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Ultrastructural Study and Redescription of Coccospora micrococcus(Léger & Hesse, 1921) Kudo, 1925 (Microspora, Thelohaniidae), a Parasite of Midge Larvae of the Genus Arctopelopia sp. (Diptera, Chironomidae) in Sweden
Arch. Protistenkd. 144 (1994): 271-282 © by Gustav Fischer Verlag Jena
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Ultrastructural Study and Redescription of Coccospora micrococcus (LEGER & HESSE, 1921) KUDO, 1925 (Microspora, Thelohaniidae), a Parasite of Midge Larvae of the Genus Arctopelopia sp. (Diptera, Chironomidae) in Sweden EVA
K. C.
BYLEN
& J. I.
RONNY LARSSON
Department of Zoology, Division of Systematics, University of Lund, Lund, Sweden
Summary: The cytology of the microsporidium Coccospora micrococcus, which is a parasite
of the adipose tissue of midge larvae, Arctopelopia sp., in Sweden, is described based on observations using light and electron microscopy. Merogonial plasmodia divide by plasmotomy and by rosette-like budding, yielding diplokaryotic merozoites. The number of merogonial cycles is unknown. The sporogony probably begins with a meiotic division. Each diplokaryotic sporont yields eight sporoblasts. A fragile sporophorous vesicle encloses each group of spores. The monokaryotic spores are spherical. Living spores measure 2.5-2.71Jm, fixed and stained spores 1.6-1.8IJm. The spore wall has a layered exospore with a double-layer. The polaroplast consists of an anterior lamellar and a posterior sac-like part. The isofilar polar filament is short, without coils. The membranes of the vacuole, produced by the Golgi apparatus, and their connection with the polar filament are visible in mature spores. There are two types of inclusions of the sporophorous vesicle: granular, initiated in the early phase of the sporogony, and tubular of exospore origin. The microsporidium is compared to the microsporidia with spherical spores, and the possible relations to the genus Pilosporella and to other Thelohania-like microsporidia are discussed. Key Words: Coccospora micrococcus: Arctopelopia sp.; Microsporidia; Ultrastructure; Redescription; Taxonomy.
Inroduction In 1921 LEGER & HESSE described four new microsporidia with spherical spores, three of them parasites of midge larvae, and created the new genus Cocconema for them (LEGER & HESSE 1921). The species were described in the manner typical for the time, with a minimum of characteristics: the shape and size of the spores, the name and geographic location of the host, and a few comments on the gross pathology. There were no illustrations. In a following paper, the descriptions were repeated together with three line drawings of Cocco-
nema spores, but without indications of the species to which they belonged (LEGER & HESSE 1924). The species described in the genus Cocconema have been treated in a series of reviews (KUDO 1924; POISSON 1953; WEISER 1961; COSTE-MATHIEZ & TUZET 1977; SPRAGUE 1977), and their taxonomic positions have been discussed. When it became apparent that the name Cocconema was preoccupied, the new name Coccospora was proposed (KUDO 1925). However, no later studies, based on new sources of material, have appeared, and we
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know as little about the species as we knew in 1921. It cannot be excluded that some of these species actually have been studied in the meantime. The diagnoses are so vague and difficult to use that it is easier for a microsporidiologist, when studying a microsporidium of this type, to find some characteristics not mentioned in the description and base the description of a new species upon them, rather than to identify the microsporidium with any of the old Cocconema species. We have studied a microsporidian parasite of midge larvae, producing spherical octospores. The small number of characteristics that can be compared suggests that it is the species originally called Cocconema micrococcus. The species is redescribed, based on characteristics derived from the light an electron microscopic cytology. It is compared to other microsporidia with spherical spores, and the taxonomic considerations are discussed.
Material and Methods The hosts were three specimens of Arctopelopia sp. (Diptera, Chironomidae) in two different samples of midge larvae collected from temporary forest pools at Finstorp, in the province of Scania, southern Sweden, on July 29, 1987 and July 31, 1991. Fresh squash preparation~ were made using the agar method of HOSTOUNSKY & ZIZKA (1979) and studied using phase contrast microscopy and dark field illumination. Permanent squash preparations were lightly air-dried, fixed in Bouin-Duboscq-Brasil solution overnight and stained using Giemsa solution or Heidenhains's iron haematoxylin (ROMEIS 1968). All permanent preparations were mounted in DePeX (BDH Chemicals Ltd England). Measurements were made with an eye-piece micrometer at x 1,000. For transmission electron microscopy pieces of infected segments were excised and fixed in 2.5 % (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 24 hand 47 h. After washing in cacodylate buffer and postfixation in 2.0% (w/v) osmium tetroxide in cacodylate buffer for 1 h in 4°C, the pieces were washed and dehydrated in an ascending series of ethanols to propylene oxide and embedded in Epon. Semithin sections for light microscopy were stained with methylen blue, and ultrathin sections for electron microscopy were stained with uranyl acetate and lead citrate according to REYNOLDS (1963).
Results Pathogenicity The posterior segments of infected larvae exhibited white spots, shining through the semitransparent cuticle. The fat body was the only tissue affected by the parasite. It was hypertrophic and filled with mostly sporulating microsporidia (Fig. 1). No syncytium was formed.
Merogony The earliest stages observed were merogonial plasmodia with diplokaryotic nuclei (Fig. 2). They divide by plasmotomy and by rosette-like budding yielding 5.3-7.4 flm wide merozoites with nuclei coupled as diplokarya, measuring 2-3 flm across the widest diameter (Fig. 3). Merogonial plasmodia and merozoites are surrounded by an approximately 8 nm thick plasma membrane. The cytoplasm is electron dense with numerous free ribosomes (Fig. 4).
Sporogony The last generation of merozoites mature to sporonts. The two components of the diplokaryon dissociate (Fig. 5), probably followed by a meiotic division, yielding lobed plasmodia with four isolated nuclei (Figs. 6, 7). The following mitosis yields totally 8 nuclei per lobed plasmodium. The plasmodium divides in a rosettelike manner, but all lobes are not always synchronous (Fig. 7). The sporogony is octosporoblastic, and the sporoblasts are enclosed by a sporophorous vesicle generated by the sporont (Fig. 5). In ultrathin sections the beginning of the sporogony is indicated by a layer of electron dense material secreted outside the plasma membrane of the sporonts, and simultaneously the cytoplasm becomes less electron dense. When the sporoblasts are budded off from the plasmodium their cytoplasm is rather lucent, but the electron density increases when they mature to spores (Fig. 8). The polar filament and the polaroplast are generated by the Golgi apparatus in the normal way. Occasionally sporoblasts develop
Figs. 1-4. Pathology and merogony of Coccospora micrococcus. Fig.1. Semithin section of infected fat body filled with spores (S). * indicates the hypoderm. Methylene blue; scale bar = 5 flm. Fig. 2. Electron micrograph of a merogonial plasmodium (MPL) with three diplokaryotic nuclei (D). Scale bar 2 flm. Fig.3. Light micrograph of merogonial plasmodia dividing by plasmotomy (arrows) and by rosette-like budding (arrow-heads) producing merozoites (M) with one diplokaryon (D) each. Giemsa; scale bar = 10 flm. Fig.4. Electron micrograph of merozoites (M) produced by plasmotomy (arrow-heads). D = diplokaryon; scale bar = 2 flm.
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into spores of aberrant shape; egg- and bean-shaped spores have been observed. These spores have the same cytology as normal spores, but the polar filaments are longer, usually making a single coil.
The mature spore The mature spores are spherical. Living spores measure 2.5-2.7 flm (Fig. 9), fixed and stained spores 1.6-1.8 flm (Figs. 10, 11). The spore wall has the normal three layers: an approximately 6 nm thick plasma membrane; a lucent endospore about 42 nm thick at the anterior pole and up to 100 nm thick over parts of the spore; and outermost a 45-55 nm thick exospore (Fig. 11). The exospore is composed of four different layers, which in direction outwards are: a c. 14 om thick, electron-dense layer with a granular border to the endospore; a distinctly less electron-dense c. 17 nm thick layer; a c. 6 nm thick double-layer; and a granular surface layer of irregular thickness, 17-35 nm, with an indistinct border (Fig. 12).
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The isofilar polar filament is c. 130 nm in diameter where it joins the anchoring disc but tapers posteriorly to 102 nm (Fig. 11). The filament is curved, and it seems to end close to the posterior vacuole or to continue and extend into the membrane system inside the vacuole (Fig. 13). The transverselly sectioned filament exhibits four layers, in direction inwards: (1) a c. 6 nm thick unit membrane; (2) an electron-dense layer, c. 3 nm; (3) a less electron-dense layer, c. 19 om; and (4) the core composed of diffuse layers of slightly different electron density, measuring c. 58 nm in diameter (Fig. 14). The anchoring apparatus is of normal construction with an up to 310 nm wide anchoring disc, composed of at least 6 layers. The polaroplast has two regions. The anterior polaroplast consists of closely packed, membrane-lined c. 5-7 nm wide lamellae. The longitudinal sectioned anterior polaroplast is about 38 nm wide and nearly ten times as long as wide (Fig. 11). The polar sac which encloses the anchoring disc, extends laterally, enclosing approximately the anterior 3/4 of the umbrella-like part of the polaroplast (Fig. 11).
Page 274 Figs. 5-8. Sporogony of Coccospora micrococcus. Fig. 5. Electron micrograph oftwo early sporonts (SP), where the two components of the diplokarya are separating and centriolar plaques (C) are visible. Arrow-heads indicate possible synaptonemal complexes that would imply a meiotic division. The sporophorous vesicle (SV) is initiated at this stage, and granular material (+) fills the episporonta1 space. Scale bar = 1.5 flm. Fig. 6. Electron micrograph of a sporont (SP) at the four-lobe stage with three lobes visible. N = nucleus; scale bar = 1.5 flm. Inset shows the sporont wall at this stage, arrow-heads indicate the primordium of the double-layer. Scale bar = 200 nm. Fig. 7. Light micrograph of sporogonial stages, showing four-lobed plasmodia (arrow) and the asynchronous division of the sporont yielding sporoblasts (SB). Note the difference in size between the sporont lobes and the merozoites in Fig. 3. Giemsa; scale bar = 10 11m. Fig. 8. Electron micrograph of a sporoblast (SB) demonstrating the developing polar filament (PF) with the anchoring apparatus (A) and the anterior (PA) and posterior (PP) parts of the polaroplast. T = tubular inclusions of the episporontal space; scale bar = 111m.
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Page 275 Figs. 9-12. Mature spores of Coccospora micrococcus. Fig. 9. Light micrograph of living spores (S). Phase-contrast microscopy; scale bar = 5 11m. Fig. 10. Light micrograph of fixed and stained spores (S). Giemsa; scale bar = 5 11m. Fig. 11. Electron micrograph of a mature spore demonstrating the spore wall (arrows) with the double-layer of the exospore visible; the anchoring disc (A), the polar sac (PS), the short polar filament (PF), the anterior lamellar polaroplast (PA), the posterior sac-like polaroplast and the nucleus (N). Scale bar = 150 nm. Inset shows how the polar filament bends without making any coils * indicates the posterior end of the filament. Scale bar = 150 nm. Fig. 12. Electron micrograph of the spore wall demonstrating the plasma membrane (arrow-heads), the lucent endospore (EN) and the exospore (EX) with four distinct layers (I-IV). S =spore; * =artifact; scale bar =50 nm.
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Page 276 Figs. 13-16. The ultrastructure of mature spores of Coccospora micrococcus. Fig. 13. The external unit membrane of the polar filament (PF) appears continuous (arrow) with the membrane system of the posterior vacuole Transversely sectioned vacuole membranes resemble tubules (arrow-head). Fig. 14. Transversely sectioned polar filament exhibiting four layers (1-4). Arrows indicate the smooth endoplasmic reticulum of the cytoplasm. Fig. 15. Transversal section through the posterior polaroplast, demonstrating the mosaic arrangement of the membrane-lined (arrow-heads) sacs PF =Polar filament. Fig. 16. Longitudinal section of the posterior vacuole demonstrating the regular arrangement of membranes (arrow-heads). Figs. 13-16: scale bars = 100 nm.
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Figs. 17-18. Electron micrographs of the inclusions and the wall of the sporophorous vesicle. Fig. 17. Tubular inclusion (T) of exospore origin in the episporontal space, arrows indicate the double layer of the exospore material. Fig. 18. Detail of adjacent sporophorous vesicles, in one of them is indicated the membrane-like wall (arrows), remainders of granular inclusions (*) and part of a mature spore (S). Figs. 17-18: scale bars = 100 nm.
The posterior polaroplast occupies about 1/3 of the spore volume. It consists of sac-like, membrane-bounded compartments, c. 0.5 11m in diameter, in a mosaic arrangement around the polar filament (Fig. 15). The compartments are filled with diffuse material, somewhat less electron-dense than the cytoplasm (Fig. 11). The single nucleus has approximately the same electron density as the cytoplasm and occupies most of the spore volume below the posterior polaroplast (Fig. 11). The largest sectioned nucleus measured approximately 604 nm. A thin strand of electron-dense cytoplasm with numerous polyribosomes separates the nucleus from the posterior polaroplast. The posterior vacuole is situated opposite to the anchoring disc, and is composed of a membrane-system (Figs. 13, 16). The longitudinally sectioned membranes lie about 8 nm apart (Fig. 16). Transverselly sectioned membranes seem to be curled up in a regularly arranged whorl, resembling tubules about 17-19 nm wide (Fig. 13). The diameter of the largest sectioned vacuole was c.0.5I1 m.
The sporophorous vesicle The wall of the sporophorous vesicle is initiated as blisters from the plasma membrane of the young sporont. The blisters are filled with granular material, and when they grow into a continuous sporophororus vesicle, the
granules are dispersed gradually in the episporontal space. When sporoblast buds are present, excess exospore material forms tubular structures, where the double-layer and surface layer of the exospore constitute the tubular wall (Fig. 8, 17). The inclusions disappear when the spores mature, and only traces remain in vesicles with mature spores. The wall of the vesicle, which resembles an about 7 nm thick unit membrane, is fragile (Fig. 18).
