ISSN 1990519X, Cell and Tissue Biology, 2013, Vol. 7, No. 5, pp. 458–464. © Pleiades Publishing, Ltd., 2013. Original Russian Text © E.V. Raikova, 2013, published in Tsitologiya, 2013, Vol. 55, No. 6, pp. 365–371.
The Nervous System of Parasitic Cnidarian Polypodium hydriforme E. V. Raikova Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia email:
[email protected] Received February 21, 2013
Abstract—The nervous system of intracellular parasitic cnidarian Polypodium hydriforme at various stages of its life cycle has been studied by the immunocytochemical method using antibodies to FMRFamide and by electron microscopy. Neurosecretory, sensory, and ganglion cells have been identified both at the parasitic stage (planula and stolon stages, when body layers are inverted) and in freeliving animals. These cells are characterized by the presence of round neurosecretory granules about 80–120 nm in diameter. Gap junctions have been detected between nerve cells. Most of the neurosecretory and sensory cells have been observed in the epidermis of sensory tentacles of freeliving animals. Sensory cells possess immobile flagella. The chains of ganglion cells are located under the epidermis and penetrate mesoglea. A centriole encircled by a fragment of nuclear envelope, which is a marker of ectodermal lineage cells in Polypodium, has been described in the cytoplasm of the sensory cells, thus proving the ectodermal nature of the nervous system. Like in most cni darians, the nervous system of Polypodium hydriforme is a network containing FMRFamidelike neuropep tides. Neither sense organs, nor ringshaped nerve concentrations have been observed. Keywords: Polypodium hydriforme, cnidarians, nerve cells, neurosecretory granules, FMRFamide, confocal microscopy, electron microscopy DOI: 10.1134/S1990519X13050118
Cnidarians are diploblastic animals possessing stinging cells known as cnidocytes. Their nervous sys tem is under intense study because it first appeared in evolution in these animals. Such topics as the origin of the cnidarian nervous system (Watanabe et al., 2009), its development at various stages of the life cycle, sense organs (Nakanishi et al., 2008), nerve ring formation (Koizumi, 2007), neuromediators (Grimmelikhuijzen et al., 1991), and the morphology of neurons (Westfall, 2004) are currently being widely discussed. Polypodium hydriforme is the only intracellular par asite among cnidarians. Most of its life cycle (several years) is spent in the developing oocytes of sturgeons; its free life in the fresh water after being released from infected eggs lasts probably only one summer season (Raikova, 1994). The embryonic stages, planula and stolon occur inside oocytes. Freeliving animals have 12 or 24 tentacles in two lateral groups on the sides of a sacshaped body. The tentacles are of two types: sen sory tentacles catch prey, while supporting tentacles are used for locomotion. The mouth is located on top of the body. Sense organs have not been revealed. The Polypodium hydriforme nervous system was first described by Lipin (Lipin, 1911) as a nerve net work. He carried out studies on macerated cells stained with methylen blue at the stage of parasitic sto lon when the cell layers are inverted; i.e., the entoderm is outside and faces the egg yolk, whereas the ectoderm is inside. Lipin believed that the nervous system of
inverted Polypodium had an entodermal origin, while other uninverted cnidarians had an ectodermal ner vous system. Lipin also described bi and tripolar neu rons and demonstrated their junction with muscle cells. However, he did not reveal sensory cells (Lipin, 1911). There are numerous data on cytological character istics of various Polypodium cells in both body layers at the sequential stages of the life cycle (Raikova, 2008). Preliminary data on its nerve cells obtained by modern methods have been reported at conferences (Raikova and Napara, 1997; Napara, 1997; Raikova et al., 2003), but no papers have been published on the sub ject so far. The morphological features of Polypodium hydri forme taken together make it possible to consider it as a separate cnidarian class (Raikova, 1973, 1988, 2005; Bouillon and Boero, 2000). Thereby, the study of its nervous system, particularly the question of whether it differs from the nervous system of freeliving cnidari ans, is of special interest. The aim of this paper is to summarize the data on the localization and cytomor phology of ganglion, neurosecretory, and sensory cells at the sequential stages of the life cycle of this intracel lular parasite.
458
THE NERVOUS SYSTEM OF PARASITIC CNIDARIAN Polypodium hydriforme
MATERIALS AND METHODS Materials were sampled in different fisheries on the Volga River in Astrakhan district. Parasitic stages (obtained mostly from sterlet oocytes) and freeliving specimens of Polypodium (grown from infected oocytes in Petri dishes) (Raikova, 1994) have been used. Electron microscopy. Samples were fixed with 2.5% glutaraldehyde and postfixed with 1% osmium tetrox ide in cacodylate or veronalacetate buffer, dehydrated with acetone and embedded into epon/araldide mix ture or Spurr. Sections were visualized under JEM 100C electron microscope (Jeol, Japan). To localize FMRFamide by indirect fluorescence (Coons et al., 1955) samples were fixed with Stefanini solution (2% paraformaldehyde, 15% picric acid in 0.1 M Naphosphate buffer, pH 7.6). Animals were stored in the fixative for several weeks, then incubated for 24–48 h in 0.1 M Naphosphate buffer with 20% sucrose, washed three times for 5 min by PBS with 0.2% Triton X100 (PBST) (Sigma, United States), and incubated with primary antibodies (rabbit anti bodies to FMRFamide, 1 : 400) (Incstar Corp., United States) for 48–72 h at 10°C with shaking. Then, samples were washed three times for 5 min with PBST and incubated for 1–2 h with FITClabeled secondary antibodies, 1 : 30 (Dako Inc., Denmark). After washing with PBST, the animals were mounted in glycerol with PBS (2 : 1) and examined with a CLSM LEICA TCS 4D confocal scanning laser microscope (Leica Mycrosystems, United States). The optical section set was used to obtain projections with maximal intensity for all serial sections and three dimensional reconstructions. RESULTS It is difficult to identify nerve cells under a light microscope; therefore, most data in this work were obtained by electron and confocal scanning micros copy. Three types of nerve cells were distinguished at sequential stages of Polypodium hydriforme life cycle: ganglion, neurosecretory, and sensory cells. The earliest lifecycle stage in which individual nerve cells are identifiable is parasitic planula. Nerve cells appear in the ectoderm and are reliably distin guished by the presence of 80 to 120nm dense cored vesicles in the cytoplasm. Later, at the stolon stage, nerve cells form a separate layer located under the ectoderm (Fig. 1a). These are ganglion cells. Neuro secretory and sensory cells are abundant in the stolon ectoderm and in the epidermis of freeliving animals. Most of them are encountered in sensory tentacles; fewer are visible in the mouth area and in supporting tentacles. No nerve cells are observed in the sac shaped body of freeliving polypodia on its aboral sur face. Individual nerve cells are revealed in the ento CELL AND TISSUE BIOLOGY
Vol. 7
No. 5
2013
459
derm. In tentacles, a nervous network was observed in the mesoglea beneath the muscle cells (Napara, 1997). Ganglion cells are elongated and they look like bi and tripolar neurons (Figs. 3a, 3b). These cells were described in detail by Lipin (Lipin, 1911). Under an electron microscope, they are recognized by a light cytoplasm with dense cored vesicles (Figs. 1a–1c) and microtubules. Gap junctions are visible between gan glion cells (Fig. 1c). Sensory and neurosecretory cells usually have a spindlelike shape and are easily visualized by confocal microscopy (Figs. 3c, 3d). Under an electron micro scope, they are visible close to cnidoblasts and recog nized by the presence of dense cored vesicles (Fig. 2b). The latter usually have equal size in each cell; their diameter ranges from 80 to 120 nm. The vesicle mem brane is not tightly adjacent to the core, therefore, an obvious “halo” is evident (Figs. 1b, 1c; Fig. 2b). Sen sory cells reach the epidermis surface but lack the acid mucopolysaccharide granules common for the epider mal cells (Figs. 2a–2d). Tight (septate) junctions bind them with neighboring cells (Figs. 2a, 2d). The nucleus is located close to the basal part of the cell (Fig. 2c). The cytoplasm has a Golgi complex, mitochondria, and microtubules (Fig. 2a). Structures resembling synaptic vesicles that have dense content or are empty are occasionally visible. Usually they are observed in contact with cnidoblasts. The cytoplasm of the nerve cells contains a centriole surrounded by fragments of the nuclear envelope (Fig. 2c). Sensory cells have sensitive filaments that are similar to fla gella, but immobile (Figs. 2c, 2d). The flagellum is located inside a pocketlike depression of the cyto plasm. It is surrounded by a sheath consisting of regu larly arranged parallel fibrils. The cytoplasm contains the daughter centriole. The cells form tight junctions with adjacent cells. Immunofluorescence with antibodies to FMRF amide reveals chains of nerve cells forming a network with strands going in longitudinal and transversal directions, entering mesoglea and reaching muscle cells (Figs. 3a, 3b). The nerve chains are most evident under ectoderm in the parasitic stolon and under the epidermis in the tentacles of freeliving polypodia (Fig. 3d). Tentacles have numerous spindlelike cells with granular content (Figs. 3c, 3d) as well as bi and tripolar neurons (Figs. 3a, 3b). Spindle cells showing a positive reaction with FMRFamide antibodies and characteristic localization in the tentacles are identified as sensory cells, which correspond to neurosecretory cells identi fied by the electron microscopy. DISCUSSION Electron and confocal microscopy have expanded our knowledge of the nervous system of Polypodium hydriforme. Along with the “neurons” described by Lipin (Lipin, 1911), which are currently classified as
460
RAIKOVA
mt
dcv
dcv cp me
m
cn n n m (a)
(b)
epr
m
cn
gj
mt dcv dcv dcv
dcv
(c) Fig. 1. Nerve cells of Polypodium hydriforme, electron microscopy. (a) Neurosecretory cell (arrow) with dense cored vesicles in stolon ectoderm under cnidoblast, (b) fragment of a neurosecretory cell, and (c) fragments of a neurosecretory cell, two ganglion cells and cnidoblast. cn—cnidoblast, cp—capsular precursor, dcv— dense cored vesicles, epr—endoplasmic reticulum, gj—gap junction, m—mitochondrion, me—mesoglea, mt—microtubule, n—nucleus. (a) 26000×, (b) 33000×, and (c) 43000×.
ganglion cells, other nerve cell types (neurosecretory and sensory) have been revealed at various lifecycle stages. Our data confirm Lipin’s main observation that the Polypodium hydriforme nervous system is a nerve net
work (Figs. 3a, 3b). However, contrary to Lipin’s con clusion that there is an entodermal origin of the ner vous system, our results indicate its ectodermal origin, as in other cnidarians. Nerve cells first appear in the planula ectoderm. Moreover, they have a reliable cyto CELL AND TISSUE BIOLOGY
Vol. 7
No. 5
2013
THE NERVOUS SYSTEM OF PARASITIC CNIDARIAN Polypodium hydriforme
461
amps
m amps
gc
dcv
m n
m cn (a)
(b) ax
f
ax c
c
n sj
ac
(d)
(c)
Fig. 2. (a, c, d) Sensory and (b) neurosecretory cells of Polypodium hydriforme, electron microscopy. (a) Sensory cells in epithelium of parasitic stolon, arrows show septate junctions; (b) neurosecretory cell, arrows show neurosecre tory granules; (c) sensory cell with centriole inside pocket of nuclear envelope and flagellum fragment; and (d) sensory cell with flagellum fragment. ac—accessory centriole, amps—acid mucopolysaccharide granules, ax—axoneme of flagellum, c—centri ole in the nuclear membrane pocket, cn—cnidoblast, dcv—dense cored vesicles, f—fibrillar cortex around flagellum, gc—Golgi complex, m—mitochondrion, n—nucleus, sj—septate junction. (a) 8800×, (b) 21000×, (c) 26000×, and (d) 63000×.
