Ultrastructural study and description of Mrazekia tetraspora Léger & Hesse, 1922 and transfer to a new genus Scipionospora n. g. (Microspora, Caudosporidae)

Europ. J. Protisto!' 32, 104-115 (1996) February 23, 1996

European Journal of

PROTISTOLOGY

Ultrastructural Study and Description of Mrazekia tetraspora Leger & Hesse, 1922 and Transfer to a New Genus Scipionospora n. g. (Microspora, Caudosporidae) Eva K. C. BylE~n and J. I. Ronny Larsson Department of Zoology, University of Lund, Lund, Sweden

SUMMARY A rod-shaped microsporidium with tetrasporoblastic life cycle has been found in the fat body of midge larvae of the genus Endochironomus (Diptera, Chironomidae) in Sweden. The nuclei are in diplokaryotic arrangement throughout the life cycle. The earliest stages observed were multinucleate merogonial plasmodia dividing by fragmentation. The sporont divides by rosette-like budding. Excess lobes without nuclei are produced beside the four sporoblasts. Fixed and stained spores measure 4.5 - 7.9 x 0.7-0.9 j.lm. The exospore includes a double-layer. The anchoring apparatus is of normal construction. The isofilar polar filament is short, without coils and exhibits seven layers in transverse sections. The polaroplast consists of an anterior lamellar part and a posterior sac-like part. A vacuole is visible at the posterior pole of the mature spores. The sporogonial stages and the mature spores are enclosed by a sporophorous vesicle which has a wall resembling a unit membrane. The inclusions of the episporontal space are granular and polygonal tubules of exospore origin. The microsporidium is compared to other rod-shaped microsporidia from midge larvae and to diplokaryotic rod-shaped microsporidia. The microsporidium is identified as Mrazekia tetraspora Leger & Hesse, 1922. A new genus, Scipionospora, is established as the species neither can be accomodated in the genus Mrazekia nor in another established genus. The new genus is provisionally included in the family Caudosporidae.

Introduction The earliest microsporidia producing rod-shaped spores were described in 1916 when Leger & Hesse established the genus Mrazekia [11]. In 1922 Leger & Hesse added two new species to the genus Mrazekia, and one of them, M. tetraspora, exhibited spores in distinct groups of four [12]. We have studied a microsporidian parasite of midge larvae that produces four rod-shaped spores in a sporophorous vesicle. The small number of characteristics that can be compared suggests that our microsporidium is the species Mrazekia tetraspora Leger & Hesse, 1922. The species is redescribed, based on characteristics derived from the light and electron microscopic cytol0932-4739-96-0032-0104$3.50-0

ogy. It is compared to other microsporidia with rodshaped spores, the taxonomic considerations are discussed, and a new genus is established. Material and Methods The microsporidium was present in two samples of midge larvae, collected on July 30,1985, from the river Hoje near the village of Bjellerup and on April 19, 1988 from a small pond, communicating with the river Kiivlinge near the village of Gardsranga, in Scania, in the south of Sweden. The host were three larvae of Endochironomus sp. Kieffer (Diptera, Chironomidae). Permanent squash preparations were lightly air-dried and fixed in Bouin-Duboscq-Brasil solution for at least one hour.

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© 1996 by Gustav Fischer Verlag, Stuttgart

Scipionospora tetraspora g. et. comb. n.. 105 For paraffin sectioning infected segments were fixed in the same fixative overnight, washed and dehydrated in an ascending series of ethanols, cleared in butanol and embedded in paraplast. Sections were cut longitudinally at 10 11m. Squash preparations and sections were stained using Heidenhain's haematoxylin and Giemsa solution. For details on the histological techniques used see the manual by Romeis l16]. Permanent preparations were mounted in DePeX (BDH Chemicals Ltd England). Measurements were made with an eye-piece micrometer at x 1,000, except for living spores which were measured in one light micrograph. For scanning electron microscopy spores were smeared on circular cover glasses, lightly air-dried, and fixed in 2.5% glutaraldehyde in 0.2 M sodium cacodylate buffer at 4°C for 25 h. After washing in buffer and critical point drying the smears were covered with gold and palladium. For transmission electron microscopy infected segments of host tissue were excised and fixed in 2.5% (v/v) glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) at 4°C for 25 - 27 hours. After washing in cacodylate buffer and post-fixation in 2% (w/v) osmium tetroxide 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. Sections were stained with uranyl acetate and lead citrate l151.

Results Prevalence and pathology. The microsporidium was found in three larvae, which exhibited a whitish discoloration of the fat body lobes. The fat body was filled with microsporidia. Infected cells were slightly hypertrophic but not degraded to syncytia (Fig. 1). Merogony. The earliest stage observed in the light microscope was merogonial plasmodia containing a large number of nuclei (Fig. 2). Using the electron microscope the nuclei of the dividing plasmodia were observed to be in diplokaryotic arrangement (Fig. 3). The resulting merozoites are rounded, 2.0-4.5 llm in diameter, with 1.2-3.5 llm wide diplokarya (Figs. 4, 7). The electron dense cytoplasm contains endoplasmic reticulum and free ribosomes (Figs. 4, 7). A membrane covered with ribosomes on the cytoplasmic side separates the merogonial stages from the host cytoplasm (Figs. 3, 4). The membrane that is enclosing the merogonial stages of the life cycle is interpreted as a parasitophorous vacuole because of the ribosomes attached to the outside. There are no signs of this membraneous structure in any later stages of the life cycle. The number of merogonial cycles is unknown. Sporogony. The last generation of merozoites matures to sporonts (Figs. 5, 6). The maturation is revealed by the electron dense material secreted from the sporont to form a cover of the plasma membrane. Simultaneously the cytoplasm of the sporont becomes less electron dense. The dense surface material is released in a blister-like manner from the plasma membrane forming a sporophorous vesicle (Fig. 5). A new layer of electron dense material is then secreted to form the primordia of the exospore covering the plasma membrane. The diplokaryon undergoes the first divi-

sion at this stage, yielding a sporogonial plasmodium with two diplokarya inside the sporophorous vesicle (Figs. 5, 7, 8). The diplokarya divide once more, and the four diplokaryotic nuclei hence produced are situated in the periphery of the rounded sporogonial plasmodium (Fig. 8). There were never more than four diplokaryotic nuclei observed and never chain-like sporogonial stages. The four sporoblasts are formed by rosette-like budding (Figs. 7, 9, 10, 11). The sporophorous vesicle is persistent, and mature spores always occur in groups of four (Figs. 9, 12). In addition to sporoblasts excess lobes of the plasmodium are budded off (Figs. 11, 12). The plasmodium fragments are enclosed by sporont wall material. We have never been able to identify nuclei in the fragments, and they are successively disintegrated to irregular tubular inclusions. Young immature spores are irregularly shaped, and they are enclosed by a complete exospore layer (Fig. 13). The Golgi apparatus, from which the polar filament with the polar sac and the polaroplast are generated, is visible in the posterior part of the immature spore (Fig. 13). The primordia of the polaroplast appear as membranes folded in lamellar manner near the anterior pole, and forming sac-like structures filled with moderately electron dense material posteriorly (Fig. 13). During the maturation of the spore an anchoring apparatus is organized. The polar sac and the two regions of the polaroplast become more regularly organized with the short lamellae anteriorly and the sac-like structures posteriorly. The lucent endospore layer appears late. It is not visible until the spores have reached a regular rod-shape and the extrusion apparatus is nearly complete (Fig. 14). The spore. Mature spores are rod-shaped, straight or lightly curved (Figs. 8, 9, 15, 18). Living spores measure 5.4-5.9 x 1.1-1.211m (x = 5.6 x 1.111m, n = 15) (Fig. 16). Fixed and stained spores measure 4.5 -7.9 x 0.7-0.911m (x = 5.9 x 0.811m, n = 40) (Fig. 17). The living spores, which were measured in one light micrograph, were too few to reveal the total variation in size. In stained spores the large, 0.9-1.411m, distended and lucent posterior vacuole is visible in the posterior fourth of the spores, and the small lucent anterior polaroplast is seen at the anterior end (Figs. 15, 17). The spore wall consists of three layers (Figs. 15, 19, 20): a plasma membrane, which is a 6 -7 nm thick unit membrane, a translucent endospore measuring 1117 nm at the anterior pole and varying between 22 and 48 nm over the rest of the spore, and a 2428 nm wide layered exospore. The internal layer of the exospore is thin and granular, the median layer is a double layer, resembling an 8-9 nm thick unit membrane. The external, 16-19 nm thick, layer is of medium electron density with more dense granulation on the surface (Figs. 19, 20, 21). The polar filament is isofilar, short and without coils (Figs. 14,15). The anterior part follows the central axis of the spore for about 2/5 of the spore length, turns

