Hirotoshi Endo

Draft Genome of the Pearl Oyster Pinctada fucata: A Platform for Understanding Bivalve Biology

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ∼1150-Mb genome at ∼40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry.

The Diversity of Shell Matrix Proteins: Genome-Wide Investigation of the Pearl Oyster, Pinctada fucata

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.

Bivalve-specific gene expansion in the pearl oyster genome: implications of adaptation to a sessile lifestyle

IntroductionBivalve molluscs have flourished in marine environments, and many species constitute important aquatic resources. Recently, whole genome sequences from two bivalves, the pearl oyster, Pinctada fucata, and the Pacific oyster, Crassostrea gigas, have been decoded, making it possible to compare genomic sequences among molluscs, and to explore general and lineage-specific genetic features and trends in bivalves. In order to improve the quality of sequence data for these purposes, we have updated the entire P. fucata genome assembly.ResultsWe present a new genome assembly of the pearl oyster, Pinctada fucata (version 2.0). To update the assembly, we conducted additional sequencing, obtaining accumulated sequence data amounting to 193× the P. fucata genome. Sequence redundancy in contigs that was caused by heterozygosity was removed in silico, which significantly improved subsequent scaffolding. Gene model version 2.0 was generated with the aid of manual gene annotations supplied by the P. fucata research community. Comparison of mollusc and other bilaterian genomes shows that gene arrangements of Hox, ParaHox, and Wnt clusters in the P. fucata genome are similar to those of other molluscs. Like the Pacific oyster, P. fucata possesses many genes involved in environmental responses and in immune defense. Phylogenetic analyses of heat shock protein70 and C1q domain-containing protein families indicate that extensive expansion of genes occurred independently in each lineage. Several gene duplication events prior to the split between the pearl oyster and the Pacific oyster are also evident. In addition, a number of tandem duplications of genes that encode shell matrix proteins are also well characterized in the P. fucata genome.ConclusionsBoth the Pinctada and Crassostrea lineages have expanded specific gene families in a lineage-specific manner. Frequent duplication of genes responsible for shell formation in the P. fucata genome explains the diversity of mollusc shell structures. These duplications reveal dynamic genome evolution to forge the complex physiology that enables bivalves to employ a sessile lifestyle in the intertidal zone.

Identification and characterization of a matrix protein (PPP-10) in the periostracum of the pearl oyster, Pinctada fucata

The periostracum is a layered structure that is formed as a mollusk shell grows. The shell is covered by the periostracum, which consists of organic matrices that prevent decalcification of the shell. In the present study, we discovered the presence of chitin in the periostracum and identified a novel matrix protein, Pinctada fucata periostracum protein named PPP‐10. It was purified from the sodium dodecyl sulfate/dithiothreitol‐soluble fraction of the periostracum of the Japanese pearl oyster, P. fucata. The deduced amino acid sequence was determined by a combination of amino acid sequence analysis and cDNA cloning. The open reading frame encoded a precursor protein of 112 amino acid residues including a 21‐residue signal peptide. The 91 residues following the signal peptide contained abundant Cys and Tyr residues. PPP‐10 was expressed on the outer side of the outer fold in the mantle, indicating that PPP‐10 was present in the second or third layer of the periostracum. We also determined that the recombinant PPP‐10 had chitin‐binding activity and could incorporate chitin into the scaffolds of the periostracum. These results shed light on the early steps in mollusk shell formation.

GSP-37, a novel goldfish scale matrix protein: identification, localization and functional analysis

A novel noncollagenous acidic protein was identified from the scales of goldfish (Carassius auratus), a freshwater teleost. Using an in vitro calcium phosphate crystallization assay, the EDTA-soluble fraction from these scales was screened for crystallization inhibitory activity, and a highly phosphorylated glycoprotein, named goldfish scale protein (GSP)-37, was isolated through 5 HPLC purification steps. The cDNA for GSP-37 has an open reading frame encoding a precursor protein, consisting of a signal peptide and GSP-37, with 19 and 137 amino acid residues, respectively. The C-terminal region of GSP-37 contains the RGD consensus sequence for cell adhesion. Although native GSP-37 strongly inhibited crystallization, alkaline phosphatase treatment dramatically reduced its inhibitory activity. Reverse transcription-PCR analysis revealed that GSP-37 is expressed only in scales but not in other calcified tissues, bones or pharyngeal teeth. In situ hybridization demonstrated that GSP-37-expressing cells were localized in the central regions of regenerating scales, where organic matrices were actively synthesized and were not stained with either alkaline phosphatase or tartrate-resistant acidic phosphatase, osteoblastic and osteoclastic cell markers, respectively. Immunohistochemical analyses showed that GSP-37 is localized in the uppermost region of the bony layer of the scale, which is thought to correspond to the enamel or enameloid layer of vertebrate teeth. All these data strongly indicate that GSP-37 is deeply associated with calcification in fish scales.

Initiating the Mollusk Genomics Annotation Community: Toward Creating the Complete Curated Gene-Set of the Japanese Pearl Oyster, Pinctada fucata

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.

Chlorophyll fluorescence control in microalgae by biogenic guanine crystals

Magnetic fields were applied to water suspensions of guanine crystals to induce changes in light scattering as a possible way to control photosynthesis in microalgae. The effect of guanine microcrystals with and without an applied magnetic field on the photosynthesis of a unicellular microalgae (plant), Pleurochrysis. carterae (P. carterae), was investigated by examining chlorophyll fluorescence. The fluorescence intensity at 600–700 nm of the photosynthetic cells increased remarkably when the concentration ratio of guanine microcrystals was 10 times larger than that of the cells. This increase in fluorescence occurred reproducibly and was proportional to the amount of guanine microcrystals added. It is speculated that the guanine microcrystals enhance the intensity of the excitation light on the cells by concentrating the excitation light or prolonging the time of light exposure to the cells. Moreover, applying a 500-mT magnetic field allowed modulation of the fluorescence intensity, depending on the direction of the fluorescence light.

Magnetically Controlled Biogenic Crystals as Photo-Bioreactors for Algae

Biogenic guanine crystal (GC) is abundantly contained in fish scales. This crystal has thin plate form and high light reflectance with the largest plane of it. Effects of magnetic fields on GC, which shows diamagnetic orientation were investigated in our previous studies. In this paper, we tried to apply the biogenic crystals to micro algae culture for the purpose of acceleration of the micro algaes photonic synthesis. Recently, micro algae were focused on the view of producing bio-ethanol or bio-fuel. In particular, crystals, which have high refractive index and contain nitrogen in their molecule, seem be to available for one of fertilizer for plants. In addition, crystals, which have high refractive index, reflect light well, so we expect that these properties help algaes photonic synthesis. Additionally, some algae have the photo-tactic characteristics, phototaxis. Therefore, we also aim to utilize these crystals for efficient culturing of algae. In this paper, microscopic observations of behaviors of the micro algae in the cell culture chamber with a buffer containing the crystals were carried out. We tried controlling of alga motions with directive light stimulation from magnetically aligned biogenic GCs.