logical marker, such as a centriole surrounded by nuclear membrane fragments, which is a common characteristic of all Polypodium cells derived from an CELL AND TISSUE BIOLOGY
Vol. 7
No. 5
2013
ectodermal germ layer (Raikova, 1984). In the ento derm, nerve cells occur rarely. As in many cnidarians, Polypodium nerve cells are located subepidermally. As,
462
RAIKOVA
cc
tpn
tpn
bn
mc mc ta (a)
(b)
s
nch s
(c)
(d)
Fig. 3. Localization of FMRFpositive cells in sensory tentacles of Polypodium hydriforme. Maximal projections of several optical sections from the confocal microscope. (a) Tripolar neuron crossing the tentacle against the background of muscle cells, (b)nerve network, (c, d) sensory cells and sub epidermal nerve chain in the tentacle anlage, and (d) magnified lower left part of Fig. 3b. bn—bipolar neuron, cc—cnidocyte, mc—muscle cells, nch—nerve cell chain, s—sensory cell, ta—tentacle axis, tpn—threepolar neuron. (a) 450×, (b) 290×, (c) 300×, and (d) 720×.
in Polypodium muscle cells are separated from epithe lial cells and are also located in mesoglea under the epidermis (Lipin, 1911; Raikova and Napara, 1999; Raikova et al., 2007), it is not surprising that nerve muscle contacts are observed here. We have also demonstrated that Polypodium nerve cells contain neuropeptides reacting with antibodies to FMRFamide. This characteristic, as well as the pres ence of a diffuse nerve net (Grimmelikhuijzen et al., 1991), supports the inclusion of Polypodium into Cni daria. However, the methods we used are insufficient to discriminate neuropeptides in ganglion and sensory cells, as well as neuropeptide expression in ecto and
entodermal cells. These differences are described, for example, in Hydra (Koizumi et al., 2004) due to appli cation of antibodies to other neuropeptides. Sensory and neurosecretory cells in Polypodium are described here for the first time. Their main feature is the presence of neurosecretory granules containing FMRFamide like neuropeptide. These structures, densecored vesicles 70–150 nm in diameter, are common for representatives of all classes of Cnidaria (Grimmelikhuijzen et al., 1991; Westfall, 1987). The differences between sensory and neurosecretory cells in Polypodium are not clearly apparent. It is even pos sible that they are the same cells, but referred to by dif CELL AND TISSUE BIOLOGY
Vol. 7
No. 5
2013
THE NERVOUS SYSTEM OF PARASITIC CNIDARIAN Polypodium hydriforme
ferent names. The presence of a flagellum, which is common for sensory cells in freeliving cnidarians (Westfall, 2004), presumably distinguishes sensory cells from neurosecretory cells. However, the flagel lum could only be detected by electron microscopy. When observed by confocal microscopy, sensory and neurosecretory cells look similar. The absence of significant differences between sen sory and neurosecretory cells has also been noted in Hydra (Westfall, 1973; Westfall and Kinnamon, 1978). The authors explained this fact by the cells being mul tifunctional and primitive compared to specialized neurons in higher organized animals. Unfortunately, we failed to find typical synapses in Polypodium nerve cells. Gap junctions only indicate electric conductiv ity between Polypodium hydriforme cells. Neither a nerve ring in adult animals common for many medusas and some Hydra species (Koizumi, 2007), nor ganglion cell clusters in planula considered as a brain precursor (Piraino et al., 2003), nor sensory organs were revealed in Polypodium, probably due to its parasitic way of life. Whereas in freeliving cnidarians the total reorga nization of nervous system occurs during planula metamorphosis (Martin, l988; Nakanishi et al., 2008), in polypodia it does not take place, probably because the planula stage is never freeliving: it develops inside the infected oocyte after the embryo gastrulation, and there it continues its development, budding and trans forming into stolon (Raikova, 1980). The lack of plan ula nervous system reorganization is, presumably, the main difference in the functioning of the nervous sys