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Ultrastructural study of Glugea cladocera pfeiffer, 1895, and transfer to the genus Agglomerata (microspora, duboscqiidae)
Europ. ]. Protisto\. 32, 412 - 422 (1996) December 31, 1996
European Journal of
PROTISTOLOGY
Ultrastructural Study of Glugea cladocera Pfeiffer, 1895, and Transfer to the Genus Agglomerata (Microspora, Duboscqiidae) J. I. Ronny Larsson1, Dieter Ebert 2 , and Jiff Vavra 3 1Department of Zoology, University of Lund, Sweden 21nstitute of Zoology, Basel University, Switzerland 3Department of Parasitology, Charles University, Prague, Czech Republic
SUMMARY The microsporidium Glugea cladocera Pfeiffer, 1895, a parasite of the microcrustacean Daphnia magna, is redescribed based on light microscopic and ultrastructural characters. All life cycle stages have isolated nuclei. Merogonial plasmodia are elongated with a small number of nuclei. Sporogonial plasmodia divide by rosette-like division, producing a variable number of sporoblasts, usually 8 (4-16). A sporophorous vesicle, initiated at the beginning of the sporogony, collects all daughter cells of the sporont. Fibril-like projections connect the primordial exospore layer of the sporont with the sporophorous vesicle. The fibrils remain as a surface coat of mature spores. When sporoblast buds are formed, a second type of projections appear in the episporontal space. These are temporary tubules of exospore material, which disappear when the spores mature. The exospore of the mature spore is layered, with an internal layer resembling a double membrane. The polaroplast has three regions: wide lamellae, narrow lamellae and tubules. The polar filament is lightly anisofilar with 1- 2 wide anterior, and 4- 3 narrow posterior coils, arranged in one layer of coils in the posterior half of the spores. The identification of the species is discussed. Otto Jirovec, who redescribed the species in 1936, transferred it to the genus Thelohania. Comparison with slides in the collection of Prof. Otto Jirovec, Prague, revealed that the microsporidium studied by us apparently is identical to Jirovec's Thelohania cladocera from Daphnia magna, but it is not identical to his T. cladocera from D. pulex. Based on life cycle characters and the ultrastructure, the species is transferred to the genus Agglomerata.
Abbreviations A
E EX
ER
F G N P1-P3
PM PS SV
- anchoring disc endospore - exospore - endoplasmic reticulum = polar filament - Golgi vesicles - nucleus = anterior (l), median (2) and posterior (3) polaroplast region = plasma membrane polar sac sporophorous vesicle
0932-4739-96-0032-0412$3.50-0
T
V
tubules of exospore material posterior vacuole
Introduction Pfeiffer (1895) described the new microsporidum Glugea cladocera II, a parasite of the fat body and hypodermis of cladocerans of the genera Daphnia and Limnetis in Germany [11]. The description is typical for its time, with a minimum of diagnostic characters. The situation today, with knowledge of a great and increasing number of microsporidian parasites © 1996 by Gustav Fischer Verlag, Stuttgart
Ultrastructure of Agglomerata cladocera comb. nov. . 413 of Cladocera, has actually made this diagnosis useless for discrimination of the species. ]irovec, the first reviser, gave a more detailed account, based on his own observations of the microsporidium from the host Daphnia magna with provenance from Moravia, and he transferred the species to the genus Thelohania [4]. In a later publication Daphnia pulex, from a locality in Bohemia, was added to the list of hosts [5]. Pfeiffer's slides could not be traced. They have probably been destroyed, and we will probably never see the microsporidian cells that were described by him. ]irovec's slides have survived in Prague and they have been used for an evaluation. Recent investigations on the ecology and interactions between microsporidia and Cladocera have revealed a number of microsporidian infections and shown that a population sometimes hosts a mixture of microsporidian species [6, 15]. Also Daphnia magna hosts several microsporidia, and in our investigations we have frequently observed two species occurring together, one using the hypodermis, the other one primarily developing in the fat body (unpublished observations). We have further studied ]irovec's slides and noticed that the microsporidium identified by him to be Thelohania cladocera (Pfeiffer, 1895) actually is two species, one in Daphnia magna, another one in D. pulex. This report treats the ultrastructural cytology of the microsporidium of the hypodermis, which we have found to be Glugea cladocera II Pfeiffer, 1895. The identification and the genus position are discussed.
Material and Methods Infected specimens of Daphnia magna were collected in October 1992 from a small farm pond in northern Oxfordshire, England, close to the village of Ambrosden (about 15 kilometres north-east of Oxford, Northern Latitude 51 °52'80", Western Longitude 1°7'20". This corresponds to the Bicester population discussed in [1]). In our collection more than 50% of all adult D. magna were infected. In contrast to other microsporidia of D. magna [9] we were not able to maintain the infection in the laboratory [1, 9]. The second sample was collected in July 1993 from the same pond. Fresh squash prepar~tions were made by the agar method of Hostounsky and Zizka [3] and studied using phase contrast microscopy and dark field illumination. Permanent squash preparations were lightly air-dried and fixed in methanol for at least 15 min for Giemsa staining, or in Bouin-Duboscq-Brasil (BDB) solution for at least one hour previous to staining with Heidenhain's iron haematoxylin. For paraffin sectioning whole animals were fixed in BDB solution overnight or longer. After washing and dehydration in a graded series of ethanols, specimens were cleared in butanol and embedded in Paraplast (Lancer St. Louis, MO, USA). Sections were cut more or less longitudinally at 10 11m and stained with haematoxylin. For details on the histological techniques used see the manual by Romeis [13]. All permanent preparations were mounted in D.P.X. (BDH Chemicals Ltd., Poole, England). Measurements were made with an eye-piece micrometer at x 1,000 or using an image analysis program (Micro Macro AB, Gothenburg, Sweden).
For transmission electron microscopy, hosts were cut in halves and fixed in 2.5% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 36 h. After washing in cacodylate buffer and post fixing in 2% (w/v) osmium tetroxide in cacodylate buffer for 1 h at 4°C, the specimens were washed and dehydrated in an ascending series of bufferacetone solutions to absolute acetone and embedded in epon. Sections were stained using uranyl acetate and lead citrate [12].
Five slides with Thelohania cladocera originating from the collection of Otto Jirovec, Prague, were used for comparison: 1. Sections stained by haematoxylin and eosin. Label: "Thelohania c1adocera D. m. Lohovec". 2. Sections stained by haematoxylin. Label: "Thelohania c1adocera D. m. Lohovec" and added in ink by Jirovec's hand: "Thelohania cladocera. Heidenh." 3. Smear stained by Giemsa. Two labels. Labell: "Thelohania cladocera D. m. Lohovec". Label2 (Jirovec's hand): "Daphnia magna Lohovec 1935 Thelohania". Added in ink on the slide: "3-411 x 2". 4. Smear stained by Giemsa. Label: "Thelohania c1adocera D. m. Lohovec". Added in ink by Jirovec's hand: "Dafnia pulex Thelohania cladocera Stary kveten 1937". 5. Smear stained by Giemsa. Label: "Thelohania c1adocera". Added in ink by Jirovec's hand: "Dafnia pulex Thelohania c1adocera Stary kveten 1937".
Results
Prevalence and pathology Hypodermic cells in all parts the body, including the appendages, were invaded by the microsporidium. In specimens with less severe infection, the cells harbouring microsporidia appeared as isolated islands (Figs. 1, 3). The head region of the host seems to become infected last, possibly an adaptation of the parasite not to kill before the rest of the body is utilized. Specimens with advanced infection had wide areas where practically every cell was filled with microsporidia (Fig. 2). Infected cells were markedly hypertrophic, often twice as large as uninfected cells and completely packed with mostly sporulated microsporidia. Hypertrophic host nuclei were not observed. In a part of the material a second microsporidium occurred in the adipose tissue (Fig. 1).
