Characteristics of the genus Bacillidium Janda, 1928 (microspora, mrazekiidae) — Reinvestigation of the type species B. criodrili and improved diagnosis of the genus

Europ.J.Protistol. 30, 85-96 (1994) February 18, 1994

European Journal of

PROTISTOLOGY

Characteristics of the Genus Bacillidium Janda, 1928 (Microspora, Mrazekiidae) - Reinvestigation of the Type Species B. criodrili and Improved Diagnosis of the Genus J. I. Ronny Larsson Department of Zoology, University of Lund, Sweden

SUMMARY The cytology of a microsporidium identified as Bacillidium criodrili Janda, 1928, the type species of the genus Bacillidium, is described with emphasis on the ultrastructure. The host was Rhyacodrilus coccineus, not the type host Criodrilus lacuum. The living spores, which measure 2.1-2.7 X 21.7-24.4 !-lm, are approximately equally long but slightly more narrow than the sizes reported in the description. The exospore of the sporoblast releases the superficial, double membrane-like layer, which forms a sac-like structure, resembling a c. 28 nm thick individual sporophorous vesicle. The remaining exospore layer is a uniform, about 35 nm thick structure. The polaroplast, which occupies approximately the anterior 1/10 of the spore, has two lamellar regions, where the lamellae of the anterior region are more closely packed. The polar filament has a 404-495 nm wide anterior part, the "manubrium", reaching approximately to the middle of the spore, a short section with decreasing width, and a final, 138-160 nm wide section ending in the posterior part of the spore. Membrane-lined rounded bodies, filled with uniform dense material, are present in the posterior region of the spore. Nuclei of all life cycle stages are coupled diplokaryotically. The cytological characteristics and the identification of the species are disc~ssed, and an emended diagnosis of the genus Bacillidium is given together with a list of species.

Abbreviations A B C D E EN ES EX F G HM M P PA PM PP PS

=

=

= =

=

= =

=

=

= =

=

=

anchoring disc dense sporal body centriolar plaque diplokaryon endoplasmic reticulum endospore exospore-derived sac exospore narrow part of polar filament Golgi apparatus host mitochondrion manubroid part of polar filament presporal stage anterior part of polaroplast plasma membrane posterior part of polaroplast polar sac

© 1994 by Gustav Fischer Verlag, Stuttgart

R S SB SR U

= rounded coelomocytic body

= spore = sporoblast sporont

= unit membrane

Introduction The microsporidia of the family Mrazekiidae Leger and Hesse, 1922 have exceptionally large, rod-shaped spores, produced by disporoblastic sporogony, and also the merogonial reproduction appears to be by binary fission. Until the present time approximately 17 species have been placed in this family, grouped in the genera Bacillidium, Hrabyeia, ]irovecia, Mrazekia and Rectispora. Hrabyeia [19J and Rectispora [16J are monotypical genera which have been defined using ultrastructural characteristics. 0932-4739/94/0030-0085 $3.50/0

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Three of the ]irovecia species have been studied at the ultrastructural level, J. caudata (Leger and Hesse, 1916), the type species of the genus [15], J. involuta Larsson, 1989 [14], and J. lumbriculi (jfrovec, 1936) [22,23], and also this genus can be defined precisely. Mrazekia contains two species: M. argoisi Leger and Hesse, 1916, the type species, and M. cyclopis (Vavra, 1962). Debaisieux was the last person to find M. argoisi [2], and the ultrastructural cytology is completely unknown. However, M. cyclopis has been studied using modern techniques, revealing fine structural characteristics of the genus [18, 20, 21, 26, 28]. Like ]irovecia, Mrazekia has clear discriminating characters visible using light microscopy, and there is no reason to doubt that M. argoisi and M. cyclopis are congeneric, which means that characteristics of M. cyclopis can be used to define the genus. The last genus, Bacillidium, has no unique characters visible at the light microscopic level, so even if some of the species, B. filiferum Larsson, 1989 [13], B. strictum (Leger and Hesse, 1916) [17], and Mrazekia brevicauda Leger and Hesse, 1916 [5], which probably is a Bacillidium species, have been studied using electron microscopy, we cannot be convinced that they are congeneric with B. criodrili Janda , 1928, the type species. This species has only beenstudied in the material originating from Janda, and the fine structure is completely unknown. In March 1992 one specimen in a sample of the freshwater oligochaete Rhyacodrilus coccineus was found to host a microsporidium of the Bacillidium-type. The light microscopic characteristics indicated that the species was B. criodrili, evenif the host differed from the type host, and the geographic provenance was different. The light microscopic and ultrastructural characteristics are described here, some aspects on the cytology and the identification of the species are discussed, and the finds are used for a more precise diagnosis of the genus Bacillidium. Material and Methods The microsporidium was obtained from one specimen of

