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The identification of novel peptides that regulate reproduction is essential for studying reproductive physiology in bivalves. Therefore, we aimed to identify peptides that affect the reproductive physiology of bivalves. We identified an oocyte maturation-, sperm motility-, and spawning-inducing peptide from the visceral ganglia of the pen shell, Atrina pectinata. The peptide consisted of 26 amino acid residues (GFDSINFPGTIDGFKDYSSNKKERLL). This peptide induced oocyte maturation and sperm motility activation at less than 1 nM upon the treatment of gonad fragments and induced spawning at 1 nmol when injected into mature individuals. Mature eggs and sperms artificially spawned by peptide administration were fertilized, and we confirmed that the development proceeded normally to veliger (D-shape) larvae. These results indicated that GFDSINFPGTIDGFKDYSSNKKERLL stimulated the gonads of pen shells and induced oocyte maturation, sperm motility activation, and spawning.

Molluscan shells are composed of calcium carbonates, with small amounts of extracellular matrices secreted from mantle epithelial cells. Many types of shell matrix proteins (SMPs) have been identified from molluscan shells or mantle cells. The pen shell Atrina pectinata (Pinnidae) has two different shell microstructures, the nacreous and prismatic layers. Nacreous and prismatic layer-specific matrix proteins have been reported in Pteriidae bivalves, but remain unclear in Pinnidae. We performed transcriptome analysis using the mantle cells of A. pectinata to screen the candidate transcripts involved in its prismatic layer formation. We found Asprich and nine highly conserved prismatic layer-specific SMPs encoding transcript in P. fucata, P. margaritifera, and P. maxima (Tyrosinase, Chitinase, EGF-like proteins, Fibronectin, valine-rich proteins, and prismatic uncharacterized shell protein 2 [PUSP2]) using molecular phylogenetic analysis or multiple alignment. We confirmed these genes were expressed in the epithelial cells of the mantle edge (outer surface of the outer fold) and the mantle pallium. Phylogenetic character mapping of these SMPs was used to infer a possible evolutionary scenario of them in Pteriomorphia. EGF-like proteins, Fibronectin, and valine-rich proteins encoding genes each evolved in the linage leading to four Pteriomorphia (Mytilidae, Pinnidae, Ostreidae, and Pteriidae), PUSP2 evolved in the linage leading to three Pteriomorphia families (Pinnidae, Ostreidae, and Pteriidae), and chitinase was independently evolved as SMPs in Mytilidae and in other Pteriomorphia (Pinnidae, Ostreidae, and Pteriidae). Our results provide a new dataset for A. pectinata SMP annotation, and a basis for understanding the evolution of prismatic layer formation in bivalves.

Molluscan shells are composed of calcium carbonates, with small amounts of extracellular matrices secreted from mantle epithelial cells. Many types of shell matrix proteins (SMPs) have been identified from molluscan shells or mantle cells. The pen shell Atrina pectinata (Pinnidae) has two different shell microstructures, the nacreous and prismatic layers. Nacreous and prismatic layer-specific matrix proteins have been reported in Pteriidae bivalves, but remain unclear in Pinnidae. We performed transcriptome analysis using the mantle cells of A. pectinata to screen the candidate transcripts involved in its prismatic layer formation. We found Asprich and nine highly conserved prismatic layer-specific SMPs encoding transcript in P. fucata, P. margaritifera, and P. maxima (Tyrosinase, Chitinase, EGF-like proteins, Fibronectin, valine-rich proteins, and prismatic uncharacterized shell protein 2 [PUSP2]) using molecular phylogenetic analysis or multiple alignment. We confirmed these genes were expressed in the epithelial cells of the mantle edge (outer surface of the outer fold) and the mantle pallium. Phylogenetic character mapping of these SMPs was used to infer a possible evolutionary scenario of them in Pteriomorphia. EGF-like proteins, Fibronectin, and valine-rich proteins encoding genes each evolved in the linage leading to four Pteriomorphia (Mytilidae, Pinnidae, Ostreidae, and Pteriidae), PUSP2 evolved in the linage leading to three Pteriomorphia families (Pinnidae, Ostreidae, and Pteriidae), and chitinase was independently evolved as SMPs in Mytilidae and in other Pteriomorphia (Pinnidae, Ostreidae, and Pteriidae). Our results provide a new dataset for A. pectinata SMP annotation, and a basis for understanding the evolution of prismatic layer formation in bivalves.

In the current method of pearl production, the mantle fragment of a donor pearl oyster is transplanted into a host pearl oyster together with an inorganic bead (pearl nucleus). After this surgical procedure, only outer epithelial cells (OEC) in the transplanted mantle survive in a host pearl oyster and form a pearl sac to begin pearl formation. Therefore, implantation of only the OEC instead of the mantle fragment would be a possible alternative to the current procedure. To examine the potential of pearl production by implanting OEC in Pinctada fucata, we developed a cell implantation method using the pearl nucleus carrying a small pit inoculated with OEC. As a result, approximately 70% of the inserted nuclei formed the nacreous layer when the OEC were inoculated at 5 × 104 cells/nucleus. Then, OEC isolated from two genetically different types of pearl oysters that significantly differed in shell nacre color (yellowness) were mixed at four different ratios, and the prepared OEC mixtures were transplanted to investigate the effects of the blend on the yellowness of pearls to be harvested. The yellowness of harvested pearls differed significantly in accordance with the mixing ratio. Similarly, OEC isolated from two types of pearl oysters that showed a significant difference in the thickness of their shell nacre aragonite tablets were mixed at four different ratios and transplanted. Mean thickness of the aragonite tablets of the harvested pearls differed according to mixing ratio. These results suggest the method to control pearl quality by blending OEC obtained from pearl oysters genetically improved by selective breeding for traits related to pearl quality.

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.

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。

Gametogenesis of one year-old induced triploid mussels, Mytilus galloprovincialis, was examined histologically and compared to sibling diploid mussels. Histological analysis revealed that triploid mussels developed a number of primary spermatocytes that were arrested at prophase I. Late in the reproductive season, triploid mussels produced an extremely small number of spermatozoa (9/10000 μm2 gonadal section) compared to diploid mussels (1072/10000 μm2 gonadal section). All triploid mussels were identified as males, whereas the sex ratio of diploid mussels was almost equal (1.12:1.0, male:female), indicating that sex determination for this species may follow a Z:W model. Sertoli cells in triploid mussels were prominent, had an enlarged cytoplasm, and were easily seen using light microscopy. In comparison, Sertoli cells in diploids were thin and could only be seen by electron microscopy. Sertoli cell hypertrophy in triploid mussels probably reflects their role in eliminating abnormal and degenerat...

The pond snailLymnaea stagnalisis capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA–LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA–LTM in theLymnaeaCNS.

In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster. Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20 degrees C in 5% CO2:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2'-deoxyuridine was added to the culture at 30 microns. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2'-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.