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.

In our previous publication, we identified novel gene candidates involved in shell formation by EST analyses of the nacreous and prismatic layer-forming tissues in the pearl oyster Pinctada fucata. In the present study, 14 of those genes, including two known genes, were selected and further examined for their involvement in shell formation using the RNA interference. Molecular characterization based on the deduced amino acid sequences showed that seven of the novel genes encode secretory proteins. The tissue distribution of the transcripts of the genes, as analyzed by RT-PCR and in situ hybridization, was mostly consistent with those obtained by the EST analysis reported previously. Shells in the pearl oysters injected with dsRNAs targeting genes 000027, 000058, 000081, 000096, 000113 (nacrein), 000118, 000133 and 000411 (MSI60), which showed expression specific to the nacreous layer forming tissues, showed abnormal surface appearance in this layer. Individuals injected with dsRNAs targeting genes 000027, 000113 and 000133 also exhibited abnormal prismatic layers. Individuals injected with dsRNAs targeting genes 000031, 000066, 000098, 000145, 000194 and 000200, which showed expression specific to prismatic layer forming tissues, displayed an abnormal surface appearance in both the nacreous and prismatic layers. Taken together, the results suggest that the genes involved in prismatic layer formation might also be involved in the formation of the nacreous layers.

Although a wide variety of proteins and genes possibly related to the shell formation in bivalve have been identified, their functions have been only partially approved. We have recently performed deep sequencing of expressed sequence tags (ESTs) from the pearl oyster Pinctada fucata using a next-generation sequencer, identifying a dozen of novel gene candidates which are possibly associated with the nacreous layer formation. Among the ESTs, we focused on three novel isoforms (N16-6, N16-7, and N19-2) of N16 and N19 families with reference to five known genes in the families and determined the full-length cDNA sequences of these isoforms. Reverse transcription-polymerase chain reaction revealed that N16-6 was expressed in gill, gonad, adductor muscle, and mantle, whereas N16-7 exclusively in mantle. N19-2 was expressed in all tissues examined. In situ hybridization demonstrated their regional expression in mantle and pearl sac, which well corresponded to those shown by EST analysis previously reported. Shells in the pearl oyster injected with dsRNAs of N16-7 and N19-2 showed abnormal surface appearance in the nacreous layer. Taken together, novel isoforms in N16 and N19 families shown in this study are essential to form the nacreous layer.

We confirmed the existence of growthpromoting substances in the conditioned media (CM) from the rotifer Brachionus plicatilis at an early exponential growth phase and isolated a novel protein with a growth-promoting activity from the crude extract (CE) of rotifer cells. CM was prepared from the culture media where rotifers had been cultured at an early exponential growth phase and filtered through a 0.22-lm filter membrane. The growth-promoting activity was determined using rotifers in CM for 5 days. As a result, the increase of rotifers added with CM was significantly higher than that of the control in artificial seawater (P \ 0.001). Moreover, the growthpromoting activity of CM was dose-dependent and inactivated by heat treatment at 80°C for 60 min. Meanwhile, CM filtered through a \10 kDa ultrafiltration membrane showed a low activity, whereas proteinase K treatment resulted in a complete inactivation. These results suggest that the rotifer secrets growth-promoting proteins into CM. CE also contained a protein with the activity and properties similar to those found in CM. Then, CE was subjected to purification of a growth-promoting protein for convenience using various types of chromatography after fractionation with 30-80% saturated ammonium sulfate. Subsequently, a protein with an approximate molecular weight of 25000 was isolated, and its N-terminal amino acid sequence was determined to be PAVVDFTAVWFGPLQMIKP. An orthologue was found in the EST database of B. plicatilis, the full sequence of which showed about 50% identity to the corresponding regions of thioredoxins from other organisms.

Many genes have been identified to participate in the shell formation so far. Nevertheless, the whole picture of the molecular mechanisms underlying the shell formation has remained unknown. In our previous study, we analyzed comprehensively genes expressed in the shell-producing tissues and identified 14 genes to be involved in the shell formation by the RNA interference (RNAi) method. In the present study, we performed further screening to find additional novel genes involved in the formation of the nacreous and prismatic layers. We here selected 80 genes from the EST data as candidates to function in the shell formation, conducted knockdown experiments by the RNAi method, and observed surface appearances on the nacreous and prismatic layers. We newly identified 64 genes that could participate in the shell formation. Taken together with our previous study, 78 genes were