Rhyacodrilus coccineus (Veydovsky, 1875) (Oligochaeta, Tubificidae), collected on March 10, 1992 , in the small river Hoje ii, close to the village of Esarp in the south of Sweden. Fresh squash preparation s were made by the modified agar method of Ho stounsky and Zizka [6] and examined using phase contrast microscopy and dark field illumination. Permanent squash preparations were air-dried lightly and fixed in BouinDub oscq-Brasil solution for at least one hour. For paraffin sectioning the posterior half of the host animal was fixed in the same fixative overnight. After wa shing and dehydration in a graded series of ethanols, the specimen was cleared in butanol and embedded in Paraplast (Lancer, St. Louis, MO, USA). Sections were cut longitudin ally at 6 urn,Squash preparation s and sections

were sta ined using Giemsa solution, Heidenhain's iron haematoxylin, Ziehl's carbol fuchsin or the modification of Noland 's sta in according to Farley [4]. For details on the general histolo gical technique s used see the manu al by Romeis [25]. All permanent preparations were mounted in D.P.X. (BDH Chemicals Ltd., Poole, England). Measurements were made with an eye-piece microm eter at X 1000. For transmission electron microscopy segments from the anterior half of the host specimen were excised and fixed in 2.5 % (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 21 h. After washing in buffer and post fixation in 2 % (w/v) osmium tetrox ide in cacodylate buffer for one hour at 4 °C the pieces were washed and dehydrated in an ascending series of buffer-acetone solutions to absolute acetone, and embedded in epon. Section s were stained using uranyl acetate and lead citrate [24]. Smears, numbered 920310-N-(1-12, 14) RL, sections numbered 920310-N1-(1-25) RL, and epo n blocs are in the collection of the author. Five slides, made by Victor Jand a, or possibly made by Otto jirovcc from material provided by Janda, kept in ColI. Jirovec at the Departm ent of Parasitology and Hydrobiology, Charles University, Prague, were used for comparison. The slides (four with serial sections and one smear) were sta ined using Heidenhain or Domin ici stain, and they were labelled: "Bacillidium criodrilli, Criodrillus sp., O. jirovec".

Results

Prevalence and Pathology The sample of oligochaetes contained several hundreds of specimens, none exhibiting external signs of infection. 35 specimens of various species, including three Rhyacodrilus coccineus, were dissected and studied using microscopy. The only specimen hosting microsporidia was one R. coccineus. The microsporidium was present in coelomocytes in all parts of the body (Fig. 1). Both coelomocytes and their nucleus were hypertrophic. An excess number of nuclei was not observed. The parasite deprived the host cellof the normal cytology, and the mitochondria became aggregated, mostly close to the cell wall (Fig. 3). Infected coelomocytes contained a few 4.9-6.7 11m wide, rounded bodies, which were intensely stained using normal histological stains (Fig. 1). They were uniform and electrondense in ultrathin sections, and they were not limited by a unit membrane (Fig. 4). It is unclear to what extent they were associated with the microsporidium, but occasionally microsporidia and dense bodies were in close contact (Fig. 8). Most spores were immature and randomly dispersed in the coelomocytes, but there were also positions with beginning dense packing of spores, arranged with their longitudinal axes in parallel (Fig. 1).

Figs. 1-4. Pathogenicity of Bacillidium criodrili. - Fig. 1. Hypertroph ic coelomocytes of Rhyacodrilus coccineus filled with ~ microspor idia (" indicates spores in regular arrangement) : dense bod ies (R) arc present in all coclomo cyres (longitudinal section 6 fAm, haematoxylin ). - Fig. 2. A squashed coclomocyte packed with mostly presporal stages (haematoxylin). - Fig. 3. Periphery of a microsporidia-filled coelomocyte, with the presporal stages close to the cell wall. - Fig. 4. Detail of a dense coelomocytic body of uniform granular material, no ext ernal membrane. Scale bars: Fig. 1 = 50 urn; Fig. 2 = 25 urn; Fig. 3 = 1 urn; Fig. 4 = 0.5 urn,

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The host specimen lacked monocystid gregarines, but some squash preparations contained spores of a myxosporidian of the genus Myxobolus. There were no myxosporidia or actinomyxidia present in the gut epithelium or the gut lumen of the sectioned half of the host, and as it was the posterior part that was longitudinally sectioned, spores could be expected to be present in the gut lumen, even if the site of origin was a part of the epithelium anteriorly to the sectioned region. The Myxobolus spores might have been present on the surface of the segments squashed.

Presporal Stages and Life Cycle As this was a case of young infection, presporal stages and immature spores were dominant (Figs. 1-3,5). The most immature stages were confined to the periphery of the coelomocyte (Figs. 1, 3). All life cycle stages had nuclei coupled as diplokarya. Two sequences of reproduction were revealed, merogony and sporogony, and as stages with more than two diplokarya never were observed, all reproduction appeared to be by binary fission. Cells of the two sequences differed in shape, size and staining properties. The earliest life cycle stage recognized was irregularly rounded merogonial plasmodia with two diplokarya (Fig.6a-b). They were up to 4.7 urn long, and each nucleus of the diplokaryon measured up to 1.8 urn wide in stained preparations. The nuclei were more intensely stained than the cytoplasm. They gave rise to nearly spherical, up to 3.0 urn wide merozoites (Fig. 6a-b). Plasmodia and cells interpreted as merozoites had identical staining properties, which indicated that they belonged to the same phase of reproduction. The spherical diplokaryotic cells outnumbered the plasmodia greatly, which suggested that they were the daughter cells. It is unknown if there is more than one sequence of merogonial reproduction. When merozoites matured to sporonts the size of the cell and the diplokaryon increased, and the shape changed slightly to become almost spherical (Fig. 6c-e). The greatest sporont observed measured 12.8 urn in diameter, the greatest diplokaryon was 7.0 urn wide. The affinity to the stainings decreased, and the difference in staining between nuclei and cytoplasm was reduced (Fig. 6d). Centriolar plaques were revealed as dark spots at the

nuclear periphery (Fig. 6c). The diplokaryon divided once, yielding a rounded plasmodium with two diplokarya (Fig. 6e). Sporoblasts were half spherical to ovoid, measuring up to 8.1 urn long, and the components of the diplokaryon were up to 4.6 urn wide (Fig. 6f). There were very small differences in staining between the cytoplasm and the diplokaryon of sporoblasts. Merogonial plasmodia were not observed in ultrathin sections, but a series of stages from merozoites (Fig. 7) to spores (Fig. 15) were present. The ultrastructural cytology of the early stages conformed with previous reports of Bacillidium-like micro sporidia, and there is no need to treat it in detail. When maturing to sporonts the endoplasmic reticulum became more prominent, arranged in concentricallayers around the diplokaryon, and the cytoplasm lost some of its density (Fig. 8). Up to 223 nm wide, uniformly electron-dense centriolar plaques were seen in shallow depressions of the nuclear envelope (Fig. 8). At the beginning of the sporogony electron-dense material, in the normal way, accumulated on the approximately 8 nm thick plasma membrane (Fig. 8). The dense material was the primordium of the future exospore, which was initiated patchily, but increased to a complete cover before the sporoblasts were released (Fig. 9). A Golgi apparatus, a rounded zone of closely packed cytoplasmic vesicles, was present in the proximity of the diplokaryon from the beginning of sporogony (Fig. 8). The newly formed sporoblasts had an approximately 18 nm thick uniform exospore layer on the plasma membrane (Fig. 9). During the maturation the exospore increased in size, and when the thickness approached 45 nm the superficial 10 nm thick zone was modified to a double membrane-like structure (Fig. lOa). Slightly later, when a distinct electron lucent endospore zone was present, the double layer lost contact with thee. 35 nm thick uniform exospore, and a granular material accumulated on the surfaces of the double layer (Fig. lOb). When the morphogenesis of the sporal organelles was about to start, the surface layer of the exospore had formed a complete sac-like structure, widely separated from the sporoblast (Figs. 10c, 11). From this stage on, including around mature spores, the sac was about 28 nm thick, with distinct granular zones on each side of the double layer (Figs. 10c, 17). The exospore thickness remained at c. 35 nm.

Figs. 5-9. Presporal development of B. criodrili. - Fig. 5. Fresh smear with spherical presporal stages, probably mostly sporonts, and ~ spores (phase contrast); inset shows one sporont at greater magnification (interference phase contrast). - Fig.6a. Merogonial plasmodium with dividing diplokarya (arrowheads indicate mitotic spindles) and merozoites, nuclei are intensely stained. b. Merogonial plasmodia with duplicated diplokarya and merozoites of the last generation. c. Merozoites maturing to sporonts; nuclei are less intensely stained (arrowheads indicate centriolar plaques). d. Sporont with elongated diplokarya in the process of dividing. e. Sporogonial plasmodium with two diplokarya. f. Two sporoblasts (haematoxylin; stages shown in Figs. a-e are taken from the same microscope slide). Fig. 7. Merozoites of the last generation; the endoplasmic reticulum is still weakly developed, plasma membrane without external reinforcements. - Fig. 8. Two sporonts with endoplasmic reticulum in concentrical arrays around the diplokaryon, electron-dense material is accumulating on the plasma membrane (arrowheads); the two sporoblasts are embedded in a dense coelomocytic body. - Fig. 9. Two elongated sporoblasts with central diplokaryon and continuous electron-dense cover on the plasma membrane. Scale bars: Fig. 5 = 25 urn, inset = 10 urn; Fig. 6 (with common bar on b) = 5 urn; Figs. 7-9 = 1 urn,

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Sporogenesis and Spores Immature spores were approximately uniformly thick cylinders with both ends equally blunt (Figs. 12, 15). The central diplokaryon, which together with the Golgi apparatus were the only structures visible in the sporoblast (Fig. 11), was in the early phase of spore maturation pushed forwards towards the front end of the spore by the developing polar filament (Figs. 12, 13). It was later pushed backwards by the developing polaroplast to a position near the centre of the spore (Fig. 15). The polar filament originated from the Golgi apparatus in the posterior half of the spore, and it grew in anterior direction, penetrating between the diplokaryon and the spore wall to reach the anterior pole of the spore (Figs. 12, 15). After that the length continued to increase in posterior direction. The zone of the immature spore posteriorly to the diplokaryon was dominated by a voluminous Golgi area (Figs. 15, 16). Except for the developing polar filament, two other structures were prominent here: a globular area of more densely interwoven vesicles and electron-dense rounded or slightly irregular bodies, limited by a c. 5 nm thick unit membrane (Figs. 15, 16). The dense bodies persisted also in mature spores, where they measured up to 733 nm in diameter (Figs. 17,25 ). The most posterior part of the immature spore was traversed by fibrous material, but there were no signs of a posterior vacuole (Figs. 15, 16). The mature spores were anteriorly wider and more blunt, while the posterior half tapered regularly toward s the more pointed posterior pole (Fig. 18). Mature living spores measured 2.1-2.7 x 21.7-24.4 urn, fixed and stained spores were 1.9-2.3 x 20.9-23.2 urn large. The sac, formed by the release of exospore material, might obscure the dimensions of the spore in stained preparations. However, carefully made haematoxylin stainings or the use of Noland's gentian violet stain (Fig. 18a) made it possible to discriminate between the spore and the sac. The small number of mature spores made it impossible to find mature spores that over their total length were perfectly sectioned longitudinally and close to the midline. However, by combining information from numerous

