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Flabelliforma ostracodae n. sp. (Microspora, Duboscqidae), a new microsporidian parasite of Candona sp. (Crustacea, Ostracoda)
European Journal of
Europ.]. Protisto!' 30,280-287 (1994) August 29, 1994
PROTISTOLOGY
Flabelliforma ostracodae n. sp. (Microspora, Duboscqidae), a New Microsporidian Parasite of Candona sp. (Crustacea, Ostracoda) Annika M. Bronnvall and J. I. Ronny Larsson Department of Zoology, University of Lund, Sweden
SUMMARY A new microsporidian parasite, Flabelliforma ostracodae n. sp., is described based on light and electron microscopy. It was found in ostracods (Candona sp.), collected on flooded meadows in southern Sweden. All life cycle stages have isolated nuclei. Unfixed spores are oval, measuring 3.8 x 2.7 f.lm. The polar filament is isofilar and arranged in 13-16 coils in the posterior half of the spore. The polaroplast is divided into two, almost equally long, well defined regions. The anterior polaroplast consists of thin, closely packed lamellae, while the posterior polaroplast has wider, more irregularly arranged lamellae. Merogonial plasmodia are initially rounded and produce up to 16 merozoites by rosette-like budding. Sporogonial plasmodia produce 16-32 sporoblasts by rosette-like budding. The spores are produced within a fragile sporophorous vesicle. The identity of the species and the taxonomic position are discussed.
Introduction Until the present time Vavraia cyclocypris, a parasite of Cyclocypris ovum, is the only microsporidian species reported from Ostracoda. It was found by Voronin and Melnikova in the proximity of St Petersburg in 1984 and it was preliminary placed in the genus Vavraia [11]. This report deals with another microsporidian parasite of Ostracoda. The host is a species of the genus Candona. The life cycle and cytology are described with emphasis on the ultrastructure. The identification of the species and its systematic position are discussed briefly. Material and Methods Ostracods of the genus Candona Baird, 1846 were collected on flooded meadows close to the river Hoje a, at Esarp in the south of Sweden on January 8th, 1989. The specimens could not be identified to species leve!. White animals were selected and punctured with a needle. The haemolymph was smeared on agar glasses according to the technique described by Hostounsky andZizka [3] and examined using phase contrast microscopy and dark field illumination. Each infected specimen was cut in two halves. One half was squashed, lightly air dried and fixed in Bouin-Duboscq-Brasil solution overnight. The slides were stained using Giemsa solution or Heidenhain's iron haematoxylin [9]. Permanent preparations were mounted in D.P.X. (BDH Chemicals Ltd). 0932-4739/94/0030-0280$3.50/0
The other half was fixed for transmission electron microscopy using 2.5 % (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 25 h. After being washed in cacodylate buffer it was postfixed in 2 % (w/v) osmium tetroxide in cacodylate buffer at 4°C for 1 h. Finally the piece was washed in buffer, dehydrated in an ascending series of buffer-acetone solutions to absolute acetone and embedded in epon. Sections were stained with uranyl acetate and lead citrate [8]. Results
Prevalence, Pathology and Life Cycle The population of ostracods harboured two microsporidian species, both undescribed, which induced identical discoloration. The prevalence of each species is unknown. No mixed infections were found. Infected tissues were seen through the transparent cuticle as anomalous white coloration. The adipose and connective tissues were the principal sites of infection. The life cycle comprised at least one merogonial and one sporogonial division, both with isolated nuclei. In the initial merogony, nuclear division gave rise to lobed plasmodia, measuring up to 16.6 f.tm in diameter in stained smears, and containing up to 16 nuclei (Figs. 1-5). The plasmodia divided by rosette-like budding into uninucleate cells, merozoites, which subsequently matured into sporonts. These underwent nuclear division resulting.in sporogonial plasmodia with 16-32 nuclei (Figs. 6-9). Reduc© 1994 by Gustav Fischer Verlag, Stuttgart
Flabelliforma ostracodae n. sp.. 281 tional division was not observed. The largest plasmodium found in squash preparations measured 19.3 !-tm in diameter. Sporoblasts, which were formed by rosette-like budding, remained within a sporophorous vesicle produced by the sporont. Buds were initiated already when the sporogonial plasmodium contained only four nuclei (Fig. 6). Mature spores were oval with a slightly pointed anterior pole (Figs. 10-12). Fresh spores were about 3.8 x 2.7 !-tm (n = 25). Fixed and stained spores measured 3.0 - 3.6 x 1.4 !-tm (n = 25). There were no obvious differences in staining properties between merogonic and sporogonic nuclei when using Heidenhain's haematoxylin, except that sporont nuclei showed a tendency to destain more easily. Using Giemsa solution the merogonic nuclei were only slightly less intensely stained than the sporogonic nuclei, but because of the darker cytoplasm of the meronts the difference was enhanced.
Presporal Stages The only merogonial stage observed in ultrathin sections was binucleate plasmodia (Fig. 13). Their cytoplasm contained a great number of free ribosomes and a weakly developed rough endoplasmic reticulum (RER). The nuclei were moderately electron dense, with areas of heterochromatin, and an approximately 18 nm thick nuclear envelope of traditional type with double unit membranes and pores. The youngest sporont observed had two distinctly separated nuclei (Fig. 14). The cytoplasm contained free ribosomes, although in a considerably smaller amount than in the binucleate meront, and RER with only a small number of unevenly attached ribosomes. At this stage the first signs of the sporophorous vesicle could be seen as tiny blisters protruding from the plasma membrane. The first sign of the thick sporont wall was seen after at least one more nuclear division. Inside the newly separated
Figs. 1-12. Light microscopical aspect of Flabelliforma ostracodae n. sp. - Figs. 1-2. Rounded merogonial plasmodia (M) with 4 and 6 nuclei (N). - Figs. 3-5. Lobed merogonial plasmodia with 8-16 nuclei. - Figs. 6-9. Rosette-like budding sporogonial plasmodia (5) with increasing number of nuclei. Budding of the plasmodium is initiated already at the four nuclei stage. - Fig. 10. Agroup of mature spores. - Fig. 11. Fresh spores. - Fig. 12. Location of the holotype (T). - Figs. 1-2,5-10,12. Haematoxylin staining. Figs. 3-4. Giemsa staining:- Scale bars: Figs. 1-10, 12 = 15 11m; Fig. 11 = 10 11m; Fig. 12 = 6 11m.
282 . A. M. Bronnvall and
J. I. R. Larsson
sporophorous vesicle, the wall was initiated as patchwise secretion of electron dense material on the plasma membrane (Fig. 15). When the thick sporont wall was almost complete, most ribosomes had become associated with the RER membranes, and the RER was organized in concentrical layers arround the nuclei (Figs. 16-17). Centriolar plaques and mitotic spindle tubules were observed in the dividing nuclei at this stage (Fig. 16). Golgi apparatuses were in close contact with the nuclei in the developing sporont (Fig. 18). Among the first structures to become distinct in the sporoblast was the polar filament with the attached polar sac, containing the primordium of the anchoring disc (Fig. 19). These structures were formed by the Golgi apparatus at the posterior end of the sporoblast, and they were pushed forwards when the length of the filament increased. Transverse sections of the developing filament showed an electron dense center surrounded by a moderately electron dense ring and a unit membrane cover (Fig. 19). Apparently the Golgi apparatus also generated a vacuole with electron dense content. It occurred in various positions in the immature spore (Figs. 20-21). However, at the end of spore maturation it became localized to the posterior pole together with the deteriorating Golgi apparatus. The polaroplast was initiated in the immature spore when the polar filament had reached the anterior pole and had begun coiling up posteriorly. As the size of the polaroplast increased, the filament coils were pushed backwards becoming closely arranged in the posterior half of the spore. The initial polaroplast was not divided into two regions, although the lamellae were somewhat wider at the posterior pole.
The Mature Spore The spore wall was three-layered with exospore, endospore and plasma membrane (Figs. 23-24).The exospore was about 30 nm thick, with an internal electron dense layer (14-18 nm) and an external double layer. The endospore was 74-96 nm thick, except at the anterior pole where the thickness was reduced to 30-40 nm. The plasma membrane was approximately 8 nm thick. The polar filament was isofilar with a diameter of 85-99 nm. It was arranged in 13-16 coils which, in the longitudinally sectioned spores, were seen as a slightly irregular single row close to the spore wall in the posterior half of the spore (Figs. 23, 25). The angle of tilt of the anterior coil to the long axis of the spore was 70°-75°. The transversely sectioned filament exhibited a series of concentricallayers (Fig. 26a-e). From the uniformly electron dense center (38-46 nm in diameter), in direction outwards, the layers were: an electron lucent, fibrillous layer (8-10 nm thick), a narrow, moderately dense layer interrupted by a narrow zone of more electron lucent material
and, finally, a narrow distinctly dense layer. These layers were together 7-9 nm thick. The unit membrane cover of the filament was about 5 nm thick. The diameter of the filament increased close to the anchoring disc, forming an approximately 110 nm long and 140 nm wide attachment section. Close to the anchoring disc the electron lucent layer of the polar filament widened slightly, forming a chalice-like structure. Inside this structure, the diameter of the electron dense center increased until it finally connected with the anchoring disc. In longitudinal sections of the spore, only one layer was distinctly seen in the anchoring disc (Figs. 23, 27). The polaroplast, which occupied the anterior third of the spore, surrounded the anterior part of the polar filament, ending at the level of the first coil (Fig. 27). It was divided into two almost equally long and well defined regions. The anterior polaroplast had 9-11 nm thick, closely packed lamellae filled with electron dense material, while the posterior part consisted of up to 27 nm wide, less densely packed and more irregularly arranged lamellae containing less electron dense material. The polar sac extended backwards for about 1/4 of the spore length (Fig. 27). It enclosed the anchoring disc and covered the anterior part of the polaroplast like an umbrella. The sac was filled with granular material, which increased in density towards the membrane. The approximately 5 nm thick polar sac membrane was continuous with the membranes of the polar filament and the lamellae of the polaroplast. The electron dense cytoplasm contained polyribosomes (Fig. 27), which were localized mainly around the nucleus and the polar filament and below the plasma membrane. The membrane-lined posterior vacuole was filled with electron dense material. The largest sectioned vacuole was 0.74 J.tm in diameter. The large nucleus was situated in the region of the polar filament coils. The shape was bent and irregular, making it difficult to measure, but at least up to 0.9 J.tm wide nuclei were seen.
