Protistology 12 (1), 3–11 (2018) Protistology

Trophic strategies in dinoflagellates: how nutrients

pass through the amphiesma

Vera Kalinina, Olga Matantseva, Mariia Berdieva and

Sergei Skarlato

Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia

| Submitted January 25, 2018 | Accepted February 20, 2018 |

Summary

Dinoflagellates, the protists of high ecological relevance, possess a very complex cell
covering, the amphiesma. In this article, we review the available information about
the structure and role of the amphiesma and discuss how nutrients overpass this
barrier, focusing on membrane transport, micropinocytosis and receptor-mediated
endocytosis. The hypothesized role of the pusule, a unique membrane organelle with
unknown functions, in dinoflagellate nutrition is discussed.

Key words: amphiesma, dinoflagellates, endocytosis, membrane transporters, nutri-

ent uptake, pusule

Introduction food webs. Nearly half of the dinoflagellate species

are obligate heterotrophs lacking chloroplasts, and
Dinoflagellates are unicellular eukaryotic many phototrophic chloroplast-containing species
organisms playing a crucial role in marine ecosystems are in fact mixotrophs which can use dissolved
as one of the main groups of primary producers. They organic compounds (osmotrophy) and/or prey
are represented mainly by free-living planktonic cells (phagotrophy) as additional nutrient sources
forms, but some of them belong to benthic, parasitic (Stoecker, 1999; Jeong et al., 2005; Matantseva
or symbiotic species (Gómez, 2012). For example, and Skarlato, 2013; Pechkovskaya et al., 2017).
members of the genus Symbiodinium are widely Diverse trophic strategies of dinoflagellates are often
known for their endosymbiotic relationships with considered as a key factor defining their success in
various invertebrates, including reef-building corals marine habitats. At the same time, nutrition of these
(Baker, 2003). Dinoflagellates are often the causative protists is not sufficiently studied from the standpoint
organisms of harmful algae blooms, or red tides of cell biology. Little information has been acquired
(Richlen et al., 2010; Glibert et al., 2012; Telesh since the time the explicit review by Schnepf and
et al., 2016; Skarlato and Telesh, 2017). Some of Elbrächter (1992) was published. Here we provide
them produce potent toxins which can be a reason of a review of the available information about cellular
human poisoning if accumulated in fish and shellfish and molecular aspects of dinoflagellate nutrition,
(Wang, 2008). Despite the ecological significance i.e. direct nutrient transport into a cell mediated
of dinoflagellates as primary producers, they are by membrane proteins, evidences for receptor-
also important as a heterotrophic component of mediated endocytosis and micropinocytosis, and

doi:10.21685/1680-0826-2018-12-1-1
© 2018 The Author(s)
Protistology © 2018 Protozoological Society Affiliated with RAS
4 · Vera Kalinina, Olga Matantseva, Mariia Berdieva and Sergei Skarlato

consider how these processes can be reconciled with This hypothesis is supported by the fact that ecdysis,
the complex cell covering of these organisms. an initial process of amphiesmal rearrangement, is
Ca2+-dependent (Tsim et al., 1997; Berdieva et al.,
AMPHIESMA AND THE ROLE OF AMPHIESMAL VESICLES 2018). In the process of ecdysis, amphiesmal vesicles
fuse with each other and a cell loses motility. Then
Dinoflagellates along with ciliates and apicom- a cell sheds its plasma membrane, thecal plates and
plexans belong to the clade Alveolata and are the outer amphiesmal vesicle membrane, while the
characterized by a similar structure of the cell inner amphiesmal vesicle membrane becomes a
covering. In general, the cell covering of these new plasma membrane (Pozdnyakov and Skarlato,
protists consists of a continuous plasma membrane 2012). Ecdysis occurs during the life cycle of
and flattened single-membrane vesicles (alveoli) many dinoflagellates and can be induced by stress,
localized underneath. These vesicles are called an e.g. mechanical perturbation (centrifugation) or
inner membrane complex (IMC) in apicomplexans application of chemical agents, such as a cellulose
or alveolar sacs in ciliates. In dinoflagellates, the synthesis inhibitor, 2,6-dichlorobenzonitrile (Mor-
entire cell covering, including a plasma membrane rill, 1984; Morrill and Loeblich, 1984; Sekida et
and alveoli, is termed amphiesma, and alveoli – al., 2001; Pozdnyakov et al., 2014). The new cell
amphiesmal vesicles, or sacs. Based on morphology covering emerges in a relatively short time. The
of the cell covering, dinoflagellates are separated into appearance of juvenile amphiesmal vesicles is
two groups: armored (thecate) and naked (athecate). observed 15 min after ecdysis in Scrippsiella hexa-
Amphiesmal vesicles of armored species contain praecingula (Sekida et al., 2001) and 75 min – in
rigid thecal plates built of cellulosic material, while Heterocapsa niei (Morrill, 1984). In 2 hours, a cell
amphiesmal vesicles of naked species lack them. usually restores motility.
Recently, it was shown that armored dinoflagellates Robust experimental evidences for amphiesmal
have a monophyletic origin and had developed from vesicles operating as intracellular stores of certain
an athecate ancestor (Orr et al., 2012). In some chemical compounds are absent; nevertheless, there
taxa, amphiesmal vesicles contain fibrous layer are some indirect observations corroborating this
(pellicle) involved in the rearrangement of the cell assumption. In the study of the ultrastructure of the
covering and/or can be underlined by microtubules naked dinoflagellate Prosoaulax lacustris, Calado
(Pozdnyakov and Skarlato, 2012). and colleagues (1998) observed cytoplasmic vesicles
The role of amphiesmal vesicles as a structure fusing with (and probably releasing their filling into)
providing rigidity to the cell covering is obvious. In the amphiesmal vesicles. However, the nature of this
addition, alveoli may be involved in cell signaling, filling was not determined.
membrane trafficking and storage of various The presence of amphiesma must complicate
ions and molecules, but these functions have not nutrient transport into a cell, because cytoplasm
been confirmed experimentally in the case of is separated from the plasma membrane by two
dinoflagellates. However, there is some information additional membranes in the major part of the cell
surface. Thus, to reach cytoplasm, nutrients have to
concerning functioning of homologous membrane
cross three membranes, as well as thecal plates in the
compartments in ciliates.
case of armored species.
The cortical sacs of ciliates function as a large
Ca2+ store resembling terminal parts of sarcoplasmic MEMBRANE TRANSPORT OF DISSOLVED NUTRIENTS
reticulum in myocytes (Stelly et al., 1991). Calcium
mobilization from the cortical sacs triggers the Nutrient transport across the plasma membrane
store-operated Ca2+ entry and subsequent cellular can be active or passive. Active transport is carried
responses, e.g. trichocyst exocytosis. Indeed, out in the direction against the gradient of chemical
a composition of proteins of the sarcoplasmic or electrochemical potential and requires the energy
reticulum involved in Ca2+ regulation is similar input, while passive transport involves movement
to that in alveolar sacs of ciliates. For instance, of substances along these gradients. There is a great
alveolar sacs contain the sarcoplasmic reticulum diversity of specific transporters, ion channels and
Ca2+-ATPase (SERCA) and Ins(1,4,5)P3 receptor porins participating in the nutrient transport in
(Plattner, 2014). living organisms (Chrispeels et al., 1999; Saier,
Similar to alveolar sacs of ciliates, amphiesmal 2000; Schubert et al., 2017). For instance, in plants,
vesicles of dinoflagellates may function as Ca2+ stores. nitrate transporters NRT2 and NPF, ammonium
 Protistology · 5

