Parasitol Res (2003) 91: 390–397 DOI 10.1007/s00436-003-0978-3

ORIGINAL PAPER

M. A. Ali Æ A. S. Abdel-Baki Æ T. Sakran R. Entzeroth Æ F. Abdel-Ghaffar Light and electron microscopic studies of stomum n. sp. (: ) infecting the blackspotted grunt Plectorhynicus gaterinus (Forsskal, 1775) in the Red Sea, Egypt

Received: 1 July 2003 / Accepted: 30 July 2003 / Published online: 18 September 2003 Springer-Verlag 2003

Abstract A new myxosporean parasite, Myxobolus sto- this effort by investigating myxosporean parasites in the mum n. sp., is described from the oral cavity and lips of Red Sea, Egypt. The present study deals with a new the blackspotted grunt Plectorhynicus gaterinus (For- species of Myxobolus infecting the blackspotted grunt sskal, 1775) in the Red Sea, Egypt. The parasite was (local name gatrina), Plectorhynicus gaterinus (Forsskal, observed as tiny aggregates of whitish cysts hardly no- 1775). The parasite is described by light and electron ticed within the muscles of the oral cavity, especially microscopy and its histological implication is also pre- within the lips. The spores were subspherical and mea- sented. sured 8.5·6.5 lm. Polar capsules were equal, pear- shaped, occupied about half of the spore length and measured 4.4·2.4 lm. Histological evaluation of the Materials and methods infection revealed no significant impact on the host. The ultrastructure of the plasmodial wall and sporogenesis of Live or freshly caught fish samples were collected from boat- the present species followed the usual pattern valid for landing sites, fishermen and sometimes from the markets of Suez and Hurghada at the Gulf of Suez and Red Sea, respectively. A most studied myxosporean species. total of 30 fish (16 from Suez, 14 from Hurghada) were examined. Descriptions and measurements of spores followed the guidelines of Lom and Arthur (1989). Measurements were based on 30 spores Introduction and data were presented as mean ±SD (range). For histology, the infected parts were fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned and stained with haematoxylin and There is considerable information on the myxosporean eosin. For the electron microscopy, small intact cysts with minimal parasites from different parts of the world. The African surrounding tissue were isolated and fixed in 3% glutaraldehyde in Myxosporea are being extensively studied in many parts 0.1% sodium cacodylate (pH 7.4), washed in the same buffer and of the continent, like Cameron, Benin and Egypt. post-fixed with 2% OsO4 in the same buffer. The tissue pieces were dehydrated in graded ethanol and embedded in araldite. Ultra- However, the marine myxosporean species are rarely sections were stained with uranyl acetate and lead citrate and investigated in Africa. Few studies have been carried out examined with a Philips (model 208) electron microscope at on marine fish, e.g. Fantham (1919, 1930), Fall et al. 80–100 kV. (1997) and Ali et al. (2001). The present work continues

Results A. S. Abdel-Baki (&) Æ T. Sakran Beni-Suef Faculty of Science, Light microscopy Zoology Department, Cairo University, Egypt E-mail: [email protected] The infection was observed as tiny aggregates of whitish cysts hardly noticed within the muscles of the oral cavity M. A. Ali National Institute of Oceanography & Fisheries, and more frequently within the lips. These aggregates Egypt were clusters of ovoid cysts of variable numbers (up to eight cysts per fish). A single plasmodium measured R. Entzeroth Institute of Special Zoology and Parasitology, 406 lm (400–410 lm) in length and 255 lm (250– Technical University of Dresden, Germany 270 lm) in width. The prevalence was 6/14 (42.8%) in F. Abdel-Ghaffar Hurghada samples and 4/16 (25%) in Suez samples. In Faculty of Science, Zoology Department, most cases, the oral cavity of the grunt fish was infested Cairo University, Egypt with pranizae or larval gnathid isopods. 391

Spores Histology

The spores were subspherical in frontal view (Figs. 1, The plasmodia were distributed throughout the muscle 11). They measured 8.5±0.8 lm (7.0–10.0 lm) in of the oral roof. The parasite formed plasmodia among length and 6.5±0.6 lm (5.5–7.5 lm) in width. Polar the muscle fibres (Fig. 2). Mature spores were located capsules were equal, pear-shaped and occupied centrally inside the plasmodia, while the developmental about half of the spore. They measured 4.4±0.5 lm stages were peripherally arranged (Fig. 4). A thin layer (4.0–5.0 lm in length and 2.4±0.4 lm (2.0–3.0 lm of the host connective tissue encapsulated the plasmodia in width. The polar filament showed mostly five and (Figs. 3, 4). No host infiltration was apparent around rarely six coils oblique to the main axis of the polar the developing parasite. capsules.

Electron microscopy

Plasmodia Fig. 1 Fresh spores of Mxyobolus stomum sp. n. with Nomarski interference contrast. Bar 10 lm The plasmodial wall of the present species was clearly Fig. 2 Longitudinal section of an infected muscle (M), showing the demarcated into an outer ectoplasm and an inner plasmodium (P) surrounded by a thin layer of connective tissue endoplasm (Fig. 5). External to this wall was a thin (CT). Bar 60 lm Fig. 3 Semithin section of an infected muscle (M), showing the layer of connective tissue in contact with the muscle plasmodium (P) surrounded with a thin layer of connective tissue of the host (Fig. 5). The plasmodia were surrounded (CT). Bar 30 lm by a single-unit membrane from which short pinocy- Fig. 4 Magnified part of a semithin section of an infected muscle totic channels and vesicles extended nearly close to (M), showing the plasmodium (P) surrounded with a thin layer of connective tissue (CT). The plasmodia contained developmental the periphery of the endoplasmic zone (Fig. 5). In stages (DS) at the periphery and mature spores (Sp) close to the the outer zone of the endoplasm, there were centre. Bar 10 lm numerous mitochondria, vegetative nuclei, generative 392

Fig. 5 A part of the plasmodium of M. stomum sp. n., showing the plasmodial wall composed of ectoplasm (Ec) and endoplasm (En). Externally, the plasmodium was surrounded by a layer of connective tissue (CT)in contact with the muscle of the host (M). Some host nuclei (HN) were observed close to the plasmodium membrane. The plasmodium was bounded by a single-unit membrane from which pinocytotic channels (arrows) and vesicles (V) originated. The periphery of the endoplasm contained numerous generative cells (GC) and pansporoblasts (Ps), surrounded by a large number of mitochondria (m). Bar 2 lm

cells and developing pansporoblasts, while the central all the stages of spore formation were observed, each part of the plasmodia contained mature spores spore developed from five cells: two peripherally ar- (Fig. 5). ranged valvogenic cells, a pair of capsulogenic cells and a binucleated sporoplasm (Fig. 10). As spores proceeded toward maturation, there was structural progress in Sporogenesis capsulogenesis, sporoplasm maturation and valvogene- Sporogenesis was asynchronous and early sporogenic sis (Figs. 8, 9, 10). stages were concentrated in the peripheral areas (Fig. 5). Generative cells, the earliest recognizable stages of spo- Capsulogenesis rogenesis, were nearly spherical, with a diameter of 4 lm and limited by a double-unit membrane. The cytoplasm Capsulogenic cells were found at the apical pole of the of the generative cells contained numerous ribosomes developing spore and, together with the sporoplasm, and a large eccentric nucleus with peripherally arranged formed a central core that was ensheathed by valvogenic chromatin material (Fig. 6). The generative cells were cells (Fig. 10). The capsulogenic cells were characterized always surrounded by numerous mitochondria. by the presence of distended cisternae of the endoplas- Sporogenesis began with the union of two generative mic reticulum (Fig. 8). The differentiation of the cap- cells. That union resulted in an early pansporoblast, the sulogenic cell started with appearance of a club-shaped pericyte containing the sporont (Fig. 7). Although not structure, which was the initial stage of the capsular 393

Fig. 6 A generative cell with large voluminous eccentric nucleus primordium (Fig. 8). Gradually, the initial stage differ- (N), which contained dark chromatin material at the periphery. entiated into the bulbous primordium and the associated The cell was bounded by a double-unit membrane (arrowheads). Bar 300 nm external tube, which was closed at the distal end. The Fig. 7 Two-cell pansporoblast consisting of a pericyte with its external tube was later internalized into the primordium nucleus (N1), enveloping the sporont cell with its nucleus (N2). Bar to make the polar filament coils with 5–6 turns (Figs. 9, 1 lm 10). The developing polar capsule had a homogenous Fig. 8 A capsulogenic cell containing capsular primordium (CP), core of medium electron-density, surrounded by an external tube (ET) and destined cisternae of endoplasmic reticulum (asterisks). Bar 300 nm electron-lucent layer and an outer layer of medium Fig. 9 Transverse section through maturing spores, showing density (Fig. 9). A stopper with a triangular cap-like almost mature polar capsules. The polar capsule was composed cover plugged the apex of each mature polar capsule of an electron-dense outer layer (arrow), a central translucent layer (Fig. 9). Maturation of the two polar capsules was (Lu) and an inner dense core with polar filament coils (PF). A stopper (Pl) with a triangular cap-like cover (Cp) plugged the apex usually synchronous (Fig. 10). of the polar capsule. Bar 300 nm Fig. 10 Longitudinal section through mature spores, showing two synchronously developed polar capsules (PC) with polar filament Sporoplasm coils, sporoplasm (Sp) with two nuclei (SN1, SN2) situated closely side by side, small dense bodies (sporoplasmosomes; arrowheads) and As soon as the polar capsule primordium appeared, the valvogenic cells (VC) with valve-forming bodies (VFB). Bar 1 lm sporoplasm could be easily recognized. In the mature 394

sutural folds and a higher number of polar filament turns (six to seven vs five). Similarly, M. meglitschus can be separated from the present species by its distinct intercapsular process, which usually extends beyond the half way point of the two polar capsules, and its thick-walled shell valves with five sutural markings. M. macroplasmodialis can be readily distinguished from the present species by having longer and wider spores with an intercapsular process. M. intrachondrealis has body dimensions close to the present species but differs in having a higher number of filament coils (nine to 11 vs five) and an intercapsular Fig. 11 Line diagram of the spore of Myxobolus stomum sp. n. Bar process. 5 lm All the compared species are described from fresh water hosts, except M. aeglefini. Additionally, the host, site of infection and locality of spore, it filled nearly all the space beneath the polar all the previously discussed species differ from the capsules (Fig. 10). The sporoplasm contained two nuclei present species. Also, no myxosporean infection is (situated closely side by side) and mitochondria and mentioned in the literature for the present fish host sometimes exhibited dense matrices known as sporo- (Plectorhynchus gaterinus). Therefore, it appears that the plasmosomes (Fig. 10). A small area of sporoplasm in present species is a new one and the name M. stomum sp. mature spores was occupied by a glycogen body that was n. is proposed and derived from the site of infection often vacuolated. (oral cavity, lips).

Valvogenesis Histology Valvogensis was initiated by the gradual envelopment of the capsulogenic cells and sporoplasm by valvogenic The plasmodia of the present species were randomly cells. Their nuclei were flattened and laterally located. distributed in the muscles of the oral cavity. The site Later in the course of development, these cells gave of infection was comparable with M. procerus Kudo, rise to the two shell valves surrounding each spore and 1934 (Cone et al. 1997), M. pseudodispar Gorbunova, the sutural ridge joining the valves. Sometimes, 1936 (Baska 1987) and M. artus Akhmerov, 1960 abnormal spores with three shell valves were observed. (Ogawa et al. 1992). There were no discernible path- In mature spores, discontinuous, electron-dense sub- ological changes in the tissue due to the presence of stances known as valve-forming bodies appeared in the the present plasmodia. The only impact of the plas- cytoplasm of the valvogenic cells (Fig. 10). At the modia was the replacement of muscle by the plasmo- spore apex, the wall of the valve formed a triangular dial masses. However, the low number of cysts and thickening. their small size would probably not pose any potential harm to the host. The presence of connective tissue encapsulating the plasmodia is one of the usual Discussion responses associated with myxosporean infections of fish (Mitchell 1977). Light microscopy

The present myxosporean matches in shape with many Electron microscopy other related species (11): Myxobolus aeglefini Auer- bach, 1906 (Lom and Dykova 1992), M. paraplintoni Li Plasmodia and Desser, 1985 (Cone and Overstreet 1998), M. ald- richetti (Su and White 1994), M. meglitschus (Sarkar The plasmodial wall of the present species consisted of a 1996), M. macroplasmodialis (Molnar et al. 1998) and single-unit membrane with pinocytotic channels, which M. intrachondrealis (Molnar 2000). is similar to that of Myxosoma funduli (Current et al. With respect to the present species, M. aeglefini dif- 1979), Myxobolus exiguus (Pulsford and Matthews fers in having wider spores and sutural markings. The 1982), Kudoa lunata (Lom and Dykova 1988) and spores of M. paraplintoni are larger in all body dimen- Myxobolus sp. (Abdel-Ghaffar et al. 1994). However, the sions, having six to eight sutural markings and a higher plasmodia of other myxosporean species may exhibit a number of polar filament coils (six to seven vs five). double-unit membrane, like Henneguya exilis (Current Although M. aldrichetti has dimensions close to the and Janovy 1976) and Myxobolus sp. (Desser and present form, it differs in having four to five triangular Paterson 1978). Current and Janovy (1978) concluded 395 that the differences in the nature of the plasmodium wall early pansporoblasts in Myxobolus plasmodia (Pulsford are tissue-dependent. and Matthews 1982; Lom et al. 1989; El-Matbouli et al. Externally, the plasmodia were surrounded by a thin 1990). layer of connective tissue similar to that of H. pinnae (Schubert 1968), H. exilis (Current and Janovy 1976), M. exiguus (Pulsford and Matthews 1982) and Tetrau- Capsulogenesis ronema deseaqualis (Azevedo and Matos 1996). The Capsulogenesis of the present species followed the usual connective tissue encapsulation of the plasmodia is one pattern observed in most myxosporeans. However, the of the usual responses associated with myxosporean origin of the polar capsule was controversial. Lom infections in fish (Mitchell 1977; Pulsford and Matthews (1969) suggested that the capsular primordium is formed 1982; Ali et al. 2001). from smooth-walled vesicles, which might originate The plasmodia of the present species contained the from smooth endoplasmic reticulum or even the Golgi generative cells, vegetative nuclei and pansporoblasts at complex. Other investigators assumed that granular the peripheral zone, while mature spores were centrally endoplasmic reticulum is involved in the formation of located. This seems to be a typical pattern for myxosp- the capsular primordium (Schubet 1968; Desser and orean plasmodia, as described in many studies (Current Paterson 1978; Pulsford and Matthews 1982; Lom and 1979; Current and Janovy 1977; Current et al. 1979; Dykova 1988) Moreover, El-Matbouli et al. (1990) Desser et al. 1983a, 1983b; Abdel-Ghaffar et al. 1994, suggested that the polar capsule originates from a 1995). spherical body formed of series of membranes. The exact origin of the polar capsules is not conclusively solved. According to the literature, the endoplasmic reticulum Sporogenesis seems to be the most likely organelle to form the polar Sporogenesis of M. stomum was essentially similar to capsules. In the present species, aggregates of rough other described species. There was one morphologically endoplasmic reticulum were observed close to the polar distinct type of generative cell, which had a double capsules, which might support this opinion. membrane and a large eccentric nucleus. More than one Maturation of the polar capsule of the present species type of generative cell has been recorded from studies of was synchronous. Similar findings were obtained by other myxosporeans. Siau (1977) and Current (1979) Hulbert et al. (1977), El-Matbouli et al. (1990), Abdel- reported two distinct types of generative cell in Ghaffar et al. (1994, 1995) and Casal et al. (1996). M. exiguus and H. adipose, respectively, while Grasse After the polar capsule was fully developed, it was and Lavette (1978) reported three types of generative cell plugged with a stopper, which was covered by a cap-like in the plasmodia of Sphaeromyxa sabrazesi. structure at its apical end. A similar structure was re- The origin of the pansporoblast is the subject of ported in the genus Myxobolus by El-Matbouli et al. argument among investigators. Two opinions have been (1990) and in other myxosporean genera, such as suggested for the formation of the pansporoblast. The Sphaerospora (Lom et al. 1985) and Kudoa (Lom and first theory of pansporoblast formation suggests Dykova 1988). endogenous cleavage of the generative cell (Hulbert et al. 1977; Uspenskaya 1984; Molnar 1994). The other theory Sporoplasm of pansporoblast formation suggests the union of two generative cells, one of which is the pericyte and envel- In most studied myxosporeans, including the present ops the other cell, the sporont (Schubert 1968; Lom species, the sporoplasm was composed of a single 1969; Current and Janovy 1977; Current 1979; Current binucleated cell, which is in accordance with Lom and et al. 1979; Pulsford and Matthews 1982; Lom et al. Puytorac (1965), Lom (1969), Current and Janovy 1983; Azevedo et al. 1989; Abdel-Ghaffar et al. 1994, (1977), Desser and Paterson (1978), Current (1979), 1995; Casal et al. 1996). The latter theory is widely ac- El-Matbouli et al. (1990, 1995), Abdel-Ghaffar et al. cepted by the majority of researchers for many my- (1994), Casal et al. (1996), Lom and Dykova (1996) and xosporean species and is confirmed by ultrastructural Canning et al. (1999). However, in other myxsporeans; it evidence. The present study revealed that sporogenesis was found that the sporoplasm was composed of two started with the union of two generative cells. That un- separated cells, as in S. renicola (Lom et al. 1983), ion resulted in an early pansporoblast and thus the S. gobioni (Lom et al. 1985), shasta envelopment theory is more accepted here. (Yamamoto and Sanders 1979) and S. epinepheli In the present study, most of the pansporoblasts were (Supamattaya et al. 1993). Stehr (1986) detected a in the early stage of development. The overall observa- third pattern in the sporoplasm. He reported that tions of sporogenesis proved that the spores develop K. paniformis differed from other myxosporeans in from a ten-cell pansporoblast (disporic pansporoblast). having a sporoplasm composed of a smaller cell with Each spore develops from five cells: two capsulogenic electron-dense cytoplasm surrounded by a large cell. cells, two valvogenic cells and a binucleated sporoplas- Sitja-Bobadilla and Alvarez-Pellitero (1995) reported mic cell. Similar studies reported the presence of only another unique sporoplasmic pattern where four to 12 396 sporoplasms were observed in the spore of Polysporo- Baska F (1987) Histological studies on the development of My- plasm sparis and P. mugilis. xobolus pseudodispar Gorbunova, 1963 in the roach (Rutilus rutilus). Acta Vet Hung 35:251–257 The plasmosomes observed in the present material Canning EU, Curry A, Anderson CL, Okamura B (1999) Ultra- were as usually reported in other studies (Pulsford and structure of trachinorum sp. nov. from the gallblad- Matthews 1982; Schmahl et al. 1989; El-Matbouli et al. der of the lesser weaver fish Echiichthys vipera. Parasitol Res 1990). In contrast, plasmosomes were missing in some 85:910–919 Casal C, Matos E, Azevedo C (1996) Ultrastructure data on the life myxosporeans, like Unicapsula muscularis (Schubert cycle stages of Myxobolus braziliensis n. sp., parasite of an et al. 1975), S. epinepheli (Supamattaya et al. 1993), Amazonian fish. Eur J Parasitol 32:123–127 Sinuolinea tetraodoni (El-Matbouli and Hoffmann Cone DK, Overstreet RM (1998) Species of Myxobolus (Myxozoa) 1994) and Myxidium trachinorum (Canning et al. from the bulbus arteriosus of centrarchid fishes in North 1999). America, with a description of two new species. J Parasitol 84:371–374 The presence of glycogen bodies in the sporoplasm is Cone DK, Eurell T, Axler R, Rau D, Beasley V (1997) Intense an essential in the myxosporean spore, which could infections with a variant of Myxobolus procerus (Myxosporea) provide the energy necessary for further developmental in muscle of trout-perch (Percopsis omiscomaycus) in Duluth stages in the spore¢s life cycle. The present sporo- Harbor, Lake Superior. Folia Parasitol 44:7–11 Current WL (1979) Henneguya adiposa Minchew (Myxosporidia) plasm showed small-sized glycogen vacuoles which in the channel catfish: ultrastructure of the plasmodium wall were similar to those reported in Myxobolus hendrick- and sporogenesis. J Protozool 26:209–217 soni (Mitchell et al. 1985), M. cotti (El-Matbouli et al. Current WL, Janovy J Jr (1976) Ultrastructure of interlamellar 1990) and Myxobolus sp. (Abdel-Ghaffar et al. 1994). Henneguya exilis in the channel catfish. J Parasitol 62:975–981 Current WL, Janovy J Jr (1977) Sporogenesis in Henneguya exilis infecting the channel catfish: an ultrastructural study. Protis- Valvogenesis tologica 13:157–167 Current WL, Janovy J Jr (1978) Comparative study of ultrastruc- ture of the interlamellar and intralamellar types of Henneguya The spores of are essentially composed of exilis from channel catfish. J Protozool 25:56–65 two shell valves. In some cases, abnormal spores might Current WL, Janovy J Jr, Knight SA (1979) Myxosoma funduli be observed, as in the present study. The typical flat Kudo (Myxosporidia) in Fundulus kansae: ultrastructure of the valve cell with its flat nucleus was observed during the plasmodium wall and of sporogenesis. J Protozool 26:574–583 Desser SS, Paterson WB (1978) Ultrastructural and cytochemical present valvogenesis. The microtubules generally found observation on sporogenesis of Myxobolus sp. (Myxosporidia: in the shell valves were not observed in the present Myxobolidae) from common shiner Notropis coronutus. materials, which conform with Stehr (1986) in K. pani- J Protozool 25:314–326 formis, Lom et al. (1989) in M. jiroveci, Abdel-Ghaffar Desser SS, Molnar K, Horvath I (1983a) Ultrastructure of sporo- genesis of the myxosporeans, Sphaerospora angulata and et al. (1994) in Myxobolus sp. and Canning et al. (1999) Sphaerospora carassii in the common carp Cyprinus carpio. in Myxidium trachinorum. J Protozool 30:415–422 Desser SS, Molnar K, Weller I (1983b) Ultrastructure of sporo- Acknowledgements The authors are grateful for the technical genesis of Thelohanellus nikolskii Akhmerov, 1955 (Myxozoa: assistance of Dr. Aimdip Mouafo and Dr. Fleig in the electron Myxosporea) from the common carp Cyprinus carpio. microscopy. We also appreciate the University of Dresden (Freude J Parasitol 69:504–518 und Fo¨ rderer) for funding the electron microscopic study. 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