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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.

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。

The pond snail Lymnaea stagnalis is an excellent model system in which to study the neuronal and molecular substrates of associative learning and its consolidation into long-term memory. Until now, the presence of cyclic AMP (cAMP)-responsive element binding protein (CREB), which is believed to be a necessary component in the process of a learned behavior that is consolidated into long-term memory, has only been assumed in Lymnaea neurons. We therefore cloned and analyzed the cDNA sequences of homologues of CREB1 and CREB2 and determined the presence of these mRNAs in identifiable neurons of the central nervous system (CNS) of L. stagnalis. The deduced amino acid sequence of Lymnaea CREB1 is homologous to transcriptional activators, mammalian CREB1 and Aplysia CREB1a, in the C-terminal DNA binding (bZIP) and phosphorylation domains, whereas the deduced amino acid sequence of Lymnaea CREB2 is homologous to transcriptional repressors, human CREB2, mouse activating transcription factor-4, and Aplysia CREB2 in the bZIP domain. In situ hybridization revealed that only a relatively few neurons showed strongly positive signals for Lymnaea CREB1 mRNA, whereas all the neurons in the CNS contained Lymnaea CREB2 mRNA. Using one of the neurons (the cerebral giant cell) containing Lymnaea CREB1 mRNA, we showed that the injection of a CRE oligonucleotide inhibited a cAMP-induced, long-lasting synaptic plasticity. We therefore conclude that CREBs are present in Lymnaea neurons and may function as necessary players in behavioral plasticity.

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.