transporter AMT and urea transporter DUR3 play a vesicles also lie very close to each other (Dodge
crucial role in the nitrogen assimilation. In addition, and Crawford, 1970). Alternatively, the same sets
aquaporins of the NIP (nodulin 26-like intrinsic of transporters could be present not only on the
protein) and PIP (plasma membrane intrinsic plasma membrane, but also on the outer and inner
proteins) subfamilies can facilitate uptake of some membranes of the amphiesmal vesicles. It is assumed
nutrients, such as urea (Gaspar et al., 2003; Wallace that thecal plates do not represent a serious obstacle
and Roberts, 2005). for small molecules and ions, because they bear
Little is known about the composition of plasma thecal pores (Klut et al., 1989; Hoppenrath and
membrane proteins responsible for the nutrient Leander, 2008). According to TEM images, there
transport in dinoflagellates. Overall, these protists is a possibility, that the outer and inner amphiesmal
are likely to express an extremely wide range of membranes fuse in the thecal pores forming
proteins involved in the transport of micro- and membrane pipes or veins (Morrill and Loeblich,
macronutrients into a cell, especially considering 1983). Thus, a thin cytoplasmic layer between the
their mixotrophic lifestyle and remarkable ability plasma membrane and outer amphiesmal vesicle
to use different inorganic and organic nutrient membrane is linked with central cytoplasm, and
sources (Lee, 2008; Zhao et al., 2017). Nevertheless, nutrients can easily pass through (Spector, 1984).
specific data on the spectrum of such proteins are However, it is commonly observed that the pores are
still scarce. Most of the information on this account often locked by tricho- and mucocysts (Hoppenrath
was obtained by the analysis of genomic and et al., 2013). In addition, it is reasonable to suggest
transcriptomic data by means of bioinformatics. that membrane transporters are localized mainly in
Several transporters involved in the uptake of the regions that lack amphiesmal vesicles: flagellar
nitrogen, such as nitrate transporters of the fami- canal and the pusule (Fig. 1).
lies NRT2 and NPF, have been found in the As mentioned above, most dinoflagellates are
transcriptomes of Prorocentrum minimum, Karenia heterotrophs or mixotrophs and can utilize organic
brevis, Lingulodinium polyedrum, Alexandrium ta- substances as a nutrient source. Small molecules,
marense, Symbiodinium sp.; moreover, ammonium such as urea and free dissolved amino acids, are
transporters AMT have been identified in K. brevis transported by the membrane proteins. But many
and Amphidinium carterae, and urea transporters molecules are too big for that and thus can be
DUR3, as well as aquaporins MIP – in P. minimum captured only by endocytosis, in particular, by
(Morey et al., 2011; Dagenais Bellefeuille and receptor-mediated endocytosis or micropinocytosis.
Morse, 2016; Matantseva et al., 2016; Lauritano et
al., 2017; Pechkovskaya et al., 2017). Experimental WHERE ENDOCYTOSIS IS POSSIBLE?
data are even more limited. It has been shown that
the expression of NRT2.1 protein in L. polyedrum Cell covering of Alveolata is complex and,
does not depend on the nitrogen source and time therefore, all three alveolate groups (Apicomplexa,
of the light/dark cycle (Dagenais Bellefeuille and Ciliata and Dinoflagellata) have evolved permanent
Morse, 2016). cytostomes and/or fixed endocytic sites. In sporo-
However, the question of cellular localization zoa, a big group of apicomplexan parasites, these
of the nutrient transport systems remains open. If sites are called micropores and represent tiny
they are localized only on the plasma membrane, plasma membrane invaginations in the IMC
nutrients still have to overcome the barrier of the openings. The number of micropores can vary
amphiesmal vesicles to reach cytoplasm. This can depending on the life cycle stage. In some species
be achieved in several potential ways, some of there is only one endocytic site, while there are
which were discussed by Schnepf and Elbrächter two or more micropores scattered sparsely on
(1992) and shown in figure 1. First, nutrients can the cell surface in the others (Scholtyseck and
pass through the sutures between the amphiesmal Mehlhorn, 1970). Interestingly, a micropore-like
vesicles. However, according to transmission structure was observed in the parasitic dinoflagellate
electron microscopy (TEM) observations, in Hematodinium sp. (Appleton and Vickerman, 1996).
armored dinoflagellates these sutures appear too Most ciliates, for example Paramecium and
tight leaving no space even for small molecules to Tetrahymena, have a permanent cytostom by which
pass (Dodge and Crawford, 1970; Hoppenrath and the food particles are engulfed. In addition, these
Leander, 2008). In naked species, the amphiesmal organisms are capable of clathrin- and dynamin-
6 · Vera Kalinina, Olga Matantseva, Mariia Berdieva and Sergei Skarlato

Fig. 1. Schematic representation of the possible endocytosis sites and hypothetical localization of membrane
transporters in naked and armored dinoflagellates.

mediated (receptor-mediated) endocytosis (Wiejak amphiesmal vesicles were detected in Amphidinium

et al., 2004; Elde et al., 2005). The process is carterae and Prosoaulax lacustris (Klut et al., 1989;
characterized by the appearance of tiny pits coated Calado et al., 1998). Some of these openings served
with clathrin on the cytoplasmic surface. These as the sites of trichocyst extrusion and the other
structures are often termed collared pits due to the represented plasma membrane invaginations ending
presence of the electron dense protein complex with collared pits, which means that in these species
around the pit neck. In ciliates, numerous clathrin- endocytosis may potentially occur not only in the
coated collared pits are observed on the bottom of region of flagellar canal. In addition, it was shown
the plasma membrane sockets, termed parasomal that some cytoplasmic vesicles fused with the plasma
sacs. Apparently, the parasomal sacs also play a role membrane and presumably released their content via
of the docking sites for recycling endosomes (Allen the pores between amphiesmal vesicles. However, it
and Fok, 2000). was noted that collared pits and fusing vesicles were
According to the transcriptomic data, the rather rare than usual on the cell surface (Calado et
clathrin-dependent endocytic pathway may be al., 1998).
also relevant for the dinoflagellate nutrition. In
particular, this assumption was made in the study THE PUSULE
of Alexandrium catenella (Zhang et al., 2014).
However, there is only one site of active endocytosis, The pusule is a cellular organelle which is only
which is the flagellar canal, where collared pits are found in dinoflagellates. In general, it represents
commonly observed in dinoflagellates. Collared pits a system of membrane vesicles, tubules and sacs
in the region of flagellar canal have been described connected with the flagellar canal by a permanent
in both naked and armored species, for example, opening. A similar structure was found in Cystodini-
in Amphidiniuin rhynchocephalum (Farmer and um bataviense, the dinoflagellate lacking flagella,
Roberts, 1989), Gymnodinium nolleri (Ellegaard and but no direct contact with external environment
Moestrup, 1999), Woloszynskia limnetica (Roberts was observed (Timpano and Pfiester, 1985). The
et al., 1995) and Ceratium furcoides (Roberts, 1989). pusule is usually surrounded by the vacuolar
In naked diniflagellates, collared pits can also be system and mitochondria. Dodge (1972) deve-
found on the cell surface. A number of pores between loped a classification of pusules based on TEM
 Protistology · 7

Table 1. Classification of pusules according to J.D. Dodge (1972).

Type of pusule Subtype Organisms

a) Simple type: vesicles Marine organisms: Amphidinium carterae, A. rhynchocephalum, the
opening directly into flagellar symbiotic stage of A. klebsii, Akashiwo sanguinea
canal
b) Pusule with collecting Marine organisms: Amphidinium herdmanii, Warnowia pulchra,
chamber which branches
Pusule with vesicles Karlodinium veneficum
from the flagellar canal
c) Pusule with internal Freshwater organism: Gymnodinium australe
collecting chamber
d) Complex tubular pusule Freshwater organism: Woloszynskia coronata
with vesicles
Marine and freshwater organisms: Lingulodinium polyedra, Tripos furca,
a) Simple tubular pusule Borghiella tenuissima, Ceratium furcoides, Prosoaulax lacustris (Calado
et al., 1998).
Pusules constructed Marine organisms: Kryptoperidinium foliaceum, Scrippsiella acuminata,
of tubules or sack b) Tubular pusule with Peridiniurn depressum, Heterocapsa rotundata, Cachonina hallii,
only invaginations Heterocapsa triquetra
Marine organisms: Prorocentrum balticum, P. minimum, P. nanum, P.
c) Sack pusule (Fig. 2) micans, P. dentatum, Peridinium cinctum (Calado et al., 1999).
Aureodinium pigmentosum, Alexandrium tamarense, Goniodoma
pseudogoniaulax, Oxyrrhis marina, Katodinium glandulum,
No pusule Crypthecodinium cohnii, Symbiodinium microadriaticum, Amphidinium
chattonii.

microphotographs of 40 dinoflagellate species from material are often observed in the lumen of flagellar
both marine and freshwater environments. He canal and the pusule, allowing to assume that big
specified two main groups: (1) pusules consisting nondigested particles may also be excreted via this
of vesicles or a collecting chamber, surrounded by region (Klut et al., 1987; Calado et al., 1999). In
vesicles, and (2) tubular or sack puseles (Table 1). addition, Loeblich and colleagues suggested that the
As an example, ultrastructure of the sack pusule of pusule might be implicated in mucilage excretion in
P. minimum is shown in Fig. 2. Prorocentrum sp. (Loeblich et al., 1979).
The function of the pusule is not completely There are the alternative hypotheses of the
understood. Table 2 summarizes the knowledge pusule function. A curious idea was proposed by
available to date. A widespread hypothesis sug- Morrill and Loeblich who inferred that the pusule
gests that the pusule plays an important role in might be a membrane source during the cell division
osmoregulation. This organelle can shrink and (Morrill and Loeblich, 1984). Since rebuilding of
swell thus resembling a contractile vacuole (CV) of the cell covering after ecdysis requires comparable
freshwater protists, such as ciliates and amoebas. amount of new membrane material, the pusule can
However, there is no experimental proof of this support this process as well.
suggestion so far. Dodge (1972) noticed that the most Furthermore, the pusule is likely to be involved
complex pusular system observed in dinoflagellates in the dinoflagellate nutrition. First, the region
was found in the freshwater species Woloszynskia where phagocytosis and uptake of big molecules
coronate, but this organelle is well developed in takes place is restricted to the sulcus and flagellar
most of the marine species too. Interestingly, the canal. Some dinoflagellates have evolved special
increased salinity (28-34 ppt NaCl) leads to the feeding organelles, such as peduncle, lobopodia, and
enlargement of the pusule in Prorocentrum micans pallium (Schnepf and Elbrächter, 1992). Second,
(Klut et al., 1987), suggesting that this organelle it is probable that the pusule also plays a role in
rather functions as a cellular “kidney” excreting the nutrient uptake being an important site of the
the excess of salt and waste molecules, but not the endocytosis and membrane transporter localization.
excess of water as it happens in the case of CV. The Klut and colleagues (1987) treated P. micans and
size of the pusule also increases with increasing A. carterae with ferritin or horseradish peroxidase
temperature, but it does not depend on pH in the (HRP). They have shown that these molecules are
range from 6.0 to 8.0 or presence/absence of light accumulated in the pusule and flagellar canal of
(Klut et al., 1987). Interestingly, disorganized both species. Vacuolar canaliculi, as well as some
bacteria, membranes, amorphous and fibrillar vesicles surrounding the pusule, also contained HRP
8 · Vera Kalinina, Olga Matantseva, Mariia Berdieva and Sergei Skarlato

Fig. 2. The sack pusule of Prorocentrum minimum. A – Transverse section of the sack pusule and two flagella;
B – a collapsed sack pusule. Abbreviations: ch - chloroplast, fl1, fl2 - longitudinal and transverse flagellum,
fv - fibrous vesicle, g - Golgi apparatus, mt - mitochondrion, pu - pusule. Scale bars: 1 µm.

reaction products and ferritin. nutrients. Future investigations should provide the
Soyer and Prevot (1981) poisoned P. micans answers to the following topical questions: (1) how
with cadmium chloride and observed that the the systems are responsible for the nutrient uptake
most affected cellular organelles are mitochondria distributed in the amphiesmal membranes, and (2)
located near the sack pusule. They assumed that do flagellar canal and the pusule that are free of
the molecules and ions from the environment, amphiesmal vesicles represent major regions of the
including Cd2+, are captured by the pusule; there- uptake and engulfment of various nutrient sources.
fore, mitochondria in the proximity of the pusule
are affected in the first place. However, taking
into account the previous hypothesis, damaged Acknowledgments
organelles could be transported there for subsequent
excretion. The research was funded by the Russian Science
Foundation, project 16-14-10116.

Concluding remarks
References
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 Protistology · 9

Table 2. Possible functions of the pusule.

Experimental
Function Pros Cons evidence
Osmoregulation 1) Changes the volume (Cachon et al., 1983) 1) Present in marine species
(contractile vacuole 2) The most complex pusule system is in the (Dodge, 1972) no
style) freshwater dinoflagellate Woloszynskia coronate 2) No regular contraction
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3) Mitochondria positioned close to a pusule are yes
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Mucilage excretion Appearance of fibrillar material in the lumen of a no
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Ingestion of molecules 1) Uptake of horseradish peroxidase, cationized
and ions entry ferritin, and lectins via the flagellar canal and the
pusules was shown by TEM in P. micans and A.
carterae (Klut et al., 1987). yes
2) Mitochondria positioned close to a pusule are
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1981)
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Membrane source for Hypothesized (Morrill and Loeblich, 1984)
cell division no

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Address for correspondence: Vera Kalinina. Institute of Cytology, Russian Academy of Sciences Tikhoretsky
Avenue 4, St. Petersburg 194064, Russia; e-mail: [email protected]