Marteilioides chungmuensis and its impact

Transcription

1 Marteilioides chungmuensis and its impact on Crassostrea gigas Naoki Itoh Laboratory of Aquaculture Biology Graduate School of Agricultural Science Tohoku University, Sendai Miyagi, Japan.

2 Abnormal enlargement of the ovary in the Pacific oyster Seki (1934) firstly reported this disease in southwestern Japan, and considered as physiological disorder.

3 Disease distribution in Japan Okayama ( ) Hiroshima (1934, -1977?) No infection (1999 to present) Fukuoka (2001) Mie (1996) Due to the unusual appearance, infected oysters lose marketability. Estimated annual damage at about half million euro in Okayama Pref.

4 Development of the disease Prevalence observed by histology 2000 transverse section healthy gonad oogenesis and maturation spawning shrunk and replaced to connective tissues affected gonad not observed Small infected gonads appeared and enlarged with continuous oogenesis parasite not observed early developmental stages sporulation

5 Seasonal transition in histological sections of infected gonads (2000) September October November to December Bar=100µm December From December-April Bar=50µm

6 Causative agent of the disease Matsusato et al. (1977) found the causative agent, a coccidian parasite in oocytes, but did not identified. Chun (1979) reported similar disease in Korea. Comps et al. (1986) described the parasite in Korea as Marteilioides chungmuensis n.sp. Imanaka et al., (2001) described eight developmental stages in the oocyte. Since some of them appeared to be different from Comps et al. (1986), classification of the parasite remained unknown. The parasite in Japan was (finally) identified as M. chungmuensis from spore structures (Itoh et al., 2002)**. Phylum Paramyxea (Desportes and Perkins, 1990)* spores in a sporont cells in a spore Paramyxa 4 4 Paramarteilia genus Marteilia Marteilioides M. chungmuensis M. branchialis Paramyxoides Spore of the ovarian parasite * Phylum Cercozoa is psoposed for their new taxonomic position (Cavalier-Smith and Chao, 2003). ** Feist et al. (2009) proposed that species be classified from number of cells within the spores.

7 Isolation of M. chungmuensis cells and identification of the parasite gene sequence Itoh et al., (2003) Dis Aquat Org Isolation procedures Isolated sporonts (secondary cells) Complete 18S rrna gene sequence of M. chungmuensis was identified.

8 Phylogenetic positions of paramyxean parasites based on small subunit rrna Entamoeba Ochromonas Arabidopsis Mucor Saccharomyces Phreatamoeba Pyrenomonas Symbiodinium Trypanosoma Gracilaria Haplosporidium Naegleria Oxytricha Dictyostelium Hexamita Giardia Marteilioides Marteilia Paramyxea Ameson Encephalitozoon Tritrichomonas Euglena Henneguya Physarum Mus Tenebrio 0.1 substitutions/site

9 Development of detection probes for in situ hybridization and PCR Nested-PCR probes Itoh et al., (2003) Fish Pathol M M: molecular marker 1: Heavy infection of M. chungmuensis 2: Light infection of M. chungmuensis 3: Uninfected oyster 4: Mytilus edulis infected with Marteilia sp. (Maruteilia maruini?)) 5: Ostrea edulis infected with Marteilia refringens 6: Saccostrea glomerata infected with Marteilia sydneyi 7: No template control In situ hybridization probes Specifically detected the parasite. 50µm 50µm Sensitively detected the parasite than ordinary histology.

10 Early developmental stages of Marteilioides chungmuensis Itoh et al., (2004) Int J Parasitol Uninfected oysters were exposed to a disease area, and 30 oysters were sampled every week Individuals with PCR (+) were then used for ISH works. sporulation female Prevalence detected by PCR male 10 µm After exposure to epidemic area M. chungmuensis invades into male oyster, but the parasite can not develop to maturation stages

11 Developmental stages detected by in situ hybridization binucleate stage 10µm 20µm 10 µm 10µm The labial palp (at 2 wks p.e.) 10µm Connective tissue in visceral mass (at 3 wks p.e.) 200µm Cell division in the gonadal epithelia (at 5 wks p.e) 10µm uninucleate stage Binary division in visceral mass connective tissue (at 3 wks p.e.) 100µm Sporulation in the oocytes (at 5 wks p.e.)

12 Predicted developmental stages of M. chugmuensis in the Pacific oyster

13 M. chungmuensis infection in another oyster species Itoh et al., (2004) J Fish Dis appearance PCR In situ hybridization C. gigas 8/30 20/30 12/30 C. nippona 0/50 20/50 2/50 wp wp Crassostrea nippona is also susceptible to M. chungmuensis, in spite of low infection level. M. chungmuensis may be able to complete maturation in C. nippona. cf. Ovarian Marteilioides is reported from the manila clam in Korea (Lee et al., 2001) and Japan (Itoh et al., 2006), and an oyster, Saccostrea echinata. in Australia (Hine & Thorne, 2000 ). Introduction of other bivalve species from epizootic areas implyes a risk to introduce M. chungmuensis.

14 Seasonal field surveys of M. chungmuensis infection Tun et al., (2007) J Invertebr Pathol nonsexual male female Oocytes (and oogenesis) are observed in infected female even in non-reproductive seasons. Older oyster is more suitable for the parasite infection.. Why prevalence was suddenly decreased after winter season?

15 Pathogenicity of M. chungmuensis on oysters effects of biopsy on oyster mortality infected biopsy control (w/o biopsy) Tun et al., (2008) Int J Parasitol mortality of experimental groups MgCl2 anesthesia infected female infected female uninfected female male nonsexual biopsy and examination uninfected female cultured in sea. Prevalence in recovered male died nonsexual M. chungmuensis forces host oysters to continue oogenesis, resulting in weakness and death in oysters.

16 Invasion period of M. chungmuensis into the Pacific oyster Prevalence during the experiment period in the field oysters from a disease-free area Tun et al. (2008) Dis Aquat Org examined by histology and PCR Examined by PCR (1+yr) exposed to an epizootic area for 1 month An infective stage of M. chungmuensis exists all year around. (more in summer to autumn than winter?)

17 Effects of water temperature on parasite development In February 2005 oysters from a disease-free area 2 wks later washed and then maintained at 24 C sampled weekly Transfer to experimental tanks exposed to an epizootic area Both gonads and the parasites were developed in oysters maintained in warm temperature. M. chungmuensis invaded into most oysters even in winter, but did not survive for a few weeks in reproductively inactive oysters. If oyster reproduction/oogenesis is controlled artificially, development of the parasite and the disease is also manipulated?

18 Tun et al. (2006) Aquaculture Oyster under low nutrients condition differentiate to male more than female intertidal group (IG) transferred group (TG) submerged group (SG) 4 mo later high tide low tide In the IG group, oysters differentiated to male more than female.

19 Prevalence of whole oysters in the group SG TG IG Oyster culture under poor nutrient condition (IG group) reduces the ratio of female oysters, resulting in decline of M. chungmuensis prevalence! Prevalence of female oysters in the group However, Growth of oyster under low nutrients condition was significantly slower than other conditions...

20 Summary The etiological agent of abnormal enlargement of ovary was identified as Marteilioides chungmuensis. Sensitive detection methods were developed. M. chungmuensis infects another oyster species (and clams as well?), and introduction of bivalves from the epizootic areas should be conducted carefully. M. chungmuensis causes not only unusual appearances but also mortality to the host oysters. Manipulation of oyster reproduction system may lead to control of the disease, but further researches are required for establishment of practical countermeasures.