transverse or oblique longitudinal sections, it was apparent that the organization of the immature spore (Fig. 15) persisted after maturation, and the size and construction of the sporal organelles could be evaluated from them. The polar filament was initiated early by the sporoblast, and already when it was expanding forwards two sections were present: the wide anterior "manubrium", and the narrow final part (Fig. 13). In the mature spore the manubrium reached approxim ately to the middle (Fig. 18c,e,f), passing close to the spore wall in the region of the diplokaryon. It was partly embraced by the lighly crescent-shaped internal surface of the diplokaryon. The final section was slightly winding, ending near the posterior pole of the spore. The manubroid part was uniformly thick, 404-495 nm (Figs. 18,22), and the diameter was gradually reduced in the transitional zone (Fig. 23) to a final part measuring 138-160 nm in diameter. Forced ejection of the polar filament was successful only in a few spores. The mature polar filament exhibited a distinct stratification, with layers of different electron density and thickness. Up to eight layers could be distinguished, which in the description and illustrations are referred to by the numbers (1-8 ) used in direction inwards. The complexity of the fine structure increased gradually with the matura tion of the filament. In the primordial filament three layers were present (Fig. 13): a dense zone (4), a narrow zone of translucent fibrous or globular components (5), and a mottled, moderately dense centre (8). When the filament had reached the apex of the spore (Fig. 14), a new moderately dense layer (3) had appeared externally to layer (4), which had increased in width, and internally to the fibres (5) moderately dense material was present (6). The mature manubrium (Figs. 22, 24) exhibited the most complex organization: an external about 5 nm thick unit membrane (1); one dense (2) and one less dense (3) each about 7 nm thick layers; a dense, rather mottled, up to 65 nm wide layer (4); the more or less lucent fibrous zone (5); a moderately dense, c. 27 nm wide layer (6); a c. 21 nm wide, dense zone (7) which looked like a double-layer when correctly sectioned; and the moderately dense centre (8) which sometimes exhibited an indistinct

Figs. 10-1 7. Sporoblasts and immature spores of B. criodrili. - Fig. lOa-c . The superficial layer of the exosporeof the sporoblast is released, forminga sac-likestructure. - Fig. 11. Sporoblastwith central diplokaryonand complete exospore-derived sac.- Fig. 12a-b. Immature spores with anterior diplokaryon, the developing polar filament is moving forwards (haematoxylin). - Fig. 13. Ultrathin sectionofthesame stage; thepolar fil amentis wider anteriorly, thepolar sacisconical, theprimodium ofthe anchoringdiscisgranular (numbers referto layersofthepolarfil ament).- Fig. 14. Anteriorpole ofan immaturespore; thepolar filamenthasreached theanterior pole, the polar sac is more flat; a girdle-likestrucrure (arrows) has been added to the granular primordium of the anchoring disc; the polaroplast has not yet been initiated. - Fig. 15. Longitudinally sectioned immature spore with narrow endospore layer and nearly completeanterior polaroplast; thediplokaryonismore central, andtheposterior zoneisdominatedbya voluminousGolgi apparatus. >Fig. 16. DetailoftheGolgi zone at greater magnification, revealing thenarrowpartofthepolar filament, membrane-lined densebodies, a fibrous posterior zone ('f), and a spherical area with closely interwoven vesicles (white arrow). - Fig. 17. Nearly mature spore, transverselysectioned close to theposterior pole, exhibiting membrane-lineddense bodies, thetransverselysectioned narrow filament, andtheGolgi apparatus; theexospore-derivedsacisdouble-layeredwithgranular material on bothsurfaces (magnified oninset). Scale bars: Figs. 10 (with common bar on b), 14 and 17 = 100 nm; Figs. 11 and 15 = 1 urn; Fig. 12 (with common bar on a) = 10 urn; Figs. 13 and 16 = 0.5 urn; inset on 17 = 50 nm.

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stratification. In the transition to the narrow final portion (Fig. 23) layers (6) and (7) disappeared. The posterior part of the filament had a simpler stratification (Figs. 23, 25): layers (1-3) continued into this part with unchanged density and dimensions, layer (4) was reduced to about one third of the thickness in the manubrium, layer (5) could only be traced, and the centre (8) was reduced to half the thickness. The polar sac was organized around the apex of the developing polar filament, initially looking like a conical, unit membrane-lined vesicle, filled with electron-dense material (Fig. 13). When reaching the front end of the immature spore, the polar sac flattened (Fig. 14). In the mature spore the sac was a comparatively flat, crescentshaped structure, up to 816 nm wide, and filled with moderately dense material (Fig. 21). The anchoring disc was initiated in the young polar sac, initially visible as an ovoid zone of granular material (Fig. 13). In a filament that had reached the apex of the immature spore, the shape and structure of the disc primordium were unchanged, but a convex, double-layered girdle had been added (Fig. 14). Layer (4) of the filament approached the girdle, but the two structures were separated by a zone of more lucent material. In the mature spore the centre of the anchoring disc was modified to an up to 319 nm wide, layered, pad-like structure (Fig. 21). The layered girdle was surrounded by lucent material. Layer (4) of the filament connected to the girdle and to the lateral surface of the pad, layer (6), and probably also layer (5), connected to the centre of the disc. Obviously layer (7) closed anteriorly, separating the filament centre from the disc (Figs. 21, 22). The polaroplast was initiated in the immature spore. There were no signs of the polaroplast at the stage when the polar filament arrived at the apex of the immature spore (Fig. 14), but slightly later, the anterior polaroplast was nearly complete (Fig. 15). The polaroplast occupied the anterior 1/10 of the mature spore (Figs. 18,20). It had two lamellar regions. In the anterior polaroplast, about one third of the polaroplast length, the lamellae were closely packed, approximately with a period of 17 nm (Figs. 20, 21). The posterior polaroplast had up to 35 nm wide, regularly arranged but less densely packed lamellae, filled with a moderately electron-dense substance

