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Marteilioides chungmuensis of Oysters

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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), initially assigned to the phylum Paramyxea (Desportes and Perkins 1990, Berthe et al. 2000), was transferred to the phylum Cercozoa and order Paramyxida (Cavalier-Smith and Chao 2003, Feist et al. 2009). During their revision of the Paramyxida, Feist et al. (2009) proposed that the genus Marteilioides Comps, Park et Desportes (1986) be suppressed and the type species of the genus, M. chungmuensis Comps, Park et Desportes (1986) in oysters from Korea and Japan, be transferred to Marteilia. This proposal was supported by Itoh et al. (2014), Carrasco et al. (2015) and Alfjorden et al. (2017). However, Ward et al. (2016) suggested retaining this parasite in the genus Marteilioides in order to keep the genus Marteilia from being paraphyletic based on molecular analysis of the 18S rDNA of M. chungmuensis in comparison to other species in the paramyxean lineages. As suggested by Ward et al. (2016), the genus name of Marteilioides will be retained on this web page. 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 secondary cells that produce a single pluricellular spore, then degenerate such that the spore was enveloped by a cytoplasmic residuum and the plasmalemma of the secondary cell. Some of the infections described as "oyster egg diseases" may be attributed to M. chungmuensis. After phylogenetic analysis of the 18S rDNA sequence of the Marteilioides sp. from clams, Ruditapes philippinarum collected on the south coast of Korea, Yanin et al. (2013) suggested that this parasite was not the same species as M. chungmuensis.

Geographic distribution

Marteilioides chungmuensis in oysters was reported from the southern and western coasts of the Korean Peninsula, Korea (Park and Chun 1989, Yanin et al. 2013) and detected widely in Japanese waters (Comps et al. 1986, Itoh et al. 2002a). Marteilioides sp. were reported in the oocytes of Ruditapes (=Tapes) philippinarum from southern coastal areas of Korea (Lee et al. 2001, Yanin et al. 2013) and in the oocytes of Ruditapes (=Venerupis) philippinarum from Iwakuni, Yamaguchi Prefecture, Japan (Itoh et al. 2005).

Host species

Crassostrea gigas. Also in Crassostrea nippona transplanted to an enzootic area of Japan (Itoh et al. 2004a) and in Crassostrea ariakensis from the south coast of Korea (Yanin et al. 2013). 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). Marteilioides-like parasites have also been reported at low prevalence (1.6%) in the oocytes of Manila clams, Ruditapes (=Tapes ) philippinarum, from southern coast of Korea (Lee et al. 2001, Yanin et al. 2013) and in the oocytes of 1 of 40 Ruditapes (=Venerupis) philippinarum from Iwakuni, Yamaguchi Prefecture, Japan (Itoh et al. 2005).

Impact on the host

Abnormal egg-masses with a nodular appearance (like multiple tumors) among cultured Crassostrea 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 the period when 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 C. gigas lose their marketability due to the unaesthetic appearance and thus cause a serious economic loss to oyster farmers (Meyers 2006). Crassostrea nippona and Crassostrea ariakensis with infections of M. chungmuensis did not exhibit apparent nodular formation on the mantle or any other noticeable clinical signs and this parasite may not have a negative impact on farming of these species of oyster (Itoh et al. 2004a, Yanin et al. 2013).

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).

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 cells 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 primary cell (stem cell) of the sporogonic stage. Sporogonic stages were observed only inside the oocytes of the host (as described below).

Choi et al. (2011) compared haemocytes from C. gigas infected with M. chungmuensis with those of healthy-looking C. gigas using a flow cytometer and determined that the total haemocyte count, especially granulocytes, were significantly increased and the number of hya-linocytes were significantly reduced in the infected oysters. Although haemocyte viability did not differ between infected and normal-looking oysters, the rate of phagocytosis was significantly higher in the infected oysters (Choi et al. 2011).

Marteilioides-like parasites were detected in 1.6% of the clams (R. philippinarum) examined from Korea in March 1996 to April 1997 (Lee et al. 2001) and in only 1 of 40 R. philippinarum sampled from Japan in September 2003 (Itoh et al. 2005). Although various developmental stages and mature spores were observed in the oocytes, the infected clams did not show any abnormal appearance and had no host responses, such as haemocyte infiltration and phagocytosis (Lee et al. 2001, Itoh et al. 2005, Yanin et al. 2013). However, as in C. gigas infected with M. chungmuensis, Itoh et al. (2005) speculated that it is possible that Marteilioides sp. infection in R. philippinarum may reduce fecundity of the clam and hence cause a resource reduction.

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.

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 primary (stem) cell and proceed with sporulation (D). Mature spores are released from the oyster via the genital canal (E).

Diagnostic techniques

Gross

Tumour-like distensions (focal nodules) of the mantle tissues of heavily infected C. gigas (Yanin et al. 2013). However, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected C. nippona (Itoh et al. 2004a) C. ariakensis or R. philippinarum (Itoh et al. 2005, Yanin et al. 2013).

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 when she was at the Pathology Division, National Fisheries Research and Development Institute (NFRDI), 408-1, Silang-ri, Kitang-up, Kitang-gum, Pusan 619-900, Republic of Korea.

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 secondary cells (sporonts), each containing one developing spore, within each degenerate primary (stem) cell. However, primary cells containing from three to six secondary cells (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 primary (stem) cells (P) located adjacent to the ova (host cell) nucleus (HN). Each primary cell contains two secondary cells (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

In the ovary of the host, primary (stem) cells containing one to three (usually two) secondary cells (sporonts), result from endogenous budding, within the cytoplasm of infected oocytes and ova. Within each secondary cell, one tertiary cell forms by endogenous budding. The tertiary cell develops into a tricellular spore by internal cleavage endogenously (characteristic 'cell within cell' development). Itoh et al. (2003b), Tun et al. (2008b), Choi et al. (2012) and Yanin et al. (2013) determined that histological examinations were less sensitive in detecting infections with M. chungmuensis than molecular assays (PCR and ISH). Histology seems to be a less sensitive technique when infections by the parasite are at low levels, in male oysters and/or in female oysters when the oocytes are immature (Choi et al. 2012). A related species, Marteilioides branchialis, from the gills of Saccostrea glomerata (=commercialis) has from two to six and rarely up to 12 secondary cell in each primary cell and each spore is bicellular (two cells, one within the other, in each spore) (Anderson and Lester 1992).

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

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

Electron microscopy

Examination of the ultrastructure is required to identify the tricellular nature of the spore (Comps et al. 1987). Inside the ova of C. gigas, the primary (stem) cell, usually produces two secondary cells by endogenous budding and within each secondary cell one tertiary cell develops and matures into a spore containing three cell produced by consecutive internal cleavage ('cell within cell'). Park and Chun (1989) indicated that the secondary cell contains haplosporosomes, the outermost cell in the spore contains membrane bound osmophilic bodies and the middle and innermost cell contain high density cytoplasmic ribosomes.

The ultrastructure features of the Marteilioides sp. in R. philippinarum were identical to those of M. chungmuensis in C. gigas as reported by Comps et al. (1986) and Itoh et al. (2002a). These features in the mature parasite include two secondary cells in a primary cell, one spore in each secondary cell each spore consisted of one outer cell and two inner cells, and contained electron dense haplosporosomes (Itoh et al. 2005). Thus, Itoh et al. (2005) suggested a high possibility that Marteilioides sp. in R. philippinarum from Japan is identical to M. chungmuensis.

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 when she was at the Pathology Division, National Fisheries Research and Development Institute (NFRDI), 408-1, Silang-ri, Kitang-up, Kitang-gum, Pusan 619-900, Republic of Korea.

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 secondary cells (sporonts, Sp), while the one on the left is unusual in having five secondary cells, two of which are producing spores (Sp*). Image provided by Mi Seon Park when she was at the Pathology Division, National Fisheries Research and Development Institute (NFRDI), 408-1, Silang-ri, Kitang-up, Kitang-gum, Pusan 619-900, Republic of Korea.

DNA Probes

A partial sequence of the 18S small subunit (SSU) ribosomal DNA (ca. 1200 base pairs listed in GenBank accession number AB089819) was identified from secondary cells (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 (Itoh et al. 2002b, 2003a). The DNA probes detected parasite cells by in situ hybridisation (ISH) and were used to elucidate the life cycle of M. chungmuensis (Itoh et al. 2004b). 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, Tun et al. 2008b, Yanin et al. 2013). Subsequently, the entire sequence of the 18S rDNA was deposited in GenBank (accession number AB110795) and was used to develop additional probes for the molecular detection of M. chungmuensis (Choi et al. 2012) and to determine the phylogenetic position of this parasite (Itoh et al. 2002b, 2003a; Ward et al. 2016). The PCR and ISH assays described by Choi et al. (2012) were also more sensitive at detecting M. chungmuensis than traditional histological techniques.

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. (2003a).

Yanin et al. (2013) identified the 18S rDNA sequence of the Marteilioides sp. in R. philippinarum from Korea. Based on BLAST analysis and a pair-wise nucleotide distance similarity calculation, Yanin et al. (2013) indicated that the Marteilioides infecting R. philippinarum was genetically different from those infecting C. gigas and suggested that it was a different species of Marteilioides.

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

Citation information

Bower, S.M., Itoh, N. (2019): 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 and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657,Tokyo Japan.

Date last revised: January 2019

Comments to Susan Bower

Date modified: