Hrabyeia xerkophora n.gen. n.sp., a NewMicrosporidian with Tailed Spores from theOligochaete Nais christinae Kasparzak, 1973
Jiff Lom and Iva DykovaInstitute of Parasitology, Czechoslovak Academy of Sciences, Geske Budejovice,Czechoslovakia
SUMMARY
Anew microsporidian, Hrabyeia xerkophora n.g., n.sp., is described from the coelomocytes ofthe body cavity of a freshwater oligochaete, Nais christinae Kasparzak, 1973 from Czechoslovakia. Infected coelomocytes are turned into xenomas 50 Itm in size. Of the incompletelyknown life cycle, diplokaryotic sporonts developing in close contact with the host cell cytoplasmand diplokaryotic spores were studied. The spores of a Nosema-type have a gnarled caudalappendage encased with and compartmentalized by the exospore.
Introduction
Thus far only two microsporidian genera form sporeswith stout caudal prolongations, i.e., Jirovecia Weiser,1977 and Caudospora Weiser, 1946 [see 6]. In 1977,Professor Sergej Hrabe, a distinguished Czech zoologist,discovered microsporidian infection in naidid oligochaetes. He kindly placed the material at our disposal. Weexamined the few species with light and electron microscope, but because of the limited amount of material, ourresults did not depict the developmental stages sufficientlyenough to give an exact idea of the fuillife cycle. Only ashort note was prepared in 1978 [8]. We hesitated topublish our observations until more infected specimenswould be available. However, since we failed to obtain anymore material, we prefer to publish our available datarather than to leave them to oblivion. The aim of thiscommunication is to describe the microsporidian as a newtaxon, Hrabyeia xerkophora n.g., n.sp.
Material and Methods
The infected oligochaetes, Nais christinae Kasparzak, 1973were collected in 1977 in the estuaries of small creeks discharginginto the Knfnicska dam lake at Brno, Czechoslovakia. The livespecimens were examined under the dissecting microscope.Advanced infection could be detected by large spore-filledxenomas rolling in the worm's body cavity. Such infected worms
were opened and smears were prepared of the xenomas. Forhistological sections, samples were fixed in 10% neutral formalinan~ paraffin sections were stained with hematoxylin andeosm.
For electron microscopy, pieces of infected animals were fixedfor 90 min in cold, 0.1 M cacodylate-buffered 2% osmic acid,and embedded in Epon-araldite mixture. Thin sections weredouble-stained in uranium acetate and lead citrate, carbon-coatedand examined in the JEM 100 Belectron microscope.
Results
Hrabyeia xerkophora n.g., n.sp.
Host: Nais christinae Kasparzak, 1973. In the first sample, 2 out of 7 worms were infected, in the secondsample, 3 out of 10 worms were infected.
Site of infection: coelomocytes (free ceils of the coelomicfluid)Locality: Knfnicska dam lake near Beno, Czechoslovakia
Vegetative stages developed in the coelomocytes. Normal coelomocytes averaged about 8 !-tm in diameter andhad a compact nucleus about 3.5 !-tm in diameter. Theparasites induced an enormous hypertrophy of the coelomocyte and of its nucleus which assumed an enlargedvesicular shape with a large nucleolus. There could be oneor two nuclei to a xenoma. The xenoma had a thin growthof hair-like surface projections about 4 !-tm long. Grown
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xenoma with mature spores had up to 50 ~m in size andseveral xenomas together might form a confluent mass(Fig. 1).
A young xenoma contained rounded merogony stages.In histological section, it was impossible to state quitedefinitely whether they were uninucleate or diplokaryotic.The earliest stage encountered in the section had twospherical meronts in the cytoplasm. The meronts 2-3 ~m
in size divided by binary fission - no chain or rosetteformation was observed - and they filled the cytoplasmaround the xenoma nucleus (Fig. 2) or around its twonuclei (Fig. 3). Then as the xenoma grew, they replaced thexenoma cytoplasm (Fig. 4). Eventually, the grown xenomas were repleted with mature spores.
We failed to observe young xenomas in ultrathinsections which only included grown xenomas with maturespores and cells of the sporogony sequence. The sporontswere in direct contact with host cell cytoplasm (Fig. 6).They revealed a diplokaryon in all sections, in which notjust one but both nuclei were visible; they were unequal,one of them was always much larger. It was difficult toimagine that a diplokaryon composed of equally largenuclei, could reveal when sectioned, consistently in allplanes of sections unequally large cross sections of bothnuclei (Figs. 5, 7, 9). Therefore we assume that the twonuclei of the diplokaryon were really of unequal size,similar to what is quite evident in mature spores.
The cytoplasm contained a more or less developedsystem of cisternae of rough endoplasmic reticulum.Sometimes a Golgi-like reticulum could be observed nearthe nuclei. A series of sporonts with increasing thickness ofopaque deposits on the cell membrane could be observed,until in a transitional stage to sporoblast a double wallcould be made out (Fig. 10). There was no proof thatsporonts divided before the spore formation.
Sporoblasts developed tail-less, the caudal appendagewas only formed in the maturing spore. Gradually, theturns of the polar tube were deposited and an empty spaceappeared around the sporoblast; two spores may share oneempty space probably as a result of fusion of two spaces.There was no sporophorous vesicle formation. At first thesporogony stages, sporoblasts in their halos and somemature spores lay in a still functional host cytoplasmcontaining mitochondria and other cell organelles. Eventually, the xenoma cytoplasm was completely degraded,turning into an almost structure-less, electron lucentsubstance, in which the spores and sporogony stages layembedded, the empty spaces having disappeared(Fig. 13).
Hrabyeia xerkophora n.g., n.sp., a New Microsporidian . 245
Spores were elongated ovoidal or rounded bullet-shape(Figs. 11, 12), slightly narrower anteriorly; the size of freshspores was 5.9 (4.3-10.9) X 2.8 (2.3-3.4) ~m, (n = 30).At the posterior end there was a thick, rigid, gnarled tail3.4 (1.1-7.8 ~m) long and mostly curved. There was asmall posterior vacuole in the hinder end of the spore.There was no mucous envelope. In the light microscope,PAS reaction for polysaccharides d~picted just a tiny dot inthe apex of the spore, and the Feulgen-stained nucleusappeared as an elongated mass, 2 X 0.5 ~m long. In a fewcases, two fragments could be seen, a small posterior oneand a larger anterior one (Fig. 8).
The electron microscope proved that there were twonuclei with lobose outlines (Figs. 15, 18), closely apposedto each other (Fig. 16) leaving a gap of only 9 nm, theirnuclear envelopes being about 14 nm thick. The posterior
.:.
Fig. 8. A diagram of a maturespore, showing the posterior vacuole as it appears in life, combinedwith the result of PAS reaction - thepositive polar cap, and with theFeulgen - stained nuclei.
.... Fig. 1. Longitudinal section through the anterior part of the body of Nais christinae showing xenomas with mature spores, separateand in aggregations. Bar = 100 [tm. - Fig. 2. Early xenomas with merogony stages around the single, hypertrophic nucleus. Fig. 3. Above, a binucleate early xenoma with its two host cell nuclei centrally located. At the bottom, a grown xenoma with maturespores. - Fig. 4. Early xenomas; at the left, a binucleated one. Arrows point at uninfected, small coelomocytes. In Figs. 2 to 4, bar =10 [tm; Figs. 1 to 4, hematoxylin-eosin stained sections. - Fig. 5. A sporont with diplokaryon surrounded by cisternae of endoplasmicreticulum (arrows) embedded in disintegrated host cell cytoplasm. Bar = 1 [tm. - Fig. 6. Two sporonts, lying in close contact with thehost cell cytoplasm, left one showing one nucleus (N) in the section, the other sectioned beyond the nucleus. Top left, a maturing spore(S) in a clear halo. Bar = 1 [!m. - Fig. 7. Sporont in the disintegrated host cell cytoplasm, showing two unequal nuclei. Bar =1 [!m.
