Light and electron microscopic description of Polysporoplasma n. g. (Myxosporea: Bivalvulida), polysporoplasma sparis n. sp. from Sparus aurata (L), and Polysporoplasma mugilis n. sp. from Liza aurata L

Europ. J. Protisto!' 31, 77-89 (1995) March 15, 1995

European Journal of

PROTISTOLOGY

Light and Electron Microscopic Description of Polysporoplasma n. g. (Myxosporea: Bivalvulida), Polysporoplasma sparis n. sp. from Sparus aurata (L.), and Polysporoplasma mugilis n. sp. from Liza aurata L. Ariadna Sltja-Bobadilla and Pilar Alvarez-Pellitero Instituto de Acuicultura de Torre de la Sal (CSIC), Ribera de Cabanes, Castell6n, Spain

SUMMARY A new myxosporean genus, Polysporoplasma, is erected and named after the numerous sporoplasms (from 4 to 12) of the spore. Two new species, P. sparis and P. mugilis, found in the teleosteans Sparus aurata and Liza aurata, respectively, are described by light and electron microscopy. They differ in the number of coils of the polar filament, number of sporoplasms and the thickness/length ratio of the spore. Both species are disporous and histozoic in the trunk kidney. Prevalence of infection is 18.3% for P. sparis and 14.8% for P. mugilis. Remarkable features of both spores are the large size of capsulogenic cells which envelope sporoplasmic cells, the pierced ornamentation of the valves and the two posterior bulgesat the end of the spore. The taxonomical and biologicalimplications of the erection of the genus are discussed.

Abbreviations cc

= capsulogenic cell

= lipidic droplet = mitochondria m MMC = melanomacrophage center = nucleus of the capsulogenic cell nc = nucleus of the sporoplasmic cell ns = nucleus of the valvogenic cell nv = sporoplasm s = valve v vc = valvogenic cell L

Introduction In the last decade, with the growing importance of fish culture, the interest for Myxosporea has spread beyond the taxonomical and protozoological boundaries. Nevertheless, the taxonomical and phylogenetical ascription of this enigmatic group is still far from © 1995 by Gustav Fischer Verlag, Stuttgart

being completed. Furthermore, the recent findings on life cycle have posed an urgent need for a major revision of the myxosporean classification. Occasionally, the absence of ultrastructural studies leads to incorrect classifications. This is the case of a myxosporea recently reported in gilthead sea bream (Sparus aurata) from the South Atlantic coast of Spain [16]. It was initially classified as Sphaerospora sp. in absence of detailed electron microscopic observations. However, further light and ultrastructural studies on new infected fish prompt us now to broach its taxonomical ascription. In addition, another new myxosporea close to the aforementioned and found in Liza aurata (Mugilidae) from the western Mediterranean Sea, is also described. Both myxosporean species are regarded as distinct members of a new genus, herein erected and named Polysporoplasma. The species found in Sparus aurata and Liza aurata show enough morphometrical and ultrastructural differences to be considered distinct, and 0932-4739-95-0031-0077$3.50-0

78 . A. Sitja-Bobadilla and P. Alvarez-Pellitero

will be named Polysporoplasma sparis and P. mugilis, respectively. In the present study, their light and electron microscopic descriptions are presented. Material and Methods Fish From April 1990 to April 1993, gilthead sea bream (Sparus aurata), ranging from 8 to 28.5 em in length, were bimonthly sampled from semiintensively cultured stocks of a fish farm located on the South East Atlantic coast. Mullets (Liza aurata), ranging from 18 to 30 ern in length, were obtained from semiintensively cultured stocks of a fish farm, which captured them in the wild at the River Ebro delta (Mediterranean coast). They were examined in October 1991 and May 1993. In addition, gilthead sea bream from the latter farm, ranging from 11 to 31 ern in length, were also examined from October 1991 to September 1993.

Sampling and Histological Procedures Fish were overexposed to the anesthetic MS-222 (Sigma Chemicals Co. St. Louis, MO for USA), weighed, measured, necropsied and their organs excised for both fresh and histological examination. Light microscopy (LM). Thin sections (1-3 um) were obtained from material fixed in 10% buffered formalin and embedded in Historesin (Leica, Spain). They were stained with toluidine blue (TB). Spore measurements were taken directly from fresh material in the light microscope. Descriptions of the myxosporea were made according to the criteria established by Lom and Arthur [6]. Unless otherwise stated, all measurements are given in urn, Transmission electron microscopy (TEM). Small tissue portions were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2, 4°C); postfixed in 1% cacodylic OS04, dehydrated through a graded ethanol series, and embedded in Spurr's resin [17]. Ultrathin sections were double stained with uranyl acetate and lead citrate [10]. Some sections were processed on golden grids and stained by the Thiery reaction for carbohydrates [18] orthe technique forlipids (OTO) [11]. They were studied in a Philips CM-200 transmission electron microscope, operating at 60 kV. Scanning electron microscopy (SEM). Tissue samples were obtained from formalin fixed-paraffin embedded material. Paraffin was removed by several xylol washings and the critical point drying technique was applied. Stubs were covered with a gold/palladium coat of 20-50 nm of thickness with a Polaron Sputter Coater-E 500. Stubs were observed in a Hitachi HS-2300, operating at 15 kV.

Results Polysporoplasma n. g. Diagnosis. Myxosporea: Bivalvulida. Mature spores subspherical. Polar capsules two, subspherical, situated in a plane perpendicular to the plane of suture. Valves two. Several uninucleate sporoplasms, at least four and up to twelve, observed in one plane of section. Pseudoplasmodia disporous.

a

lO~m

b

Fig. 1. Diagrammatic drawings of Polysporoplasma sparis (a) and Polysporoplasma mugilis (b) spores in front view.

Type species. Polysporoplasma sparis n. sp. (Fig. La), Type host. Sparus aurata (L.) Family Sparidae. Polysporoplasma sparis n. sp. Host. Sparus aurata (L.) Family Sparidae, juveniles and adults. Prevalence. 18.3% in both fish farms (18 out of 98 at the Atlantic coast and 13 out of 71 at the Mediterranean coast). Location. (Histozoic) glomeruli (Fig. 7), tubuli, or connective tissue of trunk kidney. Locality. South East Atlantic coast and Western Mediterranean (River Ebro delta), Spain. Description. Mature spores. Subspherical in front and side views, with thickness slightly higher than length (Figs. la, 3 and 5). Anterior pole slightly protruding. Sutural line straight. Valves two, very thick, specially at the vicinity of polar capsules openings (Fig. 8), displaying a pierced or indented appearance on the surface, slightly visible by LM (Figs. 3 and 6). No mucous envelope was observed in Indian Ink preparations (Fig. 4). Posterior end of spore with 2 bulges or horns (one on each valve) in front view (Fig. 3). Polar capsules slightly pyriform, of equal size, opening at the anterior end of the spore near the suture under the thickened part of the valve (Fig. 8). Polar filament with 6-7 coils (Fig. 9). Sporoplasms several (Fig. 10). Dimensions are presented in Table 1. Developmental stages. Very early stages were not seen. Ellipsoidal pseudoplasmodia containing two developing spores (Fig. 2) suggest disporous development. In immature spores, valves appeared thick and tenuous with noticeable residual nuclei, forming two prominent bulges at the posterior end of the spore (Fig. 2). Holotype. Deposited in the Museo Nacional de Ciencias Naturales (Madrid, Spain): Colecci6n Invertebrados no Insectos, with acquisition number 37.01/8.

Polysporoplasma spp. Description . 79

Figs. 2-10. Images of Polysporoplasma sparis from infected trunk kidney of Sparus aurata . - Fig. 2. Fresh smear of disporous sporoblasts. Notice the prominent posterior bulges of immature spores (arrows), the host tissue septum (arrowhead), and the melanomacrophage center. Bar =20 urn. - Fig. 3. Fresh smear with mature spores. Notice the indented ornamentation of the valves (arrowheads). Bar = 20 urn. - Fig. 4. Indian ink preparation of a mature spore. Phase contrast. Bar = 20 um, - Fig. 5. Mature spores. Notice the extended filament of a spore in lateral view (arrow). Bar = 20 urn, - Fig. 6. Mature spore showing the pierced ornamentation of the valves and the suture. Nomarski. Bar = 20 urn. - Fig. 7. SEM image of an infected glomerulus. Notice the pierced ornamentation of the spores (arrowheads). Bar = 10 um. - Fig. 8. TB stained mature spore. Notice the thickening of the valves at the suture (arrowheads) and the size of the capsulogenic cells. Bar = 20 urn. - Fig. 9. Nomarski focusing of the coiled polar filaments of a mature spore. Bar = 20 urn. - Fig. 10. Mature spore showing several sporoplasms. Bar = 20 urn,

80 . A. Sitja-Bobadilla and P. Alvarez-Pellitero Table 1. Comparison of spore dimensions (11m) of the two species of the genus Polysporoplasma Species

Min

Spore Length (L) Thickness (T) Width Lff ratio Polar capsules Length Width Polar filament Length No. Coils

18.0 19.9 17.0 0.85

Polysporoplasma sparis n = 41 Max Mean S.D.

Min

23.0 23.5 20.0

22.0 21.0 20.0

19.83 21.3 18.14

1.20 1.14 1.34

0.97

1.08

5.5

8.0

6.25

5.0

7.5

5.9

0.71 0.81

53.0 6

58.0 7

53.0

5.3

Ultrastructural Observations Spores. Subspherical. Valves two, contacting at the suture line, without overlapping junctions (Fig. 11). Valves thickened at the suture with electron-dense or finely striated material, and slightly protruding at the anterior end (Figs. 12 and 13). The cytoplasm was amorphous, with vacuoles which occasionally opened at the exterior surface, giving an indented appearance (Fig. 14). No glycogen content was detected with the Thiery reaction (Fig. 14). SEM observations confirmed this pierced ornamentation (Fig. 7). Residual pycnotic nuclei were frequently observed at the posterior end (Figs. 12 and 15), which correspond to the posterior bulges observed by LM. In immature spores valvogenic cells were thick and quite electron-dense, with valve surface nearly smooth or only slightly indented. Their cytoplasm exhibited mitochondria, ER, generally parallel to the inner membrane (Fig. 16), double-mernbraned vesicles close to the suture (Fig. 17) and fibril-like structures (Fig. 18). Desmosome-like structures were detected between valvogenic membranes (Fig. 19). The nuclei, located at the posterior

Polysporoplasma mugilis n = 14 Max Mean S.D. 25.0 22.0 21.0 1.14

6.0 6.0

7.0

4

5

7.0

23.97 21.31 20.5

0.87 0.96 0.83

6.37 6.37

0.37 0.37

part of the spore, degenerated progressively reaching a complete pycnotic appearance in mature spores. Polar capsules two, with internal polar filament coiled 6 - 7 times; apex of the capsule plugged by a granular, stopper-like structure (Fig. 26). Wall of polar capsules formed by a narrow, homogeneous dense layer and a broad more electron-lucent band, formed by concentric layers, alternatively electron-lucent and slightly electron dense. Capsulogenic cells very large, often occupying most of the spore volume (Fig. 20). Cytoplasm with numerous lipid droplets (Figs. 21 and 22) confirmed by the OTO technique (Fig. 23), abundant rough ER (Figs. 21 and 22), mitochondria (Fig. 22) and Golgi vesicles (Fig. 24). No glycogen content was detected with the Thiery reaction (Fig. 28). Nuclei large, with prominent nucleoli (Fig. 25). Capsulogenesis seemed to occur following the general pattern described for other Myxosporea. Capsular primordia and external tubuli preceded the invagination and twisting of the filament, and nuclei remained almost intact even at the end of capsulogenesis (Fig. 25). Up to twelve uninucleate sporoplasms could be observed in one plane of suture (Fig. 27), closely packed

Figs. 11-19. TEM images of Polysporoplasma sparis. - Fig. 11. Thiery staining of a transversely sectioned mature spore. ~ Notice the thickness and ornamentation of the valves, and the sutures (arrows). Bar = 5 11m. - Fig. 12. Transverse section through the posterior end of a spore showing the pycnotic valvogenic nuclei. Bar = 5 11m. - Fig. 13. Detail of the ringed, electron-dense material of the suture. Bar = 111m. - Fig. 14. Thiery-negative staining of the valves. Arrowheads point to the vesicles open towards the exterior surface. Bar =111m. - Fig. 15. Detail of a pycnotic valvogenic nucleus. Notice the debris of the nuclear envelope (arrowheads). Bar = 111m. - Fig. 16. Limiting membranes of capsulogenic and valvogenic cells. Notice the ER (arrowheads). Bar = 0.5 11m. - Fig. 17. Detail of the suture (arrowheads) of an immature spore. Notice the doublemembraned vesicles (arrows). Bar = 0.5 11m. - Fig. 18. Detail of the fibril like structures in valves (arrowhead). Bar = 0.5 11m. - Fig. 19. Detail of desmosome like structures at the suture (arrowheads). Bar = 0.5 11m.

Polysporoplasma spp. Description· 81

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Figs. 20-26. TEM images of Polysp oroplasma sparis capsulogenic cells. - Fig 20. Transversely sectioned caps ulogenic cells of a mature spore. Bar = 5 11m. - Fig. 21. Deta il of a capsulogenic cell showing the abundant lipid droplets. Bar = 111m. - Fig. 22. Detail of the lipid dropl ets and the rough ER ("), Bar = 0.5 11m. - Fig. 23. OTO staining confirming the lipidic composition of the droplets. Bar = 0.5 11m. - Fig. 24. Detail of Golgi vesicles (arrowheads). Bar = 0.5 11m. - Fig. 25. Mature capsulogenic cell showing the nucleus with a prominent nucleolu s. Bar = 111m. - Fig. 26 , Longitudinal section of a mature polar capsule, No tice the coiled pol ar filament and the plug (arrow head). Bar = 111m.

Polysporoplasma spp. Description . 83

and surrounded by capsulogenic cells (Fig. 28). Cytoplasm contained mitochondria, generally surrounded by endoplasmic reticulum (ER), and ribosomes (Fig. 29). Cytochemistry showed the presence of abundant ~-glycogen granules (Figs. 28 and 30) and electron-dense lipid inclusions (Fig. 31). Nuclei central, with clumps of heterochromatin and without nucleoli (Figs.27 and 29). Sporoplasmosomes of different shapes and sizes present (Figs. 29 and 32). No common membrane seems to envelope the ensemble of sporoplasms and each sporoplasmic cell shows well defined membrane boundaries (Fig. 32). Polysporoplasma mugilis n. sp. Host. Liza aurata L. Family Mugilidae, juveniles and adults. Location. (Histozoic) glomeruli (Fig. 36), tubuli, lumen or connective tissue of trunk kidney. Prevalence. 14.8% (4 out of 27). Locality. Western Mediterranean (River Ebro delta), Spain. Description. Mature spores. Slightly ellipsoidal or subspherical in front and side views, slightly longer than thick and wide (Figs. 1 band 33). Sutural line straight. Valves thick, specially at the anterior end (Fig. 34). Two prominent bulges (one on each valve) occasionally visible in front view. Indented valvular ornamentation slightly visible by LM (Fig. 34). Polar capsules spherical or subspherical, of equal size, opening at the anterior end of the spore, near the suture (Fig. 34). Polar filament with 4-5 coils. Sporoplasms four (Fig. 34). Dimensions are presented in Table 1. Developmental stages were not seen. Pseudoplasmodia ellipsoidal and disporous. Holotype. Deposited in the Museo Nacional de Ciencias Naturales (Madrid, Spain): Colecci6n Invertebrados no Insectos, with the acquisition number 37. 01/9.

Ultrastructural Observations Spores. Subspherical to ellipsoidal. Valves two, contacting at the suture line, without overlapping junctions. Valves clearly protruding at the anterior apex, with amorphous, electron-dense material at both sides of the suture (Fig. 41). Cytoplasmic vacuoles frequent, occasionally opening to the exterior giving an indented appearance (Figs. 36, 37, 42 and 43). Membranebound microtubule-like structures were seldom observed in the cytoplasm (Fig. 43 inset B). Pycnotic nuclei appeared at the posterior end of the spore (Fig. 42). Up to four sporoplasmic cells were counted in one plane of section, closely packed together, and enveloped by capsulogenic cells (Fig. 37). The cytoplasm contained densely packed glycogen granules (Fig. 40), sporoplasmosomes (Fig. 37 inset), and mitochondria with scarce cristae and some electron-dense bodies (Fig. 38). Nuclei central, without defined euchromatin or nucleoli (Figs. 37). The space between two sporoplasmic membranes (Fig. 38) was more electron-dense than that between sporoplasmic and capsulogenic membranes (Fig. 39). Large capsulogenic cells occupied most of the spore volume (Fig. 37). The cytoplasm contained high amounts of rough ER, forming a network in which mitochondria were embedded (Fig. 43) along with lipid droplets (Fig. 45) and microfibrils (Fig. 46). Mitochondria (Fig. 43 inset A) had numerous cristae and some electron-dense bodies, probably phospholipids. Membrane boundaries between capsulogenic cells were similar to those between capsulogenic and valvogenic cells (Fig. 44). Nuclei large, with prominent nucleoli. Polar capsules two, subspherical, with internal polar filament coiled 4-5 times (Fig. 47). Wall of the polar .capsules formed by an external electron-dense layer, and a broad electron-lucent band formed by several concentric layers which are alternatively electron-lucent and very slightly electron-dense.

Figs. 27 -32. TEM images of Polysporoplasma sparis sporoplasmic cells, Transverse section of a mature spore showing multipie sporoplasms. Bar = 5 urn, - Fig. 28. Thiery staining of a mature spore. Notice the abundance of polysaccharides in sporoplasms and the absence in valves and capsulogenic cel1s. Bar = 5 urn. - Fig. 29. Detail of a sporoplasm with its central nucleus, mitochondria and sporoplasmosomes (arrowhead). Bar = 1 urn. Inset. Detail of a sporoplasmosome. Bar = 0.25 prn. - Fig. 30. Thiery staining showing the abundant glycogen granules. Bar = 0.5 urn, - Fig. 31. OTO staining confirming the lipidic composition of the sporoplasmic inclusions. Bar = 1 urn. - Fig. 32. Detail of the membrane boundaries between sporoplasms and between sporoplasms and capsulogenic cel1s. Arrowheads point to sporoplasmosomes. Bar = 0.5 urn, Inset. Detail of a sporoplasmosome. Bar = 0.125 urn,

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Figs. 33-40. Images of Polysporoplasma mugilis from the trunk kidney of Liza aurata. - Fig. 33. Fresh smear with mature spores. Bar = 20 urn, - Fig. 34. TB staining of a longitudinally sectioned mature spore . Notice the elevated anterior apex, the indented surface of the valves, and the large size of capsulogenic cells (arrowheads). Bar = 20 urn. - Fig. 35. SEM detail of mature spores in an infected glomerulus. Arrowheads point to the pierced ornamentation. Bar = 10 urn. - Fig. 36. TEM image of a mature spore and the neighboring affected host tissues. Bar = 5 urn. - Fig. 37. Longitudinal section of a mature spore showing four sporoplasms enveloped by the two large capsulogenic cells. Bar = 1 urn. Inset. Detail of sporoplasmosomes. Bar = 0.25 urn, - Fig. 38. Detail of sporoplasmic mitochondria in two adjacent sporoplasms. Bar = 0.25 urn. - Fig. 39. Detail of membrane boundaries between capsulogenic and sporoplasmic cells. Bar = 0.25 urn. - Fig. 40. Thiery-positive staining of a sporoplasm. Bar = 0.5 urn.

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84 . A. Sitja-Bobadilla and P. Alvarez-Pellitero

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Polysporoplasma spp. Description . 87

Discussion Taxonomic Remarks

According to the revised classification of Lorn and Noble [7], the presence of one or two sporoplasms is a diagnostic feature of the class Myxosporea. Thus, the existence of clearly more than two sporoplasms in Polysporoplasma n. g. is a unique feature among Myxosporea. Nevertheless, the remaining features of this new genus fall into the diagnosis of this group. In our opinion, the aforementioned differential feature deserves, at least, the erection of a new genus, which is named Polysporoplasma after the numerous sporoplasms of the spore. The bearing of two valves prompts us to include the new genus in the order Bivalvulida. Polyporoplasma and Sphaerospora could be considered as closely related genera, as only the sporoplasmic configuration discriminates them. This resemblance explains the preliminary classification of P. sparis as Sphaerospora sp. [16]. N evertheless, we are reluctant to ascribe the new genus to the Sphaerosporidae or any other family, since the presence of numerous sporoplasms represents such a unique feature. A future, complete taxonomical revision of the group, in the light of new findings (including new life cycle data and ultrastructural studies), will allow the redefinition of higher level taxa. The two myxosporea found in gilthead sea bream and mullet, though belonging to the same genus, show morphometrical and structural differences. P. sparis spores have an inverse thickness/length ratio than that of P. mugilis (Table 1). The polar filament of P. sparis is coiled six to seven times versus four to five in P. mugilis. Moreover, up to twelve sporoplasms were observed by TEM in P. sparis, whereas a maximum of four was detected in P. mugilis. Th erefore, the two myxosporea show enough differences to ascribe them to different species, which are named Polysporoplasma sparis and P. mugilis after their fish host families. Ultrastructural Aspects

Most of the ultrastructural features of mature and immature spores of both new species are similar to other Myxosporea, but some remarkable aspects are unique and deserve special attention. Capsulogenesis seems to occur following the general pattern described for other myxosporea, i. e. Sphaero-

spora spp. of sea bass [12, 13] and carp [3]. The highly developed ER found in both Polysporoplasma species has also been reported in some Sphaerosporidae, such as Sphaerospora galinae [9], S. carassi [3], S. elegans [4] and S. testicularis [13]. Rough ER is generally accepted to be most prominent in protein-synthesizing cells [5], which suggests a proteic composition of polar capsules. Mitochondria, embedded in the rough ER network, could provide for the energy requirements. The lipid droplets of the capsulogenic cells of both Polysporoplasma species are not rare among myxosporea and much resemble the inclusions observed in S. carassi [3]. In the latter species, capsulogenic nuclei were also visible in cells bearing completely developed polar capsules. Nevertheless, the extremely large size of capsulogenic cells, even when polar capsules are mature and totally envelope the sporoplasmic cells, is unique to our knowledge. Another remarkable feature of P. mugilis capsulogenic cells are the microfibrils organized in bundles. Similar microfilaments were described in the extracellular space between the valvogenic and the capsulogenic cells of Fabespora vermicola sporoblasts [19]. In the latter authors' opinion, those filaments would explain the motility of the spores. We ignore their meaning in P. mugilis. The gradual cytoplasmic and nuclear degeneration undergone by valvogenic cells of Polysporoplasma spp. has been described in most Myxosporea [2, 12, 14, 19]. The atrophied nuclei seem to be responsible for the posterior bulges or horns of the spores of both Polysporoplasma species, which are more prominent in immature spores, and are also present in Sphaerospora elegans [4]. A prominent suture with thickened electron dense material at the anterior apex has also been reported in S. elegans [4], S. carassi and S. angulata [3]. Although the pierced or indented ornamentation of Polysporoplasma could go unnoticed to the untrained eye by LM, it was specially visible by TEM and SEM. A similar ornamentation was observed at the posterior end of the spore of S. elegans [4] and S. galinae [9]. The observation of cytoplasmic vacuoles in a gradual process of opening to the exterior in Polyporoplasma, is in accordance with the explanation of Benajiba et aI. [1] for the origin of valvular ridges in Myxidium giardi. We did not observe microtubules framing the valvogenic vesicles, but the membrane-bound groups of microtubule-like structures could contribute to the