Discussion Cytology The cytology of the microsporidium described herein is basically in accordance with the normal of microsporidia, and only a few details of the cytology of the spore need comments. The exospore of microsporidia is a more or less complex structure (VAVRA 1976; LARSSON 1986). Different types can be distinguished and the construction of the exospore clearly reveals phylogentic relationships as well as it is of practical use for taxonomy. The exospore of the microsporidium described herein has a sequence of layers which indicates that it is related with the Thelohania-like microsporidia (LARSSON 1986). One of the
Redescription of Coccospora micrococcus
layers is a double-layer, resembling a unit membrane (Fig. 12). The construction is close to the exospore of the genus Napamichum, and the only difference is found in the surface layer, the layer externally to the double-layer. The surface layer of the Napamichum spore is a moderately electron-dense, well defined layer of uniform thickness all over the spore (LARSSON 1990). The surface layer of the microsporidium studied by us is dense, amorphous and of irregular thickness, and it resembles the less well developed surface layer found in the genus Toxoglugea (LARSSON 1980). A complex system of membranes is present at the posterior pole of the spore (Figs. 13, 16). The membranes are probably the remainders of the Golgi vesicles from which the extrusion apparatus was generated. Living spores, studied using light microscopy, normally exhibit a large, distinct vacuole in this area. In ultrathinly sectioned spores this region is usually less well preserved, and it has not been revealed in all microsporidia studied ultrastructurally that this structure is a real membrane-lined vacuole (VAVRA 1976). The vacuole region of this microsporidium is composed of folded membraneous material, looking different when sectioned longitudinally (Fig. 16) or transversely (Fig. 13). The close connection between the posterior tip of the polar filament and the vacuole region suggests that the membranes belong to a continuous system (Fig. 13).
Taxonomy Cocconema micrococcus was described very briefly, and all diagnostic characters available are: the shape (spherical) and size (1.8-2 11m) of the spores, that the spores are grouped in spherical aggregates, the pathogenic action (adipose tissue affected, giving a milky appearance to the host), that schizogonic stages were observed, the host (midge larvae of the species Tanypus setiger), and the provenance (Grenoble and Montessaux in France) (LEGER & HESSE 1921). The species has not been treated in later publications. The slides in LEGER'S collection have been destroyed (DEGRANGE, pers. commun.). LEGER & HESSE (1921) did not specify how the material was handled. They only refer to living and silver impregnated spores. In the introduction to the paper, living Cocconema spores in general are reported to be 2-3 11m in diameter. In the description of the individual species more detailed measurements are given. As it is difficult to measure living spores without using monolayer techniques, it might be suspected that the detailed measurements were made on stained material. Spores of the microsporidium described herein measure 2.5-2.7 11m when alive and 1.6-1.8 11m when fixed, which is comparable to the spore sizes of C. micrococcus (1.8-2 11m),
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C. octospora (2.10 11m) and C. polyspora (2-3.20 11m). Two of these species are clearly different from the microsporidium studied by us: C. octospora (slightly greater spores, sporulation in the gut epithelium) and C. polyspora (slightly greater spores in groups of 16, 32 or more) and can be disregarded. The fourth Cocconema species, C. slavinae is a parasite of the gut epithelium of a fresh water oligochaete, and it is also clearly different. Similarities between C. micrococcus and the Swedish microsporidium are: the shape and size of the spores, the group of hosts, the tissue affinity, and the presence of rosette-like budding presporal stages together with dispersed or grouped mature spores. The type host and the Swedish host are midge larvae of different genera, but as both genera belong in the subfamily Tanypodinae, that difference appears to be of minor importance. There are no compelling reasons to doubt that the microsporidium studied by us is C. micrococcus, but as no type material exists we cannot provide the ultimate proof. We can see two ways to solve the problem. One is to discard Cocconema micrococcus for the reason that the type material has been destroyed and the description is not sufficient for identification, to apply to the International Commission on Zoological Nomenclature to have the name suppressed, and to describe our microsporidium as a new species. The other solution is to accept a more or less arbitrary identification, accept that we have studied Cocconema micrococcus, and accept that our results can be used for a redescription of the species. The recent discussion about the type concept of the coccidian genus Sarcocystis, where the Commission stated about nomina dubia that "Conventional taxonomic procedure is here generally to restrict the application of an old name arbitrarily to one of the newly differentiated taxa" (MELVILLE 1980), makes it clear to us that it is not likely that the Commission would decide to suppress the name Cocconema micrococcus. The real choice is actually between accepting arbitrary identification or describing a new taxon, leaving Cocconema micrococcus as a taxonomic ghost. There are several genera of microsporidia characterized by having small spherical spores. The three genera of Metchnikovellidae: Amphiacantha, Amphiamblys and Metchnikovella, and the Chytridiopsis-1ike microsporidia: Burkea, Buxtehudea, Chytridiopsis, Nolleria and Steinhausia have spores of aberrant characters and cytology, and they are therefore excluded for this microsporidium. We also disregard Hessea, Ovavesicula and Spherospora, with one species each. Hessea squamosa is basically diplokaryotic throughout the life cycle, and the spores are enclosed by a thick envelope (ORMIERES & SPRAGUE 1973). Ovavesicula popilliae produces 32 sporoblasts by plasmotomy inside a thickwalled sporophorous vesicle, and the spores are spheroid to ovoid (ANDREADIS &
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HANULA 1987). Spherospora andinae produces merozoites in chains of four, and the sporogony yields 8, 16 or more rarely 32 sporoblasts (GARCIA 1991). In 1975 HAZARD & OLDARCE (1975) revised the Thelohania-like microsporidia and described the new genus Pilosporella, characterized by spherical spores. Two species, the type species Pilosporella fishi and P. chapmani, both parasites of mosquito larvae in the U.S.A., were described. About a decade later BECNEL et al. (1986) made a new investigation of P. chapmani. They confirmed what HAZARD & OLDACRE (1975) had suspected, namely that the species is dimorphic, producing monokaryotic octospores in mosquito larvae and diplokaryotic dispores in adult mosquitoes. The microsporidium described herein resembles Pilosporella, but differs in two important characters: the octospores of the microsporidium treated herein are formed by rosette-like budding (Figs. 6, 7), while the octospores of P. chapmani are formed from a chain-like plasmodium, and even if both microsporidia have exospores of the Thelohania type, the sequence and the dimensions of the layers are different (BECNEL et al. 1986). Tubular inclusions of the type produced by P. chapmani do not occur in the sporophorous vesicles of the microsporidium described herein which is a further difference. The genus Coccospora KUDO, 1925 was originally named Cocconema LEGER & HESSE, 1921. None of the four species originally described in the genus Cocconema were designated as type species. The diagnosis of Cocconema is brief, but more precise than most genera of the same age: "Microsporidies a spores spheriques, tres petites, cocciformes avec filament spiral assez court, diversement groupees selon les especes, en amas spMriques ou ovo'ides non proteges par une envelope kystique propre" (LEGER & HESSE 1921). The nuclear configurations are described, but as the interpretation is influenced by the condition of the myxosporidia, they must be disregarded. The following year the family Cocconemidae, only characterized by "Spores spMriques", was established for the genus (LEGER & HESSE 1922). In his monograph on microsporidia KUDO (1924) used the brieffamily diagnosis both for the genus Cocconema and for the family. At the same time C. micrococcus was selected as type species, probably for the reason that this species appears first in the description. KUDO (1925) introduced the new name Coccospora without improving the diagnosis of the genus. Coccospora micrococcus was later transferred to the genus Nosema, and by that act the genus Coccospora was actually abolished (WEISER 1961). SPRAGUE (1977) discussed the fate of the species, and as it lacked Nosema characteristics, as well as characteristics of any other established genus, the species was transferred to the collective and unclassified genus Microsporidium. How-
ever, in the recent classification of microsporidia SPRAGUE et al. (1992) concluded that as the genus Coccospora never has been suppressed by the International Commission on Zoological Nomenclature, the name is still available. Coccospora and Coccosporidae were recognized as valid taxa, but it was remarked that data needed to classify the family are yet not obtained. In the diagnosis of Cocconema was stated that the spores were not protected by "une envelope kystique propre" (LEGER & HESSE 1921). This does not allow us to conclude that C. micrococcus lacks sporophorous vesicles. Sporophorous vesicles are normally not visible using light microscopy, exept in those cases were the envelopes are persistent, and the presence is revealed by spores occuring in groups with regular numbers of spores. In the discussion Cocconema is compared to Chytridiopsis, another genus with spherical spores. The Chytridiopsis species partly sporulate in thick-walled, durable cysts, where the original plasma membrane of the sporogonial plasmodium is included, not fragile sporophorous vesicles (LARSSON 1993). So, actually the diagnosis tells us that Cocconema lacks cysts of the Chytridiopsis-type, but it does not exclude the presence of fragile sporophorous vesicles. KUDO (1925) established the new family Coccosporidae to substitute Cocconemidae LEGER & HESSE, 1922. If Coccospora is accepted as a valid genus it should belong in the family Coccosporidae, unless transferred to a still older family. However, the microsporidium treated herein has a presporal development with nuclei coupled as diplokarya, sporogony by rosette-like budding, octosporous sporogony in fragile sporophorous vesicles generated by the sporont, and a sporal cytology of the basic Thelohania-type, including an exospore where one of the layers is a double-layer. Undoubtedly the microsporidium is related with the Thelohania-like microsporidia. HAZARD & OLDACRE (1975) established the new family Thelohaniidae for Thelohania and a great number of related genera in a well documented monographic study, using modem techniques, where the new taxa are characterized precisely and adequately. This publication is a milestone in the history of the taxonomy of microsporidia. If the genus Coccospora is acknowledged as valid, and if the species studied by us is accepted to be the type species C. micrococcus, it is obvious that the genus Coccospora is related with the Thelohania-like microsporidia and they should belong to the same family. Applying the International Code of Zoological Nomenclature (1985) strictly, Coccosporidae is the oldest available name for the family (Article 23). However, to substitute the well known and clearly characterized family Thelohaniidae with the badly known Coccosporidae only for the reason of priority cannot be sound taxonomy, and therefore we include the genus Coccospora KUDO 1925 in the family Thelohaniidae HAZARD & OLDACRE 1975.
Redescription of Coccospora micrococcus
Taxonomic Summary and Descriptions Coccospora
KUDO
1925, emended diagnosis
Merogony diplokaryotic. Number of merogonial cycles unknown. Meiosis probably occurs. Octosporoblastic sporogony. The sporont divides in rosette-like fashion, producing eight sporoblasts. Spores monokaryotic, spherical without projections. Exospore four-layered, including a double-layer. Polaroplast with a lamellar and a sac-like part. Polar filament isofilar. Sporogony in a sporophorous vesicle. Only one sporogonial sequence observed.
C. micrococcus (LEGER & HESSE, 1921) KUDO, 1925, redescription Merogony: Plurinucleate plasmodia with at least 3 diplokaryotic nuclei divide by plasmotomy and by rosettelike budding. Number of merogonial cycles unknown. Sporogony: As for the genus. Spores: Spherical. Dimensions of living spores: 2.5-2.7 /lm (Swedish material); fixed and stained spores 1.6-1.8 /lm (Swedish material), 1.8-2 /lm (French material, description). Spore wall 102-160 nm thick; exospore fourlayered, 45-55 nm. Polar filament short and curved, without coils, c. 130 nm wide anteriorly, tapers posteriorly to c. 102 nm; it ends at the level of the posterior vacuole. Four distinct layers visible in the transversely sectioned filament. The tightly compressed lamellae of the anterior part of the polaroplast form an umbrella-like structure, about ten times long as wide. Posterior polaroplast section with membrane-bound, sac-like compartments, filled with granular material, in transverse sections visible as a mosaic around the polar filament. Polar sac encloses anterior 3/4 of the anterior polaroplast region. Sectioned nucleus up to 604 nm wide. Vacuole, c. 0.5 /lm wide, with membrane system in the posterior region of the spore. Vacuole membranes in parallel arrays when sagitally sectioned, resembling tubules when transverselly sectioned. Sporophorous vesicles: A plasma membrane-like envelope, approximately 7 nm thick, encloses the microsporidium from the beginning of the sporogony. Granular inclusions at the beginning of the sporogony, tubular inclusions of exospore nature appear when sporoblasts are formed. Vesicles with mature spores fragile. Host tissues involved: Fat body.
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Type host: Tanypus setiger (Diptera, Tanypodinae), larvae. Hosts of the Swedish material: Arctopelopia sp. (Diptera, Tanypodinae), larvae. Type locality: Grenoble et Montessaux (Haute-Saone), France. Locality of the Swedish material: Temporary forest pools at Finstorp, Scania, Sweden. Type material: Destroyed. Swedish material: Slides No. 870729-A (1-12) and No. 910731-B (1-2); in the collection of the senior author. Acknowledgements: We are greatly indebted to Mrs. LINA GEFORS, Mrs. BIRGITTA KLEFBOHM, Mrs. INGER NORLING, Department of Zoology, Lund, Mrs. AGNETA PERSSON, Department of Anatomy, Lund for skiful technical assistance. A special thanks to Dr. ERIC CARLEMAN and Dr. ROLF ODSELIUS at the Electron Microscopy Unit of the Departments of Medicine, Lund for providing transmission electron microscope facilities when urgently needed, and to Prof. CHARLES DEGRANGE, Department of Zoology and Animal Biology, Scientific and Medical University, Grenoble, for the information of the fate of coll. L. LEGER. The investigation was founded by the Royal Swedish Academy of Sciences and the Swedish National Science Research Council.
References ANDREADIS, T. G. & HANULA, 1. L. (1987): Ulrastructural study and description of Ovavesicula popilliae n. g., n. sp. (Microsporida: Pleistophoridae) from the Japanese Bettie, Popillia japonica (Coleoptera: Scarabaeidae). J. Protozool. 34: 15-21. BECNEL, 1. J., HAZARD, E. I. & FUKUDA, T. (1986): Fine structure and development of Pilosporella chapmani (Microspora: Thelohaniidae) in the mosquito, Aedes triseratus (SAY). J. Protozool. 33: 60-66. COSTE-MATHIEZ, F. & TUZET, 0. (1977): Microsporidies de Chironomidae (Dipteres, Nematoceres). Ann. Sci. Nat. Zool. Paris, 12° Serie, 19: 113-135. GARCIA, J. J. (1991): Estudios sobre el cicio de vida y ultraestructura de Spherospora andinae gen. et. sp. nov. (Microspora. Thelohaniidae), un nuevo Microsporidio de Simulidos neotropicales. Neotropica 37: 15-23. HAZARD, E. I. & OLDACRE, S. W. (1975): Revision of Microsporida (Protozoa) close to Thelohania, with descriptions of one new family, eight new genera and thirteen new species. U. S. Dep. Agric. Tech. Bull. No. 1530, 104 pp. HOSTOUNSKY, Z. & ZI~KA, Z. (1979): A modification ofthe "agar cushion method" for observation and recording rnicrosporidian spores. J. Protozool. 26: 41A-42A.
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International Code of Zoological Nomenclature, 3rd ed. (1985). International Trust for Zoological Nomenclature, London. KUDO, R. (1924): A biological and taxonomic study of the Microsporidia. III. BioI. Mon. 9 (2/3): 1-268. - (1925): Microsporidia. Science 61, No. 1579: 366. LARSSON, J. I. R. (1980): Ultrastructural study of Toxoglugea variabilis n. sp. (Microsporida: Thelohaniidae), a microsporidian parasite of the biting midge Bezzia sp. (Diptera: Ceratopogonidae). Protistologica 16: 17-32. - (1986): Ultrastructure, function and classification of Microsporidia. Progress in Protistology 1: 325-390. - (1990): Description of a new microsporidium of the water mite Limnochares aquatica and establishment of the new genus Napamichum (Microspora, Thelohaniidae). J. Invertebr. Pathol. 55: 152-161. - (1993): Description of Chytridiopsis trichopterae n. sp. (Microspora, Chytridiopsidae), a microsporidian parasite of the Caddis Fly Polycentropus flavomaculatus (Trichoptera, Polycentropodidae), with comments on relationships between the families Chytridiopsidae and Metchnikovellidae. 1. Euk. Microbiol. 40: 37-48. LEGER, L. & HESSE, E. (1921): Microsporidies a spores spheriques. C. R. Acad. Sci. Paris. 173:1419-1421. - - (1922): Microsporidies bacteriformes et essai de systematique du groupe. C. R. Acad. Sci. Paris 174: 327-330. MELVILLE, R. V. (1980): Nomina dubia and available names. Z. Parasitenkd. 62: 105-109.
ORMrERES, R. & SPRAGUE, V. (1973): A new family, new genus, and new species allied to the Microsporida. J. Invertebr. Pathol. 21: 224-240. POISSON, R. (1953): Ordre des Microsporidies. In: P.-P. GRASSE (ed.), Traite de Zoologie, Vol. I, Fasc. 2. pp. 1042-1070. Paris. REYNOLDS, E. S. (1963): The use of lead citrate at high pH as an electrono-paque stain in electron microscopy. J. Cell BioI. 17: 208-212. ROMEIS, B. (1968): Mikroskopische Technik. Mtinchen und Wien. SPRAGUE, V. (1977): Systematics of Microsporidia. In: L. A. BULLA Jr. & T. C. CHENG (eds.), Comparative Pathobiology, Vol. 2. New York and London. - BECNEL, J. J. & HAZARD, E. I. (1992): Taxonomy of phylum Microspora. Crit. Rev. Microbiol. 18: 285-395. VAVRA, J. (1976): Structure of the microsporidia. In: L. A. BULLA, Jr. & T. C. CHENG (eds.), Comparative Pathobiology, Vol. 1: 1-85. New York and London. WEISER, 1. (1961): Die Mikrosporidien als Parasiten der Insekten. Monogr. Angew. Entomol. 17: 149 pp. Accepted: October II, 1993
Corresponding author: EVA BYLEN, Department of Zoology, Division of Systematics, University of Lund, Helgonav. 3, S-223 62 Lund, Sweden; Telefax: +46-46-10 4541, Telephone +46-46-107802.
ARCHIV
FOR
PROTISTEN KUNDE
Ultrastructural Study and Redescription of Coccospora micrococcus (LEGER & HESSE, 1921) KUDO, 1925 (Microspora, Thelohaniidae), a Parasite of Midge Larvae of the Genus Arctopelopia sp. (Diptera, Chironomidae) in Sweden EVA
K. C.
BYLEN
& J. I.
RONNY LARSSON
Department of Zoology, Division of Systematics, University of Lund, Lund, Sweden
Summary: The cytology of the microsporidium Coccospora micrococcus, which is a parasite
of the adipose tissue of midge larvae, Arctopelopia sp., in Sweden, is described based on observations using light and electron microscopy. Merogonial plasmodia divide by plasmotomy and by rosette-like budding, yielding diplokaryotic merozoites. The number of merogonial cycles is unknown. The sporogony probably begins with a meiotic division. Each diplokaryotic sporont yields eight sporoblasts. A fragile sporophorous vesicle encloses each group of spores. The monokaryotic spores are spherical. Living spores measure 2.5-2.71Jm, fixed and stained spores 1.6-1.8IJm. The spore wall has a layered exospore with a double-layer. The polaroplast consists of an anterior lamellar and a posterior sac-like part. The isofilar polar filament is short, without coils. The membranes of the vacuole, produced by the Golgi apparatus, and their connection with the polar filament are visible in mature spores. There are two types of inclusions of the sporophorous vesicle: granular, initiated in the early phase of the sporogony, and tubular of exospore origin. The microsporidium is compared to the microsporidia with spherical spores, and the possible relations to the genus Pilosporella and to other Thelohania-like microsporidia are discussed. Key Words: Coccospora micrococcus: Arctopelopia sp.; Microsporidia; Ultrastructure; Redescription; Taxonomy.
Inroduction In 1921 LEGER & HESSE described four new microsporidia with spherical spores, three of them parasites of midge larvae, and created the new genus Cocconema for them (LEGER & HESSE 1921). The species were described in the manner typical for the time, with a minimum of characteristics: the shape and size of the spores, the name and geographic location of the host, and a few comments on the gross pathology. There were no illustrations. In a following paper, the descriptions were repeated together with three line drawings of Cocco-
nema spores, but without indications of the species to which they belonged (LEGER & HESSE 1924). The species described in the genus Cocconema have been treated in a series of reviews (KUDO 1924; POISSON 1953; WEISER 1961; COSTE-MATHIEZ & TUZET 1977; SPRAGUE 1977), and their taxonomic positions have been discussed. When it became apparent that the name Cocconema was preoccupied, the new name Coccospora was proposed (KUDO 1925). However, no later studies, based on new sources of material, have appeared, and we
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know as little about the species as we knew in 1921. It cannot be excluded that some of these species actually have been studied in the meantime. The diagnoses are so vague and difficult to use that it is easier for a microsporidiologist, when studying a microsporidium of this type, to find some characteristics not mentioned in the description and base the description of a new species upon them, rather than to identify the microsporidium with any of the old Cocconema species. We have studied a microsporidian parasite of midge larvae, producing spherical octospores. The small number of characteristics that can be compared suggests that it is the species originally called Cocconema micrococcus. The species is redescribed, based on characteristics derived from the light an electron microscopic cytology. It is compared to other microsporidia with spherical spores, and the taxonomic considerations are discussed.
Material and Methods The hosts were three specimens of Arctopelopia sp. (Diptera, Chironomidae) in two different samples of midge larvae collected from temporary forest pools at Finstorp, in the province of Scania, southern Sweden, on July 29, 1987 and July 31, 1991. Fresh squash preparation~ were made using the agar method of HOSTOUNSKY & ZIZKA (1979) and studied using phase contrast microscopy and dark field illumination. Permanent squash preparations were lightly air-dried, fixed in Bouin-Duboscq-Brasil solution overnight and stained using Giemsa solution or Heidenhains's iron haematoxylin (ROMEIS 1968). All permanent preparations were mounted in DePeX (BDH Chemicals Ltd England). Measurements were made with an eye-piece micrometer at x 1,000. For transmission electron microscopy pieces of infected segments were excised and fixed in 2.5 % (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4 °C for 24 hand 47 h. After washing in cacodylate buffer and postfixation in 2.0% (w/v) osmium tetroxide in cacodylate buffer for 1 h in 4°C, the pieces were washed and dehydrated in an ascending series of ethanols to propylene oxide and embedded in Epon. Semithin sections for light microscopy were stained with methylen blue, and ultrathin sections for electron microscopy were stained with uranyl acetate and lead citrate according to REYNOLDS (1963).
Results Pathogenicity The posterior segments of infected larvae exhibited white spots, shining through the semitransparent cuticle. The fat body was the only tissue affected by the parasite. It was hypertrophic and filled with mostly sporulating microsporidia (Fig. 1). No syncytium was formed.
Merogony The earliest stages observed were merogonial plasmodia with diplokaryotic nuclei (Fig. 2). They divide by plasmotomy and by rosette-like budding yielding 5.3-7.4 flm wide merozoites with nuclei coupled as diplokarya, measuring 2-3 flm across the widest diameter (Fig. 3). Merogonial plasmodia and merozoites are surrounded by an approximately 8 nm thick plasma membrane. The cytoplasm is electron dense with numerous free ribosomes (Fig. 4).
