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Heterosis improves growth and survival of shellfish species. Although breeders have widely exploited heterosis, its underlying molecular mechanisms remain unclear. In this study, a 2 × 2 complete diallel cross was facilitated between two full‐sib families to produce two inbred families (A and D) and their reciprocal hybrid families (B and C) of pearl oyster Pinctada fucata martensii. Growth traits of the four families were compared at the adult stages. Transcriptome analysis was conducted on the four families using an Illumina sequencing platform. The results revealed that the growth traits of the four families significantly varied (P < 0.05). The mid‐parent heterosis values of shell length, shell height, shell width, shell weight, and total weight were 12.9%, 14.9%, 18.2%, 17.2%, and 33.2%, respectively. The B‐ and C‐inbred (A and D) triads had 79 and 68 differentially expressed genes (DEGs), respectively, which were dominantly nonadditive, including overdominance, underdominance, and low‐parent dominance. Gene ontology term analysis showed that the DEGs in the B‐ and C‐inbred triads were enriched for metabolic process, cellular process cell part, binding, and catalytic activity. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis indicated that the DEGs in the B‐ and C‐inbred triads were involved in focal adhesion, the P13K‐Akt signaling pathway, the mRNA surveillance pathway, and the focal adhesion pathway. The reliability of the sequencing data was confirmed by real‐time polymerase chain reaction analysis of six growth‐related genes. The findings of this study provide new insights into heterosis for growth traits and the design of genetic breeding programs for this species. Two inbred families (A and D) and their reciprocal hybrid families (B and C) were developed with breeders from two full‐sib families. The mid‐parent heterosis values of growth traits at adult stages ranged from 12.9% to 33.2%. The differentially expressed genes in the B‐ and C‐inbred triads were 79 and 68, respectively, which were dominantly nonadditive, including overdominance, underdominance, and low‐parent dominance. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of non-coding RNA molecules with presumed post-transcriptional regulatory activity in various biological processes, such as development and biomineralization. Pinctada martensii is one of the main species cultured for marine pearl production in China and Japan. In our previous research, 258 pm-miRNAs had been identified by solexa deep sequencing in P. martensii , while it is far from the number of miRNAs found in other species. In this study, based on the transcriptome database of pearl sac, we identified 30 candidate pm-miRNAs by computational prediction. Among the obtained 30 pm-miRNAs, 13 pm-miRNAs were generated from the complementary strand of protein-coding mRNAs, and 17 pm-miRNAs could not be annotated using blastx and tblastn analysis. Notably, 10 of the 30 pm-miRNAs, such as pm-miR-1b, pm-miR-205b and pm-miR-375b, were homologous with the reported pm-miRNAs, respectively. To validate the existence of the identified pm-miRNAs, eight randomly selected pm-miRNAs were tested by stem loop quantitative RT-PCR analyses using 5.8S as the internal reference gene. Target prediction between the obtained pm-miRNAs and biomineralization-related genes by microTar, miRanda and RNA22 indicated pm-miR-2386 and pm-miR-13b may be the key factors in the regulation network by regulating the formation of organic matrix or the differentiation of mineralogenic cell during shell formation. Thus, this study enriched miRNA databases of pearl oyster and provided a new way to understand biomineralization. [ABSTRACT FROM AUTHOR]

Abstract: Dermatopontin (DPT) is identified as a major component of the shell matrix protein. However, its exact function in the shell formation remains obscure. In this study, we described the characteristic and function of DPT gene from Pinctada martensii. DPT cDNA was 797bp long, containing an open reading fragment (ORF) of 537bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4kDa and theoretical isoelectric point of 5.97. The 5′ untranslated region (UTR) was 11bp and the 3′UTR was 249 with 18bp poly (A) tail. In the peptide, there was a signal sequence, six potential phosphorylation sites, one glycosylation site and eight cysteine residues. Moreover, a sequence motif (D-R-X-W/F/Y-X-F/Y/I/L/M-X1–2-C) was contained and repeated itself three times in the entire sequence. DPT mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the mantle, which was nacre formation-related tissue. After decreasing DPT expression using RNA interference (RNAi) technology in the mantle, the nacreous layer showed a disordered growth; whereas the prismatic layer of the shells has no significant changes. These results suggested that DPT obtained in this study was a constitutive matrix protein and participated in nacre formation in P. martensii. [Copyright &y& Elsevier]

Keratan sulfate possesses considerable amounts of negatively charged sulfonic acid groups and participates in biomineralization. In the present study, we investigated characteristics and functions of a CHST1 gene identified from the pearl oyster Pinctada fucata martensii ( PmCHST1b ) which participated in the synthesis of keratan sulfate. PmCHST1b amino acid sequence carried a typical sulfotransferase-3 domain (sulfotransfer-3 domain) and belonged to membrane-associated sulfotransferases. Homologous analysis of CHST1 from different species showed the conserved motif (5′ PSB motif and 3′ PB motif) which interacted with 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Structure analysis of sulfotransferase domain indicted that PmCHST1b showed the conserved catalytic structure character and the relationships presented in the phylogenetic tree conformed to that of traditional taxonomy. Expression pattern of PmCHST1b in different tissues and development stages showed that PmCHST1b widely expressed in all the detected tissues and development stages and showed the highest expression level in the central zone of mantle (MC). PmCHST1b expressed highly in the trochophore, D-stage larvae and spat which corresponded to prodissoconch and dissoconch shell formation, respectively. RNA interference (RNAi) successfully inhibited expression level of PmCHST1b in MC ( P <0.05), and sulfate polymer content in the extrapallial fluid significantly reduced ( P <0.05). Crystallization of shell nacre became irregular. Results above indicated that PmCHST1b may affect nacre formation by participating in synthesis of keratan sulfate in extrapallial fluid. This study provided fundamental materials for further research on the role of sulfotransferases and keratan sulfate in nacre formation. [ABSTRACT FROM AUTHOR]

In this study, we formulated five diets, namely, P1, P2, P3, P4 and P5, with Chlorella sp. powder, Spirulina platensis powder, yeast powder, soybean meal and corn gluten, respectively, as major protein sources. A feeding experiment was designed to evaluate the effects of formulated diets on the growth performance, immunity and antioxidant and biomineralization capacity of juvenile pearl oyster ( Pinctada fucata martensii ). In the experiments, the five groups were separately fed with P1, P2, P3, P4 and P5 diets. After 45 days of feeding, pearl oysters fed on P1, P2, P3 and P4 diets showed significantly higher absolute growth rate and protease and amylase activities than those fed on P5 diet (P < 0.05). Moreover, pearl oysters fed on P1, P2, P3 and P4 diets exhibited significantly higher activities of alkaline phosphatase (AKP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (P < 0.05). Significantly higher expression levels of SOD, GPx, CAT, heat shock protein (HSP) 70, HSP90, nacrein, pif177 and pearlin mRNA were observed in pearl oysters fed on P1, P2, P3 and P4 diets relative to those fed on P5 (P < 0.05). Results suggested the suitability of Chlorella sp. powder, S. platensis powder, yeast powder and soybean meal as protein sources for development of formulated diets for pearl oyster P. f. martensii . [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (LncRNAs) are abundant in the genome of higher forms of eukaryotes and implicated in regulating the diversity of biological processes partly because they host microRNAs (miRNAs), which are repressors of target gene expression. In vertebrates, miR-133 regulates the differentiation and proliferation of cardiac and skeletal muscles. Pinctada martensii miR-133 ( pm-miR-133 ) was identified in our previous research through Solexa deep sequencing. In the present study, the precise sequence of mature pm-miR-133 was validated through miR-RACE. Stem loop qRT-PCR analysis demonstrated that mature pm-miR-133 was constitutively expressed in the adductor muscle, gonad, hepatopancreas, mantle, foot, and gill of P. martensii . Among these tissues, the adductor muscle exhibited the highest pm-miR-133 expression. Target analysis indicated that pm-RhoA was the potential regulatory target of pm-miR-133 . Bioinformatics analyses revealed that a potential LncRNA (designated as Lnc133 ) with a mature pm-miR-133 could generate a hairpin structure that was highly homologous to that of Lottia gigantea . Lnc133 was also highly expressed in the adductor muscle, gill, hepatopancreas, and gonad. Phylogenetic analysis further showed that the miR-133s derived from chordate and achordate were separated into two classes. Therefore, Lnc133 hosting pm-miR-133 could be involved in regulating the cell proliferation of adductor muscles by targeting pm-RhoA . [ABSTRACT FROM AUTHOR]

Increasing evidence demonstrated that microRNAs (miRNAs) play critical roles in innate immunity in vertebrates and invertebrates. MiR-146a/b is reported as a key regulator of the immune response through mediating Toll-like receptor and cytokine signalling. In this study, a novel miR-146a was identified and characterised from Pinctada martensii (designated as pm-miR-146a), and its roles in modulating the inflammatory response after LPS stimulation were also investigated. Pm-miR-146a ubiquitously expressed in all examined tissues, with the highest level in the mantle and lowest expression in the haemolymph. Pm-miR-146a increased at 24 h after lipoplysaccharide injection, in union with up-regulated NF-κB ( P < 0.05). The over-expression of pm-miR-146a in vivo could significantly inhibit the expression of macrophage migration inhibitory factor (MIF), the potential target gene predicted by miRanda, while enforcing pm-miR-146a involved in the down-regulation of NF-κB. Thus, we propose that pm-miR-146a plays a role of negative feedback regulation to the NF-κB signal by repressing the expression of the pro-inflammatory cytokine MIF. These findings revealed that miR-146a represents a critical role in inflammatory response and offers new evidence for miRNAs in the innate immunity of molluscs. [ABSTRACT FROM AUTHOR]