(Fig. 20). The lamellae of the polaroplast, the polar sac, and the surface layer of the polar filament had identical, about 5 nm thick unit membranes. The elongate diplokaryon, which in stained spores measured 9.3-11.5 urn long, was situated approximately in the centre of the spore (Figs. 15, 18d-e). The cytoplasm was fairly dense, with strands of polyribosomes close to the spore wall (Fig. 20). There were voluminous membranelined cavities filled with electron-dense material in the posterior region of the spore, but no traditional posterior vacuole (Figs. 16, 17,25). The wall of the mature spore was 106-112 nm thick, except anteriorly where sizes down to 60 nm were measured. It had a traditional construction with an internal, c. 8 nm thick plasma membrane, a translucent endospore layer, which was reduced anteriorly, and a uniform electron-dense exospore measuring 35 ± 5 nm (Fig. 25).

Discussion

Cytology The microsporidium studied conforms with previously described Bacillidium-like microsporidia, but a few cytological details need some comments: the event of the superficial layer of the exospore, the globular posterior bodies of the spore, and the specialized area of the Golgi apparatus. The Bacillidium-like microsporidia, with the exception of Mrazekia cyclopis [18], share an identically constructed exospore, which in the sporoblast stage has a wide, uniform electron-dense internal layer and a surface layer resembling a double membrane (Fig. 10a,b). The fate of the surface layer varies from species to species. In Bacillidiumstrictum [17], Jirovecia caudata [15], and probably also in Hrabyeia xerkophora [19], the exospore remains unchanged from sporoblast to mature spore. In B. filiferum the surface layer breaks up into projecting fibrils [13]. In Rectispora reticulata [16], J. involuta [14], and in the species studied herein, the surface layer is released, forming a sac-like structure which might be mistaken for an individual sporophorous vesicle (Fig. 15). It is interesting

Figs. 18-25. Sporulated B. criodrili with provenance from Sweden (Figs. 18,20-25) and Czechoslovakia (Fig. 19). - Fig. 18. Light microscopic aspect: mature spore stainedwith gentianviolet (a)and livingspore (b, interference phasecontrast) exhibitingthe shapeof the spore and the exospore-derived sac (indicatedby arrowheads in Figs. 18-19); living spore revealing the manubrium and the dense posterior bodies (c, interference phase contrast); living spore showing the diplokaryon and the posterior bodies (d, phase contrast); haematoxylin stained spores (e-f). - Fig. 19. Haematoxylin stained spores with provenance from Janda; the exospore-derived sac is visible. - Figs. 20-21. Anterior poles of mature spores revealing the construction of the polaroplast, polar filament and anchoring apparatus (arrowhead indicates strands of polyribosomes, white arrows the girdle-like extensions of the anchoring disc, numbers indicatethe layersof the polar filament). - Fig. 22. Longitudinally sectioned manubroidpart of the polar filament. - Fig. 23. Transition between the manubroid and the narrow part of the polar filament revealing the fate of the different layers. - Fig. 24. Transversely sectionedmanubroid filament part with 8 layersvisible. - Fig. 25. Transverse sectionthrough the posterior part of the mature spore exhibitingthe layers of the spore wall, includingthe uniform exospore, the membrane-limited posterior bodies, and the layers of the narrow filament. Scale bars: Figs. 18 (with common bar on c) and 19 = 10 [tm; Fig. 20 = 0.5 urn; Figs. 21-25 = 100 nm.

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to note that the fate of the exospore coat is not a genus specific character. The surface layer behaves differently in the three ultrastructurally investigated species of the genus Bacillidium, and the same is true for ]. caudata and

J. involuta.

Janda recognized and illustrated a characteristic feature of the mature spore of B. criodrili: rounded bodies, which stained intensely using traditional histological methods, were present close to one pole (Taf. 8: Figs. 9-10 in [10]). Identical structures were apparent in the Swedish material identified as B. criodrili (Figs. 16, 18c-d). However, the dense posterior bodies are not unique to spores of B. criodrili. Dense membrane-lined posterior bodies were detected at the ultrastructural level in spores of M. cyclopis [18], R. reticulata [16], J. caudata [15], and]. involuta [14], and at least in spores of]. caudata the bodies are visible also using light microscopy (Fig. 14 in [15]). Whether these bodies are identical in all species is unknown, and it is impossible to connect them with a particular function. The Golgi apparatus of the immature spore is unusually large (Fig. 16), but the structure conforms with the normal for microsporidia [27]. However, interwoven with the lacunae of the Golgi apparatus was a spherical area with more narrow and more densely convoluted cisternae (Figs. 15, 16). This is not a usual component of the microsporidian spore. It might be identical to the clear body Gotz observed associated with the Golgi apparatus and developing manubrium in sporoblasts of Mrazekia brevicauda (Fig. 22 in [5]), even if the micrographs differ somewhat from the structure seen in B. criodrili. Like Gotz suggested [5], it might be an ageing Golgi apparatus. The structure is strikingly similar to the spherule of haplosporidian spores, like illustrated for Minchinia dentali (Fig. 33 in [3]) and Haplosporidium lusitanicum (Fig. 5 in [1]). Also the spherule has been interpreted as a stage in the development of the Golgi apparatus [3].

Identity to Bacillidium criodrili Bacillidium criodrili, the type species of the genus Bacillidium, was treated briefly in the description by Janda

in 1928 [10] and in the recent survey of Mrazekiidae [12], but covered more extensively by Jirovec in 1936 [11]. All these studies used material collected by Janda. The description was based on material cultured in Prague, but it was stated that the parasite was constantly present in Criodrilus from various locations around Bfeclav, in southern Moravia, and it was collected at least during four years between 1920 and 1925. Whether the culture also was derived from Bfeclav is unknown. Comparing the Bacillidium criodrili-like microsporidium from Sweden with Janda's slides and the published evaluations by Janda [10] and jirovec [11], the similarities are obvious, but there are also some differences. Janda characterized the spores as rod-shaped with equally rounded ends, although some of the drawings of Taf. 8: Fig. 10 in [10] suggest that the two poles might be slightly different. Jirovec described the posterior end of the spore as slightly pointed or rounded, but he exaggerated

the pointed shape in the line drawings (Abb. 6 in [11]). The Swedish material revealed clearly that the shape of the spores changed lightly during maturation. Immature spores are approximately uniformly thick cylinders with equally rounded ends (Fig. 12), while the mature spores are wider anteriorly and taper rather regularly posteriorly (Fig. 18). The shape of B. criodrili spores in Janda's slides corresponds fairly well with the Swedish material (Fig. 19). Living spores of the Swedish micro sporidium measured 2.1-2.7 X 21.7-24.4 urn. Janda reported that living spores of B. criodrili measured 1.6 X 20-22 urn, which means that the spores from Sweden and Czechoslovakia are approximately equally long, but seem to differ slightly in width. However, size differences less than one micron should not be exaggerated when an ocular micrometer was used for measurements. Jirovec stated that the size of the spores was variable. Most spores ranged in the interval 1.4-1.5 X 18-20 urn, but spores as great as 1.6 X 24-25 urn were seen [11]. It is not indicated whether the measurements refer to living spores, but as he compared the dimensions with those reported by Janda, and as he clearly stated that he had received both living and stained specimens from Janda, we might guess that living spores were measured. Stained spores originating from Janda and kept in jirovec's collection in Prague (Fig. 19) are distinctly smaller than stained Swedish spores. However, as we are unaware about the details of the techniques used (the size of microsporidia is influenced by the fixation procedure), as well as of the source of the material studied (the material used for the description and the material in jirovec's collection might be from different locations and different years) the different sizes of stained spores should not necessarily be considered more important than the obvious similarities in the fresh material. The light microscopic cytology corresponds fairly well between the Czech and the Swedish material. Janda paid little attention to the cytology. The two dark spots, or the long dark line, visible in Janda's drawings (Taf. 8: Figs. 9-10 in [10]), and the phrase "ein fadenformiges intensiv sich farbendes zentrales Gebilde (Kern?)" must denote the diplokaryon. jirovec treated the diplokaryon in detail, and he described how the diplokaryon of immature spores was situated anteriorly, while the diplokaryon of mature spores was in the centre [11]. This description was confirmed by the Swedish material, where it was seen how the diplokaryon was pushed forwards by the developing polar filament and later retracted (Figs. 12, 15). Janda did not describe the polar filament. Jirovec interpreted the filament as a rod-shaped structure, with an anterior, globular refractile body, and a manubrium reaching to the posterior vacuole. He was of the opinion that the manubrium lacked a posterior, narrow prolongation, and he believed that the absence was characteristic for Bacillidium and Mrazekia species (the genera are delimited differently nowadays). The present study revealed that the spherical polar sac is present in immature spores (Figs. 12, 13) while older spores have a cup-shaped polar sac of traditional type (Fig. 21). It was further revealed that there is a distinct size difference between the wide anterior manubrium and

Reinvestigation of Bacillidium criodrili . 95

the narrow posterior filament (Fig. 23). It is interesting to not e that jirovec found it difficult to force the spore to eject the filament, as we had the same experience with the Swedish material. Jirovec believed that the spore wall originated in the interior of the sporoblast, and illustr ated clearly how the spores were covered with a "Plasmaschicht" (Figs. 2, 3, 18 on Abb. 6 in [11]). How ever, the present study revealed that the spore wall is initiated in the norm al way, but the surface layer of the sporoblast is released, generating a sac-like cover of the spore, distinct around living spores (Fig. 18b,c), but less obvious in sta ined preparations unless special attention is paid to the structure (Fig. 18a,e,f). Ob viously Janda did not observe the unusual cytological organization of the periphery of spo roblasts and spores, but the structure is visible in stained slides originating from him (Fig. 19). Jand a described how the spo res were arranged in the coelomocytes, and mentioned that the arrangement was either at random or prominently regular [10]. Jirovec noticed the same variation and concluded that the markedly regular arrangement was characteristic for old giant cells [11]. There were only traces of regular arra ngement in the Swedish material (Fig. 1), but as it was a fairly young infection, where few of the spores had reached maturity, it is obvious that the numb er of spores per coelomocyte was not great enough to pro voke close and regular arra ngement. Th e obvious differences between B. criodrili and the Swedish microsporidium concern the geographic location and the host. Janda's material came from some populations of Criodrilus lacuum in Czechoslovakia. There are no further finds of this microsporidium as far as we kno w. However, it is not probable that the geographical distance between Czechoslovakia and Sweden is great enough to make it unlikely that the species could be present in Sweden. Th e Swedish source of material was Rhyacodrilus coccineus, an oligochaete belonging to the family Tubifi cidae, while the type host belongs to the Glossoscolecidae. We have no idea of how species specific the microsporidia of oligochaetes are. Janda reported that transmission experiments with B. criodrili to Criodrilus and LumbricuIus were unsuccessful [10]. However, j irovec received oligochaetes which Jand a had tried to infect per os, and he found that they actually were infected [11]. Th e species of oligochaetes were not specified. We are not convinced that it is correct to postulate strict host specificity, and we are therefore inclined to believe, based on identity concernin g size and cytological chara cteristics, that the micro sporidium studied is Bacillidium criodrili Jand a, 1928 , and that the results could be used for a more precise diagnosis of the genus Bacillidium.

Bacillidium sensu Issi In 1986 Issi revised the taxon om y of the microsporidi a and gave a new definition for the genus Bacillidium Janda, 1928, which in translation reads: "Microsporidia with uninucleate cylindrical spor es. Endospore weakly developed; exospore smooth. Polar tube broadened at the base, short. Polaroplast spongy, without lamellae. Sporogonial

stages surrou nded by thin membrane of par asitophorous vacuole. Spores produced in numb er of 8-fold." [8, 9]. Th e midge parasite Mrazekia bacilliforme Leger and Hesse, 1922 was selected as type species. This definition has neither been based on the descripti on Janda gave of B. criodrili [10], nor on a recent investigation of the species. As B. criodrili is a valid species, preserved in preparations originating from Jand a and with a potential of being designated as neotypes, and as this was the only species treated by Jand a in the description of the genus Bacillidium, there is no question abo ut B. criodrili being the type species of the genus Bacillidium. Issi's action was in violation of the Internation al Code of Zoological Nomenclature (Article 68 in [7]).

T axonomic Summary

Bacillidium Janda, 1928, emended diagnosis Merogony and sporogony diplok aryotic. Disporoblastic. Spores diplokaryotic, cylindrica l, witho ut tail-like ·pro longations. Polar filament with a straight, wide, anterior manub roid part, and a narrow, straig ht or coiled, posterior section. Polaroplast shor t, with two lamellar pa rts; anterior lamellae more closely and regularly arra nged. Exospore of the sporoblast with two comp onents: a surface layer resembling a double membrane and a uniform , wider basal layer. The dou ble membrane-like layer either remains as the surface layer of the exospore of the mature spore, or is transformed into fibrou s exospore projections, or is released as a sac-like envelope resembling an individual sporophorous vesicle. Sporophoro us vesicles sensu stricto absent. Onl y one sporogonial sequence observed.

Species: 1. B. criodrili Janda, 1928, type species. Type host: Criodrilus lacuum Hoffmeister, 1845 (Olig., Glossocolecidae).

2. B. filiferum Larsson, 1989 Type host: Peloscolex ferox (Eisen, 1879) (Olig., Tubifi -

cidae). 3. B. haematobium Jfrovec, 1936 Type host: Limnodrilus hoffmeisteri Claparede, 1862 (O lig., Tubificidae). 4. B. limnodrili j iro vec, 1936 Syn.: Mrazekia jiroveci Sprague, 1977 Type host: Limnodrilus claparedeianus Rarzel, 1868 (O lig., Tubificidae). 5. B. strictum (Leger and Hesse, 1916) Jfrovec, 1936 Mrazekia stricta Leger and Hesse, 1916 Type host: Lumbriculus uariegatus (Muller, 1774 ) (Olig., Lumb riculidae). 6. B. brevicauda (Leger and Hesse, 1916) comb. nov. Mrazekia brevicauda Leger and Hesse, 1916 Type host: Chironomus plumosus (Linnaeus,1758) (Dipt., Chirono midae). Remark: The ultrastructural investigation by Cotz [5] revealed that the spore of M. brevicauda exhibited a cytology of the Bacillidium type. There is no posterior

96 .

J. 1. R. Larsson

swelling of the manubrium, characteristic of the genus Mrazekia, and the exospore differs from the uniforml y layered exospore of M. cyclopis [18J. The short "tail", which inspired the species name, it not a compartmentalized tail of exospore material, characteristic for the genus Jirovecia [14, 15], but a compression of the spore in the region of the posterior vacuole, clearly visible in Fig. 1 in [5]. The species appears to conform with the genus Bacillidium in every respect, except for the untypical host. Acknowledgements The auth or is greatly indebted to Mr s. Lina Hansen, Mrs. Birgitta Klefbohm, and Mrs. Inger Norling, all at the Departm ent of Zoology, University of Lund, for excellent technical assistance, and to Dr. Josef Chalupsky, Department of Parasitology and Hydrobiol ogy, Charles University, Prague, for making slides in the collection of Otto Jfrovec available for examination. The investigation was financially supported by research grants from the Swedish Na tural Science Research Council.