Sporogony The last generation of merozoites mature to sporonts. The two components of the diplokaryon dissociate (Fig. 5), probably followed by a meiotic division, yielding lobed plasmodia with four isolated nuclei (Figs. 6, 7). The following mitosis yields totally 8 nuclei per lobed plasmodium. The plasmodium divides in a rosettelike manner, but all lobes are not always synchronous (Fig. 7). The sporogony is octosporoblastic, and the sporoblasts are enclosed by a sporophorous vesicle generated by the sporont (Fig. 5). In ultrathin sections the beginning of the sporogony is indicated by a layer of electron dense material secreted outside the plasma membrane of the sporonts, and simultaneously the cytoplasm becomes less electron dense. When the sporoblasts are budded off from the plasmodium their cytoplasm is rather lucent, but the electron density increases when they mature to spores (Fig. 8). The polar filament and the polaroplast are generated by the Golgi apparatus in the normal way. Occasionally sporoblasts develop
Figs. 1-4. Pathology and merogony of Coccospora micrococcus. Fig.1. Semithin section of infected fat body filled with spores (S). * indicates the hypoderm. Methylene blue; scale bar = 5 flm. Fig. 2. Electron micrograph of a merogonial plasmodium (MPL) with three diplokaryotic nuclei (D). Scale bar 2 flm. Fig.3. Light micrograph of merogonial plasmodia dividing by plasmotomy (arrows) and by rosette-like budding (arrow-heads) producing merozoites (M) with one diplokaryon (D) each. Giemsa; scale bar = 10 flm. Fig.4. Electron micrograph of merozoites (M) produced by plasmotomy (arrow-heads). D = diplokaryon; scale bar = 2 flm.
=
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into spores of aberrant shape; egg- and bean-shaped spores have been observed. These spores have the same cytology as normal spores, but the polar filaments are longer, usually making a single coil.
The mature spore The mature spores are spherical. Living spores measure 2.5-2.7 flm (Fig. 9), fixed and stained spores 1.6-1.8 flm (Figs. 10, 11). The spore wall has the normal three layers: an approximately 6 nm thick plasma membrane; a lucent endospore about 42 nm thick at the anterior pole and up to 100 nm thick over parts of the spore; and outermost a 45-55 nm thick exospore (Fig. 11). The exospore is composed of four different layers, which in direction outwards are: a c. 14 om thick, electron-dense layer with a granular border to the endospore; a distinctly less electron-dense c. 17 nm thick layer; a c. 6 nm thick double-layer; and a granular surface layer of irregular thickness, 17-35 nm, with an indistinct border (Fig. 12).
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The isofilar polar filament is c. 130 nm in diameter where it joins the anchoring disc but tapers posteriorly to 102 nm (Fig. 11). The filament is curved, and it seems to end close to the posterior vacuole or to continue and extend into the membrane system inside the vacuole (Fig. 13). The transverselly sectioned filament exhibits four layers, in direction inwards: (1) a c. 6 nm thick unit membrane; (2) an electron-dense layer, c. 3 nm; (3) a less electron-dense layer, c. 19 om; and (4) the core composed of diffuse layers of slightly different electron density, measuring c. 58 nm in diameter (Fig. 14). The anchoring apparatus is of normal construction with an up to 310 nm wide anchoring disc, composed of at least 6 layers. The polaroplast has two regions. The anterior polaroplast consists of closely packed, membrane-lined c. 5-7 nm wide lamellae. The longitudinal sectioned anterior polaroplast is about 38 nm wide and nearly ten times as long as wide (Fig. 11). The polar sac which encloses the anchoring disc, extends laterally, enclosing approximately the anterior 3/4 of the umbrella-like part of the polaroplast (Fig. 11).
Page 274 Figs. 5-8. Sporogony of Coccospora micrococcus. Fig. 5. Electron micrograph oftwo early sporonts (SP), where the two components of the diplokarya are separating and centriolar plaques (C) are visible. Arrow-heads indicate possible synaptonemal complexes that would imply a meiotic division. The sporophorous vesicle (SV) is initiated at this stage, and granular material (+) fills the episporonta1 space. Scale bar = 1.5 flm. Fig. 6. Electron micrograph of a sporont (SP) at the four-lobe stage with three lobes visible. N = nucleus; scale bar = 1.5 flm. Inset shows the sporont wall at this stage, arrow-heads indicate the primordium of the double-layer. Scale bar = 200 nm. Fig. 7. Light micrograph of sporogonial stages, showing four-lobed plasmodia (arrow) and the asynchronous division of the sporont yielding sporoblasts (SB). Note the difference in size between the sporont lobes and the merozoites in Fig. 3. Giemsa; scale bar = 10 11m. Fig. 8. Electron micrograph of a sporoblast (SB) demonstrating the developing polar filament (PF) with the anchoring apparatus (A) and the anterior (PA) and posterior (PP) parts of the polaroplast. T = tubular inclusions of the episporontal space; scale bar = 111m.
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Page 275 Figs. 9-12. Mature spores of Coccospora micrococcus. Fig. 9. Light micrograph of living spores (S). Phase-contrast microscopy; scale bar = 5 11m. Fig. 10. Light micrograph of fixed and stained spores (S). Giemsa; scale bar = 5 11m. Fig. 11. Electron micrograph of a mature spore demonstrating the spore wall (arrows) with the double-layer of the exospore visible; the anchoring disc (A), the polar sac (PS), the short polar filament (PF), the anterior lamellar polaroplast (PA), the posterior sac-like polaroplast and the nucleus (N). Scale bar = 150 nm. Inset shows how the polar filament bends without making any coils * indicates the posterior end of the filament. Scale bar = 150 nm. Fig. 12. Electron micrograph of the spore wall demonstrating the plasma membrane (arrow-heads), the lucent endospore (EN) and the exospore (EX) with four distinct layers (I-IV). S =spore; * =artifact; scale bar =50 nm.
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Page 276 Figs. 13-16. The ultrastructure of mature spores of Coccospora micrococcus. Fig. 13. The external unit membrane of the polar filament (PF) appears continuous (arrow) with the membrane system of the posterior vacuole Transversely sectioned vacuole membranes resemble tubules (arrow-head). Fig. 14. Transversely sectioned polar filament exhibiting four layers (1-4). Arrows indicate the smooth endoplasmic reticulum of the cytoplasm. Fig. 15. Transversal section through the posterior polaroplast, demonstrating the mosaic arrangement of the membrane-lined (arrow-heads) sacs PF =Polar filament. Fig. 16. Longitudinal section of the posterior vacuole demonstrating the regular arrangement of membranes (arrow-heads). Figs. 13-16: scale bars = 100 nm.
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Figs. 17-18. Electron micrographs of the inclusions and the wall of the sporophorous vesicle. Fig. 17. Tubular inclusion (T) of exospore origin in the episporontal space, arrows indicate the double layer of the exospore material. Fig. 18. Detail of adjacent sporophorous vesicles, in one of them is indicated the membrane-like wall (arrows), remainders of granular inclusions (*) and part of a mature spore (S). Figs. 17-18: scale bars = 100 nm.
The posterior polaroplast occupies about 1/3 of the spore volume. It consists of sac-like, membrane-bounded compartments, c. 0.5 11m in diameter, in a mosaic arrangement around the polar filament (Fig. 15). The compartments are filled with diffuse material, somewhat less electron-dense than the cytoplasm (Fig. 11). The single nucleus has approximately the same electron density as the cytoplasm and occupies most of the spore volume below the posterior polaroplast (Fig. 11). The largest sectioned nucleus measured approximately 604 nm. A thin strand of electron-dense cytoplasm with numerous polyribosomes separates the nucleus from the posterior polaroplast. The posterior vacuole is situated opposite to the anchoring disc, and is composed of a membrane-system (Figs. 13, 16). The longitudinally sectioned membranes lie about 8 nm apart (Fig. 16). Transverselly sectioned membranes seem to be curled up in a regularly arranged whorl, resembling tubules about 17-19 nm wide (Fig. 13). The diameter of the largest sectioned vacuole was c.0.5I1 m.
The sporophorous vesicle The wall of the sporophorous vesicle is initiated as blisters from the plasma membrane of the young sporont. The blisters are filled with granular material, and when they grow into a continuous sporophororus vesicle, the
granules are dispersed gradually in the episporontal space. When sporoblast buds are present, excess exospore material forms tubular structures, where the double-layer and surface layer of the exospore constitute the tubular wall (Fig. 8, 17). The inclusions disappear when the spores mature, and only traces remain in vesicles with mature spores. The wall of the vesicle, which resembles an about 7 nm thick unit membrane, is fragile (Fig. 18).
Discussion Cytology The cytology of the microsporidium described herein is basically in accordance with the normal of microsporidia, and only a few details of the cytology of the spore need comments. The exospore of microsporidia is a more or less complex structure (VAVRA 1976; LARSSON 1986). Different types can be distinguished and the construction of the exospore clearly reveals phylogentic relationships as well as it is of practical use for taxonomy. The exospore of the microsporidium described herein has a sequence of layers which indicates that it is related with the Thelohania-like microsporidia (LARSSON 1986). One of the
Redescription of Coccospora micrococcus
layers is a double-layer, resembling a unit membrane (Fig. 12). The construction is close to the exospore of the genus Napamichum, and the only difference is found in the surface layer, the layer externally to the double-layer. The surface layer of the Napamichum spore is a moderately electron-dense, well defined layer of uniform thickness all over the spore (LARSSON 1990). The surface layer of the microsporidium studied by us is dense, amorphous and of irregular thickness, and it resembles the less well developed surface layer found in the genus Toxoglugea (LARSSON 1980). A complex system of membranes is present at the posterior pole of the spore (Figs. 13, 16). The membranes are probably the remainders of the Golgi vesicles from which the extrusion apparatus was generated. Living spores, studied using light microscopy, normally exhibit a large, distinct vacuole in this area. In ultrathinly sectioned spores this region is usually less well preserved, and it has not been revealed in all microsporidia studied ultrastructurally that this structure is a real membrane-lined vacuole (VAVRA 1976). The vacuole region of this microsporidium is composed of folded membraneous material, looking different when sectioned longitudinally (Fig. 16) or transversely (Fig. 13). The close connection between the posterior tip of the polar filament and the vacuole region suggests that the membranes belong to a continuous system (Fig. 13).
Taxonomy Cocconema micrococcus was described very briefly, and all diagnostic characters available are: the shape (spherical) and size (1.8-2 11m) of the spores, that the spores are grouped in spherical aggregates, the pathogenic action (adipose tissue affected, giving a milky appearance to the host), that schizogonic stages were observed, the host (midge larvae of the species Tanypus setiger), and the provenance (Grenoble and Montessaux in France) (LEGER & HESSE 1921). The species has not been treated in later publications. The slides in LEGER'S collection have been destroyed (DEGRANGE, pers. commun.). LEGER & HESSE (1921) did not specify how the material was handled. They only refer to living and silver impregnated spores. In the introduction to the paper, living Cocconema spores in general are reported to be 2-3 11m in diameter. In the description of the individual species more detailed measurements are given. As it is difficult to measure living spores without using monolayer techniques, it might be suspected that the detailed measurements were made on stained material. Spores of the microsporidium described herein measure 2.5-2.7 11m when alive and 1.6-1.8 11m when fixed, which is comparable to the spore sizes of C. micrococcus (1.8-2 11m),
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C. octospora (2.10 11m) and C. polyspora (2-3.20 11m). Two of these species are clearly different from the microsporidium studied by us: C. octospora (slightly greater spores, sporulation in the gut epithelium) and C. polyspora (slightly greater spores in groups of 16, 32 or more) and can be disregarded. The fourth Cocconema species, C. slavinae is a parasite of the gut epithelium of a fresh water oligochaete, and it is also clearly different. Similarities between C. micrococcus and the Swedish microsporidium are: the shape and size of the spores, the group of hosts, the tissue affinity, and the presence of rosette-like budding presporal stages together with dispersed or grouped mature spores. The type host and the Swedish host are midge larvae of different genera, but as both genera belong in the subfamily Tanypodinae, that difference appears to be of minor importance. There are no compelling reasons to doubt that the microsporidium studied by us is C. micrococcus, but as no type material exists we cannot provide the ultimate proof. We can see two ways to solve the problem. One is to discard Cocconema micrococcus for the reason that the type material has been destroyed and the description is not sufficient for identification, to apply to the International Commission on Zoological Nomenclature to have the name suppressed, and to describe our microsporidium as a new species. The other solution is to accept a more or less arbitrary identification, accept that we have studied Cocconema micrococcus, and accept that our results can be used for a redescription of the species. The recent discussion about the type concept of the coccidian genus Sarcocystis, where the Commission stated about nomina dubia that "Conventional taxonomic procedure is here generally to restrict the application of an old name arbitrarily to one of the newly differentiated taxa" (MELVILLE 1980), makes it clear to us that it is not likely that the Commission would decide to suppress the name Cocconema micrococcus. The real choice is actually between accepting arbitrary identification or describing a new taxon, leaving Cocconema micrococcus as a taxonomic ghost. There are several genera of microsporidia characterized by having small spherical spores. The three genera of Metchnikovellidae: Amphiacantha, Amphiamblys and Metchnikovella, and the Chytridiopsis-1ike microsporidia: Burkea, Buxtehudea, Chytridiopsis, Nolleria and Steinhausia have spores of aberrant characters and cytology, and they are therefore excluded for this microsporidium. We also disregard Hessea, Ovavesicula and Spherospora, with one species each. Hessea squamosa is basically diplokaryotic throughout the life cycle, and the spores are enclosed by a thick envelope (ORMIERES & SPRAGUE 1973). Ovavesicula popilliae produces 32 sporoblasts by plasmotomy inside a thickwalled sporophorous vesicle, and the spores are spheroid to ovoid (ANDREADIS &
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HANULA 1987). Spherospora andinae produces merozoites in chains of four, and the sporogony yields 8, 16 or more rarely 32 sporoblasts (GARCIA 1991). In 1975 HAZARD & OLDARCE (1975) revised the Thelohania-like microsporidia and described the new genus Pilosporella, characterized by spherical spores. Two species, the type species Pilosporella fishi and P. chapmani, both parasites of mosquito larvae in the U.S.A., were described. About a decade later BECNEL et al. (1986) made a new investigation of P. chapmani. They confirmed what HAZARD & OLDACRE (1975) had suspected, namely that the species is dimorphic, producing monokaryotic octospores in mosquito larvae and diplokaryotic dispores in adult mosquitoes. The microsporidium described herein resembles Pilosporella, but differs in two important characters: the octospores of the microsporidium treated herein are formed by rosette-like budding (Figs. 6, 7), while the octospores of P. chapmani are formed from a chain-like plasmodium, and even if both microsporidia have exospores of the Thelohania type, the sequence and the dimensions of the layers are different (BECNEL et al. 1986). Tubular inclusions of the type produced by P. chapmani do not occur in the sporophorous vesicles of the microsporidium described herein which is a further difference. The genus Coccospora KUDO, 1925 was originally named Cocconema LEGER & HESSE, 1921. None of the four species originally described in the genus Cocconema were designated as type species. The diagnosis of Cocconema is brief, but more precise than most genera of the same age: "Microsporidies a spores spheriques, tres petites, cocciformes avec filament spiral assez court, diversement groupees selon les especes, en amas spMriques ou ovo'ides non proteges par une envelope kystique propre" (LEGER & HESSE 1921). The nuclear configurations are described, but as the interpretation is influenced by the condition of the myxosporidia, they must be disregarded. The following year the family Cocconemidae, only characterized by "Spores spMriques", was established for the genus (LEGER & HESSE 1922). In his monograph on microsporidia KUDO (1924) used the brieffamily diagnosis both for the genus Cocconema and for the family. At the same time C. micrococcus was selected as type species, probably for the reason that this species appears first in the description. KUDO (1925) introduced the new name Coccospora without improving the diagnosis of the genus. Coccospora micrococcus was later transferred to the genus Nosema, and by that act the genus Coccospora was actually abolished (WEISER 1961). SPRAGUE (1977) discussed the fate of the species, and as it lacked Nosema characteristics, as well as characteristics of any other established genus, the species was transferred to the collective and unclassified genus Microsporidium. How-
ever, in the recent classification of microsporidia SPRAGUE et al. (1992) concluded that as the genus Coccospora never has been suppressed by the International Commission on Zoological Nomenclature, the name is still available. Coccospora and Coccosporidae were recognized as valid taxa, but it was remarked that data needed to classify the family are yet not obtained. In the diagnosis of Cocconema was stated that the spores were not protected by "une envelope kystique propre" (LEGER & HESSE 1921). This does not allow us to conclude that C. micrococcus lacks sporophorous vesicles. Sporophorous vesicles are normally not visible using light microscopy, exept in those cases were the envelopes are persistent, and the presence is revealed by spores occuring in groups with regular numbers of spores. In the discussion Cocconema is compared to Chytridiopsis, another genus with spherical spores. The Chytridiopsis species partly sporulate in thick-walled, durable cysts, where the original plasma membrane of the sporogonial plasmodium is included, not fragile sporophorous vesicles (LARSSON 1993). So, actually the diagnosis tells us that Cocconema lacks cysts of the Chytridiopsis-type, but it does not exclude the presence of fragile sporophorous vesicles. KUDO (1925) established the new family Coccosporidae to substitute Cocconemidae LEGER & HESSE, 1922. If Coccospora is accepted as a valid genus it should belong in the family Coccosporidae, unless transferred to a still older family. However, the microsporidium treated herein has a presporal development with nuclei coupled as diplokarya, sporogony by rosette-like budding, octosporous sporogony in fragile sporophorous vesicles generated by the sporont, and a sporal cytology of the basic Thelohania-type, including an exospore where one of the layers is a double-layer. Undoubtedly the microsporidium is related with the Thelohania-like microsporidia. HAZARD & OLDACRE (1975) established the new family Thelohaniidae for Thelohania and a great number of related genera in a well documented monographic study, using modem techniques, where the new taxa are characterized precisely and adequately. This publication is a milestone in the history of the taxonomy of microsporidia. If the genus Coccospora is acknowledged as valid, and if the species studied by us is accepted to be the type species C. micrococcus, it is obvious that the genus Coccospora is related with the Thelohania-like microsporidia and they should belong to the same family. Applying the International Code of Zoological Nomenclature (1985) strictly, Coccosporidae is the oldest available name for the family (Article 23). However, to substitute the well known and clearly characterized family Thelohaniidae with the badly known Coccosporidae only for the reason of priority cannot be sound taxonomy, and therefore we include the genus Coccospora KUDO 1925 in the family Thelohaniidae HAZARD & OLDACRE 1975.
Redescription of Coccospora micrococcus
Taxonomic Summary and Descriptions Coccospora
KUDO
1925, emended diagnosis
Merogony diplokaryotic. Number of merogonial cycles unknown. Meiosis probably occurs. Octosporoblastic sporogony. The sporont divides in rosette-like fashion, producing eight sporoblasts. Spores monokaryotic, spherical without projections. Exospore four-layered, including a double-layer. Polaroplast with a lamellar and a sac-like part. Polar filament isofilar. Sporogony in a sporophorous vesicle. Only one sporogonial sequence observed.
C. micrococcus (LEGER & HESSE, 1921) KUDO, 1925, redescription Merogony: Plurinucleate plasmodia with at least 3 diplokaryotic nuclei divide by plasmotomy and by rosettelike budding. Number of merogonial cycles unknown. Sporogony: As for the genus. Spores: Spherical. Dimensions of living spores: 2.5-2.7 /lm (Swedish material); fixed and stained spores 1.6-1.8 /lm (Swedish material), 1.8-2 /lm (French material, description). Spore wall 102-160 nm thick; exospore fourlayered, 45-55 nm. Polar filament short and curved, without coils, c. 130 nm wide anteriorly, tapers posteriorly to c. 102 nm; it ends at the level of the posterior vacuole. Four distinct layers visible in the transversely sectioned filament. The tightly compressed lamellae of the anterior part of the polaroplast form an umbrella-like structure, about ten times long as wide. Posterior polaroplast section with membrane-bound, sac-like compartments, filled with granular material, in transverse sections visible as a mosaic around the polar filament. Polar sac encloses anterior 3/4 of the anterior polaroplast region. Sectioned nucleus up to 604 nm wide. Vacuole, c. 0.5 /lm wide, with membrane system in the posterior region of the spore. Vacuole membranes in parallel arrays when sagitally sectioned, resembling tubules when transverselly sectioned. Sporophorous vesicles: A plasma membrane-like envelope, approximately 7 nm thick, encloses the microsporidium from the beginning of the sporogony. Granular inclusions at the beginning of the sporogony, tubular inclusions of exospore nature appear when sporoblasts are formed. Vesicles with mature spores fragile. Host tissues involved: Fat body.
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Type host: Tanypus setiger (Diptera, Tanypodinae), larvae. Hosts of the Swedish material: Arctopelopia sp. (Diptera, Tanypodinae), larvae. Type locality: Grenoble et Montessaux (Haute-Saone), France. Locality of the Swedish material: Temporary forest pools at Finstorp, Scania, Sweden. Type material: Destroyed. Swedish material: Slides No. 870729-A (1-12) and No. 910731-B (1-2); in the collection of the senior author. Acknowledgements: We are greatly indebted to Mrs. LINA GEFORS, Mrs. BIRGITTA KLEFBOHM, Mrs. INGER NORLING, Department of Zoology, Lund, Mrs. AGNETA PERSSON, Department of Anatomy, Lund for skiful technical assistance. A special thanks to Dr. ERIC CARLEMAN and Dr. ROLF ODSELIUS at the Electron Microscopy Unit of the Departments of Medicine, Lund for providing transmission electron microscope facilities when urgently needed, and to Prof. CHARLES DEGRANGE, Department of Zoology and Animal Biology, Scientific and Medical University, Grenoble, for the information of the fate of coll. L. LEGER. The investigation was founded by the Royal Swedish Academy of Sciences and the Swedish National Science Research Council.
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Corresponding author: EVA BYLEN, Department of Zoology, Division of Systematics, University of Lund, Helgonav. 3, S-223 62 Lund, Sweden; Telefax: +46-46-10 4541, Telephone +46-46-107802.