tem during the life cycle of Polypodium hydriforme and freeliving cnidarians. As a whole, the Polypodium hydriforme nervous system is simpler than in most Cnidaria. As in other cnidarians, in Polypodium, sensory function is attributed to cnidocils of stinging cells numerous in the mouth cone and tentacles (Ibragimov and Raikova, 2004). Cindarian cnidocytes are cur rently considered as components of the nervous sys tem that have mechanosensory functions and as inde pendent effectors (MiljkovicLicina et al., 2004; Mar low et al., 2009; Watanabe et al., 2009). Morphologically, the cnidocytes of Polypodium hydri forme are more similar to mechanoreceptors than the stinging cells in other cnidarians (Raikova, 1990). However, this question is beyond the scope of this paper. ACKNOWLEDGMENTS I am kindly grateful to T.O. Kameneva for help dur ing material sampling and treatment, T.O. Napara for fruitful collaboration and sharing of some electron microscopy images (Figs. 2c, 2d), and O.I. Raikova for assistance with confocal microscope observations and the preparation of this article. CELL AND TISSUE BIOLOGY
Vol. 7
No. 5
2013
463
REFERENCES Bouillon, J. and Boero, F., The Hydrozoa: a new classifica tion in the light of old knowledge, Thalassia salientina, Uni versita degli Studi di Lecce, Dipartimento di biologia, Porto Cesario (Lecce), 2000, vol. 24, pp. 3–296. Coons, A.H., Leduc, E.H., and Connolly, J.M., Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of a hyperimmune rabbit, J. Exp. Med., 1955, vol. 102, pp. 49–60. Grimmelikhuijzen, C.J.P., Graff1, D., Koizumi, O., West fall, J.A., and McFarlan, I.D., Neuropeptides in coelenter ates: a review, Hydrobiologia, 1991, vols. 216–217, pp. 555– 563. Ibragimov, A. and Raikova, E., Nematocysts of Polypodium hydriforme, a cnidarian parasite of Acipenseriform fishes, Hydrobiologia, 2004, vol. 530–531, pp. 165–171. Koizumi, O., Nerve ring of the hypostome in hydra: is it an origin of central nervous system of bilaterian animals?, Brain Behav. Evol., 2007, vol. 69, pp. 151–159. Koizumi, O., Sato, N., and Goto, C., Chemical anatomy of hydra nervous system using antibodies against hydra neu ropeptides: a review, Hydrobiologia, 2004, vols. 530–531, pp. 41–47. Lipin, A.N., Die Morphologie und Biologie von Polypo dium hydriforme Uss., Zool. Jahrb. (Anat.), 1911, vol. 31, pp. 317–426. Marlow, H., Srivastava, M., Matus, D., Rokhsar, D., and Martindale, M., Anatomy and development of the nervous system of Nematostella vectensis, an anthozoan cnidarian, Dev. Neurobiol., 2009, vol. 69, pp. 235–254. Martin, V.J., Development of nerve cells in hydrozoan planula. II. Examination of sensory differentiation using electron microscopy and immunocytochemistry, Biol. Bull., 1988, vol. 175, pp. 65–78. MiljkovicLicina, M., Gauchat, D., and Galliot, B., Neu ronal evolution: analysis of regulatory genes in a first evolved nervous system, the hydra nervous system, Biosys tems, 2004, vol. 76, pp. 75–87. Nakanishi, N., Yuan, D., Jacobs, D., and Hartenstein, V., Early development, pattern, and reorganization of the plan ula nervous system in Aurelia (Cnidaria, Scyphozoa), Dev. Genes Evol., 2008, vol. 218, pp. 511–524. Napara, T.O., Unusual localization of the nerve net in the tentacles of Polypodium hydriforme, in Proc. 7th Int. Work shop on Hydroid Development, Evangelische Akademie Tutz ing, Germany, September 22 –25, 1997, 1997, p. 98. Piraino, S., Schmich, J., Schmid, V., and Boero, F., Further evidence of bilaterality in planula larvae, in Int. Workshop on Hydra and Evolution of Signalling Pathways, Evangelische Akademie Tutzing, Germany, September 15–18, 2003, 2003, p. 88. Raikova, E.V., Life Cycle and Systematic Position of Poly podium hydriforme Ussov (Coelenterata), a cnidarian para site of the eggs of Acipenseridae, Publ. Seto Mar. Biol. Lab., 1973, vol. 20, pp. 165–173. Raikova, E.V., Morphology, ultrastructure and develop ment of the parasitic larva and its surrounding trophamnion of Polypodium hydriforme Ussov (Coelenterata), Cell Tiss. Res., 1980, vol. 206, pp. 487–500.