Presporal stages and life cycle The presporal development was nearly complete in all specimens studied, and only in the specimens used for electron microscopy were modes of reproduction visible. All life cycle stages had isolated nuclei. Merogonial plasmodia were elongated and always had a small number of nuclei with a uniform moderately electron dense karyoplasm (Fig. 4). Sectioned merogonial nuclei measured up to 1.6!-tm (Fig. 4). Merogonial stages were delimited by an approximately 7 nm thick plasma membrane without external reinforcements. Free ribosomes gave the cytoplasm a uniformly granular appearance. There were only traces of an endoplasmic reticulum. Probably the merogonic reproduction was weak. Short chains with up to
Ultrastructure of Agglomerata cladocera comb. nov.. 415
three merozoites were observed. It is unknown jf there is more than one bour of merogony. Sporonts were uninucleate with a central nucleus and with densely granular cytoplasm where the layers of endoplasmic reticulum were arranged concentrically around the nucleus (Fig. 5). At the beginning of sporogony tubular or blister-like protrusions of thin electron-dense material appeared at the surface (Figs. 56). They were the primordia of the sporophorous vesicle. They were originally irregularly shaped, and the episporontal space contained a uniform and moderately electron-dense material. Later the primordia widened to distinct blisters, containing granular (or possibly fibrous) material resembling ribosomes but the granules were somewhat smaller than ribosomes (Figs. 5-6). Scindosome-like bodies, involved in the production of wall material, were visible in sporonts (Fig. 7). The blisters lost contact with the sporogonial plasmodium and formed a continuous sporophorous vesicle (Fig. 8). New electron-dense material appeared on the plasma membrane inside the blisters and it developed directly into a layered structure about 20 nm thick, where two electron-dense layers were separated by a lucent zone (Figs. 8-9). During the maturation from sporogonial plasmodium to sporoblasts the internal exospore layer changed into a double layer-like structure c. 6 nm thick where the external layer was somewhat more electron-dense (Figs. 9-10). A growing electron lucent gap was formed between the exospore and the plasma membrane (Figs. 9-10). It might be taken for a fixation artifact, but it was in fact the early initiation of the endospore layer. Simultaneously with the development of the sporophorous vesicle, nuclear division generated a plurinucleated plasmodium (Fig. 8). Signs of meiois were not observed. Later the nuclei accumulated at the periphery, the plasmodium split into buds in a rosette-like fashion, with one nucleus in each bud (Fig. 8), and finally it divided, usually into 8 sporoblasts (4, 8, 10, 12, 14, and 16 sporoblasts per sporont were observed). Nuclei of undivided and rosette-like plasmodia were of the same size, in sections measuring up to 1.3 lim. The granular material of the episporontal space transformed into distinct fibrils, or possibly tubules filled with electron-dense material, connecting the c. 5 nm thick envelope of the sporophorous vesicle with the exospore (Figs. 8-9). Lobed plasmodia and sporoblasts carried approximately 9 nm wide fibrils (Figs. 10-12, 14). When the sporogonial plasmodium began splitting into lobes, a second type of projection, formed from excess exospore material, appeared in the epis-
porontal space (Figs. 8, 12-13). These were approximately 43-45 nm wide tubules exhibiting the layers of the exospore (Fig. 8 inset). The tubules remained in sporophorous vesicles containing young sporoblasts but their central cavity grew wider and diameters up to 117 nm were measured. Finally the wide tubules disappeared (Fig. 14) and simultaneously the fibril-like connections between the exospore and the wall of the sporophorous vesicle were broken, which made the episporontal space almost devoid of inclusions (Figs. 14-16). Fibril-like material remained as an episporal coat. The initiation and morphogenesis of the spore organelles was normal for microsporidia.
The mature spore Live mature spores were pyriform with a pointed anterior pole (Fig. 17). The only internal structure visible was a small posterior vacuole in oblique position. Fixed and stained spores appeared more blunt (Fig. 19). Unfixed spores measured 1.5 -2.2 x 3.04.51lm (n = 25); fixed and stained spores were 1.62.1 x 2.7 -3.4llm (n = 50). Macrospores were not observed. Sporophorous vesicles were fairly persistent, and oval vesicles were observed in carefully made preparations (Fig. 18). Unfixed octosporous sporophorous vesicles were about 4 x 7 Ilm long. The wall of the mature spores measured 159165 nm, except anteriorly where it was considerably thinner, down to 55 nm (Fig. 22). It had the normal three subdivisions: exospore, a wide lucent endospore (less wide anteriorly), and an internal about 7 nm thick plasma membrane (Fig. 23). The endospore and exospore layers are built on the plasma membrane, and the three layers remain as a unit, which means that the plasma membrane forms the internal layer of the spore wall even after the sporoplasm has been ejected from the spore. The exospore, which measured approximately 24 nm, had a constant sequence of layers, in direction inwards: an electron-dense, c. 5 nm thick surface layer, a translucent median layer (11 nm), and an internal, c. 8 nm wide layer resembling a double-membrane. Diffuse granular material formed the border with the electron lucent endospore layer. The surface was covered by a coat of 5 -6 nm wide projections extending 275 nm outwards. In longitudinal sections they appeared as two electron-dense strands with lucent material in between (Fig. 23). The polar filament was lightly anisofilar (Fig. 22). It was anteriorly connected to an up to 425 nm wide, biconvex, layered anchoring disc. A polar sac of narrow
.... Fig. 1- 6. Pathogenicity and early development of Agglomerata cladocera comb. nov. - Fig. 1. Section through a part of an adult specimen of Daphnia magna with islands of A. cladocera (M1) in the hypodermis, and a second microsporidium (M2) in the fat tissue close to the gut (*). - Fig. 2. Hypodermic tissue filled with spores of A. cladocera. - Fig. 3. Section through the hypodermis exhibiting sporophorous vesicles. - Fig. 4. Chain-like formation with one uninucleate and one binucleate meront.Fig. 5. Young sporont exhibiting the initiation of the sporophorous vesicle. - Fig. 6. Intiation of the sporophorous vesicle. Figs. 1,3. Haematoxylin staining; Fig. 2. Phase contrast. Scale bars: Fig. 1 = 100 !-lm; Figs. 2-3 = 10 !-lm; Fig. 4 = l!-lmj Fig. 5 = 0.5 !-lm; Fig. 6 = 100 nm.
