Molecular cloning and characterisation of scavenger receptor class B in pearl oyster Pinctada fuctada martensii

Background: Molluscs can accumulate carotenoids in their body tissues by predominantly feeding on aquatic plant sources. Carotenoid transport and absorption are determined by the regulation of various proteins such as Scavenger receptor class B(SR-BI). We report the identification and characterisation of pearl oyster Pinctada fuctada martensii SR-BI (PmSR-BI). The correlation between total carotenoid content (TCC) and gene expression was also estimated. Results: The full-length cDNA of PmSR-BI was 1828 bp, including an open-reading frame encoding of 1518 bpwith a pI value of 5.83. PmSR-BI protein contains a hydrophobic CD36 domain and four centrally clustered cysteine residues for the arrangement of disulphide bridges. The deduced amino acid sequence had an identity of 30% to 60% with the SR-B of other organisms. Reverse transcription polymerase chain reaction analysis showed that mRNA transcripts were expressed in multiple tissues of adult pearl oyster. A higher expression of PmSR-BI gene was observed in the hepatopancreas than in the adductor muscle, gill and mantle. The TCC and gene expression of PmSR-BI were significantly correlated (P < 0.05), with a correlation coefficient of 0.978. Conclusions: The results suggested that PmSR-BI is involved in the absorption of carotenoids in the pearl oyster P. fuctada martensii

Development of SSR marker by RNA-seq and its application in genotyping pearl sac in pearl oyster Pinctada fucata martensii

Background: Pearl oyster Pinctada fucata martensii is cultured for producing round nucleated pearls. Pearl production involves a surgical operation where a mantle tissue graft from a donor oyster and a round nucleus are implanted in the gonad of a host oyster. Whether the mantle graft implanted in the gonad of a host oyster contributes to the formation of a pearl sac that secretes pearl nacre to form a pearl should be determined. In April 2012, two full-sib families were separately used as donor and host oysters for a nucleus insertion operation. The pearl sac was sampled from the host oysters at day 60 after nucleus operation. A large number of simple sequence repeat (SSR) markers were developed using Illumina HiSeq\u2122 2000 platform. The two full-sib families were also used to mine diagnostic SSR markers for genotyping donor oyster, host oyster, and pearl sac. Results: A total of 3168 microsatellite lociwere identified in 39,078 unigenes, and 1977 SSR primerswere designed by Primer 3.0. Forty-seven SSR primers were validated, and the rate of successful amplification was 72.3%. Two diagnostic SSR primers could successfully genotype pearl sac, donor oyster, and host oyster. Donor and host oysters were both homogenous, and the alleles in pearl sac were identical to those in donor and host oysters. Conclusions: The present results confirmed that themantle graft implanted in the gonad of host oyster contributed to the formation of the pearl sac in pearl oyster P. fucata martensii

Molecular characterization of CHST11 and its potential role in nacre formation in pearl oyster Pinctada fucata martensii

Background: C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells. Results: Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference. Conclusions: This study indicated thatPmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate

Genetic Diversity and Population Structure of the Peanut Worm (Sipunculus nudus) in Southern China as Inferred from Mitochondrial 16S rRNA Sequences

Genetic diversity and population structure of the peanut worm (Sipunculus nudus) were investi- gated by using 536 base-pair fragments of the mitochondrial 16S ribosomal gene. Populations were collected from three locations along the southern coast of China - Beihai, Sanya, and Xiamen. Amplified polymerase chain reaction products were sequenced in both directions and data were analyzed using ClustalX, Arlequin, and MEGA. A total of 69 polymorphic sites and 21 distinct haplotypes were revealed. The mean haplotype and nucleotide diversity of the three pop- ulations were 0.814% and 0.37%, respectively. The Beihai population had the greatest haplo- type and nucleotide diversity, followed by the Xiamen and Sanya populations. Analysis of mole- cular variance (AMOVA) showed significant genetic differentiation among the three populations (Fst = 0.0619, p<0.05) and distinct population structures among the three sites

A distinct mitogenome of peanut worm Sipunculus nudus (Sipuncula, Sipunculidae) from Beibu Gulf

Peanut worm, Sipunculus nudus is a cosmopolitan species found in inter-tidal sands. In this article, based on the samples collected from the Beibu Gulf, China, a mitochondrial DNA sequence by Illumina high throughput sequencing, was carried out on muscle and determined the complete mitogenome. This mitogenome of peanut worm is 15,331 base pairs in length (Accession number: MN481531) and comprises of 37 genes as in the typical mitochondrial gene arrangement of Sipunculus. Moreover, the phylogenetic analysis shows that our sample is a novel type in China and has a distantly genetic relationship with the known populations in South China Sea. This mitogenome will serve as the foundation for the molecular genetic studies of peanut worm