464
RAIKOVA
Raikova, E.V., Ultrastructure of the stolon of Polypodium hydriforme Ussov (Coelenterata) parasitic in oocytes of Aci penserid fishes, Monitore Zool. Ital., 1984, vol. 18, pp. 1–24. Raikova, E.V., On the taxonomic position of Polypodium hydriforme Ussov (Cnidaria), in Gubki i knidarii. Sovremen noe sostoyanie i perspektivy issledovaniya (Sponges and Cni darians. Current Status and Prospects of Research), Lenin grad: Zool. Inst. Akad. Nauk SSSR, 1988, pp. 116–122. Raikova, E.V., Fine structure of the nematocytes of Polypo dium hydriforme Ussov (Cnidaria), Zool. Scripta, 1990, vol. 19, pp. 1–11. Raikova, E.V., Life cycle, cytology, and morphology of Polypodium hydriforme, a coelenterate parasite of the eggs of Acipenseriform fishes, J. Parasitol., 1994, vol. 80, pp. 1–22. Raikova, E.V., Cytomorphological characteristics of Poly podium hydriforme and problems of myxozoan and cnidar ian phylogeny, Tsitologiia, 2005, vol. 47, no. 10, pp. 933– 939. Raikova, E.V., Cytomorphological peculiarities of Polypo dium hydriforme (Cnidaria), J. Mar. Biol. Assoc., 2008, vol. 88, pp. 133–141. Raikova, E.V. and Napara, T.O., Organization of the ner vous system in the parasitic cnidarian Polypodium, in Proc. III Mezhdunar. Konf. stran SNG po funktsional’noy neyro morfologii, posvyaschennaya 100letiyu so dnya rozhdeniya chl.korr. AN i AMN SSSR N.G. Kolosova, 7–8 aprelya 1997 (III International Conference of CIS on Functional Neu romorphology Dedicated to the 100th Anniversary of the Birth of the Corresponding Member of the Academy of Sci ences and Academy of Medical Sciences of the USSR N.G. Kolosov, April 7–8, 1997), St. Petersburg, 1997, p. 5.
Raikova, E.V. and Napara, T.O., Ultrastructural study of muscle cells of the parasitic cnidarian Polypodium hydri forme, Tsitologiia, 1999, vol. 41, no. 5, pp. 425–431. Raikova, E.V., Raikova, O.I., Reuter, M., and Gustafs son, M.K.S., Nervous system in Polypodium hydriforme: immunocytochemical observations, Proc. X Int. Workshop “Hydra and the Evolution of Signalling Pathways,” Evange lische Akademie Tutzing, Germany, September 15–18, 2003, 2003, p. 94. Raikova, E.V., Ibragimov, A.Yu., and Raikova, O.I., Mus cular system of a peculiar parasitic cnidarian Polypodium hydriforme: a phalloidin fluorescence study, Tissue Cell, 2007, vol. 39, pp. 79–87. Watanabe, H.T., Fujisawa, T., and Holstein, Th., Cnidari ans and the evolutionary origin of the nervous system, Rev. Develop. Growth Differ., 2009, vol. 51, pp. 167–183. Westfall, J.A., and Kinnamon, J.C., A second sensorymo torinterneuron with neurosecretory granules in Hydra, J. Neurocytol., 1978, vol. 7, pp. 365–379. Westfall, J.A., Neural pathways and innervation of cni docytes in tentacles of sea anemones, Hydrobiologia, 2004, vols. 530–531, pp. 117–121. Westfall, J.A., Ultrastructural evidence for a granulecon taining sensorymotorinterneuron in Hydra littoralis, J. Ultrastruct. Res., 1973, vol. 42, pp. 268–282. Westfall, J.A., Ultrastructure of invertebrate synapses, in Nervous Systems in Invertebrates, New York: Plenum Press, 1987, pp. 3–28. Translated by I. Fridlyanskaya
CELL AND TISSUE BIOLOGY
Vol. 7
No. 5
2013
