Marteilioides chungmuensis of Oysters

Category

Category 1 (Not Reported in Canada)

Common, generally accepted names of the organism or disease agent

Marteilioides of oocytes.

Scientific name or taxonomic affiliation

Marteilioides chungmuensis (Comps et al. 1987) in the phylum Paramyxea as proposed by Desportes and Perkins (1990) and supported by Berthe et al. (2000). The genus was defined by Anderson and Lester (1992) who indicated that the vegetative stages had amoeboid primary cell that cleaved internally to form secondary cells (sporonts) and sporulation consisted of sporonts that produce a single pluricellular spore, then degenerate such that the spore was enveloped by a cytoplasmic residuum and the plasmalemma of the sporont. Some of the infections described as "oyster egg diseases" may be attributed to M. chungmuensis.

Geographic distribution

Korea and Japan.

Host species

Crassostrea gigas. Also in Crassostrea nippona transplanted to an enzootic area (Itoh et al. 2004a). A similar looking parasite was reported from the ova of Crassostrea echinata from Northern Territory, Australia (Wolf 1977) and Western Australia (Hine and Thorne 2000). A Marteilioides -like parasite has also been reported at low prevalence (1.6%) from the oocytes of Manila clams, Venerupis (=Tapes ) philippinarum, in coastal areas of Korea (Lee et al. 2001).

Impact on the host

Abnormal egg-masses with a nodular appearance (like multiple tumors) among cultured Crassastrea gigas in Hiroshima Prefecture, Japan were first reported in the 1930s. Surveys in Matsushima Bay, Japan in the early 1960s revealed prevalences up to 46.2% but the intensity of infection was usually low (Imai et al. 1968). In 1974, Matsuzato et al. (1977) detected a parasite in the ova of abnormal oysters from Hiroshima Prefecture where 0 to 12% of the oysters were found affected. In Korea, M. chungmuensis was reported for the first time in 1970. Chun (1979) who called the parasite an enigmatic amoeba reported an infection prevalence of 13.3% in the Hansan area of Korea in 1978 and 1979. Park and Chun (1986) who identified the parasite as Marteilia sp. detected low prevalence (0.6%) in two oyster farms but did not detect the parasite in two others during a survey of 30 oysters from each farm per month for 1 year. Elston (1993) reported prevalences of infection up to 8.3% in the late 1980s. Apparently, the prevalence of infection in Korea has continued to increase and M. chungmuensis has been implicated as a cause of poor seed collection and high mortalities among cultured oysters since 1990 (Park 2005). Also, occurrence has extended from the spawning season (late summer-early fall) to year round with highest prevalence during spawning (from June to August) and during the gonadal regeneration period (from September to October) (Park 2002, 2005; Park et al. 2003). Tun et al. (2007) found that infected female oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive and concluded that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality of infected oysters, causing a decline in prevalence of infection within the epizootic area in autumn and the continued decrease in prevalence during the winter was attributed to recovery from infection (Tun et al. 2008a).

Marteilioides chungmuensis infects the cytoplasm of oocytes and can affect large areas of the reproductive follicles causing irregular enlargement of the infected gonadal tissues. Histological observations suggested that M. chungmuensis invades immature ova which move to the center of the follicle and the growth of the parasite was highly correlated with the growth and maturation of host gonadal cells (Itoh et al. 2002a). Infected eggs may be liberated via the genital canal or retained in the ovarian follicle and this parasite can have a significant effect on the reproductive output of an infected female oyster. Infection can also cause spawning failure by delaying spawning and destroying ripe oyster oocytes (Ngo et al. 2003). Infection also significantly reduced glycogen levels and serum protein concentrations affecting metabolic recovery after spawning (Park et al. 2003, Park 2005). Infected oysters lose their marketability due to the unaesthetic appearance and thus causes a serious economical impact.

Basic biological information pertaining to the complete life cycle of this parasite, including the method of transmission, remains unknown (Itoh et al. 2002b). However, Itoh et al. (2004b) used parasite-specific DNA probes and electron microscopy to reveal the route of infection and to identify early infective and multiplication stages in the oyster (Fig A1). Briefly, the parasite invaded the oyster through the epithelial tissues of the gills, mantle and labial palps. Extrasporogonic multiplication of the primary cell repeatedly occurred outside of host cells in the connective tissue by binary fission. In addition, internal cleavage within the primary cell resulted in the production of secondary cells which in turn occasionally contained a tertiary cell. Apparently, the secondary cell released from the primary cell migrates through the epithelium of the gonad and invades an immature oocyte where it forms the stem cell of the sporogonic stage. Sporogonic stages were observed only inside the oocytes of the host (as described below). Crassostrea gigas placed into an area enzootic for M. chungmuensis (Okayama Prefecture, Japan) in August developed gross signs of the disease within a month (Itoh et al. 2004b). In this area, Tun et al. (2008b) reported that the prevalence of infection detected by polymerase chain reaction (PCR, see below) was 70% or higher from August to October, but declined sharply in November and reached 7% or lower from February to April. However, transferring the oysters to warm seawater (from 8 to 10°C at the enzootic location to 24°C in M. chungmuensis -free experimental tanks) increased the prevalence of infection from about 7% to 87% within 3 weeks indicating that the low prevalence in winter was due to insufficient replication of M. chungmuensis at low seawater temperatures, resulting in levels not detectable by nested PCR, and not to the absence of invasion (Tun et al. 2008b).

Figure A1.Developmental cycle of Marteilioides chungmuensis in Crassostrea gigas as proposed by Itoh et al. (2004b) and copied from the International Journal for Parasitology 34:1134. An unidentified infective stage invades the epithelial tissues of the gills, mantle or labial palps (A). Extrasporogony occurs intercellularly in the connective tissue resulting in the production of secondary cells which invade the epithelium of the gonad (B). Enlargement and multiplication of the secondary cells may also occur intercellularly in the gonad epithelium (C). The secondary cells invade an oocyte, transform into a stem cell and proceed with sporulation (D). Mature spores are released from the oyster via the genital canal (E).

Itoh et al. (2004b) identified extrasporogic stages in male C. gigas but sporulation in male oysters was not confirmed. Although the prevalence of infection detected by PCR after 4 weeks of exposure in an enzootic area was similar in both male and female oysters (about 60%), the prevalence in males declined in the subsequent 3 weekly samples (down to 24%) while that of the females remained consistently high (above 60%). Itoh et al. (2004b) suggested that M. chungmuensis may be excluded from male oysters without initiating sporulation.

Diagnostic techniques

Gross: Tumour-like distensions of the mantle tissues of heavily infected oysters.

Figure 1a.The appearance of a Pacific oyster, Crassostrea gigas, infected with Marteilioides chungmuensis. Note that nodule-like structures (arrow heads) occur in the soft tissue. Image provided by N. Itoh.

Figure 1b.Large nodules on the surface of the soft tissues of C. gigas caused by infection with M. chungmuensis. Image provided by Mi Seon Park.

Smears: Dried smears of the nodules (infected gonad) stained with Wright, Wright-Giemsa or equivalent stain (e.g. Hemacolor, Merck; Diff-QuiK, Baxter) enables the rapid detection of developmental stages within and liberated from the ova. The usual form of M. chungmuensis in mature oyster oocytes is two sporonts (secondary cells), each containing one developing spore, within each degenerate stem cell. However, stem cells containing from three to six sporonts (each containing one developing spore) have been observed but are rare (Imanaka et al. 2001).

Figure 2.Smear of gonadal tissue of Crassostrea gigas infected with Marteilioides chungmuensis. Four M. chungmuensis are within intact ova of C. gigas (top of image) while others (arrows) were liberated from their host ova during the smearing process. Diff QuiK stain.

Figure 3.Magnification of two ova from Fig. 2. Degenerate stem cells (P) located adjacent to the ova (host cell) nucleus (HN). Each stem cell contains two sporonts with a nucleus (SN) and one developing spore (SP). Note that the developing spores are too darkly stained to see the tricellular nature of the spore. Diff QuiK stain.

Histology:Stem cells containing one to three (usually two) sporonts (secondary cells), resulting from exogenous budding, within the cytoplasm of infected oocytes and ova. Within each sporont, one tertiary cell forms by endogenous budding. The tertiary cell develops into a tricellular spore by internal cleavage. A related species, Marteilioides branchialis, from the gills of Saccostrea glomerata (=commercialis) has from two to six and rarely up to 12 sporonts in each primary cell and each spore is bicellular (two cells, one within the other, in each spore).

Figure 4.Remnants of an ovarian follicle of Crassostrea gigas that is filled with distorted ova containing Marteilioides chungmuensis. Three sporonts occur within the degenerating cytoplasm of a stem cell in some ova (3S) and developing spores (SP) are evident in some sporonts. The nucleus of infected ova (HN) are compressed by the parasite in most cases. Haematoxylin and eosin stain.

Figure 5 and 5a. Marteilioides chungmuensis compressing the ova nucleus (HN) of Crassostrea gigas. The stem cell of the parasite has disintegrated and appears as a vacuole around the sporonts that each contain developing spores (SP). An immature stem cell (P) is visible in an adjacent oocyte in Fig. 5. Haematoxylin and eosin stain.

Electron microscopy: Examination of the ultrastructure is required to identify the tricellular nature of the spore (Comps et al. 1987).

Figure 6. Early developmental stage of Marteilioides chungmuensis (M) adjacent to the nucleus (N) of the host cell (ova) in the gonad of Crassostrea gigas. Image provided by Mi Seon Park.

Figure 7.Ova of Crassostrea gigas containing two stem cells of Marteilioides chungmuensis adjacent to its nucleus (N). The cytoplasm of both stem cells is disintegrating but the stem cell on the right has two sporonts (Sp), while the one on the left is unusual in having five sporonts, two of which are producing spores (Sp*). Image provided by Mi Seon Park.

DNA Probes: A partial sequence of the 18s small subunit ribosomal DNA (ca. 1200 base pairs listed in GenBank accession number AB089819) was identified from sporonts isolated from infected oysters using a freeze-thaw procedure and differential centrifugation in discontinuous sucrose and Percoll gradients (Itoh et al. 2003a). The sequence was used to design three M. chungmuensis specific probes. The probes detected parasite cells by in situ hybridisation and were used to elucidate the life cycle of M. chungmuensis (Itoh et al. 2004b). The identified sequence for this parasite will also be used to determine the phylogenetic position of this parasite (Itoh et al. 2002b, 2003a). Two sets of specific primers were developed into a nested-polymerase chain reaction (PCR) that had a far greater sensitivity for detecting M. chungmuensis than traditional histological techniques and gross observations (Itoh et al. 2003b).

Figures 8 and 9.In situ hybridization of immature sporogenic stages (arrowheads) and mature spores (arrows in Fig 9.) of Marteilioides chungmuensis in the ovary of Crassostrea gigas. Images captured from the journal Disease of Aquatic Organisms in the publication by Itoh et al. (2003).

Methods of control

No known methods of prevention. Infected oysters should not be transported into areas known to be free of the disease. In Japan, the prevalence of infection increased during the summer suggesting that active multiplication of the parasite occurs in the warm water months (Imanaka et al. 2001). Tun et al. (2006) indicated that a lower prevalence of infection was detected in oysters from the intertidal zone (with a daily average of 6 hours in seawater). However, they determined that the decrease in prevalence in the intertidal oysters was not due to short-term exposure to parasitic invasion, but to factors that decreased the female population, because spore formation of M. chungmuensis (and associated pathology) occurs only in female oysters (Tun et al. 2006). The National Fisheries Research and Development Institute (NFRDI) in Korea has recommend that the oyster culture industry in affected areas grow triploid oysters which are not susceptible to infection by the parasite (Park 2005).

References

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Citation Information

Bower, S.M., Itoh, N., Choi, D.-L., Park, M.S. (2011): Synopsis of Infectious Diseases and Parasites of Commercially Exploited Shellfish: Marteilioides chungmuensis of Oysters.

Contact information for co-authors:

Naoki Itoh, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiya-machi, Tsutsumidori, Aoba-ku, Sendai 981-8555, Miyagi Japan. E-mail: nitoh@bios.tohoku.ac.jp

Dong-Lim Choi and Mi Seon Park, Pathology Division, National Fisheries Research and Development Institute (NFRDI), 408-1, Silang-ri, Kitang-up, Kitang-gum, Pusan 619-900, Republic of Korea. E-mail: dlchoi@nfrda.re.kr

Date last revised: June 2011
Comments to Susan Bower

Date modified: