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For recovery of the declining population of pen shells in the wild, the production of pen shell juveniles for transplantation or aquaculture is underway in Japan. For more stable juvenile production, artificial fertilization methods for pen shells are needed, but methods to induce oocyte maturation (meiosis resumption) used in other bivalves, which make oocytes fertilizable, were ineffective for pen shells. Here, we report evidence showing that retinoic acid (RA) has strong activity in inducing oocyte maturation and activating sperm motility in pen shells. Treatment of fully developed oocytes with 1.0 μM all-trans-RA (at-RA) induced germinal vesicle breakdown, a typical morphological sign of oocyte maturation, but 1.0 μM at-retinol and at-retinal, 2 mM ammonia, and 1.0 μM serotonin were ineffective. Treatment with at-RA for 30 min was sufficient for oocyte maturation and was more potent than its isomers, 9-cis- and 13-cis-RA. Parallel results were obtained for sperm motility activation. Oocyte responsiveness to at-RA increased during the final stage of ovary development. Artificial fertilization was successful only with the oocytes treated with at-RA, and fertilized eggs developed to D-shaped (veliger) larvae without apparent morphological abnormalities. These results indicate the possible application of RA for the artificial fertilization of pen shells.

For recovery of the declining population of pen shells in the wild, the production of pen shell juveniles for transplantation or aquaculture is underway in Japan. For more stable juvenile production, artificial fertilization methods for pen shells are needed, but methods to induce oocyte maturation (meiosis resumption) used in other bivalves, which make oocytes fertilizable, were ineffective for pen shells. Here, we report evidence showing that retinoic acid (RA) has strong activity in inducing oocyte maturation and activating sperm motility in pen shells. Treatment of fully developed oocytes with 1.0 μM all-trans-RA (at-RA) induced germinal vesicle breakdown, a typical morphological sign of oocyte maturation, but 1.0 μM at-retinol and at-retinal, 2 mM ammonia, and 1.0 μM serotonin were ineffective. Treatment with at-RA for 30 min was sufficient for oocyte maturation and was more potent than its isomers, 9-cis- and 13-cis-RA. Parallel results were obtained for sperm motility activation. Oocyte responsiveness to at-RA increased during the final stage of ovary development. Artificial fertilization was successful only with the oocytes treated with at-RA, and fertilized eggs developed to D-shaped (veliger) larvae without apparent morphological abnormalities. These results indicate the possible application of RA for the artificial fertilization of pen shells.

The genus Marteilia includes protozoan species with repeated endogenous budding sporulation and spores that corise three cells.mp All of these species are parasitic for marine invertebrates. Most species in this genus are observed in the digestive tissue of bivalves. One of the st important species, mo Marteilia refringens, has been observed in the European flat oys-ter, Ostrea edulis (Grizel et al., 1974); mussels, Mytilus edulis and Mytilus galloprovincialis (Tige and Rabouin, 1976; Villalba et al., 1993); razor clam, Solen margina-tus and the striped venus cla Chamalea gallinam,(Lopes-Flores et al., 2008a, 2008b) in Europe. Because of the econoc impact of M. refringensmi on the flat oyster industry, this species is listed as a notifiable pathogen by World Organization for Animal Health (OIE, 2012). Another species, Marteilia sydneyi, hasdetrimental impacts on the Sydney rock oyster, Saccos-trea glomerata, in Australia (Perkins and Wolf, 1976; Green et al., 2011). The recently identified species Marteilia cochillia was involved in rtality of the edible mocockle, Cerastoderma edule, in Spain (Carrasco et al., 2013). Although relatively less information is available, M. lengehi (Comps, 1976) and M. christenseni (Comps, 1985) have also been observed in Saccostrea cucullata and Scrobicularia piperata In Asian , respectively.

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.

Ovarian follicle cells are the site of yolk protein synthesis in the Ezo abalone Haliotis discus hannai. In this study, histological observations of ovarian follicle cells were conducted in H. discus hannai by immunocytochemical and in situ hybridization methods focusing on their function of yolk protein synthesis. An antibody raised against purified yolk protein (vitellin, Vn) recognized yolk granules in oocytes, and ovarian follicle cells adjacent to the oocytes under yolk accumulation were stained positively with both anti-Vn antibody and an antisense probe for vitellogenin (the precursor for vitellin, Vtg) mRNA. These results indicate that the abalone Vtg gene is transcribed and translated in the ovarian follicle cells. In oocytes in the early phase of yolk accumulation, positive reactions with the antibody appeared first in the stalk part, and the follicle cells adjacent to the stalk were also stained positively. These observations imply local transportation of yolk protein from the follicle cells to the oocyte through an extracellular space around the oocyte stalk. Ovarian follicle cells changed their morphological characteristics and reactivity to the antibody with close relationships with the stages of the adjacent oocyte, which suggests the presence of functional interactions between follicle cells and oocytes in the course of yolk protein synthesis and accumulation., エゾアワビ卵巣濾胞細胞（濾胞細胞）の卵黄タンパク質（ビテリン、Vn）合成と卵母細胞へのVn蓄積過程について、抗Vn抗体による免疫染色とVn前駆体遺伝子のin situ ハイブリダイゼーションで組織学的に検討した。その結果、前駆体遺伝子が濾胞細胞で転写、翻訳されることが示され、合成された卵黄タンパク質は濾胞細胞と卵母細胞が接する卵柄部で卵母細胞内に輸送されると示唆された。また濾胞細胞の形態と抗Vn抗体への反応性は隣接する卵母細胞の発達と密接に関連し、Vn合成と蓄積において濾胞細胞と卵母細胞間に相互作用が存在すると推定された。