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1979

The Parasitic Coelenterate, Polypodium hydriforme Ussov, from the Eggs of the American Acipenseri from Polyodon spathula

Glenn L. Hoffman US Fish and Wildlife Service

E. V. Raikova Institute of Cytology of the Academy of Sciences, Leningrad

V. Ch. Suppes Missouri Department of Conservation, Sweet Springs

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Hoffman, Glenn L.; Raikova, E. V.; and Suppes, V. Ch., "The Parasitic Coelenterate, Polypodium hydriforme Ussov, from the Eggs of the American Acipenseri from Polyodon spathula" (1979). US Fish & Wildlife Publications. 115. https://digitalcommons.unl.edu/usfwspubs/115

This Article is brought to you for free and open access by the US Fish & Wildlife Service at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in US Fish & Wildlife Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. The Journal of Parasitology, Vol. 65, No. 5 (Oct., 1979), pp. 804-810 J. Parasitol., 65(5), 1979, pp. 804-810 ? American Society of Parasitologists 1979

THE PARASITIC COELENTERATE, POLYPODIUM HYDRIFORME USSOV, FROM THE EGGS OF THE AMERICAN ACIPENSERIFORM POLYODON SPATHULA

E. V. Raikova,* V. Ch. Suppes,t and G. L. Hoffmant

ABSTRACT: No significant differences in macro- and micromorphology were found between the parasitic stolon and free-living polyps of Polypodium sp. obtained from infected eggs of the North American aci- penseriform fish Polyodon spathula and corresponding developmental stages of Polypodium hydriforme Ussov, parasitic in the Volga sterlet (Acipenser ruthenus). Therefore, both the American and the European forms of Polypodium belong to the species P. hydriforme Ussov.

For a century, the coelenterate Polypodium found in Romania (Bogatu, 1961) and in Iran hydriforme Ussov, parasitic in the eggs of (Nechat and Mokhayer, 1974). acipenserid fishes, was considered a "purely A discovery in 1970 of Polypodium on the Russian" animal. It was discovered in 1871 by American continent in the eggs of the stur- Ovsjannikov in the eggs of the Volga sterlet, geon Acipenser fulvescens inhabiting the (Acipenser ruthenus L.), and for more than 70 Great Lakes (Hoffman et al., 1974), was there- years thereafter, it appeared to be confined to fore a distinct surprise. Thereafter (in 1973), that host species and that river. Only after the Polypodium also was found in the eggs of sev- beginning of extensive ichthyoparasitological eral specimens of the American acipenseri- research in the USSR was it found in other form Polyodon spathula, a representative of hosts and other watersheds (Dogiel, 1940). the family Polyodontidae which belongs, with During the last 40 years, P. hydriforme has the family Acipenseridae, to the order Aci- been found in practically all rivers of the Eu- penseriformes (Suppes and Meyer, 1975). ropean part of the USSR inhabited by Acipen- Thus, the range of the hosts of Polypodium seridae, and also in the tributaries of the Aral has exceeded the Acipenseridae, and its area, Sea and in the Amur River of the Far East. All the Eurasian continent. We describe here the acipenserid species occurring in the USSR American form of Polypodium from Polyodon, except the Siberian sterlet (A. ruthenus ruth- compare it with the typical P. hydriforme, and enus natio marsiglii Brandt), the Siberian provide evidence that the American form is sturgeon (A. baeri Brandt), and the Atlantic probably the same species. sturgeon (A. sturio L.), proved to be hosts of MATERIALSAND METHODS P. hydriforme. It has been found in Acipenser ruthenus L. (the European sterlet), A. giil- Our material consisted of both Polypodium-in- denstiidti Brandt (the Russian sturgeon), A. fected and healthy eggs of Polyodon spathula, ob- stellatus Pallas A. nudiventris tained from 8 females caught in March, April, and (the sevrjuga), May 1973 and October 1974 in the Osage River, Lovetzky (the ship), A. schrenki Brandt (the Missouri. The infected eggs in 5 females caught in Amur sturgeon), Huso huso (L.) (the beluga), March and April belonged to the older generation and H. dauricus (Georgi) (the kaluga) (Dogiel, of oocytes. These were nearly mature and corre- 1940; Markov and Trussov, 1966; Svirsky, sponded to the Stage IV-V of Nedoshivin's (1928) scale. Two 22 and 1967). Outside the USSR, P. hydriforme was females, caught April May 11, were already in the post-spawning period and con- tained no Polypodium; one female, caught in Oc- tober 1974 and infected with Polypodium, was at Received for publication 1 November 1978. Stage IV of oocyte development (eggs due to be * Institute of Cytology of the Academy of Sciences, spawned next spring). The oocytes were fixed Leningrad 190121, USSR. either with 10% formalin or with Gilson's fluid. ? Missouri Department of Conservation, Sweet Some of the oocytes were infected with the micro- Springs, Missouri 65351, USA. sporidian Pleistophora sulci Rasin 1936, 1949 and in ad- $ Fish Farming Experimental Station, Stuttgart, (Sprague Vavra, 1977) (Cocconema sulci) Arkansas 72160, USA. dition to Polypodium.

