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Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas
International Journal for Parasitology 34 (2004) 1129–1135 www.parasitology-online.com
Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas Naoki Itoha, Hideki Komiyamab, Noriyuki Uekib, Kazuo Ogawaa,* a
Laboratory of Fish Diseases, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo Tokyo 113-8657, Japan b Fisheries Experiment Station of Okayama Prefecture, Ushimado, Okayama 701-4303, Japan Received 13 April 2004; received in revised form 4 June 2004; accepted 7 June 2004
Abstract A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Paramyxea; Marteilioides chungmuensis; Pacific oyster; Crassostrea gigas; Extrasporogony; Ovarian parasite
1. Introduction Pacific oysters, Crassostrea gigas, are cultured throughout the world. The global production of the Pacific oysters, from European, North American and Asian countries is estimated to be approximately 4 million metric tonnes per year (FAO, 2000), making this species the most important cultured bivalve. In Pacific oyster culture, there are two serious parasitic diseases. Firstly, Mikrocytos mackini (Farley et al., 1988), which causes heavy mortalities in North America (Quayle, 1961) and secondly, the ovary enlargement disease of the Pacific oyster which is caused by Marteilioides chungmuensis (Comps et al., 1986). Although high mortalities have not been reported, the oysters affected by the latter disease develop abnormal gonads, resulting in a disfigured appearance and poor market appeal. Marteilioides chungmuensis has been reported in Korea (Chun, 1979) and Japan (Seki, 1934). The parasite has been studied in several aspects such as seasonal prevalence (Imanaka et al., 2001; Ngo et al., 2003), epizootiology * Corresponding author. Tel.: þ 81-3-5841-5283; fax: þ81-3-5841-5383. E-mail address: [email protected] (K. Ogawa).
(Matsusato and Masumura, 1981), biochemical studies (Park et al., 2003) and host range (Itoh et al., 2004). In particular, the sporulation process of M. chungmuensis has been intensively studied by several authors (Chun, 1979; Comps et al., 1986; Imanaka et al., 2001; Itoh et al., 2002), however, the early developmental stages outside the oocytes have not been located in histological sections. Moreover, the inability to experimentally infect oysters under laboratory conditions makes the detection of early developmental stages difficult. Recently, it has been demonstrated that molecular detection methods such as PCR and in situ hybridisation (ISH) are useful for detecting the early developmental stages and clarify the life cycles of micro-parasites such as the Microspora (Sanchez et al., 2001; Lee et al., 2000), the Myxozoa (Longshaw et al., 2002; Antonio et al., 1999) and the Paramyxea (Kleeman et al., 2002; Audemard et al., 2002). The PCR assay and ISH techniques established by Itoh et al. (2003a,b) are likely to assist in clarifying the life cycle of M. chungmuensis. A preliminary experiment showed that Pacific oysters placed into an endemic area of M. chungmuensis infection in
0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.06.001
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August in Okayama prefecture (Japan) developed an abnormal formation in the ovary within a month. This result indicates that uncharacterised infective stages reside in the water and invade oysters during that period. This observation has provided the opportunity to conduct experimental infections in the field. During the study, uninfected Pacific oysters were placed into an endemic area at the same period as in the preliminary work and sampled weekly up to 7 weeks post placement (PP). After that, PCR and ISH techniques were applied to the oysters with the aims of identifying the initial developmental stages within the host and determining the portal of entry of M. chungmuensis into the Pacific oysters.
2. Materials and methods 2.1. Oysters and collection schedule One hundred and sixty spat of Pacific oysters, C. gigas (mean shell height ^ S.D.: 27.8 ^ 4.0 mm) were purchased from Miyagi prefecture in Japan (38.48 N, 141.38 E), where M. chungmuensis infection has not previously been reported. The spat were placed into an endemic area in Okayama prefecture (34.68 N, 131.48 E) on 13th August 2003. Before exposure, parts of the gonad were excised from 20 oysters and tested using a nested-PCR assay, which is specific for M. chungmuensis, to confirm that the oysters were not infected with the parasite. Twenty oysters were sampled weekly between 13th August and 1st October, each week a part of the gonadal tissues was excised and fixed in 100% ethanol for DNA extraction and subsequent PCR assays. The tissues from oysters showing positive by PCR were subsequently fixed in 10% phosphate buffered formalin for 48 h and processed for histology and ISH. Wet mount slides were also prepared from the gonad for gender determination by observation of gamete. Both an anterior part including the labial palps and a posterior part including the cardio-vascular system were also dissected from five oysters and each week fixed and processed for ISH to examine for parasite infection in those areas. 2.2. PCR assays PCR assays were performed as described in Itoh et al. (2003b). DNA was extracted with DNeasy Tissue Kit (QIAGEN Inc.). Nested-PCR reactions were performed using two sets of M. chungmuensis specific primers (see Itoh et al., 2003b), OPF-2/OPR-2 for the first round, and OPF-3/ OPR-3 for the second round. PCR reactions were carried out in 20 ml total volumes. PCR parameters for both rounds were as follows: 0.1 ml of Takara Ex Taq e (2.5 U/100 ml); 2 ml of 10 £ Ex Taq e Buffer; 1.6 ml of dNTP mix; 20 ng of extracted DNA as template. The first thermal cycle protocol using the primers OPF-2/OPR-2 was as follows: pre-heating at 94 8C for 5 min, 35 cycles of denaturation
94 8C for 1 min, annealing at 59 8C for 30 s, extension at 72 8C for 1 min, and a further elongation step at 72 8C for 10 min. A half of 1 ml of the first PCR product was used as template for the second PCR step. The second PCR step was the same as for the first step PCR with the exception of a lower annealing temperature of 55 8C. PCR products were electrophoresed in 1.5% agarose gels. 2.3. Conventional histology and in situ hybridisation (ISH) Tissues fixed in 10% phosphate buffered formalin were dehydrated in an ethanol series, embedded in paraffin and 5 mm sections were prepared for both conventional histology and ISH reactions. Conventional histological sections were stained with H and E. The ISH process was performed as described in Itoh et al. (2003a). In brief, three oligonucleotide probes for the parasite MCSP-01, MCSP-03 and 6-R were labeled with digoxygenin using a DIG oligonucleotide tailing Kit (Roche Inc.). Hybridisation reactions were performed with the MicroProbe Staining System (Fisher Scientific International Inc.) using manual capillary actions with modifications to the manufacture’s manual. A DIG nucleic acid detection kit (Roche Inc.) was used to visualise the hybridisation products according to the manufacture’s manual, sections were counterstained with 0.05% Bismarck brown Y (SigmaAldrich Inc.). The morphology of parasite stages in tissue sections following ISH was examined in adjacent H and E stained sections. 2.4. Transmission electron microscopic (TEM) observations Paraffin blocks, in which M. chungmuensis cells were confirmed by ISH, were prepared for TEM. Deparaffinised with xylene and rehydrated with an ethanol series (100, 90, 70 and 50%) and with distilled water (DW). The deparaffinised tissues were cut into approximate 1 mm3, fixed with 1% OsO4 solution buffered with 0.2 M cacodylate buffer for 2 h, rinsed twice with the same buffer, dehydrated through a graded ethanol series and embedded in Spurr resin. ‘Pop-off’ technique described by Kleeman (2002) was also utilised for transferring paraffin embedded tissue to resin blocks to observe the stage confirmed by ISH in the connective tissues. Ultrathin sections (, 60 nm in thickness) were stained with uranyl acetate and lead citrate. The sections were examined using an electron microscope (JEM 1010, JEOL) at 80 kV.
3. Results 3.1. PCR detection PCR assays confirmed that the experimental oysters were not infected with M. chungmuensis prior to exposure. Infection by the parasite was first confirmed at 1 week PP
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The sporulation process was observed in the ovary at 5 weeks PP by H and E stained sections (Fig. 6) and was continuously observed until the end of the experiment. ISH techniques also detected clusters of the parasites around the gonadal epithelia (Fig. 7). TEM observations successfully identified the parasite on the epithelium of the gonad (Fig. 8) which contains an oval secondary cell (5.1 £ 2.9 mm in average, n ¼ 3). The initial sporulation stage (6.3 £ 4.0 mm in average, n ¼ 2) was also found in an immature oocyte (Fig. 9). All of these pre-sporogonic stages were observed outside of the host cells, while sporogonic stages were observed only inside the host’s oocytes. Fig. 1. Prevalence of Marteilioides chungmuensis detected by PCR in Pacific oysters during the infection trial.
4. Discussion using PCR and continued until the end of the experiment (7 weeks PP) (Fig. 1). The prevalence of infection in female oysters reached 100% at 5 weeks PP and was maintained above 60% for the duration of the experiment, while parasite prevalence in male oysters recorded a maximum of 62% at 4 weeks PP and subsequently declined every week for the remainder of the experiment, finishing at only 24% prevalence by week 7 (Fig. 1). Freedom from infection and predictive value in the PCR assay were 100 and 16%, respectively. 3.2. Early development of M. chungmuensis At 2 weeks PP, M. chungmuensis was first identified in the gill, the labial palp and the mantle by ISH (Fig. 2A). H and E stained sections also showed uninucleate (Fig. 2B) and binucleate stages (Fig. 2C). TEM studies successfully identified uninucleate stages in which haplosporosomes were identified in the cytoplasm (Fig. 3). At 3 weeks PP, ISH techniques detected the parasitic infection in the connective tissues, both binucleate (Fig. 4A) and uninucleate (Fig. 4B) stages were identifiable, although H and E observations failed to characterise these stages. ‘Pop off’ preparation for TEM showed the parasite stage that contained two secondary cells in which a tertiary cell was located (Fig. 5).
Portals of entry of protozoan parasites into bivalves have been discussed by several authors. Chintala et al. (2002) considered that Perkinsus marinus invades into the Eastern oyster, Crassostrea virginica, through the mantle, the gill and the gut. Carnegie et al. (2003) detected M. mackini in the palps of apparently healthy oysters and suggested that they might be the primary portal of entry. In the case of paramyxean parasites, Grizel et al. (1974) found plasmodia of Marteilia refringens in the gill epithelium of Ostrea edulis and suggested that the gill was the location in which the infection was acquired, a theory also supported by Robledo and Figueras (1995), who found the parasite in the gill epithelial cells of a mussel, Mytilus galloprovincialis. Kleeman et al. (2002) demonstrated that Marteilia sydneyi enters through the gills and the palps of its host using ISH and TEM observations. In this study, the early developmental stages of M. chungmuensis were first observed in the organs which have direct contact with water flow, while the parasite was not detected in the digestive tubule, suggesting that the parasite does not invade into the host through the digestive system in contrast to P. marinus. The morphology of the early developmental stage in those organs was quite similar to the earliest stage of M. sydneyi as examined by Kleeman et al. (2002). These similarities
Fig. 2. Detection of early developmental stage. (A) Detection of Marteilioides chungmuensis (arrows) by in situ hybridisation in the labial palps of the Pacific oyster. The section was counterstained with Bismarck Brown. Scale bar ¼ 20 mm. (B) H and E section of M. chungmuensis uninucleate cell (arrow) in the epithelial tissue of the labial palps. Scale bar ¼ 10 mm. (C) Bicellular stage (arrow) in the gill. Scale bar ¼ 10 mm.
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Fig. 3. Electron micrograph of Marteilioides chungmuensis in the gill of the Pacific oyster. A bicellular parasite composed of a uninucleate cell (S2) contained in a stem cell (S1). N2, nucleus of S2 cell; H, haplosporosome. Scale bar ¼ 500 nm. (Inset) Haplosporosome in the stem cell. Scale bar ¼ 100 nm.
also strongly suggest that these organs may as the portal of entry for M. chungmuensis. In advanced infection, the parasite was detected in the connective tissues using ISH. Binary fission appeared to occur at this stage. ‘Pop-off’ techniques for TEM revealed this stage in the connective tissues which contained secondary cells each housing a tertiary cell. Kleeman et al. (2002) also observed similar developmental stages of M. sydneyi in which the parasite repeats cell-within-cell replication extensively,
and the authors considered the stage as an ‘extrasporogonic stage’ as defined by Lom and Dykova (1992). It is highly possible that M. chungmuensis also has the extrasporogonic stage. However, haplosporosomes were not identified clearly in TEM observations due to the low quality of ‘pop-off’ TEM. In a future study, it will be essential to examine this stage in the connective tissues by TEM. After the extrasporogonic stage, M. sydneyi has a developmental phase that migrates between the epithelial
Fig. 4. Marteilioides chungmuensis in the connective tissue of the Pacific oysters detected by in situ hybridisation. Binucleate stage (A) and uninucleate stage (B). The sections were counterstained with Bismarck Brown. Scale bars ¼ 10 mm.
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Fig. 5. Electron micrograph of ’pop off’ preparation of Marteilioides chungmuensis in the connective tissues of the Pacific oyster. C1, stem cell; C2, secondary cells; N2, nuclei of secondary cells; N3, nucleus of tertiary cell. Scale bar ¼ 500 nm.
cells of the digestive tubule. Kleeman et al. (2002) successfully detected this phase by both ISH and TEM, and defined the stem cell of the phase as a ‘nurse cell’ in the developmental process. However, a corresponding phase to that from M. sydneyi was not observed in the developmental process of M. chungmuensis. The difference between the two paramyxeans may be associated with the difference in the subsequent sporulation processes. Sporulation of M. chungmuensis occurs inside the host cell and hence the parasite may not need to migrate into intercellular space of the gonad, whilst migration between the host cells is necessary for the sporulation of M. sydneyi which occurs outside the host cell. ISH detected the clusters of the parasites around the gonadal epithelia, suggesting that
Fig. 6. H and E stained section of Marteilioides chungmuensis infection in the gonad of the Pacific oyster. Arrows indicate the parasite cells. Scale bar ¼ 20 mm.
M. chungmuensis may multiply in those areas without the ‘nurse cell’ stage. Intensive TEM observations are necessary to elucidate the details of this developmental phase. Marteilioides chungmuensis was identified both inside and outside the oocyte in the ovary. The secondary cell of the stage outside the oocyte had very similar morphology and size to that found in immature oocytes, suggesting that the secondary cell may be released from the primary cell and invade into immature oocytes. Imanaka et al. (2001) considered that the bicellular stage (described as type A in Itoh et al. (2002)) was the youngest stage involved in sporulation. However, in this study, the unicellular stage was found in an oocyte, where sporulation occurs.
Fig. 7. Clusters of Marteilioides chungmuensis cells detected by in situ hybridisation in the gonad of the Pacific oyster. Dark areas represent the parasite (arrows). The section was counterstained with Bismarck Brown. Scale bar ¼ 200 mm.
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Fig. 8. Electron micrograph of Marteilioides chungmuensis located in the epithelial tissues of the ovary of the Pacific oyster. C1, primary cell; C2, secondary cell; N1, nucleus of the stem cell; N2, nucelus of the secondary cell; H, haplosporosome. Scale bar ¼ 4 mm.
Therefore, it is suggested that the unicellular stage identified herein is the youngest sporulation stage which initiates the sporulation process as described in Itoh et al. (2002). Marteilioides chungmuensis infection has not been previously described in male oysters. In this study, PCR and ISH were used to detect the parasite in male oysters (data not shown), although sporulation in male oysters was not confirmed. The prevalence as detected by PCR gradually decreased from 4 weeks PP, indicating that
Fig. 10. Hypothetical development of Marteilioides chungmuensis in the Pacific oyster, Crassostrea gigas. Unidentified infective stage invades through the gill, the mantle and the labial palps (A). Extrasporogony repeatedly occurs in the connective tissues with internal cleavage occurring within each primary cell (B). Secondary cells are released which enlarge in size. Multiplication may occur in the gonad (C). Stem cell ( ¼ secondary cell in (C)) invade into the oocyte and sporulation starts as described in Itoh et al. (2002) (D). Mature parasites are released from the oyster via the genital canal (E).
M. chungmuensis invades the male oysters but the parasite may be excluded from the oyster without initiating sporulation. During this experiment, some of the developmental stages were described for the first time, and the portals of entry of M. chungmuensis were clarified. With these findings, a hypothetical developmental cycle of the parasite is suggested (Fig. 10). Unidentified infective stage invades through the gill, the mantle and the labial palps and subsequently extrasporogony repeatedly occurs in the connective tissues with internal cleavage occuring within each primary cell. Secondary cells are released then enlarge in size. Multiplication may occur in the gonad and then stem cell invades into the oocyte and sporulation starts as described in Itoh et al. (2002). Mature parasites are released from the oyster via the genital canal . Moreover, it was demonstrated that the ISH and PCR techniques established in Itoh et al. (2003a,b) are useful tools to elucidate the life cycle of this parasite including the stages outside the oyster. An intermediate or alternate host is likely as Perkins (1991) predicted for paramyxeans.
Acknowledgements
Fig. 9. Electron micrograph of Marteilioides chungmuensis in an immature oocyte of the Pacific oyster. P, parasite cell; NP, nucleus of the parasite cell; O, oocyte; NH, nucleus of the oocyte; H, haplosporosome. Scale bar ¼ 2 mm.
The authors sincerely appreciate the assistance of the staff of the Fisheries Experimental Station of Okayama Prefecture for supplying samples and equipment. The authors also would like to express their gratitude to Dr Mark Freeman (The University of Tokyo) and Dr Tomoyoshi Yoshinaga (The University of Tokyo) for his review of the manuscript.
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Itoh, N., Tun, K.L., Komiyama, H., Ueki, N., Ogawa, K., 2004. An ovarian infection in the Iwagaki oyster Crassostrea nippona, with the protozoan parasite Marteilioides chungmuensis. J. Fish Dis. 27, 311 –314. Kleeman, S.N., 2002. A ’pop off‘ technique for reprocessing histological sections of the paramyxean Marteilia sydneyi for electron microscopy. Parasitol. Res. 88, 439 –442. Kleeman, S.N., Adlard, R.D., Lester, R.J.G., 2002. Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis. Int. J. Parasitol. 32, 767–784. Lee, S.J., Yokoyama, H., Ogawa, K., Wakabayashi, H., 2000. In situ hybridization for detection of the microsporidian parasite Glugea plecoglossi by using rainbow trout as an experimental infection model. Fish Pathol. 35, 79–84. Lom, J., Dykova, I., 1992. Protozoan parasites of fishes. Developments in Aquaculture and Fisheries Science. Elsevier, Amsterdam. Longshaw, M., Le Deuff, R.M., Harris, A.F., Feist, S.W., 2002. Development of proliferative kidney disease in rainbow trout Oncorhynchus mykiss (Walbaum), following short-term exposure to Tetracapsula bryosalmonae infected bryozoans. J. Fish Dis. 25, 443– 449. Matsusato, T., Masumura, K., 1981. Abnormal enlargement of the ovary of oyster Crassostrea gigas (Thunberg) by an unidentified parasite. Fish Pathol. 15, 207–212. Ngo, T.T.T., Berthe, F.C.J., Choi, K.S., 2003. Prevalence and infection intensity of the ovarian parasite Marteilioides chungmuensis during an annual reproductive cycle of the oyster Crassostrea gigas. Dis. Aquat. Organ 56, 259– 267. Park, M.S., Kang, C.K., Choi, D.L., Jee, B.Y., 2003. Appearance and pathogenicity of ovarian parasite Marteilioides chungmuensis in the farmed Pacific oysters Crassostrea gigas, in Korea. J. Shellfish Res. 22, 475– 479. Perkins, F.O., 1991. ‘Sporozoa’: Apicomplexa, Microsporidia, Haplosporidia, Paramyxea, Myxosporidia, and Actinosporidia. In: Harrison, F.W., Corliss, J.O. (Eds.), Microscopic Anatomy of Invertevrates: Protozoa. Wiley, London, pp. 261 –331. Quayle, D.B., 1961. Denman Island disease and mortality, 1960. Fisheries Research Board of Canada Manuscript Report 713. Ottawa.. Robledo, J.A.F., Figueras, A., 1995. The effects of culture-site, depth, season, and stock source on the prevalence of Marteilia refringens in cultured mussels (Mytilus galloprovincialis Lmk.) from Galicia, Spain. J. Parasitol. 81, 354– 363. Sanchez, J.G., Speare, D.J., Markham, R.J.F., Wright, G.M., Kibenge, F.S.B., 2001. Localization of the initial developmental stages of Loma salmonae in rainbow trout (Oncorhynchus mykiss). Vet. Pathol. 38, 540– 546. Seki, H., 1934. The abnormal development of the oyster gonads in Hiroshima bay. Bull. Japan Sci. Soc. 9, 93–99.(in Japanese).
Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas Naoki Itoha, Hideki Komiyamab, Noriyuki Uekib, Kazuo Ogawaa,* a
Laboratory of Fish Diseases, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo Tokyo 113-8657, Japan b Fisheries Experiment Station of Okayama Prefecture, Ushimado, Okayama 701-4303, Japan Received 13 April 2004; received in revised form 4 June 2004; accepted 7 June 2004
Abstract A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Paramyxea; Marteilioides chungmuensis; Pacific oyster; Crassostrea gigas; Extrasporogony; Ovarian parasite
1. Introduction Pacific oysters, Crassostrea gigas, are cultured throughout the world. The global production of the Pacific oysters, from European, North American and Asian countries is estimated to be approximately 4 million metric tonnes per year (FAO, 2000), making this species the most important cultured bivalve. In Pacific oyster culture, there are two serious parasitic diseases. Firstly, Mikrocytos mackini (Farley et al., 1988), which causes heavy mortalities in North America (Quayle, 1961) and secondly, the ovary enlargement disease of the Pacific oyster which is caused by Marteilioides chungmuensis (Comps et al., 1986). Although high mortalities have not been reported, the oysters affected by the latter disease develop abnormal gonads, resulting in a disfigured appearance and poor market appeal. Marteilioides chungmuensis has been reported in Korea (Chun, 1979) and Japan (Seki, 1934). The parasite has been studied in several aspects such as seasonal prevalence (Imanaka et al., 2001; Ngo et al., 2003), epizootiology * Corresponding author. Tel.: þ 81-3-5841-5283; fax: þ81-3-5841-5383. E-mail address: [email protected] (K. Ogawa).
(Matsusato and Masumura, 1981), biochemical studies (Park et al., 2003) and host range (Itoh et al., 2004). In particular, the sporulation process of M. chungmuensis has been intensively studied by several authors (Chun, 1979; Comps et al., 1986; Imanaka et al., 2001; Itoh et al., 2002), however, the early developmental stages outside the oocytes have not been located in histological sections. Moreover, the inability to experimentally infect oysters under laboratory conditions makes the detection of early developmental stages difficult. Recently, it has been demonstrated that molecular detection methods such as PCR and in situ hybridisation (ISH) are useful for detecting the early developmental stages and clarify the life cycles of micro-parasites such as the Microspora (Sanchez et al., 2001; Lee et al., 2000), the Myxozoa (Longshaw et al., 2002; Antonio et al., 1999) and the Paramyxea (Kleeman et al., 2002; Audemard et al., 2002). The PCR assay and ISH techniques established by Itoh et al. (2003a,b) are likely to assist in clarifying the life cycle of M. chungmuensis. A preliminary experiment showed that Pacific oysters placed into an endemic area of M. chungmuensis infection in
0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.06.001
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August in Okayama prefecture (Japan) developed an abnormal formation in the ovary within a month. This result indicates that uncharacterised infective stages reside in the water and invade oysters during that period. This observation has provided the opportunity to conduct experimental infections in the field. During the study, uninfected Pacific oysters were placed into an endemic area at the same period as in the preliminary work and sampled weekly up to 7 weeks post placement (PP). After that, PCR and ISH techniques were applied to the oysters with the aims of identifying the initial developmental stages within the host and determining the portal of entry of M. chungmuensis into the Pacific oysters.
2. Materials and methods 2.1. Oysters and collection schedule One hundred and sixty spat of Pacific oysters, C. gigas (mean shell height ^ S.D.: 27.8 ^ 4.0 mm) were purchased from Miyagi prefecture in Japan (38.48 N, 141.38 E), where M. chungmuensis infection has not previously been reported. The spat were placed into an endemic area in Okayama prefecture (34.68 N, 131.48 E) on 13th August 2003. Before exposure, parts of the gonad were excised from 20 oysters and tested using a nested-PCR assay, which is specific for M. chungmuensis, to confirm that the oysters were not infected with the parasite. Twenty oysters were sampled weekly between 13th August and 1st October, each week a part of the gonadal tissues was excised and fixed in 100% ethanol for DNA extraction and subsequent PCR assays. The tissues from oysters showing positive by PCR were subsequently fixed in 10% phosphate buffered formalin for 48 h and processed for histology and ISH. Wet mount slides were also prepared from the gonad for gender determination by observation of gamete. Both an anterior part including the labial palps and a posterior part including the cardio-vascular system were also dissected from five oysters and each week fixed and processed for ISH to examine for parasite infection in those areas. 2.2. PCR assays PCR assays were performed as described in Itoh et al. (2003b). DNA was extracted with DNeasy Tissue Kit (QIAGEN Inc.). Nested-PCR reactions were performed using two sets of M. chungmuensis specific primers (see Itoh et al., 2003b), OPF-2/OPR-2 for the first round, and OPF-3/ OPR-3 for the second round. PCR reactions were carried out in 20 ml total volumes. PCR parameters for both rounds were as follows: 0.1 ml of Takara Ex Taq e (2.5 U/100 ml); 2 ml of 10 £ Ex Taq e Buffer; 1.6 ml of dNTP mix; 20 ng of extracted DNA as template. The first thermal cycle protocol using the primers OPF-2/OPR-2 was as follows: pre-heating at 94 8C for 5 min, 35 cycles of denaturation
94 8C for 1 min, annealing at 59 8C for 30 s, extension at 72 8C for 1 min, and a further elongation step at 72 8C for 10 min. A half of 1 ml of the first PCR product was used as template for the second PCR step. The second PCR step was the same as for the first step PCR with the exception of a lower annealing temperature of 55 8C. PCR products were electrophoresed in 1.5% agarose gels. 2.3. Conventional histology and in situ hybridisation (ISH) Tissues fixed in 10% phosphate buffered formalin were dehydrated in an ethanol series, embedded in paraffin and 5 mm sections were prepared for both conventional histology and ISH reactions. Conventional histological sections were stained with H and E. The ISH process was performed as described in Itoh et al. (2003a). In brief, three oligonucleotide probes for the parasite MCSP-01, MCSP-03 and 6-R were labeled with digoxygenin using a DIG oligonucleotide tailing Kit (Roche Inc.). Hybridisation reactions were performed with the MicroProbe Staining System (Fisher Scientific International Inc.) using manual capillary actions with modifications to the manufacture’s manual. A DIG nucleic acid detection kit (Roche Inc.) was used to visualise the hybridisation products according to the manufacture’s manual, sections were counterstained with 0.05% Bismarck brown Y (SigmaAldrich Inc.). The morphology of parasite stages in tissue sections following ISH was examined in adjacent H and E stained sections. 2.4. Transmission electron microscopic (TEM) observations Paraffin blocks, in which M. chungmuensis cells were confirmed by ISH, were prepared for TEM. Deparaffinised with xylene and rehydrated with an ethanol series (100, 90, 70 and 50%) and with distilled water (DW). The deparaffinised tissues were cut into approximate 1 mm3, fixed with 1% OsO4 solution buffered with 0.2 M cacodylate buffer for 2 h, rinsed twice with the same buffer, dehydrated through a graded ethanol series and embedded in Spurr resin. ‘Pop-off’ technique described by Kleeman (2002) was also utilised for transferring paraffin embedded tissue to resin blocks to observe the stage confirmed by ISH in the connective tissues. Ultrathin sections (, 60 nm in thickness) were stained with uranyl acetate and lead citrate. The sections were examined using an electron microscope (JEM 1010, JEOL) at 80 kV.
3. Results 3.1. PCR detection PCR assays confirmed that the experimental oysters were not infected with M. chungmuensis prior to exposure. Infection by the parasite was first confirmed at 1 week PP
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The sporulation process was observed in the ovary at 5 weeks PP by H and E stained sections (Fig. 6) and was continuously observed until the end of the experiment. ISH techniques also detected clusters of the parasites around the gonadal epithelia (Fig. 7). TEM observations successfully identified the parasite on the epithelium of the gonad (Fig. 8) which contains an oval secondary cell (5.1 £ 2.9 mm in average, n ¼ 3). The initial sporulation stage (6.3 £ 4.0 mm in average, n ¼ 2) was also found in an immature oocyte (Fig. 9). All of these pre-sporogonic stages were observed outside of the host cells, while sporogonic stages were observed only inside the host’s oocytes. Fig. 1. Prevalence of Marteilioides chungmuensis detected by PCR in Pacific oysters during the infection trial.
4. Discussion using PCR and continued until the end of the experiment (7 weeks PP) (Fig. 1). The prevalence of infection in female oysters reached 100% at 5 weeks PP and was maintained above 60% for the duration of the experiment, while parasite prevalence in male oysters recorded a maximum of 62% at 4 weeks PP and subsequently declined every week for the remainder of the experiment, finishing at only 24% prevalence by week 7 (Fig. 1). Freedom from infection and predictive value in the PCR assay were 100 and 16%, respectively. 3.2. Early development of M. chungmuensis At 2 weeks PP, M. chungmuensis was first identified in the gill, the labial palp and the mantle by ISH (Fig. 2A). H and E stained sections also showed uninucleate (Fig. 2B) and binucleate stages (Fig. 2C). TEM studies successfully identified uninucleate stages in which haplosporosomes were identified in the cytoplasm (Fig. 3). At 3 weeks PP, ISH techniques detected the parasitic infection in the connective tissues, both binucleate (Fig. 4A) and uninucleate (Fig. 4B) stages were identifiable, although H and E observations failed to characterise these stages. ‘Pop off’ preparation for TEM showed the parasite stage that contained two secondary cells in which a tertiary cell was located (Fig. 5).
Portals of entry of protozoan parasites into bivalves have been discussed by several authors. Chintala et al. (2002) considered that Perkinsus marinus invades into the Eastern oyster, Crassostrea virginica, through the mantle, the gill and the gut. Carnegie et al. (2003) detected M. mackini in the palps of apparently healthy oysters and suggested that they might be the primary portal of entry. In the case of paramyxean parasites, Grizel et al. (1974) found plasmodia of Marteilia refringens in the gill epithelium of Ostrea edulis and suggested that the gill was the location in which the infection was acquired, a theory also supported by Robledo and Figueras (1995), who found the parasite in the gill epithelial cells of a mussel, Mytilus galloprovincialis. Kleeman et al. (2002) demonstrated that Marteilia sydneyi enters through the gills and the palps of its host using ISH and TEM observations. In this study, the early developmental stages of M. chungmuensis were first observed in the organs which have direct contact with water flow, while the parasite was not detected in the digestive tubule, suggesting that the parasite does not invade into the host through the digestive system in contrast to P. marinus. The morphology of the early developmental stage in those organs was quite similar to the earliest stage of M. sydneyi as examined by Kleeman et al. (2002). These similarities
Fig. 2. Detection of early developmental stage. (A) Detection of Marteilioides chungmuensis (arrows) by in situ hybridisation in the labial palps of the Pacific oyster. The section was counterstained with Bismarck Brown. Scale bar ¼ 20 mm. (B) H and E section of M. chungmuensis uninucleate cell (arrow) in the epithelial tissue of the labial palps. Scale bar ¼ 10 mm. (C) Bicellular stage (arrow) in the gill. Scale bar ¼ 10 mm.
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Fig. 3. Electron micrograph of Marteilioides chungmuensis in the gill of the Pacific oyster. A bicellular parasite composed of a uninucleate cell (S2) contained in a stem cell (S1). N2, nucleus of S2 cell; H, haplosporosome. Scale bar ¼ 500 nm. (Inset) Haplosporosome in the stem cell. Scale bar ¼ 100 nm.
also strongly suggest that these organs may as the portal of entry for M. chungmuensis. In advanced infection, the parasite was detected in the connective tissues using ISH. Binary fission appeared to occur at this stage. ‘Pop-off’ techniques for TEM revealed this stage in the connective tissues which contained secondary cells each housing a tertiary cell. Kleeman et al. (2002) also observed similar developmental stages of M. sydneyi in which the parasite repeats cell-within-cell replication extensively,
and the authors considered the stage as an ‘extrasporogonic stage’ as defined by Lom and Dykova (1992). It is highly possible that M. chungmuensis also has the extrasporogonic stage. However, haplosporosomes were not identified clearly in TEM observations due to the low quality of ‘pop-off’ TEM. In a future study, it will be essential to examine this stage in the connective tissues by TEM. After the extrasporogonic stage, M. sydneyi has a developmental phase that migrates between the epithelial
Fig. 4. Marteilioides chungmuensis in the connective tissue of the Pacific oysters detected by in situ hybridisation. Binucleate stage (A) and uninucleate stage (B). The sections were counterstained with Bismarck Brown. Scale bars ¼ 10 mm.
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Fig. 5. Electron micrograph of ’pop off’ preparation of Marteilioides chungmuensis in the connective tissues of the Pacific oyster. C1, stem cell; C2, secondary cells; N2, nuclei of secondary cells; N3, nucleus of tertiary cell. Scale bar ¼ 500 nm.
cells of the digestive tubule. Kleeman et al. (2002) successfully detected this phase by both ISH and TEM, and defined the stem cell of the phase as a ‘nurse cell’ in the developmental process. However, a corresponding phase to that from M. sydneyi was not observed in the developmental process of M. chungmuensis. The difference between the two paramyxeans may be associated with the difference in the subsequent sporulation processes. Sporulation of M. chungmuensis occurs inside the host cell and hence the parasite may not need to migrate into intercellular space of the gonad, whilst migration between the host cells is necessary for the sporulation of M. sydneyi which occurs outside the host cell. ISH detected the clusters of the parasites around the gonadal epithelia, suggesting that
Fig. 6. H and E stained section of Marteilioides chungmuensis infection in the gonad of the Pacific oyster. Arrows indicate the parasite cells. Scale bar ¼ 20 mm.
M. chungmuensis may multiply in those areas without the ‘nurse cell’ stage. Intensive TEM observations are necessary to elucidate the details of this developmental phase. Marteilioides chungmuensis was identified both inside and outside the oocyte in the ovary. The secondary cell of the stage outside the oocyte had very similar morphology and size to that found in immature oocytes, suggesting that the secondary cell may be released from the primary cell and invade into immature oocytes. Imanaka et al. (2001) considered that the bicellular stage (described as type A in Itoh et al. (2002)) was the youngest stage involved in sporulation. However, in this study, the unicellular stage was found in an oocyte, where sporulation occurs.
Fig. 7. Clusters of Marteilioides chungmuensis cells detected by in situ hybridisation in the gonad of the Pacific oyster. Dark areas represent the parasite (arrows). The section was counterstained with Bismarck Brown. Scale bar ¼ 200 mm.
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Fig. 8. Electron micrograph of Marteilioides chungmuensis located in the epithelial tissues of the ovary of the Pacific oyster. C1, primary cell; C2, secondary cell; N1, nucleus of the stem cell; N2, nucelus of the secondary cell; H, haplosporosome. Scale bar ¼ 4 mm.
Therefore, it is suggested that the unicellular stage identified herein is the youngest sporulation stage which initiates the sporulation process as described in Itoh et al. (2002). Marteilioides chungmuensis infection has not been previously described in male oysters. In this study, PCR and ISH were used to detect the parasite in male oysters (data not shown), although sporulation in male oysters was not confirmed. The prevalence as detected by PCR gradually decreased from 4 weeks PP, indicating that
Fig. 10. Hypothetical development of Marteilioides chungmuensis in the Pacific oyster, Crassostrea gigas. Unidentified infective stage invades through the gill, the mantle and the labial palps (A). Extrasporogony repeatedly occurs in the connective tissues with internal cleavage occurring within each primary cell (B). Secondary cells are released which enlarge in size. Multiplication may occur in the gonad (C). Stem cell ( ¼ secondary cell in (C)) invade into the oocyte and sporulation starts as described in Itoh et al. (2002) (D). Mature parasites are released from the oyster via the genital canal (E).
M. chungmuensis invades the male oysters but the parasite may be excluded from the oyster without initiating sporulation. During this experiment, some of the developmental stages were described for the first time, and the portals of entry of M. chungmuensis were clarified. With these findings, a hypothetical developmental cycle of the parasite is suggested (Fig. 10). Unidentified infective stage invades through the gill, the mantle and the labial palps and subsequently extrasporogony repeatedly occurs in the connective tissues with internal cleavage occuring within each primary cell. Secondary cells are released then enlarge in size. Multiplication may occur in the gonad and then stem cell invades into the oocyte and sporulation starts as described in Itoh et al. (2002). Mature parasites are released from the oyster via the genital canal . Moreover, it was demonstrated that the ISH and PCR techniques established in Itoh et al. (2003a,b) are useful tools to elucidate the life cycle of this parasite including the stages outside the oyster. An intermediate or alternate host is likely as Perkins (1991) predicted for paramyxeans.
Acknowledgements
Fig. 9. Electron micrograph of Marteilioides chungmuensis in an immature oocyte of the Pacific oyster. P, parasite cell; NP, nucleus of the parasite cell; O, oocyte; NH, nucleus of the oocyte; H, haplosporosome. Scale bar ¼ 2 mm.
The authors sincerely appreciate the assistance of the staff of the Fisheries Experimental Station of Okayama Prefecture for supplying samples and equipment. The authors also would like to express their gratitude to Dr Mark Freeman (The University of Tokyo) and Dr Tomoyoshi Yoshinaga (The University of Tokyo) for his review of the manuscript.
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