nucleus was always much smaller. The larger anterior partof the diplokaryon was trough-shaped, embracing thepolaroplast. The nucleus was encircled by plates of welldeveloped cisternae of rough endoplasmic reticulum(Fig. 17). The polaroplast had a uniform, flocculatedstructure; no lamellar part of polaroplast could beobserved (Figs. 14,18). The polar tube fastened anteriorlyto a moderately developed anchoring disc (Fig. 19) wasslightly tapering from the disc to the end. It was not exactlyanisofilar in terms of Weiser [13] since the transitionbetween the wider proximal part and narrower distal partwas not abrupt but gradual. The widened mouth of thetube about 380 nm in diameter was covered by threevaulted electron lucent layers, 23 nm thick, separated byelectron dense boundaries. From there on, the tube ranstraight backwards through the polaroplast and wasdeposited in 7 to 10 turns beneath the walls of the posteriorspore end. Out of 25 spores, 11 had 8 turns, 9 had 9 turns,one had 7 turns and two had 10 turns of the polar tube.Transverse sections of the tube, of a usual concentricstructure, had about 100-120 nm in diameter; the last fullor only half of the turn of the tube was much thinner, about60 nm (Fig. 21).
The endospore was 76-150 nm thick; the exospore,25-35 nm thick, was covered by a glycocalyx-Iike layer21-35 nm thick (Fig. 21). The exospore split at theposterior end of the spore; the inner layer covered theendospore, the outer layer formed the envelope of thespore tail and also stratified the electron lucent matrix ofthe caudal appendage into numerous compartments. Thisarrangement suggests that the tail is formed stepwise fromthe caudal end of the spore. In the ultrathin sections, someof the large host cells contained more closely packedmature spores in various degree of digestion, suggesting aphagocyte which has ingested them rather than a xenomain which they have developed (Fig. 20).
Discussion
Although a limited material was accessible, leavingmany gaps especially in the merogony sequence, it ispossible to characterize the species sufficiently enough forfuture recognition and to establish a separate taxon forit.
A scrutiny of Larsson's [6] survey of existing genera aswell as of genera described since [e.g., 7] reveals that none
Hrabyeia xerkophora n.g., n.sp., a New Microsporidian . 247
Fig. 19. Apex of the spore showing stratified polar cap at themouth of the polar tube and adjoining anchoring disc (arrows).Bar = 0.2 !lm. - Fig. 20. Part of coelomocyte with presumablyingested spores, some of which (asteriscs) are partly digested.Bar = 2 J!m. - Fig. 21. Turns of the polar tube, the last one(arrow) having half of the diameter of the others. Small arrowspoint at the glycocalyx-like cover of the exospore. Bar =0.3 J!m.
of existing genera can accomodate the present species.Microsporidia with tailed spores bear only a superficialrelation to it. In Caudospora Weiser, 1946, the cauda is ahollow projection of the exospore [12] and in addition,there is a sporogonial plasmodium producing sporoblastsin a rosette formation. The genus ]irovecia Weiser, 1977(= Mrazekia Leger and Hesse, 1916) also producingxenomas in oligochaetes, is known to have a caudalappendage, too. In two species, however, J. brevicaudaLeger and Hesse, 1916 [see 3] and]. caudata Leger andHesse, 1916 (unpublished observations of the authors)what appears to be a true caudal appendage is in fact only anarrow constricted posterior part of the spore. These twospecies must be removed from the genus ]irovecia. A new
<IIIl Fig. 9. An early sporoblast (?) embedded in disintegrated host cell cytoplasm. Bar = 1 J!m. - Fig. 10. The double cell wall in an earlysporoblast. Bar = 0.2 J!m. - Fig. 11. Fresh spores in transmitted light. Bar = 10 J!m applies also to Fig. 12. - Fig. 12. Fresh spores inphase contrast. - Fig. 13. Part of the xenoma content with a thin, already disintegrated cytoplasm (C) with sporonts (SP), mature sporesand one maturing spore with only 3 turns of the polar tube (arrow). Bar = 5 J!m. - Fig. 14. Part of the polaroplast (P) and nucleus (N) ofthe mature spore. Bar = 0.5 !lm, applies also to Fig. 16. - Fig. 15. Posterior part of a mature spore showing the nucleus (N) with deepsurface invaginations giving it a lobose appearance; the boundary with the polaroplast (P) is not distinct, as well as the boundarybetween the two nuclei of the diplokaryon (small arrows). V = place of the posterior vacuole, T = grazing section of the tail; thickarrows point at the turns of the polar tube. Bar = 1 !lm. - Fig. 16. Enlarged part of the spore showing the partition between the twonuclei of the diplokaryon. - Fig. 17. Oblique section through a mature spore showing cisternae of rough endoplasmic reticulum(arrows) ensheathing the nucleus (N). P =polaroplast. - Fig. 18. Mature spore in an almost longitudinal section. Bar = 1 J!m, appliesalso to Fig. 17.
248 . J. Lorn and I. Dykova
genus must be established for them, or they may beaccomodated within the genus Bacillidium Janda, 1928 iffuture research of this group proves the existence of agradual transition from constricted J. caudata spores tounconstricted Bacillidium spores.
Thus we propose a new genus Hrabyeia n.g., for theNais parasite, named in honour of Dr. Sergej Hrabe,Professor of Zoology at the Purkynje University in Brno,with the following diagnosis: Merogony stages (uninucleate? diplokaryotic?) divide by binary fission to give riseultimately to isolated diplokaryotic sporonts which produce (by binary fission? directly?) diplokaryotic sporoblasts and spores. Ellipsoidal spores with slightly anisofilarpolar tube bear a gnarled caudal appendage covered byexospore and appearing to consist of endospore materialcompartmentalized by exospore layers. Induces xenomaformation in infected oligochaete coelomocytes. A single(type) species, Hrabyeia xerkophora n.g., n.sp. Type slideshave been deposited in the protozoological collection ofthe Institute of Parasitology of the Czechoslovak Academyof Sciences in Ceske Budejovice under the numbersH-PI-045 to H-PI-049.
The probable position of the genus unless future studiesof merogony prove otherwise is with the family Nosematidae Labbe, 1899 both in the conception of Weiser [13]and Sprague [11] and also Canning [l].The polaroplaststructure of H. xerkophora is rather unique, and does notfit into any of the five types listed by Larsson [5]. A slightstructural analogy may be found in Nosema eurytremae,where the posterior portion of the polaroplast is composedof small flattened, spindle-shaped sacs [2]. In a Gurleya sp.from copepods [9] the polaroplast consists of areas offloccular material, vaguely reminiscent of the polaroplastmaterial in Hrabyeia.
H. xerkophora with its rather unique features lendssupport to Sprague's [11] speculations on annelid hosts asa radiating center for microsporidian evolution, whereboth "primitive" and "progressive" lines develop and adiversity of forms can be found (e.g. Bacillidium, Burkea,Jirovecia, Pseudopleistophora). In fact H. xerkophora isreminiscent of some of the present ]irovecia in the way inwhich the two nuclei of the diplokaryon are closelyapposed in the spore (as in ]irovecia = syn. Mrdzekiabrevicauda [3]) or in the changes it produces in the hostcoelomocyte. In Bacillidium criodrili the initially compactnuclei of infected coelomocytes also turn later into vesicular nuclei with a large nucleolus [4]; the development ofhair-like projections on host coelomocytes is also similarfeature (Bacillidium limnodrili [4]). It may be the capacityof the host's cells, however, to form such xenomas ratherthan the feature of the parasite, since large (up to 100 !tm)
xenomas with a hairy surface are induced by the infectionwith a still another microsporidian, possibly a Nosema,found by Mrazek [10] in Limnodrilus.
More research is needed to understand the affinities ofthis species.
References
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