804 RAIKOVAET AL.-PARASITIC COELENTERATEFROM EGGS OF POLYODON 805

Free-living polyps, raised from stolons infecting ly the zona radiata and the follicular enve- the were cultivated in Petri dishes and oocytes lope-grow thinner (Figs. 1, 2). fixed with 10% formalin at various ages (from their emergence out of the oocytes through a period of The entodermal cells (i.e., cells of the outer 40 days of free life). layer of the stolon) are flagellated, but the fla- For histological investigation, some formalin- gella appear poorly preserved after formalin fixed and infected eggs free-living polyps were re- fixation. At places lacking the enveloping cap- fixed with Zenker's and Bouin's fluids and embed- the entodermal cells ded in paraffin.Sections (7 thick) were stained sule, contain yolk parti- with Heidenhain's "azan," iron/ln hematoxylin, May- cles, which they apparently ingested (Fig. 4). er's hemalum, or by the Feulgen technique. For- Groups of protein-secreting glandular cells malin-fixed material was also used to prepare whole also occur in the entoderm. They are usually mounts of both stolon and which were polyps, in folds of the stolon (Fig. 5) and therefore mounted in glycerin-gelatin. contact neither the capsule nor the yolk. The ectodermal RESULTS (inner layer) cells (Fig. 6) have small nuclei located in their basal ends, Unambiguous determination of the specific large vacuoles occupying the cells' middle identity of the American Polypodium proved parts, and an apical cytoplasm of reticular ap- difficult because only some stages of its de- pearance. In Volga Polypodium, the meshes velopment were present in our material. of this reticulum contained granules of acid Moreover, no species criteria distinct from mucopolysaccharide, which formed together generic ones exist for Polypodium, the genus an apical layer of the ectoderm (Raikova, always having been monotypic. Therefore, we 1960). It seems that these granules were not present here a detailed description of the preserved in the American material with the available developmental stages of the Amer- fixatives used (Fig. 6). The ectodermal epi- ican Polypodium, in order to compare them thelial cells are underlain by muscle cells, with the respective stages of P. hydriforme which are independent and not muscle parts from the type host, the Volga sterlet (Raikova, of epithelio-muscular cells (Figs. 6, 7). 1958, 1960, 1961, 1973). Sections through infected Stage IV-V eggs The parasitic stages of Polypodium found clearly show tentacles inside the stolon (Figs. in our material were represented by two-lay- 1, 3). These are everted-i.e., their ectoderm ered stolons inside oocytes of both Stages IV is inside and their entoderm is outside. The (October) and IV-V (March-April). In Stage tentacular ectoderm contains numerous cni- IV-V oocytes, the stolon occupies the entire docysts of various size classes. Both walking periperal part of an oocyte, the yolk and the tentacles and sensory tentacles can be seen- nucleus being shifted to its center (Fig. 1). the walking tentacles containing only large The two germ layers of the stolon are invert- (10 1m in diameter) and sometimes small (5 ed. The entoderm is the outermost layer, fac- 1tm) cnidocysts, and the sensory tentacles ing the surrounding yolk, and the ectodermn containing medium-sized (7 and small Itm) lines the inner cavity of the stolon, which con- enidocysts (Figs. 6-9). The ectoderm is un- tains the tentacles. An additional dense layer derlain with muscle cells, whereas the ento- covers the parasite, but not continuously (Fig. derm forms a dense axial rod in each tentacle 2); it contains elongated nuclei and inclusions (Figs. 1, 3, 7-9). In some sections this rod of ingested yolk particles. This incomplete seems to have an inner lumen (Figs. 7, 8), but layer certainly corresponds to remnants of a in most sections it is solid. syncytic envelope, the capsule, peculiar to The earliest of the parasitic stages encoun- Polypodium, which isolates the developing tered in the present study came from the eggs stolon from the yolk, assumes the function of of a Stage IV female caught in October. These yolk digestion, and degenerates when the sto- were stolons with inverted germ layers, as just lon is mature (Raikova, 1960). The nucleus of described, but with continuous capsules the infected oocyte remains visible; it is around them and with a smaller number of strongly lobulated (Figs. 1, 3), and has a tentacles inside them. Also, mature cnidocysts dense, almost fibrous karyoplasm and several were far fewer in the ectoderm of the tenta- vacuolized, frequently fused nucleoli. Usual- cles. The latest stages, found in late April, in- ly no chromosomes are visible in the nucleus. habited eggs ready to be spawned; their cap- The envelopes of the infected egg-especial- sule had, on the contrary, almost disappeared, 806 THE JOURNAL OF PARASITOLOGY,VOL 65, NO. 5, OCTOBER 1979

