Anais da Academia Brasileira de Cincias (2015) 87(4): 1981-1989

(Annals of the Brazilian Academy of Sciences)

Printed version ISSN 0001-3765 / Online version ISSN 1678-2690
http://dx.doi.org/10.1590/0001-3765201520140600
www.scielo.br/aabc

India ink induces apoptosis in the yellow clam Mesodesma mactroides (Deshayes, 1854).
Optical and ultrastructural study
Yuri B.M. Carvalho, Juan Jethro, Luis H. Poersch and Luis A. Romano
Laboratrio de Imunologia e Patologia de Organismos Aquticos, Estao Marinha de Aquicultura, Instituto de Oceanografia,
Universidade Federal de Rio Grande FURG, Rua do Hotel, 2, Querncia, 96210-030 Rio Grande, RS, Brasil
Manuscript received on December 2, 2014; accepted for publication on January 2, 2015
ABSTRACT

This paper reports on the acute inflammatory and cellular process in the yellow clam, Mesodesma
mactroides, induced by injection of India ink into the muscular foot. Histological observations with optical
and electronic microscopy were made at 24 and 48 h after injection. The induced cellular inflammatory
response consisted of a general hemocyte infiltration without necrosis and apoptotic activity. Migration
of ink-laden phagocytes across the intestinal epithelium was recorded. It appeared that the yellow clam
excreted ink particles through the gill and kidney. The positive staining for apoptosis was observed
in the digestive gland. Electronic microscopy revealed ultrastructural changes of endoplasmic reticulum
stress and apoptotic bodies in the digestive gland. The mechanism by which the India ink particles induce
apoptosis remains unknown, but might possibly be associated with the endoplasmic reticulum stress.
This work has highlighted features that require further discussion in the restricted field the inflammatory
responses of mollusks.
Key words: India ink, inflammation process, endoplasmic reticulum stress, apoptosis, Mesodesma
mactroides.
INTRODUCTION

Interest in the immunity of bivalves has been

increasing continuously over last decades due to
the catastrophic mortalitiy in aquaculture-produced
species and due to the decline of natural stocks of
economic value (Song et al. 2010).
For decades, the yellow clam Mesodesma
mactroides (Deshayes 1854) (Mesodesmatidae)
was the major shellfish resource from the sandy
beaches of the Atlantic coast of Argentina, Uruguay
and the southern part of Brazil (Castilla and Defeo
Correspondence to: Luis Alberto Romano
E-mail: [email protected]

2001). However, a combination of overexploitation

and high mortality rates along the entire distribution
range of the species culminated in the collapse of
these natural stocks (Fiori and Cazzaniga 1999).
In recent years, there has been great emphasis
on the study of parasites of the yellow clam and
the resulting pathologies (Cremonte and Figueras
2004, Carvalho et al. 2013a, b). However, basic
pathological processes, such as inflammation, have
been generally overlooked.
Classic studies using injected dyes have greatly
helped investigators describe the phagocytic cells
and their role in local and general inflammatory
processes in oysters (Pauley and Sparks 1966).
An Acad Bras Cienc (2015) 87 (4)

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Yuri B.M. Carvalho, Juan Jethro, Luis H. Poersch and Luis A. Romano

Essentially all animal cells have the ability to

undergo apoptosis by activating an intrinsic cell
suicide program, when they are no longer needed,
or have become seriously damaged. The execution
of this program leads to a morphologically distinct
form of cell death, termed apoptosis (Kerr et
al. 1972, Wyllie et al. 1980). It is now generally
accepted that apoptosis is of crucial importance
for the development and homeostasis of metazoan
animals (Bergmann et al. 1998).
Apoptosis in mollusks is involved in the larval
development process (Gifondorwa and Leise 2006)
and most likely constitutes an important immune
response that can be initiated by different inducers
(Terahara and Takahashi 2008).
The endoplasmic reticulum (ER) is a centrally
located, multifunctional, and multiprocess
intracellular organelle supporting many mechanisms
required by virtually every cell (Groenendyk et al.
2010). The membrane performs a remarkable number
of diverse functions, including protein synthesis,
translocation across the membrane, integration
into the membrane, folding, posttranslational
modification (including N-linked glycosylation,
and synthesis of phospholipids and steroids on
the cytoplasmic side of the ER membrane, and
regulation of Ca2+ homeostasis). Development and
maintenance of optimally functioning ER membrane
is essential for virtually all cellular activities, from
intracellular signaling to control of transcriptional
pathways; from ion fluxes to control of energy
metabolism; from protein synthesis to multisubunit
assembly; and from lipid synthesis to transcriptional
regulation of steroid metabolism. One of the major
advantages of the centrally located ER network
for the cell is the ability to control the composition
and the dynamics of the ER luminal environment
in an extracellular environment- independent way.
Furthermore, the ER is not an isolated organelle
because it has developed sophisticated mechanisms
of communication with many other cellular
compartments, especially the mitochondria, the
An Acad Bras Cienc (2015) 87 (4)

plasma membrane, and the nucleus (Anelli and Sitia

2008, Frischauf et al. 2008).
ER stress conditions in mammals and
humans have been observed in numerous diseases
including Alzheimer disease, Creutzfeldt
Jakob disease, Huntington disease as well as
cardiovascular diseases, indicating that ER
stress- induced apoptosis is an important factor in
pathophysiological conditions (Boya et al. 2002).
The purpose of the present study was to
describe the inflammatory and celular responses
experimentally induced after injection of a foreign
body (India ink) into the muscular foot of the
yellow clam M. mactroides
MATERIALS AND METHODS

All experimental protocols were approved by the

Federal University of Rio Grande (FURG) Animal
Care Committee.
Clams Husbandry

Specimens of yellow clam (n = 60) were collected

in December 2012 at the intertidal zone of the
Cassino Beach, (321954.2 S 0521720.4
W), Brazil, by excavating the sand with a shovel.
Specimens measured 40-60 mm in shell height
(mean = 47 mm). Once in the laboratory, the clams
were maintained for one day in tanks filled with
50 L of seawater, which was continuously aerated
(temperature = 23 C, salinity = 33 ppt), and were
fed with the microalgae Nannochloropsis occulata.
Experimental Design

Prior to injection, the clams were anesthetized

with a benzocaine solution (250 mg/L). Using a
25 gauge needle fitted to a syringe, approximately
0.05 ml of a suspension of Indian ink (Royal
Talens, Holland) or filtered seawater (30 clams for
each group) was injected into the muscular foot of
the M. mactroides,

1983

India ink induces apoptosis in Mesodesma mactroides

Following the injections, the clams of each

group were placed back in separeted tanks with 50
L of aerated seawater until the appropriate period
of time had elapsed. Water exchange, tank cleaning
and microalgae were provided daily. Ten clams
were analyzed at 24 and 48 h after the injection.
Sample Processing

Optical microscopy
Clams were shucked and the meat fixed in 20%
buffered formalin for 24 h. Sections that were
approximately 5 mm thick, including the mantle,
gills, gonad, digestive gland, kidney, and foot were
then taken from each specimen. Tissue samples
were embedded in Paraplast (Sigma, St. Louis,
MO, USA) and 5 m sections were stained with
hematoxylin and eosin (H&E).
Ultrastructural analysis

Figure 1 - Muscle tissue, 24 hours after the ink injection, with

clumps of India ink granules surrounded by massive infiltration
of hemocytes (arrow). H&E staining. (See the colors in the
online version).

mediated deoxyuridinetriphosphate nick endlabelling (TUNEL) was performed according to

the manufacturers recommendations by using
the ApogTag plus Peroxidase in situ Apoptosis
Detection Kit (S7101; Chemicon, International).
Anti-cleaved caspase-3 immunostaining was
assessed using a rabbit anti-cleaved caspase-3
polyclonal antibody (Asp175; Cell Signalling
Technology, Danvers, MA) as previously described
(Schoner et al. 2010)

For electron microscopy small fragments of the

mantle, gills, gonad, digestive gland, kidney, and
foot were cut into 1 mm blocks and immediately
fixed in phosphate buffered glutaraldehyde (pH
6.9 at 4 C), washed in Millonigs solution and
post-fixed in 1% osmium tetroxide; the tissue
blocks were then dehydrated in a graded series
of ethanol-acetone, immersed in propylene oxide
and embedded in Durcupan ACNI (Fluka Chemie
A.G., Switzerland). Ultrathin sections were cut with
a LKH ultramicrotome and doublestained with
uranyl acetate and lead citrate before examination
in a Jeol JEM-8T electron microscope (Jeol, 32,
Tokyo, Japan).

All of the experimental clams survived until the

end of the trial.

Apoptosis Analysis

Optical Microscopy

Detection of apoptosis was performed in several

tissues of both animals injected with Indian ink and
control group animals injected with physiological
solution. These samples were fixed in 20% buffered
formalin and embedded in Paraplast. For apoptosis
detection, terminal deoxynucleotidyl transferase-

Within 24 h after the India ink injection, a massive

migration of hemocytes to the location of the
injection at the muscular foot was observed. Clumps
of India ink granules were seen to be encapsulated,
and small granules were phagocytosed and
surrounded by inflammatory infiltrate (Fig. 1).

RESULTS

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1984

Yuri B.M. Carvalho, Juan Jethro, Luis H. Poersch and Luis A. Romano

Figure 2 - Gill, 48 hours after the ink injection, with India

ink encapsulated and surrounded by inflammatory infiltrate
(arrow). H&E staining. (See the colors in the online version).

Figure 3 - Kidney, 48 hours after the ink injection, with India

ink between the tubular structures (arrow). H&E staining. (See
the colors in the online version).

Within 48 h after the India ink injection, gross

examination of the experimental clams revealed
that the India ink had spread to all parts of the body.
Clumps of India ink accumulated in the gills, where
they caused severe inflammation with massive
infiltration of hemocytes, obstruction of the vessels
and hemorrhage (Fig. 2). Other organs in which
the ink particles accumulated were the kidney and
the digestive tract (Fig. 3).
Evaluation of the Apoptotic Process by Optical
Microscopy

the muscular foot of M. mactroides, it was possible

Figure 4 - Presence of apoptotic cells (arrow) in digestive

gland tubules, 24 hours after the ink injection. (See the colors
in the online version).

to observe the presence of apoptotic cells in

Electronic Microscopy

Forty-eight hours after injection of Indian ink into

digestive gland tubules and muscle cells. Apoptosis

was not observed in animals that were not injected
with India ink. Tissues were counterstained with
hematoxylin and eosin to aid in the morphologic
evaluation of normal and apoptotic cells (Fig. 4).
An Acad Bras Cienc (2015) 87 (4)

Within 24 h after the injection of the India ink, gill

and kidney cells displayed various abnormalities,
with the digestive gland being the most affected.
The shape of the nucleus was often irregular. In
some cells the mitochondria were swollen, these

1985

India ink induces apoptosis in Mesodesma mactroides

Figure 5 - Low magnification of kidney tissue, 24 hours

after the ink injection. Cells display numerous cytoplasmic
osmiophilic lamellar structures. (arrows).

Figure 6 - Gill cell, 48 hours after the ink injection, showing

dilated endoplasmic reticulum (ER) (short arrow) and
osmiophilic and electrodense lamellar structures (long arrow),
which may correspond to India ink. M: mitochondria.

cells were empty or contained osmiophilic lamellar

structures that were suggestive of india ink with
a variable density. Forty-eight h after the India
ink injection, similar condensed cell fragments
containing fragmented nuclear masses were found
in the voluminous membrane bound vacuoles in
the cytoplasm of cells. The basement membrane
appeared fragmented and had disappeared in some
areas. When present, it was not closely apposed
to the cytoplasmic membrane of the kidney
cells, which is the normal configuration. Granular
hemocytes were most frequently detected in contact
with epithelial cells and presented a rather regular,
round or oval nucleus. Their cytoplasm was usually
pale and contained scarce isolated ribosomes and
a few mitochondria. Furthermore, these cells
usually lacked a well developed endoplasmic
reticulum and a Golgi apparatus, and one or several
lysosomal vesicles of variable size were frequently
observed. The abundance of inflammatory cells in
the gill and kidney tissue, which was represented
by osmiophilic lamellar or granular electron dense
material, was often associated with india ink.
Fibroblasts predominated in the fibrotic areas, and a
basement membrane-like substance was frequently

in contact with their cell surfaces. Some fibroblasts

showed morphologic signs of great synthetic
activity, as evidenced by cytoplasm containing
numerous cisternae of rough ER that was dilated
and filled with material of medium electron density.
Within 24 h after the India ink injection, apoptotic
bodies were found in the muscle cells (Figs. 5, 6,
7, 8, 9).
DISCUSSION

Bivalve hemocytes are primarily responsible

for the defense against pathogens and foreign
particles (Ottaviani et al. 2010). Phagocytosis and
encapsulation are two major mechanisms used by
hemocytes to eliminate nonself substances and dead
cells (De Vico and Carela 2012). Phagocytosis is a
process by which nonself molecules and cell debris
are recognized and ingested, while encapsulation
is the cellular immune defense reaction against
foreign bodies that are too large to be phagocytosed
(De Vico and Carela 2012).
The inflammatory response that occurs after
pathogen invasion and/or cellular injury is a local
defense reaction from the host tissue (Cone 2001).
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Yuri B.M. Carvalho, Juan Jethro, Luis H. Poersch and Luis A. Romano

Figure 7 - Higher magnification of figure 5, osmiophilic

and electrodense lamellar structures (arrows), which may
correspond to India ink.

The functional basis of the inflammatory response

has not been substantially modified during
evolution, and the basic pattern is quite similar,
regardless of the nature of the injurious agent,
the site of its occurrence, or the injured organism
(Ottaviani et al. 2010).
According to the manufacturer, Royal Talens,
the carbon black pigment in Indian Ink is extremely
fine, less than 2 m. These particles are in constant
motion in the ink, continually bumping into one
another. The special preparation method of the
ink ensures that they do not form lumps. If a large
amount of water is added too quickly, however,
the binder (shellac) that surrounds the particles
dissolves. The particles then clot to form larger
particles and sink to the bottom.
The strong hemocyte infiltration around the
site of India ink was probably due to chemotaxis
on the part of these cells in response to a release of
chemicals from the necrotic muscle and connective
tissue cells in the area. Phagocytosis of India ink
by individual phagocytic cells in M. mactroides is
similar to the response observed in other mollusks
(Pauley and Krassner 1972).
An Acad Bras Cienc (2015) 87 (4)

Figure 8 - Higher magnification of digestive gland cell which

thickening of the membrane of the endoplasmic reticulum
(arrow) , dilated cisterns, ribosome dissociation and electronsubjects grouping the outside of the membrane (0).

Figure 9 - Digestive gland cell, 48 hours after the ink injection,

with a voluminous cytoplasmic membrane bound vacuole
containing an apoptotic body (long arrow) and electrodense
structures (short arrow), which may correspond to India ink.

Soluble substances are eliminated primarily

through the gill, kidney, and intestine. In
vertebrates, insoluble particulate material, such as
carbon, is sequestered in the liver, spleen, lungs,
and lymph nodes, all of which are part of the
mononuclear phagocyte system (MPS) (Hirsch and

Fedorko 1970). Disposal of particulate material

India ink induces apoptosis in Mesodesma mactroides

is very slow, except in the lung MPS cells, and

may persist for many years. The main pathway of
elimination in invertebrates is either the intestine or
the renal-pericardial complex. According to Pauley
and Krassner (1972), the intestine and the kidney
play an important role in the elimination of ink
particles from the sea hare Aplysia californica. The
fact that ink was injected intramuscularly in the
foot muscles may account for this result.
The electron microscopic study focused on the
abnormalities in the gill, kidney and muscle tissue.
A constant feature was the presence of conspicuous
necrosis of gill cells and several electrolucent
areas of irregular size that did not have apparent
osmiophilic material and were not limited by a
membrane, which seem to correspond to India ink
in the gill, kidney, muscle, and intestinal cells.
Abnormalities in the gill and in the kidney
epitelial basement membrane were also observed.
These included the presence of areas of rarefaction
containing osmiophilic inclusions in a basement
membrane that was otherwise thickened or
multilayered. The significance of these basement
membrane abnormalities is unknown. However,
the suggestion has been made that they result from
the deposition of India ink (Cotran 1965).
This is the first electron microscopy study of
invertebrates treated with India ink. In vertebrates,
however, a few studies have been performed
that were based on electron microscopy. Cotran
(1965) studied endothelial cells of rats and mice
overloaded with India ink, and the vascular
endothelium was examined by electron microscopy
for evidence of phagocytosis. Phagocytosis of
carbon was demonstrated in the endothelium of
small myocardial vessels, the endocardium, the
pulmonary capillary endothelium, the aorta, and
the glomerular and peritubular capillary endothelia.
Some carbon remained in the endothelium of the
heart vessels for at least 7 days after overloading.
Carbon particles were also present in circulating
mononuclear cells and in perivascular phagocytes.

1987

Apoptosis is a process with typical

morphological signatures, including plasma
membrane blebbing, cell shrinkage, chromatin
condensation and fragmentation (Kerr et al. 1972,
Wyllie et al. 1980). A family of cystein-dependent
aspartate-directed proteases, called caspases, is
responsible for the proteolytic cleavage of cellular
proteins that contribute to the characteristic
apoptotic features, such as the cleavage of caspaseactivated DNase that results in internucleosomal
DNA fragmentation. Two pathways for activating
caspases have been studied in detail. One
pathway starts with ligation of a death ligand to
its transmembrane death receptor, followed by
recruitment and activation of caspases in the deathinducing signalling complex. The other pathway
involves the participation of mitochondria, which
release caspase-activating proteins into the cytosol,
thereby forming the apoptosome, where caspases
will bind and become activated.
It is well established that prolonged ER stress
can lead to cell apoptosis. Several novel pathways
have been identified that can offer explanations
on how cells trigger programmed cell death when
faced with irreparable damages that cannot be
rescued by the unfolded protein response. Despite
these important discoveries, the in vivo molecular
mechanisms underlying ER stress induced apoptosis
are just emerging. Furthermore, it is unclear whether
observations derived from specialized cell lines
reflect tissue-specific or general mechanisms and
whether results from in vitro reconstitution assay
systems apply to endogenous cellular mechanisms
(Li et al. 2006).
The efficient functioning of the ER is essential
for most cellular activities and for survival.
Conditions that interfere with ER function lead to the
accumulation and aggregation of unfolded proteins.
If the stress is prolonged, or the adaptive response
fails, apoptotic cell death ensues. In mammals and
humans, many studies have focused on how this
failure initiates apoptosis, as ER stress-induced
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Yuri B.M. Carvalho, Juan Jethro, Luis H. Poersch and Luis A. Romano

apoptosis is implicated in the pathophysiology

of several neurodegenerative and cardiovascular
diseases (Szegezdi et al. 2006). In this study,
mollusks treated with India ink showed apoptosis,
whereas those treated with a physiological solution
did not. The cause of apoptosis in this context is
still unknown; however, India ink accumulation
within the cells may be related to ER stress.
Bordem (2012) showed ultrastructural changes
of ER stress, such as thickening of the membrane
of the endoplasmic reticulum, dilated cisterns,
ribosome dissociation and electron-subjects
grouping the outside of the membrane. Some of
these findings were observed herein, in cells studied
with electron microscopy.
Previous studies have shown that bivalve
hemocytes synthesize and secrete catecholamines
including noradrenaline and dopamine (Ottaviani
and Franceschi 1996). However, noradrenaline
has the capacity of inducing apoptosis of oyster
hemocytes (Lacoste et al. 2002) and norepinephrine
induces endoplasmic ER stress in PC12 cells.
These three factors (apoptosis, norepinephrine and
ER stress) could be related with a foreign body in
mollusks.
CONCLUSION

In conclusion, India ink proved to be a useful tool for

experimental model for inflammation in mollusks.
Lastly, note that India ink iduced apoptosis, and
the cause of this phenomenon is still unknown and
the association of apoptosis and ER in mollusks
requires further studies.
ACKNOWLEDGMENTS

L.A. Romano and Luis H. Poersch received

productivity research fellowships from the Con
selho Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq).
An Acad Bras Cienc (2015) 87 (4)

RESUMO

Este artigo reporta o processo inflamatrio agudo e

celular no marisco branco Mesodesma mactroides,
induzido atravs de injeo de tinta nanquim no p
muscular. Obervaes histolgicas com microscopia
ptica e eletrnica foram realizadas 24 e 48 h aps
a injeo. A resposta celular inflamatria induzida
consistiu de infiltrao hemocitria geral sem ocorrncia
de necrose e atividade apopttica. Migrao de fagcitos
carregados de tinta nanquim atravs do epitlio
intestinal foi registrada. Parece que o marisco branco
excretou partculas de nanquim pelas brnquias e rim. A
tino positiva para apoptose foi observada na glndula
digestiva. Microscopia eletrnica da glndula digestiva
revelou mudanas ultraestruturais de estresse do retculo
endoplasmtico e corpos apoptticos. O mecanismo pelo
qual as partculas de tinta nanquim induzem apoptose
continua desconhecido, possivelmente associado ao
estresse do retculo endoplasmtico. Este trabalho
destacou caractersticas que exigem discusses na rea
restrita das respostas inflamatrias de moluscos.
Palavras-chave: tinta nanquim, processo inflamatrio,
estresse do retculo endoplasmtico, apoptose, Mesodesma
mactroides.
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