The Sporophorous Vesicle The sporophorous vesicle was initiated like tiny blisters from the sporont already when it was binucleate (Fig. 14). When sporoblast formation was completed, the sporophorous vesicle contained a lot of debris from the sporogony. When the sporoblasts matured into spores the debris aggregated and became more organized (Fig. 19). The envelope of the mature sporophorous vesicle was an about 8 nm thick, uniformly electron dense layer which followed the outline of the spore group (Fig. 22). The vesicle was not persistent and mature spores were released already in the host.
Figs. 13-17. Presporal stages. - Fig. 13. Meront with two nuclei visible. - Fig. 14. Sporont with two nuclei visible and with protruding ~ blisters of the sporophorous vesicle. - Fig. 15. Lobed sporogonial plasmodium with the first signs of the sporont wall visible. Figs. 16-17. Lobed sporogonial plasmodia; the rough endoplasmic reticulum is organized into concentric layers around the nuclei; inset shows a centriolar plaque (arrowhead), mitotic spindle tubules (thin arrow) attached to chromatin (wide arrow). N
=
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RER = rough endoplasmic reticulum, SV = sporophorous vesicle, SW = sporont wall. Scale bars: Figs. 13-14, inset on 16 = 500 nm; Figs. 15-17 = 1000 nm.
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Flabelliforma ostracodae n. sp. . 285
Figs. 23-27. Ultrastructure of the mature spore. - Fig. 23. Longitudinally sectioned spore. - Fig. 24. The spore wall. The exospore consists of an external double layer (arrowhead) and an internal electron dense layer (arrow). - Fig. 25. Transversely sectioned polar filament coils; the anterior coil is surrounded by lamellae of the posterior polaroplast. - Fig. 26. Transversely sectioned polar filament coil, exhibiting the layers: a = electron dense center; b = electron lucent, fibrillous layer; c = moderately dense layer; d = zone of more electron lucent material followed by a narrow dense layer; e = unit membrane cover. - Fig. 27. The anterior part of the spore, showing the subdivisions of the polaroplast: thin, electron dense lamellae anteriorly, wider, more electron lucent lamellae posteriorly. AD = anchoring disc, AP = anterior polaroplast, EN = endospore, N = nucleus, PF = polar filament, PM = plasma membrane, PP = posterior polaroplast, PR = polyribosomes, PS = polar sac, SV = sporophorous vesicle, V = vacuole. Scale bars:
~ Figs. 18-22. Sporoblast and maturing spores. - Fig. 18. Sporogonial plasmodium in the final phase of releasing sporoblasts; Golgi
apparatuses (arrowheads) are visible close to the nuclei. - Fig. 19. Sporoblasts. At this stage the debris of the sporophorous vesicle appears as small granules (arrows). - Figs. 20-21. Immature spores. The electron dense vacuole occurs in various positions in the immature spore. - Fig. 22. Mature spores within a sporophorous vesicle. AD = anchoring disc, EN = endospore, EX = exospore, GA = Golgi apparatus, N = nucleus, PF = polar filament, V = vacuole. Scale bars: Figs. 18-20, 22 = 1000 nm; Fig. 21 = 250 nm.
286 . A. M. Bronnvall and]. I. R. Larsson
Discussion
It must be concluded that the microsporidium treated by us is a new species. Although the only other microsporidian species described from an ostracode, Vavraia cyclocypris, forms numerous uninucleate sporoblasts by rosettelike budding within a sporophorous vesicle, similarly to the sporogony observed by us, the differences are obvious: V. cyclocypris has smaller (about 3.4 x 1.7 !-tm in fresh smears) and more pointed spores; the exospore is described as two layered, even if Fig. 2 a in reference [11] indicates that the layering might be more complex; and the polar filament is anisofilar, with 2-3 thick coils, one intermediate coil, and 4-6 thin coils, while the polar filament of the Swedish microsporidium is isofilar [11]. However, we do not believe that it is necessary to create a new genus. Of the more than 100 microsporidian genera described until the present time, only five combine life cycles with isolated nuclei with polysporoblastic sporogony yielding 16-32 spores, like it is seen in the present microsporidium: Agglomerata, Vavraia, Tardivesicula, Duboscqia and Flabelliforma. Three of them can easily the disregarded: Agglomerata, because of the five-layered exospore, and the uniquely constructed polaroplast with three regions [6]; Vavraia, because of the thick merontogenetic, sporophorous vesicle and the simultaneous production of micro- and macrospores [1, 10]; and finally Tardivesicula, because of the rod-shaped spores and the delayed initiation of the sporophorous vesicle [5]. The genus Duboscqia is problematic. The type species D. legeri, which is a parasite of termites and hence found in terrestrial environment, was described using light microscopy, and there are no further studies providing ultrastructural data. The genus is poorly defined: spores are ovoid-ellipsoid, the sporogony yields 16 spores, and infected cells become extremely hypertrophic [4, 7]. The species studied by us produces mainly 32 spores per vesicle, and since it utilizes an aquatic host we hesitate to include it in the genus Duboscqia. The remaining genus, Flabelliforma, was created by Canning and colleagues in 1991 [2]. When comparing the ultrastructure and life cycle of the Swedish microsporidium with the type species, F. montana, it is revealed that the sporogonies are virtually identical, and so are the cytological characteristics of the spores. Our microsporidium matches the characteristics of Flabelliforma, except that the sporophorous vesicles are initiated without blister formation in F. montana, but with distinct blister formation in the species studied by us. With increasing knowledge of the ultrastructural cytology of the microsporidia, the taxonomic use of the sporophorous vesicle seems to be decreasing. Applying the diagnosis of Flabelliforma strictly, the microsporidium described herein is excluded. However, we belive it is better to provisionally rank the new species in the genus Flabelliforma than to establish a new genus, if the only difference between the two genera is the way the sporophorous vesicle is initiated. It is obvious that the species studied by us is different from F. montana, which has broader spores (2.9 x 1.9 !-tm when stained), and a short polar filament (3.5 coils). It is
also possible that the species differ in endospore thickness. The sporoblasts and spores of F. montana were distorted by the techniques used, making a detailed comparison impossible, but illustrations suggest that exo- and endospore layers of F. montana are equally thick. Our conclusion is that the microsporidium of Candona is a new species, and we believe that a ranking in Flabelliforma is the best solution of the genus problem. The most appropriate position for the genus seems to be the family Duboscqiidae Sprague, 1977.
Description
Flabelliforma ostracodae n. sp. Merogony: Rounded plasmodia with isolated nuclei produce up to 16 merozoites by plasmotomy. Sporogony: Multinucleate plasmodia with isolated nuclei produce 16-32 sporoblasts by rosette-like budding within a sporophorous vesicle. Meiosis was not observed. Spore: Oval, with a slightly pointed anterior pole. Fresh spores measure about 3.8 x 2.7 !-tm, fixed and stained spores 3.0 - 3.6 x 1.4 !-tm. The exospore is about 30 nm thick. It has an internal electron dense layer and an external layer which resembles a double membrane. The endospore is 74-96 nm thick (thinner at the anterior pole). The polar filament is isofilar with 13-16, 85- 99 nm thick, coils arranged in a single row in the posterior half of the spore. The lamellar polaroplast occupies the anterior third of the spore. It has two almost equally long, well defined regions, the posterior one with wider lamellae. Sporophorous vesicle: Uniformly electron dense, eight nm thick, following the outline of the enclosed sporegroup. No tubular inclusions. The vesicle disrupts within the host. Host tissue involved: Adipose and connective tissue. Type host: Candona sp. Baird, 1846 (Ostracoda, Cyprididae) Type locality: Flooded meadows close to the river Hoje ii, Esarp, Scania in the south of Sweden. Type series: Holotype (Fig. 12) on slide no. 890108Hl-2 RL, paratypes on slides no. 890108-Hl-(l-2) RL. Deposition of type: The slide with the holotype in the International Protozoan Type Slide Collection, Smithsonian Institution, Washington, D.C., USA. The second slide in the collection of the senior author.
Acknowledgements The authors are greatly indebted to Mrs Lina Hansen, Mrs Birgitta Klefbohm and Mrs Inger Norling, all at the Department of Zoology, and to Mrs Agneta Persson at the Department of Anatomy, University of Lund, for skilful technical assistance. The investigation was supported by research grants from the Swedish Natural Science Research Council and Helge Ax:on Johnson's Foundation.
Flabelliforma ostracodae n. sp.. 287
References 1 Canning E. U. and Hazard E. r. (1982): Genus Pleistophora Gurley, 1893. An assemblage of at least rhree genera. j. Protozool., 29, 39-49. 2 Canning E. U., Killick-Kendrick R. and Killick-Kendrick M. (1991): A new microsporidian parasite, Flabelliforma montana n.g., n.sp., infecting Phlebotomus ariasi (Diptera, Psychodidae) in France. ]. Invertebr. Pathol., 57, 71-81. 3 Hostounsky Z. and Zilka Z. (1979): A modification of the "agar cushion method" for observation and photographic recording microsporidian spores. j. Protozool., 26, 41A-42A. 4 Kudo R. R. (1942): On the microsporidian Duboscqia legeri Perez, 1908, parasite in Reticulitermes of Maryland. ]. Morphol., 71, 307-326, PI. 1-4. 5 Larsson j. I. R. and Bylen E. K. C. (1992): Tardivesicula duplicata gen. et sp. nov. (Microspora, Duboscqiidae), a microsporidian parasite of the caddis fly Limnephilus centralis (Trichoptera, Lirpnephilidae) in Sweden. Europ. j. Protistol., 28, 25-36.
6 Larsson]. I. R. and Yan N. D. (1988): The ultrastructural cytology and taxonomy of Duboscqia sidae ]IROVEC, 1942 (Microspora, Duboscqiidae), with establishment of the new genus Agglomerata gen. nov. Arch. Prot,istenkd., 135, 271-288. 7 Perez c. (1908): Sur Duboscqia legeri, Microsporidie nouvelle parasite du Termes lucifugus, et sur la classification des Microsporidies. C. R. Soc. BioI., 65, 631-633. 8 Reynolds E. S. (1963): The use oflead citrate at high pH as an electron-opaque stain in electron microscopy.]. Cell BioI., 17, 208-212. . 9 Romeis B. (1968): Mikroskopische Technik. Oldenbourg Verlag, Miinchen und Wien. 10 Weiser j. (1977): Contribution to the classification of Microsporidia. Vestn. Cs. spol. zool., 61, 308-320. 11 Voronin V. N. and Melnikova O. Y. (1984): Vavraia cyclocypris sp. n., the first find of a microsporidian parasite from Ostracoda (Crustacea). Parazitologiya, 18, 482-484.
Key words: Flabelliforma ostracodae n. sp. - Microsporidia - Ultrastructure - Taxonomy - Ostracoda Annika Bronnvall, Department of Zoology, University of Lund, Helgonaviigen 3, S-223 62 Sweden
Europ.]. Protisto!' 30,280-287 (1994) August 29, 1994
PROTISTOLOGY
Flabelliforma ostracodae n. sp. (Microspora, Duboscqidae), a New Microsporidian Parasite of Candona sp. (Crustacea, Ostracoda) Annika M. Bronnvall and J. I. Ronny Larsson Department of Zoology, University of Lund, Sweden
SUMMARY A new microsporidian parasite, Flabelliforma ostracodae n. sp., is described based on light and electron microscopy. It was found in ostracods (Candona sp.), collected on flooded meadows in southern Sweden. All life cycle stages have isolated nuclei. Unfixed spores are oval, measuring 3.8 x 2.7 f.lm. The polar filament is isofilar and arranged in 13-16 coils in the posterior half of the spore. The polaroplast is divided into two, almost equally long, well defined regions. The anterior polaroplast consists of thin, closely packed lamellae, while the posterior polaroplast has wider, more irregularly arranged lamellae. Merogonial plasmodia are initially rounded and produce up to 16 merozoites by rosette-like budding. Sporogonial plasmodia produce 16-32 sporoblasts by rosette-like budding. The spores are produced within a fragile sporophorous vesicle. The identity of the species and the taxonomic position are discussed.
Introduction Until the present time Vavraia cyclocypris, a parasite of Cyclocypris ovum, is the only microsporidian species reported from Ostracoda. It was found by Voronin and Melnikova in the proximity of St Petersburg in 1984 and it was preliminary placed in the genus Vavraia [11]. This report deals with another microsporidian parasite of Ostracoda. The host is a species of the genus Candona. The life cycle and cytology are described with emphasis on the ultrastructure. The identification of the species and its systematic position are discussed briefly. Material and Methods Ostracods of the genus Candona Baird, 1846 were collected on flooded meadows close to the river Hoje a, at Esarp in the south of Sweden on January 8th, 1989. The specimens could not be identified to species leve!. White animals were selected and punctured with a needle. The haemolymph was smeared on agar glasses according to the technique described by Hostounsky andZizka [3] and examined using phase contrast microscopy and dark field illumination. Each infected specimen was cut in two halves. One half was squashed, lightly air dried and fixed in Bouin-Duboscq-Brasil solution overnight. The slides were stained using Giemsa solution or Heidenhain's iron haematoxylin [9]. Permanent preparations were mounted in D.P.X. (BDH Chemicals Ltd). 0932-4739/94/0030-0280$3.50/0
The other half was fixed for transmission electron microscopy using 2.5 % (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 25 h. After being washed in cacodylate buffer it was postfixed in 2 % (w/v) osmium tetroxide in cacodylate buffer at 4°C for 1 h. Finally the piece was washed in buffer, dehydrated in an ascending series of buffer-acetone solutions to absolute acetone and embedded in epon. Sections were stained with uranyl acetate and lead citrate [8]. Results
Prevalence, Pathology and Life Cycle The population of ostracods harboured two microsporidian species, both undescribed, which induced identical discoloration. The prevalence of each species is unknown. No mixed infections were found. Infected tissues were seen through the transparent cuticle as anomalous white coloration. The adipose and connective tissues were the principal sites of infection. The life cycle comprised at least one merogonial and one sporogonial division, both with isolated nuclei. In the initial merogony, nuclear division gave rise to lobed plasmodia, measuring up to 16.6 f.tm in diameter in stained smears, and containing up to 16 nuclei (Figs. 1-5). The plasmodia divided by rosette-like budding into uninucleate cells, merozoites, which subsequently matured into sporonts. These underwent nuclear division resulting.in sporogonial plasmodia with 16-32 nuclei (Figs. 6-9). Reduc© 1994 by Gustav Fischer Verlag, Stuttgart
Flabelliforma ostracodae n. sp.. 281 tional division was not observed. The largest plasmodium found in squash preparations measured 19.3 !-tm in diameter. Sporoblasts, which were formed by rosette-like budding, remained within a sporophorous vesicle produced by the sporont. Buds were initiated already when the sporogonial plasmodium contained only four nuclei (Fig. 6). Mature spores were oval with a slightly pointed anterior pole (Figs. 10-12). Fresh spores were about 3.8 x 2.7 !-tm (n = 25). Fixed and stained spores measured 3.0 - 3.6 x 1.4 !-tm (n = 25). There were no obvious differences in staining properties between merogonic and sporogonic nuclei when using Heidenhain's haematoxylin, except that sporont nuclei showed a tendency to destain more easily. Using Giemsa solution the merogonic nuclei were only slightly less intensely stained than the sporogonic nuclei, but because of the darker cytoplasm of the meronts the difference was enhanced.
Presporal Stages The only merogonial stage observed in ultrathin sections was binucleate plasmodia (Fig. 13). Their cytoplasm contained a great number of free ribosomes and a weakly developed rough endoplasmic reticulum (RER). The nuclei were moderately electron dense, with areas of heterochromatin, and an approximately 18 nm thick nuclear envelope of traditional type with double unit membranes and pores. The youngest sporont observed had two distinctly separated nuclei (Fig. 14). The cytoplasm contained free ribosomes, although in a considerably smaller amount than in the binucleate meront, and RER with only a small number of unevenly attached ribosomes. At this stage the first signs of the sporophorous vesicle could be seen as tiny blisters protruding from the plasma membrane. The first sign of the thick sporont wall was seen after at least one more nuclear division. Inside the newly separated
Figs. 1-12. Light microscopical aspect of Flabelliforma ostracodae n. sp. - Figs. 1-2. Rounded merogonial plasmodia (M) with 4 and 6 nuclei (N). - Figs. 3-5. Lobed merogonial plasmodia with 8-16 nuclei. - Figs. 6-9. Rosette-like budding sporogonial plasmodia (5) with increasing number of nuclei. Budding of the plasmodium is initiated already at the four nuclei stage. - Fig. 10. Agroup of mature spores. - Fig. 11. Fresh spores. - Fig. 12. Location of the holotype (T). - Figs. 1-2,5-10,12. Haematoxylin staining. Figs. 3-4. Giemsa staining:- Scale bars: Figs. 1-10, 12 = 15 11m; Fig. 11 = 10 11m; Fig. 12 = 6 11m.
282 . A. M. Bronnvall and
J. I. R. Larsson
sporophorous vesicle, the wall was initiated as patchwise secretion of electron dense material on the plasma membrane (Fig. 15). When the thick sporont wall was almost complete, most ribosomes had become associated with the RER membranes, and the RER was organized in concentrical layers arround the nuclei (Figs. 16-17). Centriolar plaques and mitotic spindle tubules were observed in the dividing nuclei at this stage (Fig. 16). Golgi apparatuses were in close contact with the nuclei in the developing sporont (Fig. 18). Among the first structures to become distinct in the sporoblast was the polar filament with the attached polar sac, containing the primordium of the anchoring disc (Fig. 19). These structures were formed by the Golgi apparatus at the posterior end of the sporoblast, and they were pushed forwards when the length of the filament increased. Transverse sections of the developing filament showed an electron dense center surrounded by a moderately electron dense ring and a unit membrane cover (Fig. 19). Apparently the Golgi apparatus also generated a vacuole with electron dense content. It occurred in various positions in the immature spore (Figs. 20-21). However, at the end of spore maturation it became localized to the posterior pole together with the deteriorating Golgi apparatus. The polaroplast was initiated in the immature spore when the polar filament had reached the anterior pole and had begun coiling up posteriorly. As the size of the polaroplast increased, the filament coils were pushed backwards becoming closely arranged in the posterior half of the spore. The initial polaroplast was not divided into two regions, although the lamellae were somewhat wider at the posterior pole.
The Mature Spore The spore wall was three-layered with exospore, endospore and plasma membrane (Figs. 23-24).The exospore was about 30 nm thick, with an internal electron dense layer (14-18 nm) and an external double layer. The endospore was 74-96 nm thick, except at the anterior pole where the thickness was reduced to 30-40 nm. The plasma membrane was approximately 8 nm thick. The polar filament was isofilar with a diameter of 85-99 nm. It was arranged in 13-16 coils which, in the longitudinally sectioned spores, were seen as a slightly irregular single row close to the spore wall in the posterior half of the spore (Figs. 23, 25). The angle of tilt of the anterior coil to the long axis of the spore was 70°-75°. The transversely sectioned filament exhibited a series of concentricallayers (Fig. 26a-e). From the uniformly electron dense center (38-46 nm in diameter), in direction outwards, the layers were: an electron lucent, fibrillous layer (8-10 nm thick), a narrow, moderately dense layer interrupted by a narrow zone of more electron lucent material
and, finally, a narrow distinctly dense layer. These layers were together 7-9 nm thick. The unit membrane cover of the filament was about 5 nm thick. The diameter of the filament increased close to the anchoring disc, forming an approximately 110 nm long and 140 nm wide attachment section. Close to the anchoring disc the electron lucent layer of the polar filament widened slightly, forming a chalice-like structure. Inside this structure, the diameter of the electron dense center increased until it finally connected with the anchoring disc. In longitudinal sections of the spore, only one layer was distinctly seen in the anchoring disc (Figs. 23, 27). The polaroplast, which occupied the anterior third of the spore, surrounded the anterior part of the polar filament, ending at the level of the first coil (Fig. 27). It was divided into two almost equally long and well defined regions. The anterior polaroplast had 9-11 nm thick, closely packed lamellae filled with electron dense material, while the posterior part consisted of up to 27 nm wide, less densely packed and more irregularly arranged lamellae containing less electron dense material. The polar sac extended backwards for about 1/4 of the spore length (Fig. 27). It enclosed the anchoring disc and covered the anterior part of the polaroplast like an umbrella. The sac was filled with granular material, which increased in density towards the membrane. The approximately 5 nm thick polar sac membrane was continuous with the membranes of the polar filament and the lamellae of the polaroplast. The electron dense cytoplasm contained polyribosomes (Fig. 27), which were localized mainly around the nucleus and the polar filament and below the plasma membrane. The membrane-lined posterior vacuole was filled with electron dense material. The largest sectioned vacuole was 0.74 J.tm in diameter. The large nucleus was situated in the region of the polar filament coils. The shape was bent and irregular, making it difficult to measure, but at least up to 0.9 J.tm wide nuclei were seen.
The Sporophorous Vesicle The sporophorous vesicle was initiated like tiny blisters from the sporont already when it was binucleate (Fig. 14). When sporoblast formation was completed, the sporophorous vesicle contained a lot of debris from the sporogony. When the sporoblasts matured into spores the debris aggregated and became more organized (Fig. 19). The envelope of the mature sporophorous vesicle was an about 8 nm thick, uniformly electron dense layer which followed the outline of the spore group (Fig. 22). The vesicle was not persistent and mature spores were released already in the host.
Figs. 13-17. Presporal stages. - Fig. 13. Meront with two nuclei visible. - Fig. 14. Sporont with two nuclei visible and with protruding ~ blisters of the sporophorous vesicle. - Fig. 15. Lobed sporogonial plasmodium with the first signs of the sporont wall visible. Figs. 16-17. Lobed sporogonial plasmodia; the rough endoplasmic reticulum is organized into concentric layers around the nuclei; inset shows a centriolar plaque (arrowhead), mitotic spindle tubules (thin arrow) attached to chromatin (wide arrow). N
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Flabelliforma ostracodae n. sp. . 285
Figs. 23-27. Ultrastructure of the mature spore. - Fig. 23. Longitudinally sectioned spore. - Fig. 24. The spore wall. The exospore consists of an external double layer (arrowhead) and an internal electron dense layer (arrow). - Fig. 25. Transversely sectioned polar filament coils; the anterior coil is surrounded by lamellae of the posterior polaroplast. - Fig. 26. Transversely sectioned polar filament coil, exhibiting the layers: a = electron dense center; b = electron lucent, fibrillous layer; c = moderately dense layer; d = zone of more electron lucent material followed by a narrow dense layer; e = unit membrane cover. - Fig. 27. The anterior part of the spore, showing the subdivisions of the polaroplast: thin, electron dense lamellae anteriorly, wider, more electron lucent lamellae posteriorly. AD = anchoring disc, AP = anterior polaroplast, EN = endospore, N = nucleus, PF = polar filament, PM = plasma membrane, PP = posterior polaroplast, PR = polyribosomes, PS = polar sac, SV = sporophorous vesicle, V = vacuole. Scale bars:
~ Figs. 18-22. Sporoblast and maturing spores. - Fig. 18. Sporogonial plasmodium in the final phase of releasing sporoblasts; Golgi
apparatuses (arrowheads) are visible close to the nuclei. - Fig. 19. Sporoblasts. At this stage the debris of the sporophorous vesicle appears as small granules (arrows). - Figs. 20-21. Immature spores. The electron dense vacuole occurs in various positions in the immature spore. - Fig. 22. Mature spores within a sporophorous vesicle. AD = anchoring disc, EN = endospore, EX = exospore, GA = Golgi apparatus, N = nucleus, PF = polar filament, V = vacuole. Scale bars: Figs. 18-20, 22 = 1000 nm; Fig. 21 = 250 nm.
286 . A. M. Bronnvall and]. I. R. Larsson
Discussion
It must be concluded that the microsporidium treated by us is a new species. Although the only other microsporidian species described from an ostracode, Vavraia cyclocypris, forms numerous uninucleate sporoblasts by rosettelike budding within a sporophorous vesicle, similarly to the sporogony observed by us, the differences are obvious: V. cyclocypris has smaller (about 3.4 x 1.7 !-tm in fresh smears) and more pointed spores; the exospore is described as two layered, even if Fig. 2 a in reference [11] indicates that the layering might be more complex; and the polar filament is anisofilar, with 2-3 thick coils, one intermediate coil, and 4-6 thin coils, while the polar filament of the Swedish microsporidium is isofilar [11]. However, we do not believe that it is necessary to create a new genus. Of the more than 100 microsporidian genera described until the present time, only five combine life cycles with isolated nuclei with polysporoblastic sporogony yielding 16-32 spores, like it is seen in the present microsporidium: Agglomerata, Vavraia, Tardivesicula, Duboscqia and Flabelliforma. Three of them can easily the disregarded: Agglomerata, because of the five-layered exospore, and the uniquely constructed polaroplast with three regions [6]; Vavraia, because of the thick merontogenetic, sporophorous vesicle and the simultaneous production of micro- and macrospores [1, 10]; and finally Tardivesicula, because of the rod-shaped spores and the delayed initiation of the sporophorous vesicle [5]. The genus Duboscqia is problematic. The type species D. legeri, which is a parasite of termites and hence found in terrestrial environment, was described using light microscopy, and there are no further studies providing ultrastructural data. The genus is poorly defined: spores are ovoid-ellipsoid, the sporogony yields 16 spores, and infected cells become extremely hypertrophic [4, 7]. The species studied by us produces mainly 32 spores per vesicle, and since it utilizes an aquatic host we hesitate to include it in the genus Duboscqia. The remaining genus, Flabelliforma, was created by Canning and colleagues in 1991 [2]. When comparing the ultrastructure and life cycle of the Swedish microsporidium with the type species, F. montana, it is revealed that the sporogonies are virtually identical, and so are the cytological characteristics of the spores. Our microsporidium matches the characteristics of Flabelliforma, except that the sporophorous vesicles are initiated without blister formation in F. montana, but with distinct blister formation in the species studied by us. With increasing knowledge of the ultrastructural cytology of the microsporidia, the taxonomic use of the sporophorous vesicle seems to be decreasing. Applying the diagnosis of Flabelliforma strictly, the microsporidium described herein is excluded. However, we belive it is better to provisionally rank the new species in the genus Flabelliforma than to establish a new genus, if the only difference between the two genera is the way the sporophorous vesicle is initiated. It is obvious that the species studied by us is different from F. montana, which has broader spores (2.9 x 1.9 !-tm when stained), and a short polar filament (3.5 coils). It is
also possible that the species differ in endospore thickness. The sporoblasts and spores of F. montana were distorted by the techniques used, making a detailed comparison impossible, but illustrations suggest that exo- and endospore layers of F. montana are equally thick. Our conclusion is that the microsporidium of Candona is a new species, and we believe that a ranking in Flabelliforma is the best solution of the genus problem. The most appropriate position for the genus seems to be the family Duboscqiidae Sprague, 1977.
Description
Flabelliforma ostracodae n. sp. Merogony: Rounded plasmodia with isolated nuclei produce up to 16 merozoites by plasmotomy. Sporogony: Multinucleate plasmodia with isolated nuclei produce 16-32 sporoblasts by rosette-like budding within a sporophorous vesicle. Meiosis was not observed. Spore: Oval, with a slightly pointed anterior pole. Fresh spores measure about 3.8 x 2.7 !-tm, fixed and stained spores 3.0 - 3.6 x 1.4 !-tm. The exospore is about 30 nm thick. It has an internal electron dense layer and an external layer which resembles a double membrane. The endospore is 74-96 nm thick (thinner at the anterior pole). The polar filament is isofilar with 13-16, 85- 99 nm thick, coils arranged in a single row in the posterior half of the spore. The lamellar polaroplast occupies the anterior third of the spore. It has two almost equally long, well defined regions, the posterior one with wider lamellae. Sporophorous vesicle: Uniformly electron dense, eight nm thick, following the outline of the enclosed sporegroup. No tubular inclusions. The vesicle disrupts within the host. Host tissue involved: Adipose and connective tissue. Type host: Candona sp. Baird, 1846 (Ostracoda, Cyprididae) Type locality: Flooded meadows close to the river Hoje ii, Esarp, Scania in the south of Sweden. Type series: Holotype (Fig. 12) on slide no. 890108Hl-2 RL, paratypes on slides no. 890108-Hl-(l-2) RL. Deposition of type: The slide with the holotype in the International Protozoan Type Slide Collection, Smithsonian Institution, Washington, D.C., USA. The second slide in the collection of the senior author.
Acknowledgements The authors are greatly indebted to Mrs Lina Hansen, Mrs Birgitta Klefbohm and Mrs Inger Norling, all at the Department of Zoology, and to Mrs Agneta Persson at the Department of Anatomy, University of Lund, for skilful technical assistance. The investigation was supported by research grants from the Swedish Natural Science Research Council and Helge Ax:on Johnson's Foundation.
Flabelliforma ostracodae n. sp.. 287
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6 Larsson]. I. R. and Yan N. D. (1988): The ultrastructural cytology and taxonomy of Duboscqia sidae ]IROVEC, 1942 (Microspora, Duboscqiidae), with establishment of the new genus Agglomerata gen. nov. Arch. Prot,istenkd., 135, 271-288. 7 Perez c. (1908): Sur Duboscqia legeri, Microsporidie nouvelle parasite du Termes lucifugus, et sur la classification des Microsporidies. C. R. Soc. BioI., 65, 631-633. 8 Reynolds E. S. (1963): The use oflead citrate at high pH as an electron-opaque stain in electron microscopy.]. Cell BioI., 17, 208-212. . 9 Romeis B. (1968): Mikroskopische Technik. Oldenbourg Verlag, Miinchen und Wien. 10 Weiser j. (1977): Contribution to the classification of Microsporidia. Vestn. Cs. spol. zool., 61, 308-320. 11 Voronin V. N. and Melnikova O. Y. (1984): Vavraia cyclocypris sp. n., the first find of a microsporidian parasite from Ostracoda (Crustacea). Parazitologiya, 18, 482-484.
Key words: Flabelliforma ostracodae n. sp. - Microsporidia - Ultrastructure - Taxonomy - Ostracoda Annika Bronnvall, Department of Zoology, University of Lund, Helgonaviigen 3, S-223 62 Sweden