The initial, manual annotation analysis of the pearl oyster genome is reported in the present issue of Zoological Science. Contributors represent a wide array of research fields, including bioinformatics, molecular and cellular biology, fisheries science, biochemistry, biomineralogy, molluscan biology, evolutionary and developmental biology, and paleobiology, reflecting the pearl oyster's broad biological and economic importance. The annotated pearl oyster genome paves the way for future studies in diverse areas including pearl aquaculture, biomineralization, and lophotrochozoan biology.

Pearl is the most valuable form of gem made by organisms and its beauty has been considered attractive and precious by people all over the world. Pearl oyster aquaculture system, in which a graft from the mantle pallium is transplanted with a nucleus into mother pearl oysters, is currently conducted at the industrial level. However, it is unclear what molecular mechanisms are involved in this system of pearl formation, which produces brilliant and beautiful nacreous layers. Since it takes long time to look for the proteins and genes possibly participating in the molluskan shell formation, another sophisticated strategy has been expected to screen candidate genes. One breakthrough in the latter area was the development of next-generation sequencing (NGS) in place of the automated Sanger method, the first generation sequencing technique. Using NGS, expressed sequence tag (EST) libraries were constructed from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in the Akoya pearl oyster Pinctada fucata. Subsequently, the total genome sequence of the Akoya pearl oyster was analyzed. This database, together with the EST database mentioned above, is useful for identifying genes responsible for superior phenotype characters for pearl oyster aquaculture such as fast growth, disease resistance, easy domestication, and ability to produce high quality pearls. Subsequent marker-assisted breeding of the Akoya pearl oyster should establish strains with superior genotypes, producing even higher quality Akoya pearls.

During the 18th and 19th centuries, studies of how pearls are formed were conducted mainly in Europe. The subsequent pearl culturing experiments conducted worldwide in the early 20th century, however, failed to develop into a pearl industry. In Japan, however, Kokichi Mikimoto succeeded in culturing blister pearls in 1893 under the guidance of Kakichi Mitsukuri, a professor at Tokyo Imperial University (now the University of Tokyo) and the first director of the Misaki Marine Biological Station, Graduate School of Science, University of Tokyo. This success and subsequent developments laid the foundation for the pearl farming industry, developed new demand for cultured pearls in the European jewelry market, and initiated the full-scale industrialization of pearl culturing. In addition, research at the Misaki Marine Biological Station resulted in noteworthy advances in the scientific study of pearl formation. Today, pearls are cultured worldwide, utilizing a variety of pearl oysters. The pearl farming industry, with its unique origins in Japan, has grown into a global industry. Recently, the introduction of genome analysis has allowed cultured pearl research to make rapid progress worldwide in such areas as the dynamics of mother-of-pearl layer formation and biomineralization. This signals another new era in the study of pearls.

The genome sequence of the Japanese pearl oyster, the first draft genome from a mollusk, was published in February 2012. In order to curate the draft genome assemblies and annotate the predicted gene models, two annotation Jamborees were held in Okinawa and Tokyo. To date, 761 genes have been surveyed and curated. A preparatory meeting and a debriefing were held at the Misaki Marine Biological Station before and after the Jamborees. These four events, in conjunction with the sequence-decoding project, have facilitated the first series of gene annotations. Genome annotators among the Jamboree participants added 22 functional categories to the annotation system to date. Of these, 17 are included in Generic Gene Ontology. The other five categories are specific to molluskan biology, such as “Byssus Formation” and “Shell Formation”, including Biomineralization and Acidic Proteins. A total of 731 genes from our latest version of gene models are annotated and classified into these 22 categories. The resulting data will serve as a useful reference for future genomic analyses of this species as well as comparative analyses among mollusks.

We constructed a web-based genome annotation platform, MarinegenomicsDB, to integrate genome data from various marine organisms including the pearl oyster Pinctada fucata and the coral Acropora digitifera. This newly developed viewer application provides open access to published data and a user-friendly environment for community-based manual gene annotation. Development on a flexible framework enables easy expansion of the website on demand. To date, more than 2000 genes have been annotated using this system. In the future, the website will be expanded to host a wider variety of data, more species, and different types of genome-wide analyses. The website is available at the following URL:  http://marinegenomics.oist.jp.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

The mechanisms of contraction of molluscan striated and smooth muscles differ from those in vertebrates. Molluscan striated muscles adopt a myosin-linked regulation, unlike vertebrates. Smooth muscles in these species show a unique form of contraction, in which the tension is maintained for a long time with little energy consumption, called catch. The available gene information is insufficient to elucidate the mechanism of contraction of molluscan muscles at the molecular level. BLAST searching was thus used to annotate genes encoding proteins related to muscle contraction in the completely determined genome of the pearl oyster Pinctada fucata using partial nucleotide sequences obtained by 3′ RACE. We identified genes that encode components of the thick-filament, such as myosin heavy chain, myosin essential and regulatory light chains, paramyosin and twitchin; of the thin-filament, such as actin, tropomyosin, troponin-T, troponin-I, troponin-C and calponin; and the PKA catalytic subunit, which is a key player in the regulation of catch contraction. The analysis indicated that isoforms of myosin heavy chain, paramyosin, and calponin are produced by alternative splicing.

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.

Homeobox genes are involved in various aspects of the development of multicellular animals, including anterior-posterior patterning of the body plan. We performed a genomic survey of homeobox genes in the Japanese pearl oyster, Pinctada fucata, and annotated 92 homeobox-containing genes and five homeobox-less Pax genes. This species possesses 10 or 11 Hox genes. We annotated another homeobox genes that cover 77 out of the 111 gene families identified in the amphioxus genome. Investigation of these repertoires of homeobox genes will shed new light on the comparatively less well-understood lophotrochozoan development.

To gain a better understanding of molluscan development and its relation to the evolution of their unique body plan, we performed a genomic survey of genes encoding transcription factors, such as Tbx, Fox, Ets, HMG, NFκ;B, bZIP, and C2H2 zinc finger proteins in the Japanese pearl oyster, Pinctada fucata. We annotated 133 transcription factor genes. Together with the orthologs of known deuterostome genes, we found several orphan genes in each class of transcription factor. Some possessed clear orthologs in other species of lophotrochozoans, while no counterpart genes were found in the deuterostomes or ecdysozoans. These observations suggest that a number of transcription factor genes are unique to lophotrochozoans, and thus additional research frontiers remain to be explored with regard to such transcription factors.

Basic helix-loop-helix (bHLH) transcription factors play significant roles in multiple biological processes in metazoan cells. In recent work, we showed that three orthologous HLH families, pearl, amber, and peridot, have apparently been lost in the Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens lineages. To further address the gain and loss of bHLH proteins during bilaterian evolution, we examined the genome of the pearl oyster, Pinctada fucata, which has recently been sequenced. We characterized the putative full set 65 bHLH genes and showed that genes previously categorized into the orthologous family PTFb, actually fall into two distinct orthologous families, 48-related-1 and 48-related-2. We also identified a novel orthologous family, clockwork orange. Based on these newly identified orthologous family members and on orphan bHLH factors, we propose that genes encoding bHLH factors in bilaterians are not as evolutionarily stable as previously thought.

The pearl oyster Pinctada fucata has great potential as a model system for lophotrochozoan developmental biology research. Pinctada fucata is an important commercial resource, and a significant body of primary research on this species has emphasized its basic aquaculture biology such as larval biology and growth, aquaculture, pearl formation and quality improvement, shell formation, and biomineralization. Recently, a draft genome sequence of this species was published, and many experimental resources are currently being developed, such as bioinformatics tools, embryo and larva manipulation methods, gene knockdown technique, etc. In this paper, we report the results from our genomic survey pertaining to gene families that encode developmental signaling ligands (Fgf, Hedgehog, PDGF/VEGF, TGFβ, and Wnt families). We found most of the representative genes of major signaling pathways involved in axial patterning, as well as copies of the signaling molecule paralogs. Phylogenetic character mapping was used to infer a possible evolutionary scenario of the signaling molecules in the protostomes, and to reconstruct possible copy numbers of signaling molecule-coding genes for the ancestral protostome. Our reconstruction suggests that P. fucata retains the ancestral protostome gene complement, providing further justifications for the use of this taxon as a model organism for developmental genomics research.