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FIGURES 1-4. Sections of infected eggs of Polyodon spathula containing stolons of Polypodium. 1. Orientation photomicrograph showing egg envelopes (E), yolk (Y), oocyte nucleus (N), and the stolon (S). x30. 2. Wall of the stolon with ectoderm (EC) inside and (EN) outside, showing remnants of entodermn the capsule (C) separating the stolon from the yolk (Y). x280. 3. Top left part of Figure 1, showing oocyte nucleus (N), yolk (Y), and parts of the stolon (S) with internal tentacles (T). x83. 4. Stolon wall lacking a capsule, with entodermal (EN) cells directly ingesting yolk (Y); EC, ectoderm. x330. Staining: 1 and 3, azan; 2 and 4, hemalum. RAIKOVAET AL.-PARASITIC COELENTERATEFROM EGGS OF POLYODON 807

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FIGURES5-8. Details of the structure of the parasite's stolon. 5. A fold of the stolon wall showing glandular cells (G) in its entodenn (EN); EC, ectoderm. x 1,800. 6. Oblique section of a sensory tentacle containing medium-sized cnidocysts (MC) in its ectoderm (EC), and muscle cells (M) beneath it. x700. 7. Cross section of a sensory tentacle near its base, showing small enidocysts (SC) in its ectoderm (EC), muscle cells (M), and entodermal axis (EN). x630. 8. Cross section of a walking tentacle near its base, showing entoderm (EN) and large cnidocysts (LC) in the ectoderm (EC). x630. Staining: 5 and 7, iron hematoxylin; 6 and 8, hemalum. 808 THEJOURNALOFPARASITOLOGY,VOL.65,NO.5, OCTOBER 1979 and the entodermal cells of the stolon were degenerating specimens with incomplete sets full of yolk (Fig. 10). of tentacles (3, 4, 7, 10, etc.) were present in The following forms have been encoun- the fixed samples; some lacked a mouth. But tered among the free-living polyps (grown in the polyps continued to live, and some normal Petri dishes) after they had hatched from in- 12-tentacled animals with mouths, and even fected eggs. dividing ones, were observed in the fixed The stolon becomes evaginated with its ec- samples as late as the 24th and the 30th days. toderm outside while still in the egg. Very The last sample (40 days after hatching) con- soon after its emergence into water, the stolon tained degenerating specimens only. fragments into a number of 24-tentacled pol- DISCUSSION yps, each about 1 mm long (tentacles exclud- ed), and lacking mouth. The histological or- The gross morphological and histological ganization of such polyps is simple. Their organization of the available parasitic stages entoderm is not yet differentiated into regions of the American Polypodium, as well as the and consists of cells replete with yolk, which structure, mode of division, histology, and be- makes the nuclei inconspicuous (Fig. 11). The havior of its free-living polyps-at least at ear- ectoderm of the tentacle tips contains cnido- ly stages of their life-exactly correspond to cytes with mature cnidocysts, whereas that of the respective stages of the life cycle of the the tentacle bases contain cnidoblasts with Volga Polypodium from the sterlet (Acipenser immature cnidocysts. ruthenus), which is the type form of the During the first and second days of the free species P. hydriforme. All anatomical and his- life, 12-tentacled polyps with primordia of 12 tological features, including the structure, the new tentacles also have been observed, which size, the classes, and the distribution of the indicated longitudinal division of the polyps cnidocysts, are the same in the American coupled with neoformation of tentacles, ex- Polypodium and in P. hydriforme (Raikova, actly as it is known to occur in the Volga ma- 1960, 1961); in the type form of P. hydriforme, terial Also some six-tentacled the larger cnidocysts are 10 the medium, (Raikova, 1961). /m, were seen. The of all 7 ,m, and the small ones, 4-5 Also similar specimens histology /m. these forms is exactly the same as that of new- in both forms is the correspondence of the ly formed 24-tentacled polyps. stages of parasite development to the stages Within 48 hours after hatching, some spec- of the host's oocyte development and matu- imens with formed mouths appear. Simulta- ration. In both, the polypodia have the same neously, their entoderm begins to differen- adaptations to intracellular parasitism, in the tiate into a buccal and a gastral region. The form of inversion of the germ layers of the cells of the buccal region usually contain no stolon and formation of a capsule around the yolk particles, whereas the gastral region cells parasite. Even the reaction of the host oocyte still have yolk inclusions in their cytoplasm. to the presence of a parasite is similar and Therefore, the cells of the buccal region ap- involves fusion of the nucleoli into one or two pear narrower and higher than those in the large vacuolized bodies and partial reduction gastral region (Fig. 12). Eight days after hatch- of the oocyte's envelopes (cf. Raikova, 1963). ing, the gastral region entodermal cells also Both the American and the Russian free-living lack yolk inclusions; all the yolk captured in polyps lack sensory organs. Finally, some of the gastral cavity during evagination of the the eggs of Polyodon spathula were infected stolon thus seems to be digested by this time with the microsporidian, Pleistophora sulci, (Fig. 13). the same as in the European acipenserids. On the 15th day of free life, all the polyps Thus, a comparison of the available devel- investigated had mouths, but their entoderm opmental stages of the American Polypodium contained only some pigment granules com- from Polyodon spathula with those of the Vol- ing from the egg. No yolk was present. Most ga Polypodium from Acipenser ruthenus in- polyps had 12 tentacles and some tentacular dicates with a high degree of probability, that primordia. On the 17th day, the polyps were both forms can be attributed to a single still in good condition, but because they were species, Polypodium hydriforme Ussov. unfed (Suppes and Meyer, 1975), they soon However, a final verification of this determi- began to degenerate. On the 21st day, only nation will be possible only when other stages RAIKOVAET AL.--PARASITIC COELENTERATEFROM EGGS OF POLYODON 809