Ultrastructure of Agglomerata cladocera comb. nov. . 417 umbrella-like construction enclosed the anchoring disc and the anterior half of the anterior polaroplast (Figs. 22,24). The most proximal part of the filament was a short attachment section, about 100 nm long. The diameter in this region was about 205 nm. The uncoiled part of the filament, measuring about 125 nm in diameter, proceeded backwards in the midline of the spore, then turned lightly to the side touching the spore wall about 1/3 from the posterior pole of the spore. The final section was arranged as 5 -6 coils in one row of coils close to the spore wall in the posterior half of the spore. The coils were tilted in anterior direction, and the angle of tilt of the most anterior coil to the long axis of the spore was 50-55°. The 1-2 most anterior coils were slightly wider, 84-117 nm, and the 3-4 posterior coils were 55 -79 nm in diameter. The coiled part occupied about 1/3 of the spore length. The filament was composed of layered material where the following subdivisions could be discriminated (Fig. 26: 1-6). An external about 5 nm thick unit membrane (1) was followed by a c. 3 nm distinct electron dense layer (2), a c. 6 nm layer of moderately dense material (3), an approximately equally wide layer of dense material, which was reduced in the narrow coils (4), a moderately dense zone, approximately twice as wide, in which a fibrous structure could be observed (5), and the centre, which was clearly wider in the wide coils, with indistinct layering (6). The very centre of the wide coils was electron-dense (Figs. 22, 26). The polaroplast had three subdivisions, where each region was composed of compartments of specific shape, delimited by approximately 5 nm thick unit membranes (Fig. 22). The polaroplast enclosed the uncoiled part of the polar filament. The anterior part was composed of wide chambers of variable height (anterior-posterior distance, up to 163 nm), filled with a uniform moderately electron-dense material (Fig. 24). In this part, which constituted about one half of the polaroplast, anastomoses were frequent. The following section was shorter, about 1/4 of the polaroplast length, with closely packed lamellae (Fig. 25). The periodicity was about 13 nm. There was a distinct gap between the last lamellae and the final 1/4 of the polaroplast (Fig. 25). In the last section the compartments were tubular, measuring 23 - 25 nm in diameter (Fig. 26). The surface layer of the polar filament, the compartments of the polaroplast, and the polar sac were all parts of the same membrane system. The rounded nucleus occupied the posterior half of the spore, inside the filament coils (Fig. 22). The largest
section of nucleus measured 1.1 11m. The cytoplasm was dense with free ribosomes. There were also strands of polyribosomes in the proximity of the nucleus and the polaroplast, but the layers were less prominent than usual in microsporidian spores. Discussion Microcrustaceans host a wide variety of microsporidian species and many of these belong to the early history of the study of microsporidia. The descriptions are too superficial to allow clear identification and the species are difficult, or sometimes impossible, to compare with more recently described taxa. Type material can rarely be traced. There are only two solutions to the taxonomic problem caused by these early species, either to leave them as nomina nuda, or to use the names arbitrarily and make redescriptions based on new material. The last alternative is preferred by the International Commission on Zoological Nomenclature [10]. Pfeiffer described Thelohania cladocera in 1895, using the name Glugea cladocera II [11]. The hosts were microcrustaceans of the genera Daphnia and Limnetis with provenance from Greifswald, Germany. It is unclear if the microsporidium was found in more than one Daphnia species. The species mentioned is D. pulex. We have not been able to trace Pfeiffer's slides, which might have solved the problems with the hosts. The pathogenesis was described and illustrated (in Limnetis). The spore size was indicated to be 2-3 11m, and it was mentioned that the spores were formed in sporophorous vesicles ("Sporoblasten") measuring 3-4 11m in diameter, and containing 8, 16 or more spores. It was also mentioned that simultaneously with this microsporidium another species, Glugea leydigii, occurred in the fat body. Jirovec gave a more detailed description, with illustrations, of Glugea cladocera, based on parasites of D. magna collected in Moravia [4]. Later D. pulex collected in Bohemia was added to the list of hosts [5]. He used Thelohania as the genus name [4] and described the spores as pyriform, lightly pointed anteriorly, and recorded the size of li¥ing spores to be 1.5 -1.8 x 33.5 11m. Illustrations of spores (Figs. 1 e-k in [4]) raise the question whether there were two different microsporidia present in the samples. The spores in Fig. 1 f appear more blunt than the spores of Figs. 1 g-k. Sporophorous vesicles contained only 8 spores. Using Giem-
... Fig. 7-13. Sporogony of A. cladocera - Fig. 7. Periphery of a sporont showing one scindosome (arrow). - Fig. 8. Undivided (bottom) and rosette-like dividing sporogonial plasmodia; exospore-derived tubules in the vesicle with dividing plasmodium (inset shows longitudinally sectioned detail of tubules; arrow indicates episporontal space devoid of tubules). - Figs. 9-10. Cell periphery of the sporogonial plasmodium and the sporoblast. - Fig. 11. Sporogonial plasmodium in the process of forming lobes; exospore layer not complete (arrows); only fibril-like material in the episporontal space. - Fig. 12. Sporophorous vesicle with 8 sporoblasts visible; wide exospore-derived tubules in the episporontal space (arrow). - Fig. 13. Exospore-derived tubules at the sporoblast stage; arrowheads indicate connected fibrils. Scale bars: Figs. 7, 12-13, and inset on 8 = 100 nm; Figs. 8,11 = 11lm; Figs. 9-10 (with commom bar on 9) = 50 nm.
Ultrastructure of Agglomerata cladocera comb. nov. . 419
sa solution the spores exhibited one dark granule. Using the "Nuklealreaktion" (obviously Feulgen technique) two small spot-like nuclei were seen. The only other illustration existing of this species is a micrograph of living spores, revealing the presence of a prominent mucous coat [8]. Hazard and Oldacre revised the Thelohania-like microsporidia, and they considered T. glugea to be a dubious Thelohania species [2]. Sprague accepted their interpretation and transferred the species to the collective and unclassified genus Microsporidium [14]. Pfeiffer's type material has probably been destroyed. Jirovec;s collection has survived in Prague, and slides of the microsporidium that he identified as Thelohania cladocera have been studied. He found T. cladocera in two hosts: D. magna (Fig. 20) and D. pulex (Fig. 21). On one of the slides the host name and location written on the label do not correspond with later additions in ink written by himself. If the slides are compared, it is easily seen that the spores of the microsporidium of D. pulex are larger than the spores from D. magna and from the microsporidium treated herein. Stained nuclei were not visible in the slides from Jirovec's collection. The size of the spores, the number of sporoblasts per sporont, and the site of infection in the host correspond between Pfeiffer's description of Glugea cladocera II and the microsporidium treated herein. Pfeiffer reported that the hosts were cladocerans of two genera, Daphnia and Limnetis [11]. One host species, D. pulex, is mentioned in the text. No illustrations show microsporidia from this host. It is obvious that Jirovec's Thelohania cladocera of D. magna and D. pulex are different species. Size differences reveal that clearly. One of them, T. cladocera of D. magna, might be identical to the species treated herein. The spores are of the same size and approximately of the same shape. Octosporous sporophorous vesicles are also similar. However, two differences are obvious. Sporophorous vesicles in Jirovec's material were always octosporous, and the spores were said to be binucleate [4]. As the sporophorous vesicles are fairly fragile and break when smears are made, other spore configurations might have been overlooked. As it is not easy to make the nuclei of microsporidian spores distinctly visible in light microscopic preparations, one nucleus with grouped chromatin might give the impression of two nuclei. Thus, considering the short-
~
comings of Jirovec's techniques, too much emphasis should not be placed on the apparent difference in number of nuclei. We have reasons to believe that the microsporidium studied by us is the species Pfeiffer described as Glugea cladocera II, and it is obviously identical to Jirovec's Thelohania cladocera of D. magna. If we combine life cycle characters (all developmental stages with isolated nuclei, polysporoblastic sporogony by rosette-like budding, pyriform spores, sporontogenetic sporophorous vesicles uniting all daughter cells of the sporont) with ultrastructural characters (layered exospore, anisofilar polar filament, a polaroplast composed of three structurally different regions where the final region is tubular) one genus will be available: Agglomerata Larsson and Yan, 1988. This genus was established for a microsporidium considered identical to Duboscqia sidae Jirovec, 1942, which is a parasite of the same group of hosts [7]. We, therefore, propose to that T. cladocera should be transferred to the genus Agglomerata as Agglomerata cladocera comb. nov. A more detailed comparison between A. cladocera and A. sidae (AS), until now the only species of the genus, reveals further similarities: two kinds of projections present in the episporontal space, early initiation of the endospore, identical layering of the exospore and the polar filament and a uniquely constructed polaroplast. Even if the two species produce spores of similar shape and size, some differences, except for the different hosts, clearly tell that the species are different. A. cladocera has basically 8-sporoblastic sporogony (AS: 16-sporoblastic). The sporophorous vesicles are oval and the spore-groups break easily (AS: spherical sporophorous vesicles where the spores are tightly connected and difficult to separate). The projections present in the episporontal space are narrow fibrils, or possibly tubules, and wide exospore-derived tubules without particular arrangement (AS: uniform wider tubule-like structures and exospore-derived tubules in characteristic configurations). The spore is coated with fibrils, or tubules, which have lost their contact with the sporophorous vesicle (AS: tubule-like structures connect the spores to each other and to the sporophorous vesicle). The infection is restricted to the hypoderm (AS: spreading from the hypoderm to become a generalized infection).