References 1 Azevedo C. (1984): Ultrastructure of the spore of Haplosporidium lusitanicum n. sp. (Haplosporida, Haplosporidiid ae), parasite of a marine mollusc. J. Parasit., 70, 358-37l. 2 Debaisieux P. (1931): Etude cytologique du Mrazekia argoisii. La Cellule, 40, 147-1 7l. 3 Desport es 1. and Na shed N. N. (1983): Ultrastructure of sporulation in Minchinia dentali (Arvy), an haplosporean para site of Dentalium entale (Scaphopoda, Mo llusca); taxonomic implications. Protistologica, 19,435-460. 4 Farley C. A. (1965): A modification of Noland's stain for perman ent smears of protozoan flagella, cilia and spore filaments. J. Parasit., 51, 834. 5 Cotz P. (1981): Hom ology of the manubrium of Mrazekia brevicauda and the polar filament of other microsporidia. Z. Parasitenkd., 64, 321-333. 6 Hostounsky Z. and Zizka Z. (1979): A modification of the "agar cushion method" for observation and recording microsporidian spores. J. Protozool., 26, 41A-42A. 7 Intern at ional Code of Zoological Nomenclatu re, 3rd ed. (1985). Internation al Tru st for Zoological No menclature, London. 8 Issi 1. V. (1986): Microspor idia as a phylum of para sitic protozoa. Protozool ogy (Leningrad), 10, 6-13 6 (in Russian). 9 Issi 1. V. (1991): Microsporidia as a phylum of parasitic protozoa. Translated from Russian by Jerzy J. Lipa. Division on Microsporidia, Society for Invertebrate Pathology. 10 Janda V. (1928): Uber Mikro organismen aus der Leibeshohle von Criodrilus lacuum Hoffm. und eigenartige Neubildun gen in der Korp erwand dieses Tieres. Arch. Protistenkd., 63, 84-93, Taf. 8.

11 Jfrovec O. (1936): Zur Kenntnis von in Oligochaten par asitierenden Microsporidien aus der Familie Mrazekidae. Arch. Protistenkd., 87, 314-344. 12 Larsson J. 1. R. (1986 ): On the taxonomy of Mrazek idae: resurrection of the genus Bacillidium Jand a, 1928. J. Protozool., 33, 542-546. 13 Larsson J. 1. R. (1989): The light and electron microscopic cytology of Bacillidium (ili(erum sp. nov. (Microspora, Bacillidiidae). Arch. Prot istenkd., 137, 345-355. 14 Larsson J. 1. R. (1989 ): Light and electron microscope studies on ]irovecia involuta sp. nov. (Microspora, Bacillidiidae), a new microsporidian par asite of oligochaetes in Sweden. Europ. J. Protistol., 25, 172-1 8l. 15 Larsson J. 1. R. (1990a): On the cytology of ]irovecia caudata (Leger and Hesse, 1916) (Micros pora, Bacillidiidae). Europ.]. Protisto!', 25, 321-330. 16 Larsson]. 1. R. (1990b ): Rectispora reticulata gen. et sp. nov. (Microspora, Bacillidiidae), a new microsporidian paras ite of Pomatothrix hammoniensis (Michaelsen, 1901 ) (Oligochaeta, Tub ificidae). Europ.] . Protisto!', 26, 55-64. 17 Larsson ] . 1. R. (1992): The ultrastructural cytology of Bacillidium strictum (Leger and Hesse, 1916) Jfrovec, 1936 (Microspora, Bacillidiidae). Europ. J. Protistol., 28, 175-1 83. 18 Larsson J. 1. R., Vavra ]. and Schrcvel ] . (1993): Bacillidium cyclopis Vavra, 1962 - Description of the ultrastructural cytology and tran sfer to the genus Mrazekia Leger and Hesse, 1916 (Microspora, Mrazekiidae). Europ. J. Protisto!', 29, 49-60. 19 LornJ. and Dykova 1.(1990): Hrabyeia xerkophora n. gen. n. sp., a new microsporidian with tailed spores from the oligochaete Nais christinae Kasparzak, 1973. Europ . ]. Protisrol., 25, 243-248. 20 Lorn]. and Vavra ] . (1962): Contribution to the knowledge of microspor idian spore. 1. Int. Congr. Protozool. Prague, Czechoslovakia, pp. 487-489. 21 Lorn]. and Vavra J. (1963): Fine morph ology of the spo re in Microsporidia. Acta Protozool., Warszawa, 1, 279-283. 22 Puytorac P. de (1961 ): L'ultrastru cture du filament polaire invagine de la Microsporidie Mrazekia lumbriculi [ Irovec 1936. C. R. Acad. Sci., Paris, 253, 2600-2602. 23 Puytorac P. de (1962): Observations sur I'ultrastructure de la Microsp oridie Mrazekia lumbriculi, Jfrovec. J. Microscopie, 1,39-46. 24 Reynolds E. S. (1963): The use of lead citra te at high pH as an electron-opaque stain in electron microscopy. J. Cell BioI., 17, 208-2 12. 25 Romeis B. (1968): Mikroskopische Technik. Oldenbou rg Verlag, Miinchen-Wien. 26 Vavra ] . (1962): Bacillidium cyclopis n. sp. (Cnidospora, Microsporidia), a new parasite of copepods. Vesrn. Cs. spol. zool., 26, 295-299. 27 Vavra]. (1976): Structure of the microsporidia. In: Bulla Jr. 1.. A. and Cheng T. C. (eds): Comparative pathobiology, vol. 1, pp. 1-85. Plenum Press, New York, London . 28 Vavra J., Joyon 1.. et Puytorac P. de (1966): Observation sur l'ultrastructure du filament polaire des Microsporidies. Protistologica, 2, 109-11 2.

Key words: Bacillidium eriodrili - Diagnosis of Bacillidium - Microspo ridia - Oligochaeta - Ultra structure J.1. Ronny Larsson, Department of Zoology, University of Lund, Helgonav. 3, S-223 62 Lund, Sweden