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FIGURES9-13. Structural details of the stolon and of free-living polyps. 9. Cross section of a walking tentacle near its end, showing large (LC) and small (SC) enidocysts in its ectoderm (EC), and a solid entodermal axis (EN). x760. 10. Stolon's wall showing entoderm (EN) full of ingested yolk (Y); EC, ectoderm. x760. 11. Body wall of a 48-hour-old polyp in its gastral region, the entodenr (EN) contains much yolk (Y); EC, ectoderm. x540. 12. Same, but in the mouth region; the entoderm (EN) is free of yolk; EC, ectoderm. x540. 13. Body wall of an 8-day-old polyp; EN, entoderm, EC, ectoderm. x800. Staining: 9, hemalum; 10, azan; 11-13, iron hematoxylin. 810 THEJOURNALOFPARASITOLOGY,VOL.65,NO.5, OCTOBER 1979 of the life of the American ruthenus L.) din Danare. Bul Inst de Cercetari cycle Polypodium si Projectari Piscicola 20:.54-59. are investigated--especially the sexually ma- DOGIEL, V. A. 1940. New finding places and new ture polyps. hosts of Polypodium hydriforme (In Russian). If we accept, provisionally, that the Amer- Zool Zhurnal (Moscow) 19: 321-323. ican Polypodium is the same species, P. hy- HOFFMAN, G. L., E. V. RAIKOVA,AND W. G. YOD- ER. 1974. found driforme, as the Eurasian one, a question aris- Polypodium sp. (Coelenterata) in North American sturgeon. J Parasitol 60: es whether it existed there ever since the 548-550. separation of the respective continents, or was MARKOV,G. S., AND V. Z. TRussov. 1966. Some introduced into America artificially. Poten- problems of parasitology of acipenserid fishes Nauchnoi Sessii ZNIORH tially, Polypodium may be inadvertently in- (In Russian). Tezisy Sci Session of Central Research Inst for troduced into a new water basin when either (Abstr Sturgeon Fisheries) Astrakhan, p. 59-61. adult fish or eggs of Acipenseridae are trans- NECHAT, H. A., AND B. MOKHAYER. 1974. Para- ported into it (Dogiel, 1940; Raikova, 1958). sites de l'appareil genital des esturgeons. Rev We are not aware of any records of the trans- Med Vet 125: 355-360. NEDOSHIVIN,A. YA. 1928. Materials to the study portation of living sturgeons or their eggs from of the Don fisheries (In Russian). Trudy Azovo- Eurasian countries to the American continent, Chernomorsk Nauchno-promyslovoi Exped 4: and would be grateful to hear from any person 1-175. having such information. In the meantime, it RAIKOVA, E. V. 1958. The life cycle of Polypodium seems reasonable to accept the first alterna- hydriforme Ussov (Coelenterata) (In Russian, Zool Zhurnal 37: in English summary). (Moscow) tive, i.e., that P. hydriforme is indigenous 345-358. American waters, as it is in Eurasian ones. If . 1960. Morphological and cytochemical in- so, P. hydriforme appears to be very ancient. vestigation of parasitic stages of the life cycle It must have existed before separation of the of Polypodium hydriforme Ussov (Coelenter- ata) (In Russian) Tsitologiya (Leningrad) 2: two continents and changed little ever since 235-251. of its in the two now widely separated parts 1961. Cytological pecularities of free-living range. This seems the more surprising be- stages of development of Polypodium hydri- cause no species of Acipenseriformes is com- forme Ussov (Coelenterata) (In Russian). Tsi- mon to both America and Eurasia. The tologiya (Leningrad) 3: 396-408. 1963. and of the thus must be Morphological cytochemical species stability parasite changes in the oocytes of the sterlet and the greater than that of its hosts. The eggs of Po- sturgeon under the effect of parasitizing of lyodon were lightly infected, ca. 0.01% al- Polypodium hydriforme Ussov (Coelenterata) there was a incidence of infected (In Russian). Doklady Akad Nauk SSSR 152: though high 985-988. fish (up to 88%, according to Suppes and Mey- and 1973. Life cycle systematic position of This incidence was than that er, 1975). higher Polypodium hydriforme Ussov (Coelenterata), for the Russian sterlet (78%; Raikova, 1958). a cnidarian parasite of the eggs of Acipenseri- Consequently, it is possible that in America dae. Proc 2nd Int Symp Cnidaria, Seto Marine Biol Lab 20: 165-174. the principal host of Polypodium hydriforme AND V. 1977. is and not SPRAGUE, V., J. VAVRA. Systematics Polyodon spathula Acipenser ful- of the Microsporidia. In: L. A. Bulla and T. C. vescens, of which only one infected specimen Cheng (eds.), Comparative Pathobiology. Vol. had thus far been found (Hoffman et al., 1974). 2. Plenum Press, New York and London. 510 p. In any case, a further investigation of the mor- SUPPES, V. C., AND F. P. MEYER. 1975. Polypodi- um sp. (Coelenterata) infection of the paddle- phology and the systematic position of poly- fish (Polyodon spathula) eggs. J Parasitol 61: podia from various species and genera of Aci- 772-774. penseridae and Polyodontidae is likely to be SVIRSKY,V. G. 1967. Infection of the Amur stur- productive and interesting. geon and kaluga with Polypodium hydriforme Ussov (In Russian). In Okhrana Prirody na Dal- in the Far LITERATURE CITED nem Vostoke (Nature Protection East), Vladivostok 4. BOGATU,D. 1961. Un caz infestatie cu Polypodium hydriforme Ussov 1885 la cega (Acipenser