MicroRNA expression signature in the striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis.

Increasing evidences point to the potential role of microRNAs (miRNAs) in muscle growth and development in animals. However, knowledge on the identity of miRNAs and their targets in molluscs remains largely unknown. Scallops have one large adductor muscle, composed of fast (striated) and slow (smooth) muscle types, which display great differences in muscle fibers, meat quality, cell types and molecular components. In the present study, we performed a comprehensive investigation of miRNA transcriptomes in fast and slow adductor muscles of Yesso scallop Patinopecten yessoensis. As a result, 47 differentially expressed miRNAs representing ten miRNA families were identified between the striated and smooth adductor muscles. The KEGG enrichment analysis of their target genes were mainly associated with amino acid metabolism, energy metabolism and glycan biosynthesis. The target genes of miR-133 and miR-71 were validated by the dual-luciferase reporter assays and miRNA antagomir treatment in vivo. The identification and functional validation of these different miRNAs in scallops will greatly help our understanding of miRNA regulatory mechanism that achieves the unique muscle phenotypes in scallops. The present findings provide the direct evidences for muscle-specific miRNAs involved in muscle growth and differentiation in molluscs.

Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro.

The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright-Giemsa staining, micro-examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell-surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response.

A manganese superoxide dismutase (MnSOD) from ark shell, Scapharca broughtonii: Molecular characterization, expression and immune activity analysis.

Manganese superoxide dismutase (MnSOD) is one of the key members of the antioxidant defense enzyme family, however, data regarding to the immune function of MnSOD in mollusks still remain limited now. In this study, a full-length MnSOD cDNA was identified by rapid amplification of cDNA ends (RACE) method from cDNA library of ark shell Scapharca broughtonii (termed SbMnSOD). The cDNA contained an open reading frame (ORF) of 696 bp which encoded a polypeptide of 232 amino acids, a 5'-UTR with length of 32 bp and a 3'-UTR of 275 bp. Four putative amino acid residues (His-57, His-105, Asp-190 and His-194) responsible for manganese coordination were located in the most highly conserved regions of SbMnSOD and the signature sequence (DVWEHAYY) also existed in SbMnSOD. The deduced amino acid sequence of SbMnSOD shared high homology to MnSOD from other species. All those data revealed that the SbMnSOD was a novel member of the MnSOD family. The mRNA expression profiles of SbMnSOD in tissues of foot, gill, mantle, adductor muscle, hemocytes and hepatopancreas analyzed by quantitative real-time PCR (qRT-PCR) suggested the mRNA transcripts of SbMnSOD distributed in all the examined tissues. Importantly, Vibrio anguillarum challenge resulted in the increased expression of SbMnSOD mRNA with a regular change trend in all examined tissues, indicating SbMnSOD actively participated in the immune response process. What's more, further analysis on the antibacterial activity of the recombinant SbMnSOD showed that the fusion protein could remarkably inhibit growth of both Gram-positive and Gram-negative bacteria. The present results clearly suggested that SbMnSOD was an acute phase protein involved in the immune reaction in S. broughtonii.

Development of EST-SSRs from the ark shell (Scapharca broughtonii) transcriptome and their application in genetic analysis of four populations.

BACKGROUND: The ark shell (Scapharca broughtonii) is one of the most economically important mollusks in the Bohai Sea and Yellow Sea of China. In recent years, ark shells from the Korean population were introduced to China for seed propagation and culture. OBJECTIVE: To explore the impact of the introduction of Korean ark shell on the genetic diversity of native population in China. METHODS: Simple sequence repeat (SSR) is effective and widely used tool for genetic analysis. In this study, 180 EST-SSRs were selected and verified by polymerase chain reaction and polyacrylamide gel electrophoresis. Subsequently, five polymorphic EST-SSRs were screened and their primers were modified by fluorescein for use in the genetic analysis of four populations. RESULTS: Genetic analysis showed that 361 alleles amplified by five SSR loci were detected in the four populations. The number of alleles for the five SSRs ranged from 8 to 30, with a mean of 18.05 (standard deviation, SD = 6.492). The effective number of alleles varied from 2.253 to 22.222, with a mean of 10.596 (SD = 4.713). Observed heterozygosity and expected heterozygosity were 0.167-0.833 and 0.566-0.971, with average values of 0.520 (SD = 0.177) and 0.891 (SD = 0.062), respectively. Polymorphic information content ranged from 0.521 to 0.953, with a mean of 0.865 (SD = 0.070). The pairwise genetic differentiation coefficient (FST) of the four populations ranged from 0.0267 to 0.0477, showing low genetic differentiation. The phylogenetic tree constructed by neighbor-joining method showed that the genetic distance between the Chinese Dalian native population and three Korean populations was relatively more far than that among those Korean populations. CONCLUSION: The results indicated that the genetic structure of the Dalian wild population was less affected by the introduced Korean wild populations.

Integrated transcriptome and metabolome analysis reveals molecular responses of the clams to acute hypoxia.

Mudflat shellfish have evolved well-adapted strategies for coping with dynamic environmental fluxes and stressful conditions, including oxygen availability. The Manila clams Ruditapes philippinarum are worldwide cultured shellfish in marine intertidal zone, which usually encounter great risk of acute hypoxia exposure in coastal habitats. To reveal the effects of acute hypoxia on metabolic changes of the clams, we performed the integrated analysis of transcriptomics and metabolomics to investigate the global changes of genes and metabolites during acute hypoxia stress at the whole-organism level. The comparative transcriptome analysis reveals that the clams show the remarkable depression in a variety of biological performance, such as metabolic rates, neuronal activity, biomineralization activity, and cell proliferation and differentiation at the hypoxic condition. The metabolomic analysis reveals that amino acid metabolism plays a critical role in the metabolic changes of the clams in response to acute hypoxia. A variety of free amino acids may not only be served as the potential osmolytes for osmotic regulation, but also may contribute to energy production during the acute hypoxia exposure. The metabolite analysis also reveals several important biomarkers for metabolic changes, and provides new insights into how clams deal with acute hypoxia. These findings suggest that clams may get through acute hypoxia stress by the adaptive metabolic strategy to survive short-period of acute hypoxia which is likely to occur in their typical habitat. The present findings will not only shed lights on the molecular and metabolic mechanisms of adaptive strategies under stressful conditions, but also provide the signaling metabolites to assess the physiological states of clams in aquaculture.

Developmental dynamics of myogenesis in Yesso Scallop Patinopecten yessoensis.

The development of bivalves has been extensively studied over the last 150 years. