Ultrastructural description of the spore maturation stages of the clam parasite Minchinia tapetis (Vilela, 1951) (Haplosporida: Haplosporidiidae)

Systematic Parasitology 49: 189–194, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Ultrastructural description of the spore maturation stages of the clam parasite Minchinia tapetis (Vilela, 1951) (Haplosporida: Haplosporidiidae) Carlos Azevedo Department of Cell Biology, Institute of Biomedical Sciences and CIIMAR, University of Oporto, 4099.003 Porto, Portugal Accepted for publication 4th October, 2000

Abstract The fine structure of maturing spores of a haplosporidian parasite found in the gill, mantle and foot tissues of Ruditapes decussatus L. (Mollusca, Bivalvia), a species of commercial importance in Portugal, is described. When observed free in suspension, immature spores exhibit one or two epispore cytoplasmic extensions (ECE) which constitute a projection of a portion of the exosporoplasm, sometimes without ultrastructural organisation, surrounded by the plasmalemma. Free spores observed by light microscopy (LM) after 3–5 days of incubation in filtered sea-water exhibit no ECE attached to the spore wall. The mature spore is ovoid to ellipsoid, operculate, uninucleate and measures c. 4.8 µm long and c. 3.9 µm wide. The spore shape and size and the identity of the host living in the same geographical region suggest that this species is the same as previously described using LM observations as Haplosporidium tapetis Vilela, 1951 and later transferred to Minchinia Labbé, 1896.

Introduction Numerous haplosporidian pathogens have been found in different marine species (see reviews by Perkins, 1988, 1989; Bower et al., 1994). A state of nomenclatural confusion between two genera of the Haplosporidiidae, Minchinia Labbé, 1896 and Haplosporidium Lühe, 1900 (=Aplosporidium Caullery & Mesnil, 1899) (see Sprague, 1963), has resulted from the lack of precise definitions of the terms used to describe what are collectively called ‘ornaments’. These may be taxonomically specific characters, which can be used to distinguish the two genera (Perkins & van Banning, 1981; Azevedo, 1984; Azevedo et al., 1999). The arbitrary use of designations such as ornaments (Perkins 1975; 1979), tails (van Banning, 1977; Comps & Tigé, 1997), filaments (Marchand & Sprague, 1979; Ormières, 1980; Azevedo, 1984; Azevedo & Corral, 1987; Azevedo et al., 1999), tapelike filaments (Azevedo, 1984) and projections (van Banning, 1977, 1979; Ball, 1980), as well as ribbons and epispore cytoplasm (EC) (Perkins & van Banning, 1981), strands (Perkins, 1975; Marchand & Sprague, 1979; La Haye et al., 1984), epispore cytoplasm extensions (ECE) (McGovern & Burreson, 1990; Comps

& Tigé, 1997; Azevedo et al., 1999) and wrappings (Perkins, 1975) are frequently employed to describe the same or similar structures directly or indirectly attached to the spore wall. The number, position and insertion in the spore wall of these structures should be re-examined as they are the focus of some taxonomic confusion. The present study contains a detailed description of the ephemeral ECE observed in spores of Minchinia tapetis (Vilela, 1951) Chagot, Bachère, Ruano, Comps & Grizel, 1987, previously described as Haplosporidium tapetis Vilela, 1951.

Materials and methods Infected gaping clams Ruditapes decussatus L. (Mollusca, Bivalvia) were obtained in the estuarine region of southern Portugal (the Algarve) from a population with high mortalities. For transmission electron microscopy (TEM), small fragments of the heavily infected gills, mantle and foot tissues were fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate buffer (in filtered sea-water-FSW), pH 7.6, for 3 hr at 4 ◦ C, washed for 2 hr in the same buffer and post-fixed in

190 buffered 2% OsO4 for 3 hr at 4 ◦ C. All pieces were dehydrated in a graded series of ethanol and embedded in Epon. Semithin sections for light microscopy (LM), stained with methylene blue-azur II, were used to locate the stages of the spore maturation. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined in a JEOL 100CXII TEM operated at 60 kV. Isolated immature and mature spores obtained from homogenised parasitised tissues were rinsed in FSW, centrifuged and then incubated in FSW for 3 and 5 days. The spores were prepared for LM, scanning electron microscopy (SEM) and TEM by techniques described previously (Azevedo, 1984).

Results Our observations were concentrated on the structure of maturing and mature spores. The spores observed by LM among the host cells did not exhibit ornaments (Figures 1, 2). Only free immature spores observed using LM (Figure 3), in TEM whole-mounts (Figure 4) and using SEM (Figures 5, 6) possessed 1, 2 and, more rarely, 3 ECE, which were not in continuity with the spore wall or showed any internal cytoskeletal structures (Figures 10, 11). On the other hand, as observed using LM, SEM and TEM, the wall of mature spores possessed neither ornamentations nor any attached cytoskeletal structure (Figures 7, 8, 11, 12). This morphological feature was elucidated by intensive study of serial ultrathin sections made of the sucessive maturation stages of isolated spores (Figures 10, 11). The ECE were observed only in free immature spores and were formed exclusively by the plasmalemma with a small amount of exosporoplasmic material (epispore cytoplasm) of the immature spore (Figure 10). Projections of the wall or ECE with skeletal structures have never been observed in fully mature spores (Figures 7, 8, 12). Stages of spore maturation were observed within the sporocyst, which was delineated by a thick membranous structure with an irregular outline (Figure 9). Each spore was formed by a clearly evident exosporoplasm membrane, EC (or exosporoplasm) and endosporoplasm completely surrounded by a membrane in close contact with the internal face of the wall (Figures 10,11). The uninucleate endosporoplasm contained the typical haplosporidian structures, an apical spherulosome and several haplosporosomes (Figure 10). With further maturation, the endosporo-

plasm became denser and the wall and the operculum became denser and thicker. Simultaneously, the exosporoplasm degenerated (Figure 11) and disappeared (Figure 12). The endosporoplasm of mature spores was highly electron-dense and their internal structures were hardly visible. At the final maturation stage the exosporoplasm was lysed and the external plasmalemma and ECE could no longer be distinguished (Figure 2). There was no significant variation in the morphology or size of the spores when they were observed using LM (Figures 2, 8), SEM (Figures 5–7) or TEM (Figures 11, 12). Spores had an ellipsoidal shape and were c. 4.8 µm long and c. 3.9 µm wide (n = 30). The apical zone of the wall was differentiated into a typical opercular system (Figures 7, 13). The ellipsoidal operculum was c. 3.9 µm wide and connected to the spore wall by a hinge. The spore wall consisted of 3 layers: an electron-dense outer layer 25 nm thick; an electron-light middle layer 10 nm thick; and an electron-dense inner layer 15 nm thick that contacted with the endosporoplasm membrane internally (Figures 11, 13). Observed 3 and 5 days after incubation in FSW, the free mature spores seemed viable and devoid of any ECE (Figures 7, 8). The gills of some specimens of clams infected by M. tapetis were also parasitised with a few trophozoites of Perkinsus atlanticus Azevedo, 1984 (Phylum Apicomplexa). Morphological differences were not apparent in the spores of M. tapetis found in R. decussatus also infected with P. atlanticus.

Discussion Unornamented mature spores of Haplosporidium tapetis Vilela, 1951, measuring 12 × 5–7 µm and infecting the clam Tapes decussatus (=Ruditapes decussatus) from southern Portugal, were described using LM (Vilela, 1951). Later, based on the absence of spore ornamentation, Chagot et al. (1987) transferred this species to the genus Minchinia, as M. tapetis, despite the fact that the specimens they studied had smaller spores (5–6 µm long) than those described by Vilela (1951). In our observations the morphology of the spores seems to be similar to previous descriptions of Minchinia sp. (see Azevedo & Corral, 1989) and M. tapetis (see Chagot et al., 1987). They have a similar spore structure and size, and parasitise the same host living in the same geographical area.

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Figure 1–8. Photomicrographs and electron micrographs of the spores of Minchinia tapetis found in the gills of Ruditapes decussatus. 1. Semithin section showing spores (S). × 900. 2. Unstained fresh spores observed by LM. × 2,500. 3. Fresh immature spores each showing an ECE (E) at the posterior end. × 2,800. 4. Whole-mount of an isolated immature spore observed by TEM showing the epispore (E) and the operculum (O). × 5,500. 5. SEM of immature spore showing an ECE (E) at the posterior end and the operculum (O). × 6,500. 6. SEM of immature spore showing the operculum (O) and two opposite ECE (E). × 7,500. 7. Mature spore observed by SEM without ECE. × 7,000. 8. Living spore obtained after 5 days of incubation (lacking ornaments) and showing the spore devoid of ECE. Abbreviations: O, operculum; W, spore wall. × 5,300.

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Figure 9–13. Ultrathin sections containing maturing spores. 9. Ultrathin section of the host tissue showing spores (S) in different phases of maturation. × 4,420. 10. Ultrathin section of an immature spore showing the plasmalemma (arrows) adjoined to the operculum (Op) and the exosporoplasm (∗ ) in close contact with the spore wall (W). The endosporoplasm (∗∗ ) shows the spherulosome (S) and haplosporosomes (H). × 11,800. 11. A spore with dense endosporoplasm. The epispore cytoplasm (∗ ) appears degraded: O, operculum; W, wall. × 10,100. 12. Ultrathin transverse section of a free spore, showing the exosporoplasm (∗ ) and epispore membrane (arrows), both with signs of lysis. × 13,500. 13. Ultrathin section showing details of the lateral portion of operculum (O) and wall (W) from an immature spore with well-preserved exosporoplasm (∗ ). × 44,500.

193 The Phylum Haplosporidia consists of three genera (Perkins, 1989). The status of the two major genera, Haplosporidium and Minchinia, continues to be confused and is a source of disagreement between some authors. In an attempt to solve this problem, an important observation appears to be a knowledge of when the spores attain full maturation, which is morphologically characterised by a complete absence of ECE and, consequently, no epispore membrane. In addition, there are often incomplete ultrastructural descriptions of the ornaments, which are also known by several different terms (see Introduction), and their attachment points to the spore wall (Azevedo et al., 1999). The absence of an exact definition of the ultrastructural organisation of the ornaments and the lack of knowledge of the precise point of insertion of the tail in the spore wall have resulted in a state of taxonomic confusion between the genera Haplosporidium and Minchinia. According to some authors, Haplosporidium has two opposite filaments (tails) attached to the base of the spore wall (Ormières, 1980; Azevedo, 1984; La Haye et al., 1984; Azevedo et al., 1985) or spores ornamented with filaments (Hine & Thorne, 1998). Their observations agree with the description of the type-species of Haplosporidium, H. scolopli (Caullery & Mesnil, 1899) Lühe, 1900, which refers to two filaments attached to the posterior end of the spore (Caullery & Mesnil, 1905; plate XI, figure 17b and c). On the other hand, in Haplosporidium armoricanum (Azevedo et al., 1999), two long filaments are attached to the spore wall by a bundle of 9–13 fibres each. Some species of Minchinia have been described without tails (Rosenfield et al., 1969; Marchand, 1974; Cahour et al., 1980; Desportes & Nashed, 1983; Azevedo & Corral, 1989). Likewise, some authors described Haplosporidium without tails (Ormières & De Puytorac, 1968), whereas others claim that Minchinia has tails or an eccentrically attached ECE attached to the spore wall at opposite ends (van Banning, 1977; Ball, 1980, 1981; Perkins & van Banning, 1981; Comps & Tigé, 1997; Hine & Thorne, 1998). In a footnote, Lauckner (1983) considered that Minchinia is a nomen nudum and placed all haplosporidian species in Haplosporidium. This decision has not gained wide acceptance, because the spores of these two genera are morphologically distinct (Sprague, 1963; Desportes & Nashed, 1983; Chagot et al. 1987; Azevedo & Corral, 1989; Perkins, 1989; McGovern & Burreson, 1990; Azevedo et al., 1999).

After reviewing Ball’s publications in which the type-species of Minchinia, M. chitonis (Lankester, 1895) Labbé, 1896, was described, we confirm that his observations were obtained from a mature spore (Ball, 1980, 1981). In the type-species, the entire spore was invested by a membrane with extensions that formed two long projections (epispore cytoplasm), one at either end (Ball, 1980). Our observations show that the extensions of the spore wall are similar to those of M. chitonis (see Ball, 1980), which are composed entirely of epispore cytoplasm and never attached to the spore wall, as indicated for another species of Minchinia (see van Banning, 1977). Based on the presence of ECE, rather than spore wall filaments, a haplosporidian of Teredo spp. was placed in Minchinia by McGovern & Burreson (1990). On the other hand, Sprague (1982) described Minchinia as containing spores ornamented either with an anterior and a posterior tail, and Haplosporidium spores with threads wound around them. According to present observations and the studies on different species of Minchinia (van Banning, 1977; Ball, 1980; Azevedo & Corral, 1989), the presence of the ECE in fully mature spores has never been recorded (Azevedo et al., 1999). We believe that these structures are visible only in immature spores isolated from the sporocysts and that they seem to be artefacts. ECE appear not to occur in mature spores (Azevedo et al., 1999). The disappearance of the epispore membrane and, consequently, the ECE in the final stage of spore maturation appears to be the most important argument to justify that these are ephemeral and visible only in maturing spores (McGovern & Burreson, 1990; Azevedo et al., 1999). In the present study, in which several microscopical methods were used, we reinforce the suggestions of some authors that future studies of the haplosporidian spores should include TEM and SEM imaging (Desportes & Nashed, 1983; McGovern & Burreson, 1990; Perkins, 1991; Azevedo et al., 1999).

Acknowledgements This work was supported partially by grant from the A. Almeida Foundation (Porto-Portugal). We would like to thank the excellent technical assistence of Laura Corral and João Carvalheiro.

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