animals in captivity, as it has been observed that severe
e-mail: [email protected] ulcers can lead to death if untreated (Hanlon et al. 1984;
P. Imperadore Mather and Anderson 2007; Polglase 1980). It has also been
Stazione Zoologica Anton Dohrn, Biology and Evolution of noticed that undamaged squids adapt better than injured
Marine Organisms, Naples, Italy

https://doi.org/10.1007/978-3-030-11330-8_14
194 L. Zullo and P. Imperadore
animals to temperature and salinity changes (Hanlon et al. mantle and on the eyeballs skin are reported in Fig. 14.1aI
1983). Skin plays also an important role in cephalopods and aII where several small scratches are clearly visible.
survival in their natural environment as they use body pat- Arms can present unhealed wounds (Fig. 14.1bI) or regen-
terning, obtained through changes in color and shape of the erating arms (Fig. 14.1bII and bIII) at various stages of
skin, for concealment and communication (Messenger 2001; regeneration depending on the time the traumatic event
Packard 1988; Packard and Hochberg 1977). Hence, wounds occurred. Lesions can be localized at any point along the arm
and lesions have to be carefully monitored in captive animals although they are mostly found at its mid-terminal portion.
to ensure a full recovery of their function and appearance
and to avoid transferring possible infections to other animals
(this can be exacerbated in closed water systems), as also 14.2 Skin and Tissue Damage After
suggested in the Guidelines for the Care and Welfare of Experimentally Induced Injury
Cephalopods in Research (Fiorito et al. 2015).
In this chapter, we will attempt at describing in details the Wound healing in cephalopods has been investigated after
healing and regeneration processes occurring at mantle and experimentally induced mantle injury (Bullock et al. 1987;
arms of Octopus vulgaris, a Cephalopods representative. Polglase et al. 1983) and arm transection during the initial
stages of arm regeneration (Féral 1988; Lange 1920; Shaw
et al. 2016). In both cases, similar phenomena are observed.
14.1.1 Skin and Tissue Damage in Wild Animals: Indeed, even though arm regeneration involves recovery and
From Healing to Regeneration regrowth of several tissues and structures (i.e., nerves, mus-
cles, vessels, skin, etc.) it first requires healing of the wound.
Traumatic events occurring in wild animals can cause Healing starts with muscular contraction and epidermis
alterations of several aspects of the body appearance. Ani- in-folding (mainly connective tissue of the dermis) soon after
mals can manifest from simple superficial skin lesions to injury (30 min). This phenomenon usually lasts for the first
partial or complete structure loss, as often occurs in the case 12 h, contributing to reducing the size of the lesion
of arms (Fig. 14.1). (Fig. 14.2). Here we present an exemplary case where a
As injured arms normally regenerate, it is not rare to see complete mantle wound closure is established as soon as
animals with one or more regenerating arms. Superficial 4 days after injury (Fig. 14.2aI and aII). Indeed, at 4 days only
wounds can have various extensions and can be very a thin line (red arrow), due to intense muscular contraction
localized or distributed over the body, although small lesions and epidermis in-folding, is still visible around the site of
are more frequently observed. Examples of wounds on injury.
Fig. 14.1 Skin lesions in wild

caught animals. a Wounds local-
ized at the mantle (I) and around
the eyeballs (II) (red asterisks);
b examples of injured
non-regenerating arm tip (I, red
asterisk, scale bar 5 mm) and
regenerating arms (II, red asterisk,
scale bar 6 mm; III, red arrow).
Regeneration can occur at any
location of the arm depending
only on the site of injury and will
proceed towards the regeneration
of a fully functional arm (II: early
regenerating arm, III: late regen-
erating arm)
14 Regeneration and Healing 195
Fig. 14.2 Experimentally induced injury. a An animal showing a skin (III) after lesion. Scale bars: 2 mm. Soon after cut, the arm tip is
wound on the dorsal side of the mantle soon after experimental lesion interested by wound closure. 24 h’ post injury, the majority of the
(red arrow in I, Day 0). Four days later (II) the wound is almost exposed area is covered by skin (II). Two days post surgery (III) the
completely closed, only a thin line (red arrow), due to intense muscular wound is still visible but extremely reduced in size and suckers close to
contraction and epidermis in-folding, is still visible around the site of the lesion are brought toward the wound
injury. b Appearance of an arm tip uninjured (I), 24 h (II) and 48 h
At the tissue level, hemorrhagic areas and hemocytes infected wound. The majority of these cells become necrotic
invade the wound, producing swelling in the central part of in proximity of the bacteria likely due to the effect of toxins
the wound which is mainly observed 12 h post injury as a released by the pathogens (Bullock et al. 1987).
result of increased hemocytes diapedesis. Hemocytes Healing of the arm starts soon after the cut. In the
aggregates on the injury site (between day 1 and 3) and example reported here, already 24 h’ post injury, the
temporary protect the wound, also containing abundant majority of the exposed area is covered by skin and 2 days
extracellular matrix and likely vesicles and/or mucous. Later, post surgery the wound is still visible but extremely reduced
blood cells stratify in several layers and change their shape in size; suckers close to the lesion are brought toward the
from round to fusiform, providing a first dermal plug cov- wound (compare Fig. 14.2bI, bII, bIII). The time required for
ering the whole surface of the wound. In the arm, this rep- the complete healing in the arm can go from less than 24 h
resents the primary blastema that is supposed to supply up to 2 weeks on the base of several factors, mainly water
material for the regenerating tip. Epidermal cells round up 2 temperature, animals’ age and health status of the animals
weeks post lesion, while 3 weeks later the epidermis thick- (Féral 1988; Lange 1920). In addition, healing appears also
ness increases (for a detailed description see (Shaw et al. to depend on animal “self-abilities,” as two different popu-
2016)). Time of healing can be greatly affected by bacterial lations of healers have been identified for the common
infection that determines delays in muscle contraction and octopus, named slower and faster healers: in the latter case
epidermal migration; this eventually results in an incomplete 80% of the wound is covered in 6 hours, 50–60% is
closure of the wound. In addition, an increase in hemocytes observed instead for the slower group with modest increase
release and in their activity is observed compared to not in the following 18 h (Shaw et al. 2016).
14.3 Arm Regeneration and well-organized (Fig. 14.3bII) (Fossati et al. 2011, 2013;
Lange 1920; Zullo et al. 2017).
We will now describe more in details the histological A special case of regeneration is represented by the
modifications occurring at tissue level following arm injury aberrant arm regeneration where the injured arm forms bi- or
and regeneration (Fig. 14.3). trifurcation. It is not uncommon in literature to find exam-
Immediately after healing, the process of arm regenera- ples of aberrant regeneration of cephalopods arms and ten-
tion starts and already 3 days after injury a little knob at the tacles (Alejo-Plata and Méndez 2014; González and Guerra
cutting point is visible. This later elongates forming a small 2008; Toll and Binger 1991). Here we report a case where
protrusion. The first well-identifiable structure has the shape the regenerating stump developed a tripartite arm composed
of a hook and appears around 17 days after the injury by structurally normal arms (Fig. 14.4).
(Fig. 14.3aI). From histological analysis, a very thin layer of
undifferentiated cells composes the first observed knob.
A more defined and intensely proliferating “blastema” (bl) is 14.4 Pallial Nerve Degeneration
then visible together with diffuse vascular components (v) at and Regeneration
the arm tip (Fig. 14.3aII). This structure then disappears,
tissues enter in a differentiation state and the process of A pair of pallial nerves, connecting the subesophageal mass
histogenesis starts. At later stages (between 50 and 60 days of the CNS to the mantle, is involved in the control of
usually) a complete structure is visible with the restoration of breathing and in the mantle skin pattering. If both nerves are
a typical non-injured arm (Fig. 14.3bI). At this stage both damaged, the animal dies soon after as breathing is com-
nervous (g) and muscle elements (m) become clearly visible pletely abolished (Fredericq 1878); however, if only one of
Fig. 14.3 Arm regeneration process. a Regenerating arm 17 days post post injury. I, macroscopic image of the arm tip. Scale bar: 5 mm. II:
injury. I: macroscopic image of the arm tip. Scale bar 5 mm. II: Hematoxylin and eosin stained longitudinal section showing the well
Hematoxylin and eosin stained longitudinal section showing the developed nervous (g: ganglia) and muscle (m) elements. Scale bar
blastemal areas (bl) at the arm tip rich of vascular components (v). 100 µm. At this stage of regeneration, a normal arm structure is fully
Scale bar 50 µm. At this stage of regeneration, a hook-like structure is restored. Images AII, BI and BII by courtesy of Dr. F. Carella
visible and the tip is much unorganized. b Regenerating arm 55 days
Complete functional recovery requires several months,

but usually already after 3 to 4 days post surgery the den-
ervated skin undergoes some peculiar changes (Sanders and
Young 1974). First we can observe skin paling due to
chromatophore muscles relaxation, as neural control
between CNS and periphery is lost (Sereni and Young
1932). This is followed by dark color waves randomly and
quickly crossing the skin of the affected side of the mantle.
These have been named “wandering clouds” and are due to a
state of chromatophores hyperexcitability (Imperadore et al.
2017; Packard 1992; Sanders and Young 1974). Three to
five days after injury, the skin on the side of lesion appears,
at rest, homogeneously colored and, seven to fourteen days
later it is able to match the color pattern of the contralateral
side. However, paling immediately returns on the injured
side during rapid pattern changes, such as prey attack or
escape. This effect cannot be the result of a lack in functional
regeneration but it is more likely driven by local effects
involving skin photoreceptors (Imperadore et al. 2017).
At the microscopical level, lesions of the nerves induce
inflammation followed by the formation of a scar between
the two nerve stumps, hemorrhagic areas, and additional
cicatricial tissues in the muscles damaged. These scars are
mainly formed by hemocytes, which rush to the site of lesion
and invade the nerve stumps. The scar separating the stumps
does not represent a barrier, as fibers of the central stump
(still connected to the CNS) start immediately to regenerate,
reaching the opposite stump, with a growth rate between 7
and 18 µm/h (Fig. 14.5c). Peripheral stump regeneration is
observed after 10–14 days post lesion, it appears initially
Fig. 14.4 Aberrant regeneration. Aberrant regeneration of an arm after characterized only by degenerative phenomena, with axons
lesion in the wild resulted in the formation of three morphologically swelling and fragmentation (Imperadore et al. 2017; Sereni
normal tips. Scale bar 1 cm. Source Dr. Panagiotis Grigoriou
(CretAquarium, Heraklion, Greece)
and Young 1932). Connective tissue in the nerve appears
also to be involved in the process, sealing the cut stumps
the two is subjected to injury, the animal is not only able to initially, shaping and driving them toward each other a few
survive, but it also regenerates the nerve, recovering the lost days later. Both, hemocytes and connective tissue actively
functions (Imperadore et al. 2017, 2018; Sanders and Young proliferate at the cut nerve and interestingly neural elements
1974; Sereni and Young 1932). This lesion has never been have also been suggested start differentiating as soon as two
described as a naturally occurring phenomenon, such as arm weeks after lesion (Imperadore et al. 2017).
regeneration. The few accounts available depict it as the
effect of surgical operations, through pallial nerve cut or
crush. This might be due to the particular location of the 14.5 Concluding Remarks
nerves, which are well-protected inside the mantle cavity.
When one of these nerves is experimentally transected, two Arms and mantle skin wounds of various severity and even
immediate effects are obtained: paling of the mantle skin and arm loss are common events experienced by cephalopods in
paralysis of the respiratory muscles on the side ipsilateral to nature. Upon injury, animals can manifest a series of
the lesion (Fig. 14.5a, b) (Fredericq 1878; Imperadore et al. responses going from mild aberration of the body coloration
2017; Sanders and Young 1974; Sereni and Young 1932). and patterning to severe behavioral modifications. Injury is
Despite this loss of functions, the animal appears healthy often only the “first hit” while “second hits” are determined
and active soon after recovery from anesthesia. An increase in by a variety of processes such as inflammation and infection
self-grooming actions close to the injured area is the only of the wound occurring hereinafter that can alter not only the
alteration that might occur after lesion (Imperadore et al. aspect but also the functionality of the damaged tissues.
2017). Body skin appearance have been listed within the potential
Fig. 14.5 Pallial nerve degeneration and regeneration. An animal c Pallial nerve seven days post lesion presents a scar dividing the two
before pallial nerve lesion (a) shows full ability in changing body pattern nerve stumps (Central and Peripheral stumps), however the central
(I) and in controlling contraction of respiratory muscles on both sides of stump is able to regenerate with fibers reaching the opposite side toward
the mantle (II). Soon after lesion (b) the two functions are lost on the the stellate ganglion. Scale bar 0.2 mm
denervated side, which becomes completely pale (I) and flaccid (II).
indicators of health and welfare in cephalopods (Fiorito et al. might take place even days after experimentation. For most
2015) and thus represents a feature that have to be constantly of these events, no special treatment has yet been estab-
monitored especially in captive animals. Moreover, it is lished. It is therefore important, in the context of animal
fundamental to provide a constant assessment of experi- welfare, to early identify the occurrence and progress of
mental animals undergoing surgical procedures as the regeneration/degeneration processes to avoid the possible
occurrence of secondary effects induced by tissue incisions occurrence of unhealthy conditions in captive animals.
References Imperadore P, Shah SB, Makarenkova HP, Fiorito G (2017) Nerve

degeneration and regeneration in the cephalopod mollusc Octopus
vulgaris: the case of the pallial nerve. Sci Rep 7
Alejo-Plata MDC, Méndez OV (2014) Arm abnormality in Octopus Imperadore P, Uckermann O, Galli R, Steiner G, Kirsch M, Fiorito G
hubbsorum (Mollusca: Cephalopoda: Octopodidae). Amer Mala- (2018) Nerve regeneration in the cephalopod mollusc Octopus
colo Bull 32:217–219 vulgaris: label-free multiphoton microscopy as a tool for investi-
Budelmann BU (1998) Autophagy in octopus. S Afri J Mar Sci gation J R Soc Interface 15 https://doi.org/10.1098/rsif.2017.0889
20:101–108 Lange MM (1920) On the regeneration and finer structure of the arms
Bullock AM, Polglase JL, Phillips SE (1987) The wound healing and of the cephalopods. J Exp Zool 31:1–57
haemocyte response in the skin of the lesser octopus Eledone Mather JA, Anderson RC (2007) Ethics and invertebrates: a cephalopod
cirrhosa (Mollusca: Cephalopoda) in the presence of Vibrio perspective. Diseas Aqua Org 75:119–129
tubiashii. J Zool 211:373–385 Messenger JB (2001) Cephalopod chromatophores: neurobiology and
Bush SL (2006) Autotomy as a deep-sea squid defense. Integr Comp natural history. Biol Rev 76:473–528
Biol 46:E19–E19 Packard A (1988) The skin of cephalopods (coleoids): general and
Bush SL (2012) Economy of arm autotomy in the mesopelagic squid special adaptations. In: Trueman ER, Clarke MR (eds) The
Octopoteuthis deletron. Mar Ecol Progr Ser 458:133–140 Mollusca, form and functions, vol 11. Academic Pres, pp 37–67
Féral JP (1988) Wound healing after arm amputation in Sepia officinalis Packard A (1992) A note on dark waves (wandering clouds) in the skin
(Cephalopoda: Sepioidea). J Invert Pathol 52:380–388 of Octopus vulgaris. J Physiol 446:40
Fiorito G, Affuso A, Basil J et al (2015) Guidelines for the Care and Packard A, Hochberg FG (1977) Skin patterning in Octopus and other
welfare of cephalopods in research—a consensus based on an genera. Symp Zool Soc Lond 38:191–231
initiative by CephRes, FELASA and the Boyd Group. Lab Anim Polglase JL (1980) A Preliminary Report on the Thraustochytrid(s) and
49:1–90 Labyrinthulid(s) Associated with a Pathological Condition in the
Fossati SM, Benfenati F, Zullo L (2011) Morphological characteriza- Lesser Octopus Eledone cirrosa. Bot Mar XXIII: 699–706
tion of the Octopus vulgaris arm. Vie Milieu 61:197–201 Polglase JL, Bullock AM, Roberts RJ (1983) Wound healing and the
Fossati SM, Carella F, De Vico G, Et AL (2013) Octopus arm haemocyte response in the skin of the Lesser octopus Eledone
regeneration: role of Acetylcholine Esterase during morphological cirrhosa (Mollusca: Cephalopoda). J Zool 201:185–204
modification. J Exp Mar Biol Ecol 447:93–99 Sanders GD, Young JZ (1974) Reappearance of specific colour patterns
Fossati SM, Candiani S, Nodl MT et al (2015) Identification and after nerve regeneration in Octopus. Proc R Soc London B, Biol Sci,
expression of Acetylcholinesterase in Octopus vulgaris arm devel- p 186
opment and regeneration: a conserved role for ACHE? Mol Sereni E, Young JZ (1932) Nervous degeneration and regeneration in
Neurobiol 52:45–56 Cephalopods. Pubbl Staz Zool Napoli 12:173–208
Fredericq L (1878) Recherches sur la Physiologie du poulpe commun. Shaw TJ, Osborne M, Ponte G, Fiorito G, Andrews PL (2016)
Arch Zool Exp Gen 7:535–583 Mechanisms of wound closure following acute arm injury in
González AF, Guerra A (2008) First observation of a double tentacle Octopus vulgaris. Zool Letters 2:8
bifurcation in cephalopods. JMBA2—Biodiversity Records: 1–6 Toll RB, Binger LC (1991) Arm anomalies: cases of supernumerary
Hanlon RT, Hixon RF, Hulet WH (1983) Survival, growth, and development and bilateral agenesis of arm pairs in Octopoda
behavior of the loliginid squids Loligo plei, Loligo pealei, and (Mollusca, Cephalopoda). Zoomorphol 110:313–316
Lolliguncula brevis (Mollusca: Cephalopoda) in closed sea water Tressler J, Maddox F, Goodwin E, Zhang Z, Tublitz NJ (2014) Arm
systems. Biol Bull 165:637–685 regeneration in two species of cuttlefish Sepia officinalis and Sepia
Hanlon RT, Forsythe JW, Cooper KM, Dinuzzo AR, Folse DS, pharaonis. Invert Neurosci 14:37–49
Kelly MT (1984) Fatal penetrating skin ulcers in laboratory-reared Zullo L, Fossati SM, Imperadore P, Nodl MT (2017) Molecular
octopuses. J Invert Pathol 44:67–83 determinants of cephalopod muscles and their implication in muscle
Imperadore P, Fiorito G (2018) Cephalopod tissue regeneration: regeneration. Front Cell Dev Biol 5:53
consolidating over a century of knowledge. Front Physiol 9:593.
https://doi.org/10.3389/fphys.2018.00593
made.
Other Disorders
15
Camino Gestal, Santiago Pascual, and Sarah Culloty
Abstract
Neoplasia are growth disturbances characterized by excessive, abnormal proliferation of cells,
independent of normal-regulating mechanisms of the animal and persisting after termination
of the stimulus that initiated growth. Over lasts years there has been an increase in research
into tumors of invertebrates. In cephalopods, reports on the incidence of the tumors or
neoplasia are scarce. They have been described as hard compact and homogeneous nodules of
connective tissue located in the mantle or at the base of suckers. The aetiological origin of
tumors observed in cephalopods is unknown, but it could be related to aquarium maintenance.
Other injurious agents, including infectious (virus, bacteria, or parasites) and xenobiotics,
may also have produced the lesions. In occasions inflammatory processes have been
associated with tumors with severe oedema associated. Inflammation is part of the biological
response to body tissues to harmful stimuli such as pathogens, damaged cells or effect of
xenobiotics. The inflammatory focus is characterized by exudation with interstitial fluid
changes and hemocytic migration. Some inflammatory lesions include fibrosis and necrotic
cells in the affected area, with loss of histological features and even organ architecture. All
this changes have been observed in cephalopods in inflammatory reactions originated by
infection with different pathogens. The chapter covers a selection of the reported cases of
disorders related to neoplasia and inflammation.

Keywords
Neoplasia Tumors Inflammation Haemocytic infiltration Aedema
C. Gestal (&)
15.1 Introduction
Aquatic Molecular Pathobiology Group, Institute of Marine
Research, Spanish National Research Council (CSIC), 36208 Neoplasia are growth disturbances characterized by exces-
Vigo, Pontevedra, Spain sive, abnormal proliferation of cells, independent of
normal-regulating mechanisms of the animal and persisting
S. Pascual after termination of the stimulus that initiated growth (Sparks
1985). Similarly, tumors are ectopic masses of tissue formed
Vigo, Pontevedra, Spain by due to an abnormal cell proliferation. Over lasts years there
e-mail: [email protected] has been an increase in research into tumors of invertebrates,
S. Culloty since previously it was thought that tumors only occurred in
Aquaculture & Fisheries Development Center, School of vertebrates and invertebrates were not susceptible to devel-
Biological, Earth and Environmental Sciences, University College oping neoplasia (Engel 1930). Nowadays the research in the
Cork, The Cooperage, Distillery Fields, North Mall, Cork, Ireland
area has increased and increasing number of tumors has been

https://doi.org/10.1007/978-3-030-11330-8_15
202 C. Gestal et al.
detected in these organisms. Thus, histological and molecular

research has provided a better understanding of the nature of
these abnormal growths (Tascedda and Ottaviani 2014). In
marine invertebrates, different types of tumors have been
described, namely those emerged spontaneously, those due to
hereditary phenomena, and those due to a wide range of
environmental factors. Among those induced by environ-
mental factors, different injurious agents including chemical
toxins, physical stress, biological infections and potential
carcinogenic substances, may produce that lesions (Hanlon
and Forsythe 1990a). In molluscs, tumors, tumor-like growths
or neoplasia have been described in gastropods, bivalves, and
in some cases in cephalopods. In bivalves, sarcomas of
hematopoietic origin such as disseminated neoplasia,
or gonad neoplasia have been identified as causative of
important mortalities (Carballal et al. 2015). Very few reports Fig. 15.1 Macroscopic aspect of nodules located at internal mantle
have been published in cephalopods. side of Octopus vulgaris
Other disorder commonly observed in cephalopods is
inflammation. It is part of the biological response to body
tissues to harmful stimuli such as pathogens, damaged cells, unknown, especially in those observed in wild individual.
or effect of xenobiotics. Inflammation is a protective However, different authors suggest that it could be the result
response with a function of eliminates the initial cause of the of traumatic episodes related to aquarium maintenance
cell injury, clear out necrotic cells and tissues damaged and (Hanlon and Forsythe 1990b). Other injurious agents,
initiate tissue repair. The description of the tissue modifi- including infectious (virus, bacteria or parasites) and xeno-
cations occurred in relation to infectious or non-infectious biotics, may also have produced the lesions (Hanlon and
agents by histopathological analysis is essential for an Forsythe 1990a).
accurate diagnosis of the disease.
15.3 Inflammation
15.2 Neoplasia and Tumors Histologically haemocytic infiltration with eosinophilic

staining is observed in the intercellular space of a tissue with
In cephalopods, the incidence of the tumors or neoplasia is inflammation. The inflammatory focus is characterized by
extremely low. In 1951 Jullien and Jullien (1951) reported exudation with interstitial fluid changes and hemocytic
cuttlefish with hard compact and homogeneous whitish migration. Some inflammatory lesions include fibrosis and
nodules of connective tissue with loss of normal stratified necrotic cells in the affected area, with loss of histological
appearance and highly vascularized at the periphery. In some features and even organ architecture. All this changes have
cases, haemocytic infiltration is observed between the con- been observed in cephalopods in inflammatory reactions
nective tissue and muscular fibers (Wauttier and Wautier originated by bacteria, parasites such as the coccidian
1955). Similar nodules were also observed in Octopus vul- Aggregata or metazoans such as Anisakis infections
garis (Figs. 15.1 and 15.2). Nigmatullin provided morpho- (Fig. 15.5). In occasions, inflammatory processes have been
logical data on tumors observed in O. vulgaris off the associated with tumors with severe oedema associated.
northwest coast of Africa with a prevalence of 1.6% of Hanlon and Forsythe (1990a) described the presence of
caught octopus. Pascual et al. (2006) described lesions severe edema of the mantle and arms of O. joubini and
characterized by consistent swelling of smooth surface O. maya, where epidermis and dermis are separated from the
nodules located at the sucker’s base and mantle of Octopus underlying muscle layers, or even the entire dermis of the
hubbsorum (Figs. 15.3 and 15.4). At histological level, the mantle was separated from the muscle layers by a watery,
lesions appear as mass proliferations of dense fibrous tissue almost gelatinous, layer of fluid. This condition was always
between the dermis and muscle layers (Fig. 15.4a–c). fatal within 48 h. Similar lesions associated to inflammation
Muscular tissue surrounding the lesions was degenerated process have been observed in Octopus vulgaris maintained
and necrotic foci were observed (Figs. 15.2 and 15.4). The for long period in aquarium installations also with fatal result
aetiological origin of tumors observed in cephalopods is within 48–72 h (pers. obs.) (Fig. 15.6).
15 Other Disorders 203
Fig. 15.2 Histological sections

of nodules in O. vulgaris (a–g).
a–e Detail of mass proliferations
of dense fibrous tissue between
the dermis and muscle layers.
f–g Detail of necrotic foci also
observed in a and b. Scale bars
a, d, 200 µm; b, f, g, 100 µm;
c, e, 500 µm
Fig. 15.3 Macroscopic aspect of (a) (b)

nodules located at the sucker’s
base (a) and mantle (b) of
Octopus hubbsorum

of nodules of O. hubbsorum.
(a–c) Mass proliferations of dense
fibrous tissue between the dermis
and muscle layers. Scale bars
a, 1 mm; b, c, 500 µm
15 Other Disorders 205
Fig. 15.5 Inflammatory focus in the mantle of O. vulgaris showing loss of histological features (epithelium) (c–d) and even organ
exudation with interstitial fluid and hemocytic migration (a–b). architecture (a). Scale bars a, 500 µm; b, 200 µm; c, d, 100 µm
Fibrosis and necrotic cells is observed in the affected area (b), with
(a) (b)
Fig. 15.6 Aedema in the arm of O. vulgaris maintained for long showing the epidermis and dermis separated from the underlying
period in aquarium installations (a–b). a Macroscopic aspect of muscle layers, by a watery, almost gelatinous, layer of fluid close to the
aedema focus at arm level characterized by exudation with interstitial sucker. Scale bar b, 1 mm
fluid accumulation and hemocytic migration. b Histological detail
harmful stimuli such as inflammatory reactions. The causes

15.4 Concluding Remarks and effects of these disorders are not well studied. However, it
is known that at least some of them could be the result of
While the study and identification of neoplasia has been traumatic episodes, both in the wild or related to aquarium
increased in invertebrates and specifically in molluscs in the maintenance. Therefore, in the context of animal welfare,
last years, the incidence of tumors in cephalopods is extremely further research is needed in order to identify and to analyze
low. Similarly occurs with histopathological descriptions of the progress of those processes to avoid fatal results and to
biological response or reactive processes of body tissues to provide the best welfare conditions to the animals in captivity.
References Jullien A, Jullien AP (1951) Sur un type de tumeur non provoquée

expérimentalement et observée chez la Seiche. Acad Sci Paris
210:608–610
Carballal MJ, Barber BJ, Iglesias D, Villalba A (2015) Neoplastic Pascual S, Rocha A, Guerra A (2006) Gross lesions in the Hubb
diseases of marine bivalves. J Invert Pathol 131:83–106 octopus Octopus hubbsorum. Mar Biol Res 2:420–423
Engel CS (1930) Warum erkraken wirbellose tiere nicht an krebs? Sparks AK (1985) Synopsis of invertebrate pathology exclusive of
Ztschr F Krebsforsch 32:531–543 insects. Elsevier, Amsterdam
Hanlon RT, Forsythe JW (1990a) Diseases of Mollusca: Cephalopoda. Tascedda F, Ottaviani E (2014) Tumors in invertebrates. Int Sci J
Structural abnormalities and neoplasia. In: Kinne O (ed) Diseases of 11:197–203
marine annimals, vol III. Biologische Anstalt Helgoland, Hamburg, Wauttier V, Wautier J (1955) Le cancer et les invertébrés, 1ére
pp 47–227 partie: réactions tumorales naturelles. Bull Mens Soc linnéde Lyon
Hanlon RT, Forsythe JW (1990b) Diseases of Mollusca: Cephalopoda. 3:76–96
Diseases caused by protistans and metazoans. In: Kinne O
(ed) Diseases of marine annimals, vol III. Biologische Anstalt
Helgoland, Hamburg, pp 47–227
made.
Cephalopod Senescence and Parasitology
16
Katina Roumbedakis and Ángel Guerra
Abstract
In the majority of the shallow-water cephalopod species, senescence is a short stage of their
lifespan, which takes place at the end of sexual maturity. Senescence is not a disease,
although senescent cephalopods can be frequently mistaken with diseased animals.
Senescence is accompanied by physiological, immunological and behavioural changes,
which are briefly exposed in this chapter. A suppressed immune system may increase
susceptibility to parasite infection in senescent cephalopods. High prevalence of infection by
Aggregata octopiana was observed in Octopus vulgaris and Aggregata sp. in Octopus maya.
In both cases, the infection was found in post-spawned females and was infecting different
organs. Cestode larvae Prochristianella sp. were found in the buccal mass of post-spawned O.
maya females.

Keywords
Senescence Physiological changes Immunological changes Parasitology
Generally, males die approximately the same time as the

16.1 Introduction females (Hanlon 1983; Mangold 1983; Van Heukelem
1983), but in some species, males may live longer than
In the majority of the shallow-water cephalopod species, females, such as Enteroctopus dofleini (Hartwick 1983) or
senescence is a short stage of their lifespan, which takes Octopus vulgaris (Mangold 1983).
place at the end of sexual maturity (Tait 1986, 1987; The physiological process by which senescence occurs is
Anderson et al. 2002). After mating, female octopuses adopt not completely understood (Tait 1986, 1987; Anderson et al.
an ‘extreme pattern of maternal care’ (Wang and Ragsdale 2002). Optic gland secretions, probably activated by envi-
2018). The mother abstains from food and exclusively takes ronmental factors (e.g. light, temperature) and nutrition (Van
care of the eggs, which will cost its own life; death usually Heukelem 1979), are involved in gonad maturation and in
occurs by the time of the offspring hatching (Wells 1978; the inhibition of feeding (Tait 1986, 1987; Wodinsky 1977).
Hanlon and Messenger 1996; Anderson et al. 2002). The Although a relatively small amount of information is
senescence is reached still while the female breeds the eggs available regarding the mechanisms that lead to senescence,
or soon after their hatching, while in males it usually begins it has been hypothesized that in species with terminal
after mating (Pascual et al. 2010). spawning (see Rocha et al. 2001 for a review of the
reproductive strategies), the levels of hormonal secretions
K. Roumbedakis (&)
are high which would trigger the inhibition of feeding and
Association for Cephalopod Research (CephRes), Naples, Italy
e-mail: [email protected] lead to death. In species with intermittent spawning, the
levels of hormonal secretions are lower, which would ini-
Á. Guerra
Ecology and Biodiversity Department, Institute of Marine tiate spawning without inhibiting food intake and growth
Research, Spanish National Research Council (CSIC), Eduardo (Jackson and Mladenov 1994). Moreover, an attractive
Cabello 6, 36208 Vigo, Pontevedra, Spain genetic mechanism of ageing, and therefore, senescence,

https://doi.org/10.1007/978-3-030-11330-8_16
208 K. Roumbedakis and Á. Guerra
was hypothesized by Guerra (1993) for all living cephalo- 1986, 1987; Pollero and Iribarne 1988; Castro et al. 1992;
pods, except Nautilus species. Cortez et al. 1995; Zamora and Olivares 2004; Estefanell et al.
When sexual maturity occurs, the digestive gland ceases 2010; Roumbedakis et al. 2018). Changes in gonads and the
to function properly, leading to a gradual decrease in food digestive gland appearance are also observed: ovaries and
intake until its cessation, consequently stopping growth digestive glands became pale and opaque and oviductal
(Mather 2006). A reduction of digestive enzymes in the glands darken (Fig. 16.1a–c), a reduction in size also occurs.
posterior salivary glands and digestive gland observed in Mature and spent females of the deepwater squid Moro-
post-spawning octopuses is also assumed to contribute to teuthis ingens showed advanced tissue breakdown with
starvation, body weight loss, and consequently, death individuals having a tin mantle wall with inelastic, gelati-
(Sakaguchi 1968). Signs of degeneration in the nervous nous appearance. Histological examination of the mantle
system and deterioration in the long-term memory process wall revealed that tissue breakdown was due to a drastic
can also occur in senescent cephalopods (Chichery and histolysis of muscle tissue and to a lesser extent, collagen
Chichery 1992a). fibres (Jackson and Mladenov 1994). These features are
considered by these authors in relation to the processes
contributing to terminal maturation in M. ingens. Thus,
16.2 Other Signs of Cephalopod Senescence because all the examined individuals had empty caecums,
senescence and post-spawning death may be associated with
Senescence is accompanied by physiological, immunologi- starvation, as found in captive Illex illecebrosus (Rowe and
cal and behavioural changes, which may include: (i) reduced Mangold 1975). Moreover, O’Dor and Wells (1978) noted
or loss of appetite and feeding; (ii) retraction of the skin that the tissue breakdown and death in Octopus vulgaris is
around the eyes; (iii) cloudy eyes; (iv) loss of coordination; not simply due to starvation and the energy demand of a
and (iv) occurrence of skin lesions (Chichery and Chichery developing ovary, because males also cease eating, show
1992a, b; Dumont et al. 1994; for reviews also see Anderson similar tissue degeneracy and die at approximately the same
et al. 2002; Mather 2006). time as their mates. Therefore, they concluded that tissue
Probably, one of the most remarkable consequences in degeneracy and death should be under the control of the
senescent cephalopods is the loss of body weight as a con- optic hormone and that this hormone acted to inhibit protein
sequence of starvation. A loss of body weight was observed in synthesis. Nevertheless, it is probably that a developing
both male and female senescent octopuses: Enteroctopus ovary also produces a hormone that increases the release of
dofleini (Cosgrove 1993; Anderson et al. 2002); Octopus muscle amino acids into the blood (O’Dor and Wells (1978).
cyaneae (Van Heukelem 1976); Octopus maya (Roumbe- The information on the effects of senescence in males is
dakis et al. 2018); Octopus mimus (Cortez et al. 1995); much scarcer than in females. Taitt experience (2013) with a
Octopus vulgaris (O’Dor and Wells 1987; Hernández-García senescent male Caribbean Reef Octopus (O. briareus)
et al. 2002) and Octopus rubescens (Anderson et al. 2002). showed that its behaviour at the end of their life cycle is
According to these studies, in males the total loss of body markedly different than that of females. Senescent males
weight varied between 4.3 and 32.1%, while in females larger tend to be quite active, often at odd/uncharacteristic times of
percentages were observed, varying between 25 and 71%. the day. Like females, males will generally refuse food
A decrease in both gonadosomatic and hepatosomatic offerings when they have approached the end of their life
indexes associated with the deterioration of general physio- cycle. The male also exhibited a decline in his ability to
logical condition occurs in post-spawned cephalopods (Tait control his chromatophores. The movement of both the male
(a) (b) (c)
Fig. 16.1 Physiological changes in the gonad and in the digestive gland of post-spawning Octopus maya. a Gonad of a female octopus
immediately after spawning; and b 40 days after spawning; c digestive gland of a female octopus 40 days after spawning
16 Cephalopod Senescence and Parasitology 209
and the female are significantly less coordinated than in early

stages of experimentation.
Parallel to body deterioration, a possible reduction in the
immunological responses can occur during senescence (Pas-
cual et al. 2010). However, studies evaluating the immuno-
competence and health status of senescent cephalopods are
rare (e.g. Roumbedakis et al. 2018). Although an immuno-
logical compensation through the cost of using energy reserves
in female octopuses in the first period after spawning (i.e., until
at least 40 days post-spawning) can occur (Roumbedakis et al.
2018), there is some evidence of an impairment of the immune
system in senescent cephalopods. For instance, arm regener-
ation may not occur in injured senescent cuttlefish (Féral 1988) Fig. 16.2 Prevalence of infection of Aggregata spp. in post-spawned
and host defence against parasite infection may be impaired in female octopuses: Octopus vulgaris (n = 4; compiled from Pascual
senescent octopuses (Pascual et al. 2010). The presence of et al. 2010) and O. maya (n = 22; original data)
‘unhealed wounds’ in senescent female octopuses may be
possibly related to an impairment of the immune responses
(Wang and Ragsdale 2018). signs of inflammation were observed. A suppressed immune
Behavioural changes are also observed in senescent system may increase susceptibility to parasite infection in
cephalopods. A recent study with sexually matured female senescent cephalopods.
Octopus bimaculoides described four stages of reproductive As commonly observed in adult octopuses, post-spawned
behavioural stages: (i) non-mated; (ii) feeding; (iii) fasting; female octopuses also present high prevalence of infection
and (iv) declining (Wang and Ragsdale 2018). The most by Aggregata spp. (Apicomplexa, Aggregatidae) (Fig. 16.2).
remarkable characteristics commonly observed in senescent In post-spawned female O. vulgaris, large numbers of
animals (e.g. skin lesions, retraction of skin around the eyes, sporogonial stages (infective forms) of Aggregata octopiana
missing arm tips or suckers) were seen in the fourth stage. were highly spread in host tissues, probably to ensure the
The authors also identified that multiple signalling systems completion of parasite’s life cycle (Pascual et al. 2010). In
of the optic glands are involved in these behaviours. post-spawned female O. maya infections by Aggregata
sp. were observed in the caecum (Fig. 16.3a), intestine, and
gills of the hosts (Roumbedakis et al. 2017).
16.3 Parasitology and Senescence In addition, post-spawned female O. maya was also par-
asitized with cestode larvae Prochristianella sp. (Cestoda,
Senescence is not a disease, although senescent cephalopods Trypanorhyncha). This parasite was found in the buccal mass
can be frequently mistaken with diseased animals. For that of the female octopuses (Fig. 16.3B), with 90.9% of preva-
reason, in order to be able to differentiate these conditions, it lence and mean intensity of infection of 155.4 ± 107.73.
is important to recognize its signs (Anderson et al. 2002). Both parasites, the coccidian Aggregata sp. and the thry-
Senescence-like symptoms due to poor animal welfare (e.g. panorhynch cestode Prochristianella sp., are transmitted by
changes in water quality parameters) can also occur the food chain. In this study, female octopuses were captured
(Budelmann 1998). Skin lesions caused by hits against the in the wild and acclimatized in the laboratory for approxi-
tank walls also are a common cause for bacterial infections mately 1–2 months until sampling, which occurred immedi-
(Hanlon and Forsythe 1990), which may eventually become ately after or until 40 days after spawning. In the beginning of
a more likely occurrence due to the loss of coordination in the acclimation period octopuses fed on frozen crabs or on an
senescent cephalopods. artificial diet and then feeding was gradually reduced until the
Parasitological studies in senescent cephalopods are rare, spawning. This suggests that these animals were already
however, they may provide important insights into host– infected with both parasites before capture.
parasite interactions. In senescent female cephalopods, the
transmission of parasites through the food chain (e.g.
trematodes digenea, cestodes, nematodes) might be reduced 16.4 Concluding Remarks
or inexistent, due to the reduction or lack of feeding in this
period. Moreover, senescent cephalopods parasitized may The degenerative process and death are so rapid in the
eventually not properly activate immunological reactions, as majority of cephalopods that quantitative and qualitative
observed in Octopus vulgaris infected with Aggregata changes are very important. For this and other reasons (e.g.
(Pascual et al. 2010), in which low host reaction and no excellent adaptation of some species to confinement
210 K. Roumbedakis and Á. Guerra
Fig. 16.3 Parasites of (a) (b)

post-spawning female O. maya.
a Caecum infected with the
coccidian Aggregata
sp. b Trypanorhynch cestode
Prochristianella sp. collected
from the buccal mass
conditions, and their capacity to tolerate well surgical Dumont E, Chichery MP, Nouvelot A, Chichery R (1994) Variations of
operations) cephalopods are an excellent material for the the lipid constituents in the central nervous system of the cuttlefish
(Sepia officinalis) during aging. Comp Biochem Physiol Part A:
study of mechanisms regulating ageing in laboratory models, Physiol 108(2–3):315–323
and therefore in bio-gerontology. As pointed out by Nussey Estefanell J, Socorro J, Roo FJ, Fernández-Palacios H, Izquierdo M
et al. (2012), the recent emergence of long-term field studies (2010) Gonad maturation in Octopus vulgaris during ongrowing,
presents irrefutable evidence that senescence is commonly under different conditions of sex ratio. ICES J Mar Sci 67:1487–
1493
detected in nature. These authors found such evidence in 175 Féral JP (1988) Wound healing after arm amputation in Sepia officinalis
different animal species. The bulk of this evidence comes (Cephalopoda: Sepioidea). J Invert Pathol 52(3):380–388
from birds and mammals, but senescence was also evidenced Guerra A (1993) Ageing in cephalopods. In: Okutani T. et al (eds) The
in other vertebrates and insects. It could surprise the absence recent advances in cephalopod fisheries biology. Tokai University
Press, pp 684–687
of the cephalopods in this comprehensive review, but this Hanlon RT (1983). Octopus briareus. In: Boyle PR (ed) Cephalopod
may be due to the fact that senescence has hardly been life cycles: species accounts, vol 1. London Academic Press,
observed in nature in these organisms. Parasitological stud- pp 251–256
ies in senescent cephalopods are rare, however, they may Hanlon RT, Forsythe JW (1990) Diseases caused by microorganisms.
In: Kinne O (ed) Diseases of mollusca: cephalopoda. Biologische
provide important insights into host–parasite interactions. Anstalt Helgoland, Hamburg, pp 23–46
For the above reasons, we consider that both the study of Hanlon RT, Messenger JB (1996) Cephalopod Behaviour. Cambridge
senescence in cephalopods and the relationship between this University Press, Cambridge
critical period of the life cycle and the pathological processes Hartwick B (1983) Octopus dofleini. In: Boyle PR (ed) Cephalopod life
cycles: species accounts, vol. 1. London Academic Press, pp 277–291
caused by infections is still in its infancy, and should be a Hernández-García V, Hernández-López JL, Castro-Hernández JJ
field of research to be developed. (2002) On the reproduction of Octopus vulgaris off the coast of
the Canary Islands. Fish Res 57:197–203
Jackson GD, Mladenov PD (1994) Terminal spawning in the deepwater
squid Moroteuthis ingens (Cephalopoda: Onychoteuthidae) J Zool
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made.
Pathogens and Related Diseases
in Non-European Cephalopods: Central 17
and South America
Yanis Cruz-Quintana, Jonathan Fabricio Lucas Demera,

Leonela Griselda Muñoz-Chumo, Ana María Santana-Piñeros,
Sheila Castellanos-Martínez, and Ma. Leopoldina Aguirre-Macedo
Abstract
Despite the economic and ecologic importance of Mexican four-eyed Octopus maya and the
jumbo squid Dosidicus gigas, the pathogens and diseases in both species remain largely
unknown. This chapter covers the pathogens and diseases, mainly protozoan and metazoan
parasites, of these two non-European cephalopods.

Keywords
Mexico Ecuador Fisheries Parasites Octopus maya Dosidicus gigas
spanning from Ciudad del Carmen in Campeche State to Isla

17.1 Introduction
Mujeres in Quintana Roo State (Van Heukelem 1983). It is
one of the most important commercially exploited species in
The Mexican four-eyed Octopus maya and the jumbo squid
Mexican fisheries, generating over 27 million dollars in rev-
Dosidicus gigas are important fishery sources from Mexico
enue annually, with a high potential for aquaculture due to
and Ecuador, respectively. Octopus maya is an endemic
their good adaptability to captive conditions. On the other
species of the Yucatan peninsula, with a distribution range
hand, D. gigas is an endemic species of the eastern Pacific,
with a distribution range going from Alaska, USA, to Tierra
Y. Cruz-Quintana (&) J. F. Lucas Demera del Fuego, Chile (Roper et al. 2010). It is commercially caught
L. G. Muñoz-Chumo A. M. Santana-Piñeros by the USA, México, Perú, and Chile. However, in early 2014,
Grupo de Investigación en Sanidad Acuícola, Inocuidad y Salud
Ambiental. Escuela de Acuicultura y Pesquería, Facultad de the Ecuador Government decided that D. gigas must be a new
Ciencias Veterinarias, Universidad Técnica de Manabí, Calle fishery target and the collection of biological data to improve
Gonzalo Loor Velasco, S/N., Ciudadela Universitaria, CP 130104 the fisheries management began (Morales-Bojórquez and
Bahía de Caráquez, Ecuador Pacheco-Bedoya 2016). In this chapter, an overview of the
parasites and pathological conditions in two wild cephalo-
J. F. Lucas Demera pods, the Octopodidae Octopus maya from the Mexican
Caribbean coast and the Ommastrephidae Dosidicus gigas
L. G. Muñoz-Chumo from the Ecuadorian Pacific coast, is provided. Tissue slides
collected from both species were stained with hematoxylin
A. M. Santana-Piñeros and eosin (H&E). The figures include the scale bar in
micrometers, except in the majority of the macroscopic ima-
S. Castellanos-Martínez ges which include the scale in millimeters. In this chapter, we
Instituto de Investigaciones Oceanológicas UABC, Ensenada,
Mexico will not go into detail on the anatomy and normal histology of
e-mail: [email protected] Octopus maya or Dosidicus gigas, as these general aspects of
Ma. L. Aguirre-Macedo cephalopods have been addressed previously in Chaps. 3 and
Laboratorio de Patología Acuática y Parasitología, CINVESTAV 4. However, we will describe some anatomical and histolog-
Unidad Mérida, Carretera Antigua a Progreso, Km 6, Cordemex, ical aspects of the affected organs or tissues, to facilitate the
97310 Mérida, Yucatán, Mexico understanding of the tissue alterations.

https://doi.org/10.1007/978-3-030-11330-8_17
214 Y. Cruz-Quintana et al.
17.2 Pathogens and Related Diseases Larval stages of the cestoda Prochristianella hispida
in the Mexican Four-Eyed Octopus infect the anterior salivary glands of O. maya, destroying the
Octopus Maya secretory tissue and producing fibrosis. In hosts with low
infection (< 5 parasites), the cestodes cause focal necrosis of
The digestive tract of O. maya, like other octopuses, is rather the tubules’ epithelial tissue, with a slight proliferation of
short, consisting of a buccal mass, esophagus, crop, stom- connective tissue (Fig. 17.1d). In moderate infections (6–20
ach, caecum, intestine, and digestive gland. The buccal parasites), damage is more noticeable, compromising larger
mass, the first organ of the digestive system, is a roughly areas (Fig. 17.2a), whereas in heavy infections (>20 para-
spherical structure that lies just in front of the brain, within a sites per host), the secretory epithelium is replaced by par-
sinus formed by the base of the arms (Fig. 17.1a). It is asites and fibrous connective tissue (Fig. 17.2b), which
loosely attached within the sinus by the esophagus and the reduces the functionality of the gland. The parasites also
enclosing buccal membrane, a pigmented web of folded skin infect, although in less number, the scarce connective tissue
that is attached to the arms. The buccal membrane sur- between the internal and external epithelium and muscles of
rounding the beaks is folded into inner and outer lips. the buccal mass. The high prevalence of cestode larvae in the
Among other components like muscles, radula, and upper anterior salivary glands of O. maya and the severe damages
and lower beaks, the buccal mass involves the anterior associated with the infection level could cause reproductive
salivary glands (Fig. 17.1b), a simple branched tubular gland and nutritional alterations, considering the functions of sal-
surrounded by a thin capsule of lax connective tissue with iva in these two physiological processes.
scarce muscular fibers. The secretory epithelium of this Continuing through the digestive tract and posterior to the
gland is supported by a connective tissue membrane which buccal mass, esophagus, crop, and stomach, the caecum is
forms trabecules within the tissue. Acinar spaces are formed located; a tubular organ spirals around an axis or columella
and lined with secretory cells that vary in height, from (Fig. 17.3a). The caecum shows longitudinal folds protrud-
cuboidal to columnar depending on the stage of secretion ing into the lumen, increasing the surface area of the organ.
(Fig. 17.1c). The function of the mucus produced by the In the external wall of the caecum, these folds are larger and
anterior salivary glands is not only limited to the provision more abundant than those in the internal wall near the col-
of lubrication necessary for sliding and predigestion of the umella. These primary folds that arise from the inner side of
food but also provides support for eggs in spawning females. the external wall give way to secondary folds. They are all
Fig. 17.1 a Octopus maya (a) (b)

dissected, showing the digestive
system: buccal mass (arrow),
esophagus, posterior salivary
glands, intestine, stomach, and
caecum. The digestive gland
(DG) was removed to facilitate
the organs’ visibility. Scale bar:
50 mm. b Buccal mass with
anterior salivary glands (arrow).
c Anterior salivary glands
showing: cylindrical cells
(arrows), lumen (l). Scale bar:
50 µm. d Anterior salivary glands
with low cestode infection. Note (c) (d)
the normal appearance of the
secretory tissue (lower right)
despite the presence of two
cestodes (c). Scale bar: 500 µm
17 Pathogens and Related Diseases in Non-European Cephalopods … 215
Fig. 17.2 Anterior salivary glands (a) (b)

with cestode (c) infection. a Moder-
ate infection, observing the prolif-
eration of connective tissue (arrow)
and loss of secretory tissue (lower
left). Scale bar: 500 µm. b Heavy
infection where secretory tissue has
been replaced by cestodes and
fibrous connective tissue. Scale
bar: 500 µm
lined by a simple, cylindrical, and ciliated epithelium, and As found in Octopus vulgaris (Estévez et al. 1996; Gestal
have a central axis of loose connective tissue (or corion) with et al. 1999, 2002), coccidian of the genus Aggregata infects
high vascularization (Fig. 17.3b). The mucosa is underlined the wall and primary folds in the caecum and intestine of
by scarce connective tissue and thin fascicles of longitudinal Octopus maya. Small and whitish cysts containing Aggre-
muscle fibers. A similar architecture is shown in the intes- gata sporocysts are easily distinguished in the external wall
tine, which has longitudinal folds protruding into the lumen of both organs by gross observation (Fig. 17.3a). Histolog-
mainly in the proximal and medial zones. The mucosa is ically, gamogony and sporogony of Aggregata sp. are
lined by a pseudostratified and ciliated epithelium that lies observed in the loose connective tissue and epithelium of the
on a loose, highly vascularized connective tissue with some primary folds. The presence of macro- and microgametes
muscle fibers. Behind the connective tissue, a thin muscular cause enlargement, not only in the infected cells but also in
wall comprised by an external circular layer and an internal the primary folds (Fig. 17.4a). Frequently, these marked
longitudinal one provides the support and elasticity to the enlargements cause rupture of the basal membrane with the
intestine. detachment of the epithelium.
Fig. 17.3 a Dorsal view of the (a) (b)

stomach(s) and caecum coiled
(ca) of O. maya. Oocyst of
Aggregata sp. (arrow) in the
external wall of the caecum
coiled. b Normal histology of the
caecum coiled showing the
external wall with primary
longitudinal folds, with different
heights (arrows), and secondary
folds (arrowhead). (m) Muscle of
external wall. Scale bar: 200 µm
(a) (b)
(c) (d)
Fig. 17.4 a Histological section of macrogametes on an infected shown in Fig. 17.4b. Note the sporocysts in the loose connective tissue
caecum of O. maya. Scale bar: 200 µm. b Histological section of of the primary folder and throughout the epithelium. Scale bar: 50 µm.
sporocyst located on the loose connective tissue of an infected caecum d Histological section of sporocysts located on the loose connective
of O. maya. The presence of sporocysts causes hemocytic infiltration tissue of an infected intestine of O. maya. Note the destruction of the
and distension of the primary folder. Scale bar: 200 µm. c The tissue organ architecture and loss of intestinal epithelium. Scale bar:
histological section at a higher magnification of the infected caecum as 50 µm
Individual sporocysts not enclosed in cysts are located smooth muscular fibers. Each tubule represents a functional
within epithelial cells of the mucous membrane and in the unit and is composed of highly vacuolated polyhedral cells,
submucosal connective tissue of the caecum and intestine of which are seated in a thin basal lamina and defined apically
O. maya (Fig. 17.4b–d). Pericyst reactions of connective the lumen of the tubule (Fig. 17.5b). The organ’s function is
tissue and hemocytic infiltration produce marked distension related to the digestive process, supplying most of the
in the affected area (Fig. 17.4b). In the mucous membrane of digestive enzymes (Domíngues et al. 2007; Aguila et al.
the digestive tract, the sporocysts containing the infective 2007).
stages (sporozoites) migrate through the epithelium causing The presence of Aggregata sp. in the digestive gland of
degenerating and the death of the invaded cell (Fig. 17.4c). O. maya is less frequent than in the caecum and intestine.
Periodically, necrotic portions of the caecum and infected However, sporogony stages can be seen in the epithelial cells
intestinal epithelium are sloughed off and eliminated. In of the hepatopancreatic tubes. Focal necrosis of the tubular
heavily infected hosts, most of the tissue is replaced by epithelium and hemocytic infiltrations in the intertubular
parasites, and loss of caecum, intestinal epithelium, and spaces are caused by the presence of sporogony stages. The
destruction of the organ tissue architecture can be observed severity of the damage is related to the infection level, which
(Fig. 17.4d). The damage may have detrimental effects on can cause degeneration of epithelial cells and atrophy of the
gastrointestinal function and a malabsorption syndrome is organ (Fig. 17.5c, d).
probably the result in a heavily infected host, which agrees As in other coleoids, a single pair of well-vascularized
with the observations by Gestal et al. (2002). gills suspended in the mantle cavity permits the respiratory
The digestive gland, the most prominent organ of O. exchange of O. maya with the environment (Fig. 17.6a). The
maya, is brown in color and is located in the anterior half of gill has primary lamellae that extend perpendicularly to the
the body (Fig. 17.5a). This is a branched tubular organ gill axis, forming inner and outer demibranches supported by
enclosed in a capsule of loose connective tissue with scarce cartilaginous rods. Each primary lamella is folded to form
Fig. 17.5 a Digestive gland (a)

(arrow) in O. maya. Scale bar:
50 mm. b Histological section of
the digestive gland on a healthy
O. maya. The lumen (l) of the
tubules is lined by the epithelium
(e) that it is supported by the basal (b)
lamina. Scale bar: 100 µm.
c Histological section of the
digestive gland showing a focal
necrosis (arrow) in the tubular
epithelium. Scale bar: 100 µm.
d Histological section of the
digestive gland on an infected O.
maya with Aggregata sp. Note the
focal necrosis and uninucleate
sporoblasts on the epithelium.
Scale bar: 50 µm
(c) (d)
secondary lamellae that are partially partitioned by a septum. frequent parasite that infects the gills of O. maya is an
A branched arrangement of the secondary lamellae origi- unidentified larval cestode. The presence of plerocercoids in
nates the tertiary lamellae (Fig. 17.6b). The secondary and the gill tissue is more frequent in primary lamellae and less
tertiary lamellae are lined by a thin epithelium in a concave frequent in secondary lamellae, where the cestodes induce
inner and convex outer side. Between inner and outer parts, connective tissue proliferation with hemocytic infiltration
the hemolymph runs in lacunae facilitating the gas exchange (Fig. 17.7c, d). The compromised area depends on the
(Fig. 17.6c). number of cestodes and the magnitude of host tissue reac-
The gills are the target organs of most parasite species in tion, but in heavily infected hosts the hemolymph flow may
O. maya. Copepods of the genus Octopicola are frequent be reduced considerably, affecting physiological processes.
parasites of the gill tissue. The number of parasites varies A Cryptogonimidae trematode has been identified in gills
according to locality and climatic season, but generally, the from O. maya. This parasite is less frequent than copepods
abundance is high. Infections by these copepods cause and cestodes, and the histological damage associated with it
hemocytic infiltration between the inner and outer layers of is more focal. The metacercariae show a preference for the
the secondary and tertiary lamellae, with connective tissue primary lamellae, producing distention and hemocytic infil-
proliferation and lamella fusion (Figs. 17.7a, b). The damage tration (Fig. 17.8a, b).
is greater when the abundance of parasites increases, com- Extraintestinal sporogonia of Aggregata sp. have been
promising large areas of gill tissue. The second more observed in the gills of O. maya. The infected hosts show
Fig. 17.6 a Gills in O. maya (a) (b)

(arrow). b Histological section of
gill on a healthy O. maya. Note
the branching arrangement of the
lamellae. Scale bar: 500 µm.
c Histological section of a gill of a
healthy O. maya showing the
epithelium (arrow). Scale bar:
100 µm
(c)
Fig. 17.7 a Gill section of O. (a) (b)

maya with an Octopicola
copepod (Co) producing
distention of lacunae (la) and
hemocytic infiltration. Scale bar:
100 µm. b Higher magnification
of Fig. 17.7a. Note the
arrangement of the host tissue to
encapsulate the copepod (Co).
Scale bar: 50 µm. c Gill section
of Octopus maya with a strong
hemocytic reaction around an
unidentified larval cestode (c).
Scale bar: 200 µm. d Higher
magnification of the gill tissue, (c) (d)
showing proliferation of
connective tissue (Ct) around
unidentified larval cestode. Scale
bar: 50 µm
Fig. 17.8 a Gill section of O. (a) (b)

maya with a Cryptogonimidae
gen. sp. (arrows) producing
distention of primary lamellae
and hemocytic infiltration. Scale
bar: 500 µm. b Higher
magnification of Fig. 17.8a
showing the sucker (su) of
Cryptogonimidae trematode (t).
The tissue shows autolysis by
poor fixation. Scale bar: 50 µm.
c Gill section of Octopus maya
with oocysts (Oo) of Aggregata
sp. Scale bar: 200 µm. d Higher
magnification of Fig. 17.8c (c) (d)
showing distention of the lamellar
epithelium (arrow) caused by the
presence of numerous uninucleate
sporoblasts inside the oocyst
(Oo). Scale bar: 500 µm
numerous oocysts in the gills’ secondary and tertiary of idiopathic necrosis in the renal appendages of O. maya are
lamellae (Fig. 17.8c). Each oocyst containing high numbers still needed.
of uninucleate and less binucleate sporoblasts is surrounded
by a thin wall and grows among inner and outer epithelium
of secondary and tertiary lamellae (Fig. 17.8d). The larger 17.3 Pathogens and Related Diseases
oocysts disrupt the normal branched pattern of the gill and in the Jumbo Squid Dosidicus Gigas
can cause mechanical compression in adjacent lamellae,
likely affecting hemolymph flow. The impact of Aggregata The jumbo flying squid Dosidicus gigas, the other
sp. infections in O. maya has not been yet studied. However, non-European cephalopod treated in this chapter, also harbors
the histological damages observed in the intestine and gills some parasites and diseases that affect tissues and organs
suggest a high pathogenic capability of Aggregata sp. that (Pardo-Gandarillas et al. 2009; Iannacone and Alvariño
may affect the physiology and body condition on the 2009). The buccal mass, the first organ of the digestive
infected hosts. system, is heavily infected by an unidentified larval cestode.
Like other cephalopods (Hochberg 1990), the excretory Plerocercoids can be observed among the acini of anterior
organs of O. maya exhibit dicyemids attached principally to salivary glands and under the mucosal epithelium
the renal appendages (Fig. 17.9a, b). The presence of these (Fig. 17.10a, d). The damages on affected tissue are related to
vermiform stages is not associated to excretory tissue dam- the infection level. At low parasite numbers, the damages are
ages (Fig. 17.9c), and it is generally accepted that the local, consisting of focal modifications of tissue architecture
dicyemids are symbionts that may facilitate host excretion of and proliferation of connective tissue (Fig. 17.10b, c). Higher
nitrogenized compounds by contributing to the acidification parasite numbers can cause degeneration and necrosis in the
of the urine. secretory epithelium of anterior salivary glands, which is
Idiopathic focal necrosis in the epithelium of renal replaced by parasites and fibro-connective tissue (Fig. 17.10
tubules has been observed in O. maya at very low prevalence d). In severe infections (>20 parasites per host), the secretory
(Fig. 17.9d). However, no pathogen has been observed in tissue of the salivary gland may be severely affected, greatly
the histological sections of affected organs, and there are no reducing saliva production. This could have a negative effect
precedent studies pointing to a pathogen responsible for the on nutrition and therefore on the body weight and general
latter. Therefore, further investigations to clarify the etiology physiological condition of the host.
Fig. 17.9 a Renal organs in O. (a) (b)

maya (arrow). Scale bar: 50 mm.
b Section of renal tissue of
healthy O. maya showing
dicyemids symbionts (arrow) in
the lumen of the tubules.
(Ep) Epithelium of the renal
tubules. Scale bar: 250 µm.
c Higher magnification of
Fig. 17.9b showing the nucleus of
epithelial cells (Ep) of the renal
tubules and the presence of
dicyemids (Di) in the lumen.
Scale bar: 50 µm. d Section of
renal tissue with idiopathic focal (c) (d)
necrosis (arrow) in the excretory
epithelium. Scale bar: 200 µm
Fig. 17.10 Histological section (a) (b)

of buccal mass from Dosidicus
gigas. a Acini of the anterior
salivary gland (Sg); one of them
is discharging the mucous
secretion through the epithelium
(arrow). Scale bar: 250 µm.
b Anterior salivary gland with
moderate infection by larval
cestode (c). Scale bar: 250 µm.
c Anterior salivary gland showing
proliferation of connective tissue
(arrow) around a larval cestode.
Scale bar: 100 µm. d Anterior
salivary gland (Sg) heavily (c) (d)
infected with larval cestodes.
Secretory tissue is strongly
reduced and has been replaced by
parasites and fibro-connective
tissue (left). Scale bar: 500 µm

of buccal mass from male D. gigas
(a) (b)
showing spermatophores (Sp) at-
tached to the mucosa. a The epithe-
lium and lamina propria are
damaged and a hemocytic reaction
(*) is visible in the affected area.
Scale bar: 250 µm. b Hemocytic
infiltration (*) around the epithe-
lium, lamina propria and adjacent
acinus, affected. Scale bar: 250 µm
In males of D. gigas, some spermatophores can be found The secondary folds stem from both sides of the primary
attached to the mucosa of the buccal mass (also in the gills, folds and perpendicularly to their axes (Fig. 17.12a). Two
see below) causing ulcers that penetrate into the submucosa kinds of parasites, nematode and cestode, infect the caecum.
(Fig. 17.11a, b). The epithelium and basal lamina are dam- Anisakidae nematodes have been found encysted in the
aged at the site of attachment, and hemocytic infiltration can caecum wall, where a hemocytic infiltration causes disten-
be seen around the injury. A plausible explanation for this tion of the connective tissue (Fig. 17.12b). In contrast, ple-
interesting and unusual condition is that the spermatophores rocercoids of an unidentified cestode are very frequent in the
probably belong to another male and act as foreign bodies basal, medial, and apical zones of the primary folds of the
when coming into contact with the tissues of the receptor host. caecum causing degeneration and focal necrosis
The general morphology of the caecum in D. gigas is (Fig. 17.12c, d).
similar to that of O. maya. Primary folds arise from the inner In the intestine, two morphotypes of an unidentified
margin of the caecum wall and are protruded into the lumen. cestode were found (Fig. 17.13a, b). One large morphotype

of the coiled caecum of D. gigas.
a Healthy tissue showing the
external wall with primary
longitudinal folds (Pf) and
secondary folds (arrow). Scale
bar: 250 µm. b A larvae of
Anisakidae nematode (arrow)
encysted in the wall of the coiled
caecum. Scale bar: 250 µm.
c Coiled caecum showing
unidentified larval cestodes
(arrow) infecting the loose
connective tissue of primary fold.
Scale bar: 250 µm. d Primary (c) (d)
fold showing degenerated tissue
and focal necrosis (arrow) in the
loose connective tissue. Scale bar:
250 µm
(a) (b)
(c) (d)
Fig. 17.13 Wet mount and histological sections of intestine from D. cestodes. Scale bar: 500 µm. c Larval cestode of the smaller
gigas. a Fresh intestine infected with two morphotypes of unidentified morphotype infecting the intestinal submucosa. Note the protruded
larval cestodes. One larger morphotype (c) inside the intestine, and intestinal epithelium (arrow) by expansion of subjacent connective
second smaller morphotype (arrows) encysted in the intestine wall. tissue. Scale bar: 250 µm. d Higher magnification of Fig. 17.13c
Scale bar: 500 µm. b Histological section of the intestine from showing the flattened nuclei of fibroblastic cells organized around the
Fig. 17.13a showing the two morphotypes of unidentified larval larval cestode. Scale bar: 100 µm
can be found in the lumen of the intestine, while a smaller similar basic morphology. The primary lamellae form the
and more abundant morphotype infects not only the secondary ones, and these form the tertiary ones on a con-
intestinal wall and folds but also the caecum primary folds tinuous fold pattern. The main morphological difference is
and digestive gland. No damage has been associated with the the fanlike fold pattern in D. gigas in comparison to the
presence of the larger morphotype cestode in the intestinal branched arrangement pattern in O. maya (Fig. 17.15a, b).
lumen. The smaller morphotype produces a protrusion of the As mentioned above, spermatophores were found attached to
intestinal epithelium by an expansion of a subjacent con- the gills of male specimens (Fig. 17.15c). The number of
nective tissue (Fig. 17.13c). The presence of plerocercoids spermatophores in the gills varies greatly but is generally
in the corion of the intestinal fold triggers the host’s cellular high, causing compression and modification of the fanlike
response, and the fibroblastic cells come to organize them- folded pattern of lamellae (Fig. 17.15d).
selves around the parasite to encapsulate it, causing disten- Nodular lesions in the epithelium of secondary and ter-
tion (Fig. 17.13d). Like in the caecum, focal necrosis and tiary lamellae were observed and associated with the pres-
degenerated tissue can be seen near the plerocercoid, prob- ence of coccidian (Fig. 17.16a). Aggregata sp. sporocysts
ably caused by the secretions of the parasite. The presence of are phagocytized by hemocytes and surrounded by strong
this smaller morphotype of plerocercoid in digestive gland hemocytic infiltration (Fig. 17.16b). The space between
was infrequent, and no damage or alteration of the normal inner and outer epithelium in the infection sites is distended
tissue architecture was related to the presence of parasite and filled with proliferative connective tissue, reducing its
(Fig. 17.14a, b). gas exchange capability. In addition, oocysts containing
Despite the macroscopic differences in gill morphology numerous uninucleate sporoblasts are present and produce
between D. gigas and O. maya, microscopically they share a distention of the lamellae (Fig. 17.16c). Multiple oocysts of
Fig. 17.14 Histological sections (a) (b)

of digestive gland from D. gigas.
a Healthy tissue showing the
tubular epithelium (arrow) and
the lumen of the tubules (l). Scale
bar: 250 µm. b Unidentified
larval cestode (arrow) among
tubules of the digestive gland.
Scale bar: 250 µm
Fig. 17.15 Macroscopic view (a) (b)

and histological section of gill
from D. gigas. a Macroscopic
view of fold pattern and primary
efferent vessel (arrow) in gill.
b Histological section showing
the fold pattern and primary
efferent vessel (lower view). Scale
bar: 500 µm. c Spermatophores
(arrow) attached to the gill in
mature male of D. gigas. Scale
bar: 5 mm. d Histological section
showing the modified architecture
of the gill caused by the attached
spermatophore (Sp). Scale bar: (c) (d)
250 µm
different sizes are present in the same lamella, separated by a containing Anisakidae nematodes are visible by gross
thin wall (Fig. 17.16d). observation in the mantle of D. gigas (Fig. 17.17a). A con-
The mantle of D. gigas, as in other cephalopods, is nective tissue reaction and hemocytic infiltration surround
mainly a muscular layer lined with a delicate yet sophisti- the nematodes (Fig. 17.17b), but the organization of the
cated skin, which functions as the primary barrier against host’s tissue response may be associated to the time of
pathogens and environmental stressors. For this reason, permanence of the larva. A less organized inflammatory
some pathogens settle in this organ as the first site of response could be associated with newly migrating larvae
infection, facilitating the entry of opportunistic pathogens. (Fig. 17.17c).
This is the case of Anisakidae nematodes, whose larvae In the musculature of the mantle, and to some extent in
penetrate through the skin and encyst in the mantle muscu- that of the tentacles, there are also nodules of variable size,
lature until the final host devours the squid and the life cycle well delimited, and brown in color (Fig. 17.18a). These
is completed. Whitish and slightly protuberant nodules nodules contain a homogeneous hyaline material, with very

of gill from D. gigas. a Nodular
lesion (arrow) and oocysts of
Aggregata sp. (arrowhead) in gill.
Scale bar: 500 µm. b Proliferative
reaction around the free
sporocysts of Aggregata
sp. (arrow). Scale bar: 30 µm.
c Oocysts (Oo) of Aggregata
sp. among inner and outer
epithelium (arrow). Scale bar:
100 µm. d Higher magnification
of Oocysts (Oo) in gill. Each
oocyst, containing many
uninucleate and some binucleate (c) (d)
sporoblasts, are delimited by a
thin wall (arrow). Scale bar:
30 µm

and histological sections of
mantle from D. gigas. a A larva
of Anisakidae nematode (arrow)
encysted in the mantle
musculature. Note the host’s
tissue response forming a white
ring around the nematode. Scale
bar: 100 µm. b Histological
section of the tissue that was
showed in Fig. 17.17a. The
nematode (central view),
sectioned transversally, is
rounded by an organized
connective tissue and hemocytic
infiltration. Scale bar: 500 µm.
c Degeneration of muscular fibers
and focal necrosis with hemocytic
infiltrate in the mantle muscle (c)
(M) caused by the presence of
Anisakidae nematodes (arrow).
Scale bar: 250 µm

and histological sections of
mantle from D. gigas.
a Macroscopic view of a nodule
in the mantle (arrow), just below
the epithelium. The tissue has
been cut transversally to facilitate
the view. Scale bar: 500 µm.
b Histological section of the
mantle showing a hyaline nodule
(arrow). Scale bar: 500 µm.
c Higher magnification of
Fig. 17.18b showing the hyaline
material (left) and focal necrosis
(n) surrounded by fibroblasts and (c) (d)
pigmented cells (arrow). Scale
bar: 100 µm. d Giant cells with
phagocytized bacteria (*) and
colonies of bacteria (arrow) free
among connective tissue fibers.
Note the organization of the tissue
around the bacterial colonies.
Scale bar: 100 µm
few cells inside of them (Fig. 17.18b), which could be some avoid reinfections that are undoubtedly happening in natural
type of degraded residue generated during nematode environments. With cestodes, most metazoan parasites
migration. The nodules are covered by a layer of occurring in the digestive system any other host tissue can be
fibro-connective tissue and pigmented cells that give it the controlled following the same practices.
brown color (Fig. 17.18c). In some areas, foci of necrosis Coccidians of the genus Aggregata were also common to
and connective tissue proliferation can be observed around both cephalopod species, similarly to cestodes, the severity
bacterial colonies, suggesting a subsequent infection after of the damage will depend on the infection degree. In O.
nematode larvae migration (Fig. 17.18d). maya, the most affected organ was the intestine while in D.
gigas it was the gills. Aggregata are common to most
cephalopods studied till now and the diseases associated
17.4 Concluding Remarks with its presence have been addressed in several hosts.
Although it is known that in captivity an ectoparasitic
It is evident that there are severe pathological affections in protozoan Ichthyobodo necator can infect the skin of O.
wild cephalopods, with the most serious being those maya and several species of bacteria including Vibrionaceae
occurring in the digestive system, particularly in the buccal also represent a major threat, we did not find histopatho-
mass. In both cephalopods studied here, heavy infection of logical evidence of their presence in wild octopus O. maya
cestodes was identified as the etiological origin. Although or the jumbo squid Dosidicus gigas.
the severity of infection depends directly on the infection
degree, higher parasite numbers can cause degeneration and
necrosis in the secretory epithelium and salivary glands, so References
that nutrients absorption can be affected and in consequence
the entire host condition. This affection, nonetheless, can be Aguila J, Cuzon G, Pascual C, Domingues PM, Gaxiola G, Sánchez A,
easily avoided in captive cephalopods. The cephalopods in Maldonado T, Rosas C (2007) The effects of fish hydrolysate
these infections are playing as second intermediate host of (CPSP) level on Octopus maya (Voss and Solis) diet: digestive
enzyme activity, blood metabolites, and energy balance. Aquacul-
the cestodes; being copepods or small crustaceans the first ture 273:641–655
intermediate hosts and sharks or rays the definitive host, so Boyle P, Rodhouse P (2005). Cephalopods: ecology and fisheries.
that water quality control and captivity food control will Blackwell Science, p 467
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Rosas C (2007) Effects of a dry pelleted diet on growth and survival flying squid Dosidicus gigas (Cephalopoda) in the north of Peru.
of the Yucatan octopus, Octopus maya. Aquacul Nutr 13:273–280 Neotropl Helminthol 3:89–100
Estévez J, Pascual S, Gestal C, Soto M, Rodríguez H, Arias C (1996) Morales-Bojórquez E, Pacheco-Bedoya J (2016) Jumbo squid Dosidi-
Aggregata octopiana (Apicomplexa: Aggregatidae) from Octopus cus gigas: a new fishery in Ecuador. Rev Fish Sci Aquacul
vulgaris off NW Spain. Dis Aquat Org 27:227–231 24:98–110
Gestal C, Abollo E, Pascual S (2002) Observations on associated Pardo-Gandarillas M, Lohrmann K, Valdivia A, Ibañez C (2009) First
histopathology with Aggregata octopiana infection (Protista: Api- record of parasites of Dosidicus gigas (d’Orbigny, 1835) (Cepha-
complexa) in Octopus vulgaris. Dis Aquat Org 50:45–49 lopoda: Ommastrephidae) from the Humboldt Current system off
Gestal C, Pascual S, Corral L, Azevedo C (1999) Ultrastructural aspects Chile. Rev Biol Mar Oceanogr 44:397–408
of the sporogony of Aggregata octopiana (Apicomplexa, Aggre- Roper C, Nigmatullin C, Jereb P (2010) Family Ommastrephidae. In:
gatidae), a coccidian parasite of Octopus vulgaris (Mollusca, Jereb P, Roper CFE (eds) Cephalopods of the World. An annotated and
Cephalopoda) from NE Atlantic Coast. Eur J Protistol 35:417–425 illustrated catalogue of species known to date. FAO Species catalogue
Hochberg FG (1990) Diseases of Mollusca: Cephalopoda. Diseases for Fisheries Purposes. No 4, vol 2, FAO Rome, pp 269–347
caused by protistans and metazoans. In: Kinne O (ed) Diseases of Van Heukelem WF (1983) Octopus maya. In: Boyle PR
marine annimals, vol III. Biologische Anstalt Helgoland, Hamburg, (ed) Cephalopod Life Cycles, vol I. Academic Press, London,
pp 47–227 pp 311–323
made.
Pathogens and Related Diseases in
Non-European Cephalopods: Asia. 18
A Preliminary Review
Jing Ren, Xiaodong Zheng, Yaosen Qian, and Qingqi Zhang
Abstract
Parasitic diseases and other abnormalities play critical roles in causing morbidity in the majority
of Cephalopoda. However, to date, reports of cephalopod diseases from Asia are scarce and lack
detailed information on the description of specific characters. This paper presents a brief
overview of various pathogens and produced diseases in Asian cephalopods, including
coccidiosis by Aggregata, Anisakiasis, infection by the copepods Octopicola, and other
abnormalities such as edema and broken skin. The coccidian Aggregata sp. that infects the
definitive host Amphioctopus fangsiao is a heteroxenous parasite transmitted through the food
web. Anisakids play an important role in Asia as parasitic disease for cephalopods and it is even
transmitted to humans. Concerning the infection by copepods, Octopicola sp. is the only species
of the family Octopicolidae reported from North Pacific waters. Other abnormalities like edema
or broken skin may have been the result of bacterial infections, so that abnormalities could cause
the degeneration and death observed in A. fangsiao.

Keywords
Cephalopoda Pathology Diseases Amphioctopus fangsiao Aggregata Anisakis
Octopicola Asian waters
J. Ren X. Zheng (&) 18.1 Introduction

Institute of Evolution & Marine Biodiversity, Ocean University
of China, Qingdao 266003, China Cephalopoda is the most complex in the invertebrate phyla.
Cephalopods include exclusively marine animals that live in
J. Ren all oceans of the world except the Black Sea. In Asia, many
cephalopods are important economic species for human
J. Ren X. Zheng beings. Octopods were reported the highest production for
Key Laboratory of Mariculture, Ministry of Education, Ocean
University of China, Qingdao 266003, China
2010, at 217,506 tones, while European production of
octopuses was only 42,945 tones (Norman et al. 2014),
Y. Qian
Ganyu Institute of Fishery Science, Lianyungang 222100, China
which are also one of the most important commercial
e-mail: [email protected] cephalopod groups in China (Zheng et al. 2014; Xu and
Q. Zhang
Zheng 2018). Cuttlefish, such as Sepia pharaonis and
Ganyu Jiaxin Fishery Technical Development Co., Ltd., Sepiella japonica, were also an important fishery in the
Lianyungang 222100, China northern part of the Indian Ocean and southeastern Asia

https://doi.org/10.1007/978-3-030-11330-8_18
228 J. Ren et al.
Histological sections of intestinal tissue revealed

destruction of the organ architecture and substitution of the
tissue by parasite cysts (Fig. 18.2a). Through histological
sections observation, the oocysts were spherical, in the range
of 249.75–501.75 lm (mean 360.76 ± 70.39 lm) and
116.84–350.87 lm (mean 231.67 ± 74.89 lm) (n = 20)
with plenty of sporocysts in each oocyst. The size
of sporocysts was measured as follows: 17.69–20.72 lm
(mean 19.20 ± 0.93 lm) by 15.97–20.00 lm (mean
18.31 ± 1.19 lm) (n = 20). The surface of sporocysts was
smooth. The histological results are shown in Fig. 18.2b
(unpublished data).
Fig. 18.1 Distribution of parasites on Amphioctopus fangsiao. Arrows 18.3 Anisakidae

showing disseminated cysts in the epidermis
The life cycle of Anisakidae is rather complex involving

(Nesis, Yin et al. 2018). However, to date, reports of small crustaceans as the first intermediate host; fishes and
cephalopod diseases from Asia are scarce and lack of cephalopods as the second host; and marine mammals which
detailed information on the description of specific act as the definitive host (Nesis et al. 1987; dos Santos and
characters. Howgate 2011; Sangaran and Sundar 2016). Thus, anisaki-
asis is a parasitic disease of cephalopods in Asia. In Japan,
where raw squids are consumed as an integral part of the
18.2 Aggregata Sp. Japanese diet, Anisakid transmission to humans has been
reported, producing a strong pathology (Oshima 1972;
Cephalopods are specifically infected by coccidians of the Tomoo and Kliks 1987; Sakanari and Mckerrow 1989). In
genus Aggregata, which were heteroxenous parasites trans- China, natural infection of Anisakidae larvae was investi-
mitted through the food web. Sexual stages (including gated in squids (2 species, 29 specimens) caught in the
gamogony and sporogony) occur inside the digestive tract of yellow sea and the East China Sea (Koyama 1969).
the definitive cephalopod host (Dobell 1925; Gestal et al.
2002). Coccidian infection at pathological level has not been
previously reported in Asia and data of its prevalence and 18.4 Octopicola
distribution are currently scarce. Protozoan parasites of the
genus Aggregata affecting Amphioctopus fangsiao in natural To date, the genus Octopicola contains five species para-
environment have been associated with large-scale concen- sitizing octopuses (Humes 1957, 1963, 1974; Du et al.
trated deaths occurred in the process of artificial temporarily 2018). Octopicola huanghaiensis (Fig. 18.3) is the first
culture facility in China. White cysts were found in the body species reported in A. fangsiao and Octopus minor, and the
surface representing the 43% of the total number of A. fang- only species of the family Octopicolidae known in Asia (Du
siao inspected (95/220) (unpublished data) (Fig. 18.1). et al. 2018).
Fig. 18.2 Histological sections of

A. fangsiao intestines infected by (a) (b)
Aggregata sp. a Oocysts infecting
the intestinal tissue. b Sporocysts
inside the oocyst. Scale bars: A,
50 lm B, 20 lm
18 Pathogens and Related Diseases in Non-European Cephalopods: Asia. A Preliminary Review 229
Fig. 18.3 Octopicola huanghaiensis (Scale-bar: 100 lm) (Du et al. 2018)
18.5 Other Abnormalities
There are some other abnormalities recorded in Asian

octopuses (Fig. 18.4). During our study, wild specimens of
A. fangsiao were caught in March 2017 in the Yellow Sea
(off Lianyungang, Jiangsu Prov.). The specimens were
temporarily reared indoors of the Ganyu Jiaxin Fishery
Technical Development Co., Ltd. Individuals were placed in
concrete tanks, under the conditions of 11.8 °C, natural
photoperiod, as well as plastic suspensors for shelter.
From March 16 to April 2, 1303 dead A. fangsiao Fig. 18.4 Abnormalities of A. fangsiao. Arrows showing exfoliation
of the mantle and edema
individuals were sampled in order to separate males and
females. The total number of females was 836, accounting
for 64% of all deaths. There were 467 males, accounting analyze the death situation. Eventually, it was found that
for 36% of the total deaths; the female death rate was skin surface of dead individuals includes mainly edema
much higher than that of the male in breeding season, and edema with coccidian (individuals of edema only
which was extremely weird. Therefore, further examina- amount to 16% of the total number of inspection), broken
tion by gross pathology of 220 dead individuals (body skin (37% of the total number of inspection), and broken
surface) was conducted from March 24 to April 2 to arms (4% of the total number of inspection). Those
230 J. Ren et al.
abnormalities may have been the results of bacterial Gestal C, Guerra A, Pascual S, Azevedo C (2002) On the life cycle of
infection so that abnormalities could cause degeneration Aggregata eberthi and observations on Aggregata octopiana
(Apicomplexa, Aggregatidae) from Galicia (NE Atlantic). Eur J
and death of the species. Protistol 37(4):427–435
Humes AG (1957) Octopicola superba ng, n. sp., Copepode
Cyclopoide parasite d’un Octopus de la Mediterranee. Vie et
Milieu Env 8:1–8
Humes AG (1963) Octopicola stocki n. sp. (Copepoda, Cyclopoida),
18.6 Concluding Remarks associated with an octopus in Madagascar. Crustaceana 5(4):271–
280
As summarized above, only handful studies have been Humes AG (1974) Octopicola regalis, n. sp. (Copepoda, Cyclopoida,
documented for cephalopod diseases from Asia currently, Lichomolgidae) associated with Octopus cyaneus from New
Caledonia and Eniwetok Atoll. Bull Mar Sci 24(1):76–85
including coccidiosis by Aggregata sp., Anisakiasis,
Koyama T (1969) Morphological and taxonomical studies on Anisaki-
Octopicola, edema, and broken skin. However, further dae larvae found in marine fishes and squids. Jap J Parasitol 18
characterization and functional studies are needed to confirm (5):466–487
the effect of diseases on the cephalopods health. Finally, the Nesis KN, Burgess LA, Levitov BS (1987) Cephalopods of the world:
squids, cuttlefishes, octopuses, and allies. TFH, p 351
results obtained in the chapter provide a brief overview of
Norman MD, Finn JK, Hochberg FG, Jereb P, Roper CFE (2014)
parasitic diseases in Asian waters. Cephalopods of the world. An annotated and illustrated catalogue of
cephalopod species known to date. FAO Species Catalogue for
Acknowledgements This work was supported by research grants Fishery Purposes, vol 3(4), pp 1–22
from National Natural Science Foundation of China (No. 31672257) Oshima T (1972) Anisakis and anisakiasis in Japan and adjacent area.
and the Fundamental Research Funds for the Central Universities Prog Med Parasit, Japan, pp 301–393
(No. 201822022). Sakanari J, Mckerrow JH (1989) Anisakiasis. Clin Microbiol Rev 2
(3):278–284
Sangaran A, Sundar S (2016) Fish and shell fish borne parasitic
infections-a review. Int J Sci Environ Technol 5(5):2954–2958
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Commons Attribution 4.0 International License (http://creativecommons. the chapter’s Creative Commons licence, unless indicated otherwise in a
org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution credit line to the material. If material is not included in the chapter’s
and reproduction in any medium or format, as long as you give Creative Commons licence and your intended use is not permitted by
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Camino Gestal · Santiago Pascual ·

Ángel Guerra · Graziano Fiorito ·

Ángel Guerra Graziano Fiorito

ISBN 978-3-030-11329-2 ISBN 978-3-030-11330-8 (eBook)

Dr. Erica A. G. Vidal

Part I Functional Anatomy and Histology

Part II Pathogens and Related Diseases

15 Other Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

Xiaodong Zheng Key Laboratory of Mariculture, Ministry of Education, Institute of Evo-

Knowledge of pathologies of cephalopod mollusks in the

© The Author(s) 2019 1

© The Author(s) 2019 5

Palaeontologists have identiﬁed three distinct fossil clades

© The Author(s) 2019 11

Class: Cephalopoda Cuvier, 1797

Order: Octopoda Leach, 1818

SSFs; however, IF are supported by a few species, mainly

distance between the antero-dorsal margin of the mantle and

Fig. 3.10 Sepia ofﬁcinalis

Fig. 3.12 Octopus vulgaris

generally paralysed by the saliva of the posterior salivary

3.4.2.4 Excretory System 3.4.2.5 Reproductive System and Reproduction

Fig. 3.14 Sepia ofﬁcinalis

and is composed of a chain of three giant nerve cells on each

statocysts to study of the damaging effects of the impacts of 3.5.3 Euthanasia

resolution methods such as the transmission electron

© The Author(s) 2019 39

Fig. 4.1 Sections of the skin of (a) (b)

Fig. 4.3 Muscle ﬁbers of the

Fig. 4.4 a Section of the squid

Fig. 4.7 MA1. Sections of the

Fig. 4.8 D1. Sections of the

Fig. 4.9 D2. Sections of the

Fig. 4.10 D3. Transverse

Fig. 4.12 Sections of the

Fig. 4.13 Sections of the

Fig. 4.14 Transverse sections of

Fig. 4.16 Sections of the

Fig. 4.18 Transverse sections of

Fig. 4.19 Panoramic views and

Fig. 4.20 a Panoramic view of

is covered with connective tissues, and in the equatorial

Fig. 4.25 Transverse section of

Fig. 4.26 Sections of nerves of

(a) (b) (c)

Fig. 4.33 a Section of the

Fig. 4.34 a Transverse section

Needham’s (Spermatophoric) Sac, Spermatophores and Terminal Organ or Penis

animals while undergoing strong morphological changes

© The Author(s) 2019 87

(a) (b) (c)

(a) (b) (c)

(c) (d) (e)

(a) (b) (c)

5.8.4 Ink Gland Complex (Fig. 5.13) 5.8.5 Ink Sac

(c) (d) (e)

(a) (b) (b)

(a) (b) (c)

commonly molluscs) and the labyrinthulids (not considered

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