Scipionospora tetraspora g. et. comb. n.. 107 aside to touch the spore wall at the level of the diplokaryon and terminates close to the posterior vacuole (Figs. 14, 15, 22). Close to the anchoring apparatus the polar filament is 120-160 nm wide, forming an up to 106 nm long, somewhat funnel-like part, which joins the anchoring disc (Fig. 20). The diameter of the filament then tapers successively, measuring about 110 nm over most of its length, to 80 nm posteriorly. The mature polar filament exhibits a distinct stratification, in transverse sections visible as seven concentrical layers of different electron density and thickness (Fig. 19). The filament is covered by a c. 5 nm thick unit membrane (I), and in direction inwards follows a c. 5 nm thick and electron dense layer (II); a c. 10 nm thick layer (III) of lucent, spiralized fibril-like structures; a 2-3 nm thick moderately electron dense layer (IV); layer (V) the least electron dense 515 nm thick layer; a more electron dense 10-20 nm thick layer (VI); and the innermost layer (VII), 3034 nm in diameter, with electron dense centre. The polar filament is attached to an anchoring apparatus of normal construction. The up to 310 nm wide anchoring disc is composed of superimposed layers of different electron density. At least six layers were revealed. The strata become more conspicuous the more the longitudinal sections of the disc deviate from the mid-line of the spore. The polar sac encloses approximately 2/3 of the anterior polaroplast like an umbrella. The unit membrane wall of the sac is continuous with the surface layer (I) of the polar filament (Figs. 20, 21) and the membranes of the polaroplast. The polaroplast, which occupies approximately the anterior half of the spore, is composed of two parts where the compartments are lined with c. 5 nm thick unit membranes (Figs. 19, 20, 21). The anterior part consists of regularly arranged lamellae which appear so closely packed that they seem to lack a lumen (Fig. 21). This region is in longitudinal sections about half as thick as the diameter of the polar filament and it extends for about 1/20 of the spore length. The posterior region of the polaroplast occupies most of the volume in the anterior part of the spore, and it extends for about 1/4 of the spore length, ending in the middle of the spore close to the diplokaryon. It consists of irregularly arranged, sac-like compartments (Fig. 20). The two nuclei lie close together, coupled as a diplokaryon, in the centre of the spore. The diplokaryon

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measures 1.4-2.0 11m when sectioned longitudinally, which equals about 1/3 of the spore length. The nucleoplasm is electron dense with areas of less dense material close to the nuclear membranes (Fig. 15). The external nuclear membrane is sometimes continuous with the endoplasmic reticulum. The cytoplasm is electron dense with numerous free ribosomes and with a rough endoplasmic reticulum. A membrane-lined vacuole, filled with electron dense material, is situated in the posterior end of the spore (Figs. 15, 17, 22). The vacuole sometimes collapsed due to fixation techniques. Some sporophorous vesicles contained one large anomalous spore together with remainders of plasmodium lobes (Figs. 1,8,17). Anomalous spores are much larger than normal spores, and their shape is more oval and irregular. Their exospore layer appears normal, but no typical spore organelles are developed (Fig. 1). The sporophorous vesicle. The wall is c. 8 nm thick and has a structure similar to a unit membrane (Fig. 21). Sectioned sporophorous vesicles holding mature spores measured 2.2-3.4 x 5.0-8.5 11m. The vesicle is secreted by the young sporont, and a large amount of granular inclusions are observed (Fig. 5). This material disappears during the sporogonial development, and in vesicles with mature spores only traces of the granular inclusions persist (Figs. 1,15). Tubuluslike inclusions, 70- 90 nm wide and often shaped as polygons in transverse sections, appear when the sporoblasts are formed (Figs. 12,23,24). The layers of their walls equal the layers of the exospore, which reveals that they are formed by superfluous exospore material. Most of the tubulus-like inclusions persist in vesicles holding mature spores (Fig. 1).

Discussion

Cytology. The microsporidium studied conforms to some extent to other rod-shaped microsporidia from fresh-water hosts, but a few details need comments: the exospore, the short polar filament, the development and ultrastructure of the sac-like posterior polaroplast, the diplokaryotic arrangement of the nuclei, the inclusions of the sporophorous vesicle and the excess lobes produced during the sporogony.

Figs. 1-4. Pathology and presporal development of Scipionospora tetraspora gen. et comb. nov. - Fig. 1. Ultrathin section of the infected fat body. The fat cells are slightly hypertrophic, the compressed nucleus and some mitochondria are visible. Sporophorous vesicles with mature spores and tubular inclusions and with anomalous macrospores ('f) occur together with vesicles with earlier stages of the microsporidium. The arrows indicate the remainders of excess material from the dividing sporonts. Fig. 2. Light micrograph of a merogonial plasmodium with several diplokarya. Heidenhain's haematoxylin. - Fig. 3. Electron micrograph of a dividing (") merogonial plasmodium with nuclei in diplokaryotic arrangement. The double arrowheads indicate the parasitophorous vacuole covered with ribosomes on the cytoplasmic side. Arrows indicate the plasma membrane of the plasmodium. - Fig. 4. Electron micrograph showing a merozoite with diplokaryotic nuclei and a sporophorous vesicle with sporoblasts. D =diplokaryon, H =host cell nucleus, MI = mitochondria, S =spores, SB =sporoblasts, SV =sporophorous vesicle, T =tubular inclusions. Scale bars: Fig. 1 =2 !-lm, Fig. 2 = 10 !-lm, Figs. 3 and 4 = l!-lm.

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Scipionospora tetraspora g. et. comb. n.. 109

The exospore of the present microsporidium has a double-layer. This is typical of the Thelohania-like microsporidia [3, 19] but is also described from other microsporidian species which have a different life cycle like Cougourdella polycentropi [7] and Pernicivesicula gracilis Bylen & Larsson, 1994 [1]. An isofilar polar filament without coils is characteristic for a number of microsporidia with rod-shaped spores like Baculea daphniae Loubes & Akbarieh, 1978 [13], Cylindrospora fasciculata Larsson, 1986 [6], Helmichia aggregata Larsson, 1982 [5] and Pernicivesicula gracilis Bylen & Larsson, 1994 [1]. However, other rod-shaped microsporidia, like Octosporea muscadomesticae Flu, 1911 [14] and Rectispora reticulata Larsson, 1990 [8], have a long, coiled isofilar polar filament, which means that rod-shaped spores must not necessarily have short polar filaments. The ultrastructure and the development of the polaroplast of this microsporidium conforms with the polaroplast arrangement described for Pernicivesiculagracilis Bylen & Larsson, 1994 [1]: small vesicular structures in the sporoblast fuse to form the large saclike compartments that constitute the polaropJast of the mature spore. The primordia of the posterior polaroplast are also similar to polaroplast primordia in sporoblasts of Helmichia aggregata, but in this species the vesicles do not fuse. They only aggregate to form the spongious part of the polaroplast. In longitudinal sections of immature and mature spores the two elements of the diplokaryotic nuclei might appear not to be coupled. However, in those cases the nuclei are sectioned tangentia lly, outside the contact area of the two rounded nuclei. The inclusions of the sporophorous vesicle are of the normal construction for Thelohania-like microsporidia and for Pernicivesicula gracilis [1]. Tubular inclusions constructed of exospore material is the normal condition for these microsporidia, but the polygonal shape of the transversely sectioned tubular inclusions are unique for the microsporidium described herein. The excess lobes that are budded off from the sporont could be mistaken for aborted sporoblasts when observed in electron micrographs. However, sporogonial plasmodia in light microscopic preparations had

never more than four diplokarya, and there were never structures resembling nuclei in these lobes. The conclusion must be that they are not sporoblasts but excess material from the dividing plasmodium. Taxonomy. There are similarities between the microsporidium treated herein and other microsporidia of midges. The electron density and the dimension of each individual subdivision of the exospore of our microsporidium are the same as described for Helmichia aggregata Larsson, 1982 [5], another microsporidium with rod-shaped spores produced by rosette-like budding inside sporophorous vesicles. The major differences between these two species are the number of nuclei and the number of sporoblasts produced. The microsporidium treated herein is tetrasporoblastic and has two nuclei throughout the life cycle, while H. aggregata conforms with the typical life cycle of the Thelohania-like microsporidia [19] with diplokaryotic merogony, diplokaryotic sporont and a sporogony involving meiosis that yields eight sporoblasts with one nucleus each. There are three microsporidian species with rodshaped spores isolated from midge larvae that have approximately the same spore dimensions as the microsporidium of this study: Microsporidium orthocladii (Coste-Mathiez & Manier, 1968) Sprague, 1977 [2] (fresh spores = 5 x 1.8 !lm), Mrazekia bacilliformis Leger & Hesse, 1922 [12] (spores = 5 x 0.8 !lm) and Mrazekia tetraspora Leger & Hesse, 1922 [12] (spores = 6.5 x 0.8 !lm). The dimensions of M. tetraspora are nearly identical to the microsporidium described herein where fixed and stained spores measure 4.5 - 7.9 x 0.7-0.9 !lm, and accordingly to the description the spores are always arranged in groups of four inside sporophorolls vesicles. M. tetraspora was isolated from the fat body of midge larvae of the genus Tanytarsus in France. Leger and Hesse described the spores to have a short hyaline prolongation, 1.2 ~Lm, in the posterior end of the spores and they indicated that this was illustrated in a drawing of the paper [12]. This drawing only reveals a fixed and stained rod-shaped spore of normal appearance with the two lucent areas normally observed in preparations: the polaroplast area in the anterior end and the vacuole in the posterior end.

.... Figs. 5 -11. Sporogony of Scipionospora tetraspora. - Fig. 5. Ultrathin section of an early sporont exhi biting dividing diplokaryotic nuclei, arrowheads indicate the plane of division. The thin sporophorous vesicle (thick arrows) is released from the plasma membrane (thin arrows) of the sporont. The thick sporont wall is formed by the material secreted from the sporont, excess material forms the granular inclusions of the episporontal space. - Fig. 6. Light micrograph of an early sporont with the diplokaryon indicated. Heidenhain's haematoxylin. - Fig. 7. Light micrograph of merogonial plasmodia nand merozoites with the diplokarya indicated by arrowheads. An early sporont with dividing nuclei is visible, and a later sporont dividing by rosette-like budding is indicated by an arrow. Giemsa stain. - Fig. 8. Light micrograph of two early sporonts with dividing diplokarya and two older sporonts, one with three of the four diplokarya visible. The two components of the diplokaryon are clearly demonstrated. An anomalous macrospore (,:.) is indicated. Heidenhain's haematoxylin. - Fig. 9. Light micrograph of spores grouped in four. Heidenhain's haematoxylin. - Fig. 10. Electron micrograph of a lobed sporogonial plasmodium exhibiting one of the diplokarya. Parallel arrays of microtubuli (,.) follow the zone where the two components of the diplokaryon are attached. The microrubuli are associated with electron dense patches of chromatin (arrows). - Fig. 11. Electron micrograph of a sporogonial plasmodium with four sporoblast buds. D =diplokaryon, G =granular inclusions, M =merozoites, S =spores, SP =sporogonial plasmodium, W =sporont wall. Scale bars: Figs. 5, 10 and 11 = 1 !Jm, Figs. 6, 7, 8 and 9 =5 !Jm.

Scipionospora tetraspora g. et. comb. n. . 111

We interpret the hyaline prolongation to correspond to a large posterior vacuole. The size ratio between the prolongation and the entire spore described for M. tetraspora (1.2/6.5~ liS) corresponds to the microsporidium of this paper where the vacuole also occupies c. 1/5 of the total spore length when studied by the light microscope (Figs. 15, 17). Leger's collection of slides has been destroyed (Degrange, personal communication), study of type material of M. tetraspora is excluded, and the species has not been reexamined in later publications. The few characteristics of M. tetraspora that can be compared correspond quite well with the microsporidium treated herein, so we see ~o serious reason to doubt that they are identical speCIes. The genus Mrazekia has recently been re-evaluated and the diagnosis emended [10]. The life cycle is disporoblastic, lacking sporophorous vesicles. The spores have a unique manubroid polar filament, which is prominently enlarged in the posterior end. The exospore is three-layered and the polaroplast has two lamellar parts. Consequently our microsporidium cannot be included in this genus. There are about 16 microsporidian genera where tetrasporoblastic sporogony has been observed, alone or in combination with other numbers of sporoblasts. Only one of them, Baculea Loubes & Akbarieh, 1978, has rod-shaped spores. However, no life cycle stage of B. daphniae, so far the only species of the genus, has nuclei in diplokaryotic configurations [13]. It is apparent that neither Baculea nor any other tetrasporoblastic genus can accomodate the microsporidium of this paper. Three genera of microsporidia have nuclei in diplokaryotic arrangement throughout the life cycle together with rod-shaped spores without prolongations: Bacillidium Janda, 1928 [4, 9], Rectispora Larsson, 1990 [8], and Octosporea Flu, 1911 [14]. The emended diagnosis of Bacillidium describes the genus to be disporoblastic and to lack sporophorous vesicles sensu stricto. The spores have a manubroid polar filament and an exospore where the external layer is released from the internal homogenous layer forming a sac [9]. The genus Rectispora is described as disporoblastic

without sporophorous vesicles. The shape of the spores differs somewhat from the present microsporidium, as the spores are wider in the mid-region. The ultrathinly sectioned spore exhibits a uniform exospore layer enclosed by a net-like, exospore derived, surface layer. Major differences between the genus Octosporea and our microsporidium are: the more curved shape of the spores and the layers of the exospore, where the succession of the layers does not conform with the exospore described herein. Furthermore the construction of the polaroplast is different with an anterior part described as granular and a posterior part with closely packed lamellae. The differences listed above exclude our microsporidium from the genera Bacillidium, Rectispora and Octosporea. As the microsporidium treated herein cannot be included in any established genus, we see no alternative to create a new genus for M. tetraspora and establish the new combination Scipionospora tetraspora (Leger & Hesse, 1922) comb. n. There is one family erected for microsporidia that produce four sporoblasts in sporophorous vesicles: Gurleyidae Sprague, 1977. The three genera included by Sprague [17] are characterized by their oval, pyriform or lageniform and monokaryotic spores. Consequently Gurleyidae cannot be used for this microsporidium. The microsporidium described herein resembles the Thelohania-like microsporidia in the construction of the exospore and the sporogonial division, but as the nuclei are associated as diplokarya throughout the life cycle, also the family Thelohaniidae, where the diplokaryotic condition breaks at the beginning of the sporogony, is excluded. Octosporea muscadomesticae, the type species of Octosporea, is diplokaryotic, producing eight sporoblasts by rosette-like budding inside a sporophorous vesicle [14]. Sprague [17] included the genus Octosporea in the family Caudosporidae Weiser, 1958 [20] together with Caudospora Weiser, 1946 and Weiseria Doby & Saguez, 1964. These genera were originally reported to produce eight or sixteen sporoblasts, but more thorough investigations revealed that the Caudospora-species are able to produce four, eight or six-

... Figs. 12-14. The ultrastructure of the spores of Scipionospora tetraspora. - Fig. 12. Transversely sectioned sporophorous vesicle with four, not fully mature, spores; excess material (") from the sporogony and polygonal tubular inclusions is visible in the episporontal space. One component of the diplokaryon and the transversely sectioned polar filament are visible in the spores. - Fig. 13. An irregularly shaped immature spore with a posterior Golgi apparatus, the primordia of the polar sac, the lamellar part of the polaroplast, the posterior part of the polaroplast and the polar filament are exhibited. The granular inclusions of the episporontal space are almost disintegrated at this stage of the life cyle. - Fig. 14. Longitudinally sectioned immature spore exhibiting the ultrastructural cytology. The layered anchoring apparatus and the polar sac have reached complete development. The anterior part of the polaroplast is still irregular, and the posterior part still consists of a large number of small sac-like compartments. The internal organisation of the polar filament is complete, but the filament has not reached its full length. The filament ends (white arrow) near the posterior pole of the diplokaryon. The membrane-layered posterior vacuole is smaller than in a mature spore. The layers of the exospore are complete, but the endospore layer is still under construction. The undulating spore wall is an artifact. D =diplokaryon, EN =endospore, EX =exospore, G =granular inclusions, GA = Golgi apparatus, PA = anterior polaroplast, PF = polar filament, PP = posterior polaroplast, S = spores, T = tubular inclusions, V = vacuole. All scale bars =
Scipionospora tetraspora g. et. comb. n. . 113

teen sporoblasts [18]. This means that the family which has Caudospora as type genus actually can be characterized by the production of spores in multiples of four (with eight as the most common number). We hesitate to establish a new family for the genus Scipionospora, and we dislike to place the genus in an incertae sedis position. As the family Caudosporidae can accomodate this microsporidium, it seems best to include Scipionospora tetraspora provisionally in this family. Taxonomic Summary and Description

Scipionospora n. g. Merogony diplokaryotic. Number of merogonial cycles unknown. Meiosis not observed. The diplokaryotic sporont divides in a rosette-like manner producing four sporoblasts. Spores diplokaryotic, rod-shaped without projections. Exospore three-layered, including a double-layer. Polar filament isofilar. Polaroplast twoparted, lamellar and sac-like. Sporogony in sporophorous vesicle. Only one sporogonial sequence observed. Etymology. The genus name Scipionospora alluding to the shape of the spores, scipio (lat.) = rod, stick.

Scipionospora tetraspora (Leger & Hesse, 1922) comb. n. Merogony. Plurinucleate plasmodia divide by fragmentation. Merozoites measure 2.0-4.5 /lm in diameter, sectioned diplokarya measure 1.2-3.5/lm. Merogonial stages enclosed in a parasitophorous vacuole. Sporogony. As for the genus. Excess lobes without nuclei are budded off from the sporont during the sporoblast formation, and they are disintegrated when the spores mature. Anomalous sporogony yielding macrospores observed. Spores. Fixed and stained spores measure 4.5 - 7.9 x 0.7-0.9/lm. Spore wall 41-83 nm thick; 24-28 nm

thick exospore, three-layered including a double-layer. Polar filament isofilar, without coils; 120-160 nm in diameter close to the anchoring disc and tapering to 80-110 nm in diameter posteriorly; seven layers visible in transverse sections. The tightly packed lamellae of the anterior part of the polaroplast form an umbrella-like structure 4 times as long as wide. Posterior polaroplast region with sac-like compartments extends for about 1/4 of the spore length. Polar sac encloses most of the anterior polaroplast region. Sectioned nuclei in diplokaryotic arrangement measure about 1/3 of the spore length. Membrane-lined vacuole in posterior region of the spore measures 0.9-1.4 /lm. Sporophorous vesicle. Rounded in squash preparations. The wall of the vesicle is c. 8 nm thick, resembling a unit membrane. It encloses the microsporidium from the beginning of the sporogony. Granular inclusions formed at the beginning of the sporogony disintegrate when the spores mature. Tubular inclusions of exospore nature, in transverse sections shaped as polygons, appear together with sporoblasts and they persist to some extent in vesicles with mature spores. Vesicles persistent also around mature spores. Host tissues involved. Fat body. Syncytia are not formed. Type host. Tanytarsus sp. (Diptera, Chironomidae), larva. Type locality. Grenoble, France. Type material. Destroyed. Host of the Swedish material. Endochironomus sp. Kieffer (Diptera, Chironomidae), larva. Localities of the Swedish material. The river Hoje ii, near the village of Bjellerup, and a small pond connected to the river Kivlinge ii, near the village of Gardstilnga, Scania. Swedish material. Slides No. 850730-B-( 1-11); 850730-L-(1-6); and 880418-C-(1-5) in the collection of the senior author.