Fig. 14-21. Sporoblasts and spores. - Fig. 14. Sporophorous vesicle with two sporoblasts visible; some radiating fibrils from the exospore (arrow); only fibrils in the episporontal space. The sporoblasts are sectioned close to the irregularly shaped surface, * indicates episporontaJ space visible through holes in the sporoblasts. - Fig. 15. Electron-dense immature spores; more dense layer of exospore fibrils. - Fig. 16. Sporophorous vesicle with mature spores; episporontal space almost empty. - Fig. 17. Unfixed spores. - Fig. 18. Unfixed sporophorous vesicles. - Fig. 19. Stained spores. - Fig. 20. Spores and sporophorous vesicles of Thelophania cladocera from Daphnia magna (from the collection of]irovec). - Fig. 21. Spores of T. cladocera from D. pulex (from the collection of ]irovec). Figs. 17-18. Phase contrast. Figs. 19-21. Giemsa stain. Scale bars: Figs. 14-16 = 0.5 /lm; Figs. 17-21 (with common bar on 19) = 5/lm.
Ultrastructure of Agglomerata cladocera comb. nov.. 421 Redescription
Agglomerata cladocera (Pfeiffer, 1895) comb. nov. Synonyms: Glugea cladocera II Pfeiffer, 1895; Thelohania cladocera (Pfeiffer, 1895) ]Irovec, 1936 of Daphnia magna, but not of D. pulex. (P) and (J) indicate characteristics taken from Pfeiffer's description [11] and Jirovec's redescription [4], (R) from this reinvestigation. Merogony. (R) Elongated merogonial plasmodia with isolated nuclei give rise to a small number of merozoites. Sporogony. (R) Sporont uninucleate. Sporogonial plasmodia with isolated nuclei divide by rosette-like division. Spore yield: (R) 4-16, usually 8; (P) 8, 16 or more; (J) 8. Spores. (R) Pyriform with pointed anterior pole. Living spores measure 1.5 - 2.2 x 3.0 -4.5 11m, fixed and stained 1.6-2.1 x 2.7-3.4 11m. Spore wall 159165 nm thick, with an approximately 24 nm thick, layered exospore, coated by densely arranged fibrillike material. Polar filament lightly anisofilar with 1-2, 84-117nm wide, anterior coils and 3-4, 5579 nm wide, posterior coils in one row of coils in the posterior half of the spore. The angle of tilt is 50-55°. The polaroplast, which surrounds the uncoiled part of the polar filament, has three subdivisions: wide lamellae, narrow lamellae and, separated from them by a gap, tubules. A single nucleus inside the filament coils. (P) Spore size 2-3 ~lm. (J) Spores pyriform with pointed anterior pole. Living size 1.5 -1.8 x 3-3.5 11m. " ... wir sehen nach der Nuklealreaktion in reifenden und auch reifen Sporen in ihrer Mine zwei deutliche punktformige Kerne". Sporophorous vesicle. (R) Round to ovoid, measuring approximately 4 x 711m when living, unites all sporoblasts in a common vesicle. Episporontal space traversed by narrow fibrous material, which remains as the exospore coat of the mature spores, and wide tubules with walls of exospore material, which are formed when the plasmodium splits into lobes and disappear during the maturation of the spores. (P) 3 -4 11m in diameter. Host tissues involved. Hypodermal cells all over the body. Hosts. (P) Daphnia and Limnetis, (J) and (R) Daph-
nta magna. Type locality: Type locality not specified by Pfeiffer,
but material from Greifswald (Germany) is used for illustration. Locations of the material studied: (J) A village pond in Lohovec (Moravia), the pond Stary (Bohemia); (R).
A farm pond near the village of Ambrosden, about 15 km north-east of Oxford (UK). Deposition of studied material: (P) The collection of Pfeiffer has not been traced. (J) The collection of ]irovec is kept at the Department of Parasitology, Charles University, Prague, (R) The material used for the redescription is kept in the collection of Larsson, Department of Zoology, University of Lund: No. 921218-(A-C) and 930914-(T-V).
Acknowledgements The authors are greatly indebted to Mrs. Lina Gefors, Mrs. Brigitta Klefbohm and Mrs. Inger Norling, all at the Department of Zoology, University of Lund, for excellent technical assistance, and to Dr. Josef Chalupsky for making the slides in the collection of Jirovec available for study. The investigation was supported by research grants from the Royal Society, London (to D. E.), the Swedish Natural Science Research Council (to R. L.), and from the Royal Physiographic Society, Lund (to R. L.).
References 1 Ebert D. (1995): Virulence and local adaptation of a horizontally transmitted parasite. Science, 265, 1084-1086. 2 Hazard E. I. and Oldacre S. W. (1975): Revision of Microsporidia (Protozoa) close to Thelohania, with descriptions of one new family, eight new genera, and thirteen new species. U. S. Dept. Agri. Tech. Bul!. No. 1530, U. S. Department of Agricultl,lIe, Washington, D. C. 3 Hostounsky Z. and Zizka Z. (1979): A modification of the "agar cushion method" for observation and photographic recording microsporidian spores. ]. Protozool., 26 (supp!.), 41A-42A., Abstr. No. 117. 4 Jirovec O. (1936): Dber einige in Daphnia magna parasitierenden Mikrosporidien. Zoo I. Anz., 116, 136 -142. 5 Jirovec O. (1943);. Cizopasnici nasich perloocek. I. Mikrosporidie. Yest. Cz. spo!. zoo!., 23 (1942), 1-11. 6 Larsson]. I. R., Ebert D., Vavra J., and Voronin V. N. (1995): Redescription of Pleistophora intestinalis Chatron, 1907, a microspori dian parasite of Daphnia magna and Daphnia pulex, with establishment of the new genus Glugoides (Microspora, Glugeidae). Europ. J. Protisto!., 31,251-261. 7 LarssonJ. I. R. and Yan N. D. (1988): The ultrastructural cytology and taxonomy of Duboscqia sidae Jirovec, 1942 (Microspora, Duboscqiidae), with establishment of the new genus Agglomerata gen. nov. Arch. Protistenkd., 135,271-288. 8 Lorn J. and Vavra J. (1963): Mucous envelopes of spores of the subphylum Cnidospora (Doflein, 1901). Yest. Cz. spo!. zoo!., 27, 4-6.
.... Fig. 22 - 26. Ultrastructure of the mature spore. - Fig. 22. Longitudinally sectioned spore with dense fibril-like exospore coat. Fig. 23. Close-up of the spore wall (arrows indicate the double-layer of the exospore, black arrowheads structural details of the fibrils). - Figs. 24-25. Longitudinally sectioned details of the two anterior polaroplast regions (arrow heads indicate the gap separating P2 and P3). - Fig. 26. Posterior region of the polaroplast and transversely sectioned polar filament coils (layers indicated 1-6; arrow heads indicate differences in the centre). Scale bars: Fig. 22 = 0.5 11m; Figs. 23-26 = 100 nm.