.... Figs. 41-47. TEM images of Polysporoplasma mugilis. - Fig. 41. Detail of the thickened,electron-dense materialof the valves at the apical suture. Bar = 1 urn. - Fig. 42. Transverse section of a spore at the posterior end showing a pycnotic valvogenic nucleus and the pierced ornamentation (arrows). Bar =I urn. - Fig. 43. Detail of a capsulogenic cell.Notice the mitochondria embedded in a network of rough ER (*). Bar = 0.5 urn. Inset A. Detail of a mitochondria with several cristae and a phospholipidic inclusion (arrowhead).Bar = 0.25 urn, Inset B.Detail of a membranebound group of microtubule-like structures in the valve. Bar =0.25 um, - Fig. 44. Detail of membrane boundaries between capsulogenic and valvogenic cells. Bar =0.5 urn, Fig. 45. Detailof lipiddroplets of a capsulogenic cell.Bar = 0.5 urn, - Fig. 46. Detail of microfibrils in a capsulogenic cell.Bar = 0.25 urn. - Fig. 47. Coiled polar filament in a transverse section of a mature polar capsule. Bar = l um.

88 . A. Sitja-Bobadilla and P. Alvarez-Pellitero

firmness of the valve as part of the cytoskeleton. The double-rnembraned cytoplasmic vesicles of P. sparis could transport substances contributing to the formation of the suture and the neighboring electron-dense material. The sporoplasmic cells of Polysporoplasma, in spite of their plurality, displayed most of the features of other myxosporea, such as the gradual enrichment in glycogen, in opposition to the decrease in capsulogenic cells and the absence in valvogenic ones. Sporoplasmosomes, of different shape and size, were present in sporoplasmic cells of both new species. Such structures, although described in several myxosporea [8], have a still unknown function, and not to mention a taxonomical value. The taxonomical importance of numerous sporoplasms is undoubted, as stated above. However, many questions arise about the biological meaning of such a phenomenon. Are all the sporoplasms capable of initiating a new life cycle? If so, is this a strategy to enhance the possibility of infecting a new host or does each pair of sporoplasms suffer cytogamy and karyogamy to restore diploidy, as suggested for other Myxosporea? Are some sporoplasms vegetative and other generative? The absence of structural differences among them seems to refute this latter hypothesis. Which is the origin of the sporoplasms? Is sporogenesis different from other Myxosporea? These and other questions cannot be answered without further investigations on the life cycle and transmission of this new genus. The disporous development of Polysporoplasma spp. is a common feature among Sphaerosporidae [15] and other Bivalvulida. The initial stages leading to sporogenesis could not be ascertained in our study. Nevertheless, the observation of a single membrane surrounding each spore of a pseudoplasmodium would suggest that spores are formed within secondary cells, as suggested for Sphaerospora testicularis [13]. The "conspicuous vacuole", in words of Feist et al. [4], surrounding the posterior part of the spore of S. elegans could be interpreted as the membrane of the secondary cell. The septa which seem to separate some spores (Fig. 2) are in fact residual host tissues, which separate different pseudoplasmodia. More information concerning the host/parasite interphase and the pathogenic action of these new myxosporea will be presented elsewhere. Acknowledgements This work was supported by research grants MARJ89/ 0557 from Spanish CICYT and FARJAQ/1.178 from the European Community. We are grateful to technicians from the Electron Microscopic Service of the University of Barcelona, and to M. J. San Cleto and to J. Monfort for the LM histological processing.

References 1 Benajiba M. H., Marques A., and Bouix G. (1993): Ultrastructural data on sporogenesis of Myxidium giardi, Cepede 1906 (Myxzoa, Myxosporidia), parasite of Anguilla anguilla (Teleostea). Europ. J. Protistol., 29, 254261. 2 Desser S. S. and Patterson B. (1978): Ultrastructural and cytochemical observations on sporogenesis of Myxobolus sp. (Myxosporidia: Myxobolidae) from the common shinner Notropis cornutus.]. Protozool., 25,314-326. 3 Desser S. S., Molnar K., and Horwath I. (1983): An ultrastructural study of the myxosporeans, Sphaerospora angulata and Sphaerospora carassii, in the common carp, Cyprinus carpio L. J. Protozool., 30, 415 -422. 4 Feist S. W., Chilmonczyk S., and Pike A. W. (1991): Structure and development of Sphaerospora elegans Thelohan 1892 (Myxozoa: Myxosporea) in the sticklebacks Gasterosteus aculeatus L. and Pungitius pungitius L. (Gasterosteidae). Europ. J. Protistol., 27, 269-277. 5 Lentz T. L. (1971): Cell fine structure. An atlas of drawings of whole-cell structure. W. B. Saunders Co., Philadelphia; London, Toronto. 6 Lorn J. and Arthur J. R. (1989): A guideline for preparation of species descriptions in Myxosporea. J. Fish Dis., 12, 151-156. 7 Lorn J. and Noble E. R. (1984): Revised classification of the class Myxosporea Biitschli, 1881. Folia Parasitol., 31, 13-205. 8 Lorn]., Feist S. W., Dykova I., and Kepr T. (1989): Brain myxoboliasis of bullhead, Cottus gobio L., due to Myxobolus jiroveci sp. nov.: light and electron microscope observations. J. Fish Dis., 12, 15 -27. 9 Lorn]., Korting W., and Dykova I. (1985): Light and electron microscopic redescription of Sphaerospora tin cae Plehn, 1925 and S. galinae Evlanov, 1981 (Myxosporea) from the tench, Tinea tinea L. Protistologica, 21, 487-497. 10 Reynolds E. S. (1963): The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. 11 Seligman A. M., Wasserhrug H. L., and Hanker J. S. (1966): A new staining method (OTO) for enhancing contrast of lipid droplets in osmium tetroxide-fixed tissue osmiophilic thiocarbohydrazide (TCH). J. Cell Biol., 30, 424-432. 12 Sitja-Bobadilla A. and Alvarez-Pellitero P. (1992): Light and electron microscopic description of Sphaerospora dicentrarchi n. sp. (Myxosporea: Sphaerosporidae) from wild and cultured sea bass, Dicentrarchus labrax L.]. Protozool., 39, 273-281. 13 Sitja-Bobadilla A. and Alvarez-Pellitero P. (1993a): Ultrastructural and cytochemical observations on the sporogenesis of Sphaerospora testicularis (Protozoa: Myxosporea) from Mediterranean sea bass, Dicentrarchus labrax (L.). Europ. J. Protistol., 29, 219-229. 14 Sitja-Bobadilla A. and Alvarez-Pellitero P. (1993b): Zschokkella mugilis n. sp. (Myxosporea: Bivalvulida) from mullets (Teleostei: Mugilidae) of Mediterranean waters: light and electron microscopic description. J. Euk. Microbiol., 40, 755 -764. 15 Sitja-Bobadilla A. and Alvarez-Pellitero P. (1993c): Revised classification and key species of the genus Spbaerospora (Protozoa: Myxosporea). Research and Reviews in Parasitology, 53 (in press).

Polysporoplasma spp. Description . 89

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Key words: Polysporoplasma - Myxosporea - Teleostei - Ultrastructure - Taxonomy Ariadna Sitja-Bobadilla, Instituto de Acuicultura de Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Caste1l6n, Spain