Evolution of nacre- and prisms-related shell matrix proteins in the pen shell, Atrina pectinata.

The molluscan shell is a good model for understanding the mechanisms underlying biomineralization. It is composed of calcium carbonate crystals and many types of organic molecules, such as the matrix proteins, polysaccharides, and lipids. The pen shell Atrina pectinata (Pterioida, Pinnidae) has two shell microstructures: an outer prismatic layer and an inner nacreous layer. Similar microstructures are well known in pearl oysters (Pteriidae), such as Pinctada fucata, and many kinds of shell matrix proteins (SMPs) have been identified from their shells. However, the members of SMPs that consist of the nacreous and prismatic layers of Pinnidae bivalves remain unclear. In this study, we identified 114 SMPs in the nacreous and prismatic layers of A. pectinata, of which only seven were found in both microstructures. 54 of them were found to bind calcium carbonate. Comparative analysis of nine molluscan shell proteomes showed that 69 of 114 SMPs of A. pectinata were found to have sequential similarity with at least one or more SMPs of other molluscan species. For instance, nacrein, tyrosinase, Pif/BMSP-like, chitinase (CN), chitin-binding proteins, CD109, and Kunitz-type serine proteinase inhibitors are widely shared among bivalves and gastropods. Our results provide new insights for understanding the complex evolution of SMPs related to nacreous and prismatic layer formation in the pteriomorph bivalves.

A novel peptide identified from visceral ganglia induces oocyte maturation, spermatozoa active motility, and spawning in the pen shell Atrinapectinata.

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.

Evolution of Biomineralization Genes in the Prismatic Layer of the Pen Shell Atrina pectinata.

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.

Trivalent Iron Is Responsible for the Yellow Color Development in the Nacre of Akoya Pearl Oyster Shells.

The gold and cream colors of cultured Akoya pearls, as well as natural yellow nacre of pearl oyster shells, are thought to arise from intrinsic yellow pigments. While the isolation of the yellow pigments has been attempted using a large amount of gold pearls, the substance concerned is still unknown. We report here on the purification and characterization of yellow pigments from the nacre of Akoya pearl oyster shells. Two yellow components, YC1 and YC2, were isolated from the HCl-methanol (HCl-MeOH) extract from nacreous organic matrices obtained by decalcification of the shells with ethylenediaminetetraacetic acid (EDTA). Energy-dispersive X-ray and infrared spectroscopy analyses suggested that YC1 and YC2 precipitated under basic conditions are composed of Fe-containing inorganic and polyamide-containing organic compounds, respectively. YC1 solubilized under acidic conditions exhibited positive reactions to KSCN and K4[Fe(CN)6] reagents, showing the same ultraviolet-visible absorption spectrum as those of Fe(III)-containing compounds. In addition, X-ray absorption fine structure analysis supported the compound in the form of Fe(III). The total amount of Fe was approximately 2.6 times higher in the yellow than white nacre, and most Fe was fractionated into the EDTA-decalcifying and HCl-MeOH extracts. These results suggest that Fe(III) coordinated to EDTA-soluble and insoluble matrix compounds are mainly associated with yellow color development not only in the Akoya pearl oyster shells but also in the cultured Akoya pearls.

Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in Pinctada fucata.

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.

Reproduction-related genes in the pearl oyster genome.

Molluscan reproduction has been a target of biological research because of the various reproductive strategies that have evolved in this phylum. It has also been studied for the development of fisheries technologies, particularly aquaculture. Although fundamental processes of reproduction in other phyla, such as vertebrates and arthropods, have been well studied, information on the molecular mechanisms of molluscan reproduction remains limited. The recently released draft genome of the pearl oyster Pinctada fucata provides a novel and powerful platform for obtaining structural information on the genes and proteins involved in bivalve reproduction. In the present study, we analyzed the pearl oyster draft genome to screen reproduction-related genes. Analysis was mainly conducted for genes reported from other molluscs for encoding orthologs of reproduction-related proteins in other phyla. The gene search in the P. fucata gene models (version 1.1) and genome assembly (version 1.0) were performed using Genome Browser and BLAST software. The obtained gene models were then BLASTP searched against a public database to confirm the best-hit sequences. As a result, more than 40 gene models were identified with high accuracy to encode reproduction-related genes reported for P. fucata and other molluscs. These include vasa, nanos, doublesex- and mab-3-related transcription factor, 5-hydroxytryptamine (5-HT) receptors, vitellogenin, estrogen receptor, and others. The set of reproduction-related genes of P. fucata identified in the present study constitute a new tool for research on bivalve reproduction at the molecular level.

Involvement of insulin-like peptide in long-term synaptic plasticity and long-term memory of the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is 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 the Lymnaea CNS.

Partial characterization and ontogenetic development of pancreatic digestive enzymes in Japanese eel Anguilla japonica larvae.

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.

Distribution of pepsinogen- and ghrelin-producing cells in the digestive tract of Japanese eel (Anguilla japonica) during metamorphosis and the adult stage.

Pepsinogen is the precursor form of the gastric-specific digestive enzyme, pepsin. Ghrelin is a representative gastric hormone with multiple functions in vertebrates, including the regulation of growth hormone release, stimulation of food intake and gastrointestinal motility function. We investigated chronological changes in the distribution of pepsinogen-expressing cells by in situ hybridization and ghrelin-immunoreactive cells by immunohistochemistry in the Japanese eel (Anguilla japonica) during metamorphosis from the leptocephalus sage to the elver stage. The ghrelin-producing cells first appeared in the gastric cecum and pyloric portion of the stomach in the late phase of metamorphosing leptocephali, whereas the pepsinogen-producing cells were first detected in the early phase of the glass-eel stage. These suggest that endocrine cells differentiated earlier than exocrine cells in the eel stomach. Accompanying eel development, the distribution of ghrelin-producing cells spread to the esophagus and other regions of the stomach, but not to the intestine. These results may be related to the changes in dietary habits during metamorphosis in the Japanese eel.

Novel member of the PAR domain bZIP gene family isolated from Pacific oyster, Crassostrea gigas.

A cDNA encoding the first mollusk member of the PAR subfamily of bZIP transcription factors has been characterized in the Pacific oyster, Crassostrea gigas. The sequence of the cDNA predicts a protein of 260 amino acids that has 41-50% identity with the PAR domain, 72-79% identity with the basic DNA-binding domain and 34-56% identity with the leucine zipper domain of other members of the PAR subfamily. Polymerase chain reaction with reverse transcription indicates that this gene is expressed during developmental stages from an unfertilized egg to a juvenile. It was demonstrated by northern hybridization that the gene is also expressed on several adult tissues. The identification of a novel member of the PAR subfamily bZIP genes in mollusks may help to identify common functions that have been conserved through evolution and to elucidate evolutionary relationships within this subfamily of proteins.

CREB in the pond snail Lymnaea stagnalis: cloning, gene expression, and function in identifiable neurons of the central nervous system.

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.