422 . ]. I. R. Larsson, D. Ebert, and]. Vavra 9 Mangin K. L., Lipsitch M., and Ebert D. (1995): Virulence and transmission modes of two microsporidia in Daphnia magna. Parasitology (in press). 10 Melville R. V. (1980): Nomina dubia and available names. Z. Parasitenkd., 62, 105 -109. 11 Pfeiffer L. (1895): Die Protozoen als Krankheitserreger. II. Nachtrag, pp. 35 -74. Jena. 12 Reynolds E. S. (1963): The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. ]. Cell Bio!., 17,208-212.
13 Romeis B. (1968): Mikroskopische Technik. Oldenbourg Verlag, Miinchen-Wien. 14 Sprague V. (1977): Classification and phylogeny of the Microsporidia. In: Bulla Jr. L. A. and Cheng T. C. (eds.): Comparative pathobiology, vol. 2, pp. 1-510. Plenum Press, New York and London. 15 Stirnadel H. A. (1994): The ecology of three Daphnia species-their microparasites and epibionts. Diploma thesis, University of Base!.
Key words: Agglomerata cladocera comb. nov. - Microspora - Ultrastructure - Taxonomy - Daphnia magna Ronny Larsson, Department of Zoology, University of Lund, Helgonav. 3, S-223 62 Lund, Sweden, Fax: +46-46-2224541, e-mail: [email protected];DieterEbert.InstituteofZoology.BaselUniversity.Rheinsprung9.CH-4051 Basel, Switzerland; Jifi Vavra, Department of Parasitology, Charles University, Vinicna 7, 128 44 Prague 2, Czech Republic
European Journal of
PROTISTOLOGY
Ultrastructural Study of Glugea cladocera Pfeiffer, 1895, and Transfer to the Genus Agglomerata (Microspora, Duboscqiidae) J. I. Ronny Larsson1, Dieter Ebert 2 , and Jiff Vavra 3 1Department of Zoology, University of Lund, Sweden 21nstitute of Zoology, Basel University, Switzerland 3Department of Parasitology, Charles University, Prague, Czech Republic
SUMMARY The microsporidium Glugea cladocera Pfeiffer, 1895, a parasite of the microcrustacean Daphnia magna, is redescribed based on light microscopic and ultrastructural characters. All life cycle stages have isolated nuclei. Merogonial plasmodia are elongated with a small number of nuclei. Sporogonial plasmodia divide by rosette-like division, producing a variable number of sporoblasts, usually 8 (4-16). A sporophorous vesicle, initiated at the beginning of the sporogony, collects all daughter cells of the sporont. Fibril-like projections connect the primordial exospore layer of the sporont with the sporophorous vesicle. The fibrils remain as a surface coat of mature spores. When sporoblast buds are formed, a second type of projections appear in the episporontal space. These are temporary tubules of exospore material, which disappear when the spores mature. The exospore of the mature spore is layered, with an internal layer resembling a double membrane. The polaroplast has three regions: wide lamellae, narrow lamellae and tubules. The polar filament is lightly anisofilar with 1- 2 wide anterior, and 4- 3 narrow posterior coils, arranged in one layer of coils in the posterior half of the spores. The identification of the species is discussed. Otto Jirovec, who redescribed the species in 1936, transferred it to the genus Thelohania. Comparison with slides in the collection of Prof. Otto Jirovec, Prague, revealed that the microsporidium studied by us apparently is identical to Jirovec's Thelohania cladocera from Daphnia magna, but it is not identical to his T. cladocera from D. pulex. Based on life cycle characters and the ultrastructure, the species is transferred to the genus Agglomerata.
Abbreviations A
E EX
ER
F G N P1-P3
PM PS SV
- anchoring disc endospore - exospore - endoplasmic reticulum = polar filament - Golgi vesicles - nucleus = anterior (l), median (2) and posterior (3) polaroplast region = plasma membrane polar sac sporophorous vesicle
0932-4739-96-0032-0412$3.50-0
T
V
tubules of exospore material posterior vacuole
Introduction Pfeiffer (1895) described the new microsporidum Glugea cladocera II, a parasite of the fat body and hypodermis of cladocerans of the genera Daphnia and Limnetis in Germany [11]. The description is typical for its time, with a minimum of diagnostic characters. The situation today, with knowledge of a great and increasing number of microsporidian parasites © 1996 by Gustav Fischer Verlag, Stuttgart
Ultrastructure of Agglomerata cladocera comb. nov. . 413 of Cladocera, has actually made this diagnosis useless for discrimination of the species. ]irovec, the first reviser, gave a more detailed account, based on his own observations of the microsporidium from the host Daphnia magna with provenance from Moravia, and he transferred the species to the genus Thelohania [4]. In a later publication Daphnia pulex, from a locality in Bohemia, was added to the list of hosts [5]. Pfeiffer's slides could not be traced. They have probably been destroyed, and we will probably never see the microsporidian cells that were described by him. ]irovec's slides have survived in Prague and they have been used for an evaluation. Recent investigations on the ecology and interactions between microsporidia and Cladocera have revealed a number of microsporidian infections and shown that a population sometimes hosts a mixture of microsporidian species [6, 15]. Also Daphnia magna hosts several microsporidia, and in our investigations we have frequently observed two species occurring together, one using the hypodermis, the other one primarily developing in the fat body (unpublished observations). We have further studied ]irovec's slides and noticed that the microsporidium identified by him to be Thelohania cladocera (Pfeiffer, 1895) actually is two species, one in Daphnia magna, another one in D. pulex. This report treats the ultrastructural cytology of the microsporidium of the hypodermis, which we have found to be Glugea cladocera II Pfeiffer, 1895. The identification and the genus position are discussed.
Material and Methods Infected specimens of Daphnia magna were collected in October 1992 from a small farm pond in northern Oxfordshire, England, close to the village of Ambrosden (about 15 kilometres north-east of Oxford, Northern Latitude 51 °52'80", Western Longitude 1°7'20". This corresponds to the Bicester population discussed in [1]). In our collection more than 50% of all adult D. magna were infected. In contrast to other microsporidia of D. magna [9] we were not able to maintain the infection in the laboratory [1, 9]. The second sample was collected in July 1993 from the same pond. Fresh squash prepar~tions were made by the agar method of Hostounsky and Zizka [3] and studied using phase contrast microscopy and dark field illumination. Permanent squash preparations were lightly air-dried and fixed in methanol for at least 15 min for Giemsa staining, or in Bouin-Duboscq-Brasil (BDB) solution for at least one hour previous to staining with Heidenhain's iron haematoxylin. For paraffin sectioning whole animals were fixed in BDB solution overnight or longer. After washing and dehydration in a graded series of ethanols, specimens were cleared in butanol and embedded in Paraplast (Lancer St. Louis, MO, USA). Sections were cut more or less longitudinally at 10 11m and stained with haematoxylin. For details on the histological techniques used see the manual by Romeis [13]. All permanent preparations were mounted in D.P.X. (BDH Chemicals Ltd., Poole, England). Measurements were made with an eye-piece micrometer at x 1,000 or using an image analysis program (Micro Macro AB, Gothenburg, Sweden).
For transmission electron microscopy, hosts were cut in halves and fixed in 2.5% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 36 h. After washing in cacodylate buffer and post fixing in 2% (w/v) osmium tetroxide in cacodylate buffer for 1 h at 4°C, the specimens were washed and dehydrated in an ascending series of bufferacetone solutions to absolute acetone and embedded in epon. Sections were stained using uranyl acetate and lead citrate [12].
Five slides with Thelohania cladocera originating from the collection of Otto Jirovec, Prague, were used for comparison: 1. Sections stained by haematoxylin and eosin. Label: "Thelohania c1adocera D. m. Lohovec". 2. Sections stained by haematoxylin. Label: "Thelohania c1adocera D. m. Lohovec" and added in ink by Jirovec's hand: "Thelohania cladocera. Heidenh." 3. Smear stained by Giemsa. Two labels. Labell: "Thelohania cladocera D. m. Lohovec". Label2 (Jirovec's hand): "Daphnia magna Lohovec 1935 Thelohania". Added in ink on the slide: "3-411 x 2". 4. Smear stained by Giemsa. Label: "Thelohania c1adocera D. m. Lohovec". Added in ink by Jirovec's hand: "Dafnia pulex Thelohania cladocera Stary kveten 1937". 5. Smear stained by Giemsa. Label: "Thelohania c1adocera". Added in ink by Jirovec's hand: "Dafnia pulex Thelohania c1adocera Stary kveten 1937".
Results
Prevalence and pathology Hypodermic cells in all parts the body, including the appendages, were invaded by the microsporidium. In specimens with less severe infection, the cells harbouring microsporidia appeared as isolated islands (Figs. 1, 3). The head region of the host seems to become infected last, possibly an adaptation of the parasite not to kill before the rest of the body is utilized. Specimens with advanced infection had wide areas where practically every cell was filled with microsporidia (Fig. 2). Infected cells were markedly hypertrophic, often twice as large as uninfected cells and completely packed with mostly sporulated microsporidia. Hypertrophic host nuclei were not observed. In a part of the material a second microsporidium occurred in the adipose tissue (Fig. 1).
Presporal stages and life cycle The presporal development was nearly complete in all specimens studied, and only in the specimens used for electron microscopy were modes of reproduction visible. All life cycle stages had isolated nuclei. Merogonial plasmodia were elongated and always had a small number of nuclei with a uniform moderately electron dense karyoplasm (Fig. 4). Sectioned merogonial nuclei measured up to 1.6!-tm (Fig. 4). Merogonial stages were delimited by an approximately 7 nm thick plasma membrane without external reinforcements. Free ribosomes gave the cytoplasm a uniformly granular appearance. There were only traces of an endoplasmic reticulum. Probably the merogonic reproduction was weak. Short chains with up to
Ultrastructure of Agglomerata cladocera comb. nov.. 415
three merozoites were observed. It is unknown jf there is more than one bour of merogony. Sporonts were uninucleate with a central nucleus and with densely granular cytoplasm where the layers of endoplasmic reticulum were arranged concentrically around the nucleus (Fig. 5). At the beginning of sporogony tubular or blister-like protrusions of thin electron-dense material appeared at the surface (Figs. 56). They were the primordia of the sporophorous vesicle. They were originally irregularly shaped, and the episporontal space contained a uniform and moderately electron-dense material. Later the primordia widened to distinct blisters, containing granular (or possibly fibrous) material resembling ribosomes but the granules were somewhat smaller than ribosomes (Figs. 5-6). Scindosome-like bodies, involved in the production of wall material, were visible in sporonts (Fig. 7). The blisters lost contact with the sporogonial plasmodium and formed a continuous sporophorous vesicle (Fig. 8). New electron-dense material appeared on the plasma membrane inside the blisters and it developed directly into a layered structure about 20 nm thick, where two electron-dense layers were separated by a lucent zone (Figs. 8-9). During the maturation from sporogonial plasmodium to sporoblasts the internal exospore layer changed into a double layer-like structure c. 6 nm thick where the external layer was somewhat more electron-dense (Figs. 9-10). A growing electron lucent gap was formed between the exospore and the plasma membrane (Figs. 9-10). It might be taken for a fixation artifact, but it was in fact the early initiation of the endospore layer. Simultaneously with the development of the sporophorous vesicle, nuclear division generated a plurinucleated plasmodium (Fig. 8). Signs of meiois were not observed. Later the nuclei accumulated at the periphery, the plasmodium split into buds in a rosette-like fashion, with one nucleus in each bud (Fig. 8), and finally it divided, usually into 8 sporoblasts (4, 8, 10, 12, 14, and 16 sporoblasts per sporont were observed). Nuclei of undivided and rosette-like plasmodia were of the same size, in sections measuring up to 1.3 lim. The granular material of the episporontal space transformed into distinct fibrils, or possibly tubules filled with electron-dense material, connecting the c. 5 nm thick envelope of the sporophorous vesicle with the exospore (Figs. 8-9). Lobed plasmodia and sporoblasts carried approximately 9 nm wide fibrils (Figs. 10-12, 14). When the sporogonial plasmodium began splitting into lobes, a second type of projection, formed from excess exospore material, appeared in the epis-
porontal space (Figs. 8, 12-13). These were approximately 43-45 nm wide tubules exhibiting the layers of the exospore (Fig. 8 inset). The tubules remained in sporophorous vesicles containing young sporoblasts but their central cavity grew wider and diameters up to 117 nm were measured. Finally the wide tubules disappeared (Fig. 14) and simultaneously the fibril-like connections between the exospore and the wall of the sporophorous vesicle were broken, which made the episporontal space almost devoid of inclusions (Figs. 14-16). Fibril-like material remained as an episporal coat. The initiation and morphogenesis of the spore organelles was normal for microsporidia.
The mature spore Live mature spores were pyriform with a pointed anterior pole (Fig. 17). The only internal structure visible was a small posterior vacuole in oblique position. Fixed and stained spores appeared more blunt (Fig. 19). Unfixed spores measured 1.5 -2.2 x 3.04.51lm (n = 25); fixed and stained spores were 1.62.1 x 2.7 -3.4llm (n = 50). Macrospores were not observed. Sporophorous vesicles were fairly persistent, and oval vesicles were observed in carefully made preparations (Fig. 18). Unfixed octosporous sporophorous vesicles were about 4 x 7 Ilm long. The wall of the mature spores measured 159165 nm, except anteriorly where it was considerably thinner, down to 55 nm (Fig. 22). It had the normal three subdivisions: exospore, a wide lucent endospore (less wide anteriorly), and an internal about 7 nm thick plasma membrane (Fig. 23). The endospore and exospore layers are built on the plasma membrane, and the three layers remain as a unit, which means that the plasma membrane forms the internal layer of the spore wall even after the sporoplasm has been ejected from the spore. The exospore, which measured approximately 24 nm, had a constant sequence of layers, in direction inwards: an electron-dense, c. 5 nm thick surface layer, a translucent median layer (11 nm), and an internal, c. 8 nm wide layer resembling a double-membrane. Diffuse granular material formed the border with the electron lucent endospore layer. The surface was covered by a coat of 5 -6 nm wide projections extending 275 nm outwards. In longitudinal sections they appeared as two electron-dense strands with lucent material in between (Fig. 23). The polar filament was lightly anisofilar (Fig. 22). It was anteriorly connected to an up to 425 nm wide, biconvex, layered anchoring disc. A polar sac of narrow
.... Fig. 1- 6. Pathogenicity and early development of Agglomerata cladocera comb. nov. - Fig. 1. Section through a part of an adult specimen of Daphnia magna with islands of A. cladocera (M1) in the hypodermis, and a second microsporidium (M2) in the fat tissue close to the gut (*). - Fig. 2. Hypodermic tissue filled with spores of A. cladocera. - Fig. 3. Section through the hypodermis exhibiting sporophorous vesicles. - Fig. 4. Chain-like formation with one uninucleate and one binucleate meront.Fig. 5. Young sporont exhibiting the initiation of the sporophorous vesicle. - Fig. 6. Intiation of the sporophorous vesicle. Figs. 1,3. Haematoxylin staining; Fig. 2. Phase contrast. Scale bars: Fig. 1 = 100 !-lm; Figs. 2-3 = 10 !-lm; Fig. 4 = l!-lmj Fig. 5 = 0.5 !-lm; Fig. 6 = 100 nm.
Ultrastructure of Agglomerata cladocera comb. nov. . 417 umbrella-like construction enclosed the anchoring disc and the anterior half of the anterior polaroplast (Figs. 22,24). The most proximal part of the filament was a short attachment section, about 100 nm long. The diameter in this region was about 205 nm. The uncoiled part of the filament, measuring about 125 nm in diameter, proceeded backwards in the midline of the spore, then turned lightly to the side touching the spore wall about 1/3 from the posterior pole of the spore. The final section was arranged as 5 -6 coils in one row of coils close to the spore wall in the posterior half of the spore. The coils were tilted in anterior direction, and the angle of tilt of the most anterior coil to the long axis of the spore was 50-55°. The 1-2 most anterior coils were slightly wider, 84-117 nm, and the 3-4 posterior coils were 55 -79 nm in diameter. The coiled part occupied about 1/3 of the spore length. The filament was composed of layered material where the following subdivisions could be discriminated (Fig. 26: 1-6). An external about 5 nm thick unit membrane (1) was followed by a c. 3 nm distinct electron dense layer (2), a c. 6 nm layer of moderately dense material (3), an approximately equally wide layer of dense material, which was reduced in the narrow coils (4), a moderately dense zone, approximately twice as wide, in which a fibrous structure could be observed (5), and the centre, which was clearly wider in the wide coils, with indistinct layering (6). The very centre of the wide coils was electron-dense (Figs. 22, 26). The polaroplast had three subdivisions, where each region was composed of compartments of specific shape, delimited by approximately 5 nm thick unit membranes (Fig. 22). The polaroplast enclosed the uncoiled part of the polar filament. The anterior part was composed of wide chambers of variable height (anterior-posterior distance, up to 163 nm), filled with a uniform moderately electron-dense material (Fig. 24). In this part, which constituted about one half of the polaroplast, anastomoses were frequent. The following section was shorter, about 1/4 of the polaroplast length, with closely packed lamellae (Fig. 25). The periodicity was about 13 nm. There was a distinct gap between the last lamellae and the final 1/4 of the polaroplast (Fig. 25). In the last section the compartments were tubular, measuring 23 - 25 nm in diameter (Fig. 26). The surface layer of the polar filament, the compartments of the polaroplast, and the polar sac were all parts of the same membrane system. The rounded nucleus occupied the posterior half of the spore, inside the filament coils (Fig. 22). The largest
section of nucleus measured 1.1 11m. The cytoplasm was dense with free ribosomes. There were also strands of polyribosomes in the proximity of the nucleus and the polaroplast, but the layers were less prominent than usual in microsporidian spores. Discussion Microcrustaceans host a wide variety of microsporidian species and many of these belong to the early history of the study of microsporidia. The descriptions are too superficial to allow clear identification and the species are difficult, or sometimes impossible, to compare with more recently described taxa. Type material can rarely be traced. There are only two solutions to the taxonomic problem caused by these early species, either to leave them as nomina nuda, or to use the names arbitrarily and make redescriptions based on new material. The last alternative is preferred by the International Commission on Zoological Nomenclature [10]. Pfeiffer described Thelohania cladocera in 1895, using the name Glugea cladocera II [11]. The hosts were microcrustaceans of the genera Daphnia and Limnetis with provenance from Greifswald, Germany. It is unclear if the microsporidium was found in more than one Daphnia species. The species mentioned is D. pulex. We have not been able to trace Pfeiffer's slides, which might have solved the problems with the hosts. The pathogenesis was described and illustrated (in Limnetis). The spore size was indicated to be 2-3 11m, and it was mentioned that the spores were formed in sporophorous vesicles ("Sporoblasten") measuring 3-4 11m in diameter, and containing 8, 16 or more spores. It was also mentioned that simultaneously with this microsporidium another species, Glugea leydigii, occurred in the fat body. Jirovec gave a more detailed description, with illustrations, of Glugea cladocera, based on parasites of D. magna collected in Moravia [4]. Later D. pulex collected in Bohemia was added to the list of hosts [5]. He used Thelohania as the genus name [4] and described the spores as pyriform, lightly pointed anteriorly, and recorded the size of li¥ing spores to be 1.5 -1.8 x 33.5 11m. Illustrations of spores (Figs. 1 e-k in [4]) raise the question whether there were two different microsporidia present in the samples. The spores in Fig. 1 f appear more blunt than the spores of Figs. 1 g-k. Sporophorous vesicles contained only 8 spores. Using Giem-
... Fig. 7-13. Sporogony of A. cladocera - Fig. 7. Periphery of a sporont showing one scindosome (arrow). - Fig. 8. Undivided (bottom) and rosette-like dividing sporogonial plasmodia; exospore-derived tubules in the vesicle with dividing plasmodium (inset shows longitudinally sectioned detail of tubules; arrow indicates episporontal space devoid of tubules). - Figs. 9-10. Cell periphery of the sporogonial plasmodium and the sporoblast. - Fig. 11. Sporogonial plasmodium in the process of forming lobes; exospore layer not complete (arrows); only fibril-like material in the episporontal space. - Fig. 12. Sporophorous vesicle with 8 sporoblasts visible; wide exospore-derived tubules in the episporontal space (arrow). - Fig. 13. Exospore-derived tubules at the sporoblast stage; arrowheads indicate connected fibrils. Scale bars: Figs. 7, 12-13, and inset on 8 = 100 nm; Figs. 8,11 = 11lm; Figs. 9-10 (with commom bar on 9) = 50 nm.
Ultrastructure of Agglomerata cladocera comb. nov. . 419
sa solution the spores exhibited one dark granule. Using the "Nuklealreaktion" (obviously Feulgen technique) two small spot-like nuclei were seen. The only other illustration existing of this species is a micrograph of living spores, revealing the presence of a prominent mucous coat [8]. Hazard and Oldacre revised the Thelohania-like microsporidia, and they considered T. glugea to be a dubious Thelohania species [2]. Sprague accepted their interpretation and transferred the species to the collective and unclassified genus Microsporidium [14]. Pfeiffer's type material has probably been destroyed. Jirovec;s collection has survived in Prague, and slides of the microsporidium that he identified as Thelohania cladocera have been studied. He found T. cladocera in two hosts: D. magna (Fig. 20) and D. pulex (Fig. 21). On one of the slides the host name and location written on the label do not correspond with later additions in ink written by himself. If the slides are compared, it is easily seen that the spores of the microsporidium of D. pulex are larger than the spores from D. magna and from the microsporidium treated herein. Stained nuclei were not visible in the slides from Jirovec's collection. The size of the spores, the number of sporoblasts per sporont, and the site of infection in the host correspond between Pfeiffer's description of Glugea cladocera II and the microsporidium treated herein. Pfeiffer reported that the hosts were cladocerans of two genera, Daphnia and Limnetis [11]. One host species, D. pulex, is mentioned in the text. No illustrations show microsporidia from this host. It is obvious that Jirovec's Thelohania cladocera of D. magna and D. pulex are different species. Size differences reveal that clearly. One of them, T. cladocera of D. magna, might be identical to the species treated herein. The spores are of the same size and approximately of the same shape. Octosporous sporophorous vesicles are also similar. However, two differences are obvious. Sporophorous vesicles in Jirovec's material were always octosporous, and the spores were said to be binucleate [4]. As the sporophorous vesicles are fairly fragile and break when smears are made, other spore configurations might have been overlooked. As it is not easy to make the nuclei of microsporidian spores distinctly visible in light microscopic preparations, one nucleus with grouped chromatin might give the impression of two nuclei. Thus, considering the short-
~
comings of Jirovec's techniques, too much emphasis should not be placed on the apparent difference in number of nuclei. We have reasons to believe that the microsporidium studied by us is the species Pfeiffer described as Glugea cladocera II, and it is obviously identical to Jirovec's Thelohania cladocera of D. magna. If we combine life cycle characters (all developmental stages with isolated nuclei, polysporoblastic sporogony by rosette-like budding, pyriform spores, sporontogenetic sporophorous vesicles uniting all daughter cells of the sporont) with ultrastructural characters (layered exospore, anisofilar polar filament, a polaroplast composed of three structurally different regions where the final region is tubular) one genus will be available: Agglomerata Larsson and Yan, 1988. This genus was established for a microsporidium considered identical to Duboscqia sidae Jirovec, 1942, which is a parasite of the same group of hosts [7]. We, therefore, propose to that T. cladocera should be transferred to the genus Agglomerata as Agglomerata cladocera comb. nov. A more detailed comparison between A. cladocera and A. sidae (AS), until now the only species of the genus, reveals further similarities: two kinds of projections present in the episporontal space, early initiation of the endospore, identical layering of the exospore and the polar filament and a uniquely constructed polaroplast. Even if the two species produce spores of similar shape and size, some differences, except for the different hosts, clearly tell that the species are different. A. cladocera has basically 8-sporoblastic sporogony (AS: 16-sporoblastic). The sporophorous vesicles are oval and the spore-groups break easily (AS: spherical sporophorous vesicles where the spores are tightly connected and difficult to separate). The projections present in the episporontal space are narrow fibrils, or possibly tubules, and wide exospore-derived tubules without particular arrangement (AS: uniform wider tubule-like structures and exospore-derived tubules in characteristic configurations). The spore is coated with fibrils, or tubules, which have lost their contact with the sporophorous vesicle (AS: tubule-like structures connect the spores to each other and to the sporophorous vesicle). The infection is restricted to the hypoderm (AS: spreading from the hypoderm to become a generalized infection).
Fig. 14-21. Sporoblasts and spores. - Fig. 14. Sporophorous vesicle with two sporoblasts visible; some radiating fibrils from the exospore (arrow); only fibrils in the episporontal space. The sporoblasts are sectioned close to the irregularly shaped surface, * indicates episporontaJ space visible through holes in the sporoblasts. - Fig. 15. Electron-dense immature spores; more dense layer of exospore fibrils. - Fig. 16. Sporophorous vesicle with mature spores; episporontal space almost empty. - Fig. 17. Unfixed spores. - Fig. 18. Unfixed sporophorous vesicles. - Fig. 19. Stained spores. - Fig. 20. Spores and sporophorous vesicles of Thelophania cladocera from Daphnia magna (from the collection of]irovec). - Fig. 21. Spores of T. cladocera from D. pulex (from the collection of ]irovec). Figs. 17-18. Phase contrast. Figs. 19-21. Giemsa stain. Scale bars: Figs. 14-16 = 0.5 /lm; Figs. 17-21 (with common bar on 19) = 5/lm.
Ultrastructure of Agglomerata cladocera comb. nov.. 421 Redescription
Agglomerata cladocera (Pfeiffer, 1895) comb. nov. Synonyms: Glugea cladocera II Pfeiffer, 1895; Thelohania cladocera (Pfeiffer, 1895) ]Irovec, 1936 of Daphnia magna, but not of D. pulex. (P) and (J) indicate characteristics taken from Pfeiffer's description [11] and Jirovec's redescription [4], (R) from this reinvestigation. Merogony. (R) Elongated merogonial plasmodia with isolated nuclei give rise to a small number of merozoites. Sporogony. (R) Sporont uninucleate. Sporogonial plasmodia with isolated nuclei divide by rosette-like division. Spore yield: (R) 4-16, usually 8; (P) 8, 16 or more; (J) 8. Spores. (R) Pyriform with pointed anterior pole. Living spores measure 1.5 - 2.2 x 3.0 -4.5 11m, fixed and stained 1.6-2.1 x 2.7-3.4 11m. Spore wall 159165 nm thick, with an approximately 24 nm thick, layered exospore, coated by densely arranged fibrillike material. Polar filament lightly anisofilar with 1-2, 84-117nm wide, anterior coils and 3-4, 5579 nm wide, posterior coils in one row of coils in the posterior half of the spore. The angle of tilt is 50-55°. The polaroplast, which surrounds the uncoiled part of the polar filament, has three subdivisions: wide lamellae, narrow lamellae and, separated from them by a gap, tubules. A single nucleus inside the filament coils. (P) Spore size 2-3 ~lm. (J) Spores pyriform with pointed anterior pole. Living size 1.5 -1.8 x 3-3.5 11m. " ... wir sehen nach der Nuklealreaktion in reifenden und auch reifen Sporen in ihrer Mine zwei deutliche punktformige Kerne". Sporophorous vesicle. (R) Round to ovoid, measuring approximately 4 x 711m when living, unites all sporoblasts in a common vesicle. Episporontal space traversed by narrow fibrous material, which remains as the exospore coat of the mature spores, and wide tubules with walls of exospore material, which are formed when the plasmodium splits into lobes and disappear during the maturation of the spores. (P) 3 -4 11m in diameter. Host tissues involved. Hypodermal cells all over the body. Hosts. (P) Daphnia and Limnetis, (J) and (R) Daph-
nta magna. Type locality: Type locality not specified by Pfeiffer,
but material from Greifswald (Germany) is used for illustration. Locations of the material studied: (J) A village pond in Lohovec (Moravia), the pond Stary (Bohemia); (R).
A farm pond near the village of Ambrosden, about 15 km north-east of Oxford (UK). Deposition of studied material: (P) The collection of Pfeiffer has not been traced. (J) The collection of ]irovec is kept at the Department of Parasitology, Charles University, Prague, (R) The material used for the redescription is kept in the collection of Larsson, Department of Zoology, University of Lund: No. 921218-(A-C) and 930914-(T-V).
Acknowledgements The authors are greatly indebted to Mrs. Lina Gefors, Mrs. Brigitta Klefbohm and Mrs. Inger Norling, all at the Department of Zoology, University of Lund, for excellent technical assistance, and to Dr. Josef Chalupsky for making the slides in the collection of Jirovec available for study. The investigation was supported by research grants from the Royal Society, London (to D. E.), the Swedish Natural Science Research Council (to R. L.), and from the Royal Physiographic Society, Lund (to R. L.).
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.... Fig. 22 - 26. Ultrastructure of the mature spore. - Fig. 22. Longitudinally sectioned spore with dense fibril-like exospore coat. Fig. 23. Close-up of the spore wall (arrows indicate the double-layer of the exospore, black arrowheads structural details of the fibrils). - Figs. 24-25. Longitudinally sectioned details of the two anterior polaroplast regions (arrow heads indicate the gap separating P2 and P3). - Fig. 26. Posterior region of the polaroplast and transversely sectioned polar filament coils (layers indicated 1-6; arrow heads indicate differences in the centre). Scale bars: Fig. 22 = 0.5 11m; Figs. 23-26 = 100 nm.
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Key words: Agglomerata cladocera comb. nov. - Microspora - Ultrastructure - Taxonomy - Daphnia magna Ronny Larsson, Department of Zoology, University of Lund, Helgonav. 3, S-223 62 Lund, Sweden, Fax: +46-46-2224541, e-mail: [email protected];DieterEbert.InstituteofZoology.BaselUniversity.Rheinsprung9.CH-4051 Basel, Switzerland; Jifi Vavra, Department of Parasitology, Charles University, Vinicna 7, 128 44 Prague 2, Czech Republic