... Figs. 15 - 20. The mature spores of Scipionospora tetraspora. - Fig. 15. Two spores lying head to tail in a sporophorous vesicle (arrowheads) with more or less dispersed granular inclusions and disintegrated plasmodium fragments (arrow). Nucleoplasm with two electron densities, the less dense material close to the nuclear membranes (white arrow). The split plasma membrane is an artifact. The end of the polar filament is visible close to the collapsed posterior vacuole (*). - Fig. 16. Light micrograph of living spores tightly packed in groups of four enclosed by a sporophorous vesicle. - Fig. 17. Light micrograph of fixed and stained spores with anchoring apparatus and posterior vacuole (") visible. The arrow indicates an anomalous macrospore. Heidenhain's haematoxylin. - Fig. 18. Scanning electron micrograph of a group of four spores; prolongations and exospore sculpture are absent. - Fig. 19. Transverse section of the anterior part of a spore exhibiting the different layers of the polar filament (I-VII), where layer I is a unit membrane and layer III is fibril-like, the anterior lamellar polaroplast and the polar sac. The different layers of the spore wall are indicated: the plasma membrane, the lucent endospore, the thin granular layer (arrows), the double-layer (arrowheads) and the outermost layer (.,.) of the exospore. - Fig. 20. Longitudinal section of a spore demonstrating the anchoring apparatus and the two parts of the polaroplast. The diameter of the polar filament is somewhat broader where it attaches to the anchoring disc (arrows). The unit membranes of the polar sac, the compartments of the anterior and the posterior polaroplast and the cover of the polar filament (layer (I)) belong to the same system. The posterior polaroplast extends to the level of the diplokaryon. The endospore and the double-layer of the exospore (arrowheads) are indicated. A = anchoring apparatus, D =diplokaryon, EN =endospore, EX =exospore, PA =anterior polaroplast, PF = polar filament, PM = plasma membrane, PP =posterior polaroplast, PS =polar sac, S =spores. Scale bars: Fig. 15 =500 nm, Fig. 16 =10 11m, Figs. 17 and 18 =5 11m, Figs. 19 and 20 = 100 nm.

114 . E. K. C. Bylen and

J. 1.

R. Larsson

Figs. 21-24. The ultrastructure of the two poles of the spore and the sporophorous vesicle. - Fig. 21. Longitudinal section of the anterior pole of a spore demonstrating the details of the anterior polaroplast and the polar sac. The unit membrane (layer I) of the polar filament is indicated by a white arrow. The double-layer of the exospore is indicated by black arrows. The thin wall of the sporophorous vesicle resembles a unit membrane. - Fig. 22. Longitudinal section of the posterior pole of a spore exhibiting a part of the diplokaryon and the last portion of the polar filament ending close to the posterior vacuole «'). - Figs. 23, 24. Transverse and longitudinal sections of polygonal tubular inclusions of the episporontal space revealing their exospore origin (arrows indicate the double-layer). D = diplokaryon, PA = anterior polaroplast, PF = polar filament, PP = posterior polaroplast, PS = polar sac, SV = sporophorous vesicle, T = tubular inclusions. All scale bars = 100 nm.

Acknowledgements We wish to thank Ms Lina Gefors, Ms Birgitta Klefbohm, and Ms Inger Norling, Department of Zoology, University of Lund, and Ms Agneta Persson, Department of Anatomy, University of Lund, for skilful technical assistance. We are also greatly indebted to the staff of the Electron Microscopy Unit of the Departments of Medicine, Lund. Our special thanks are due to Prof. Charles Degrange, Scientific and Medical Univer-

sity of Grenoble, for information about the fate of Louis Leger's collection and to Mikael S6rensson, Department of Zoology, Lund, for helping out with the conjugation of the Latin for the genus name. The investigation was financially supported by grants from the Royal Swedish Academy of Sciences, the Swedish Natural Science Research Council and the foundation "Lars Hiertas minne".

Scipionospora tetraspora g. et. comb. n. . 115

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Key words: Endochironomus larvae - Scipionospora tetraspora - Microsporidia - Ultrastructure Eva Bylen, Department of Zoology, Division of Systematics, Helgonav. 3, S-223 62 Lund, Sweden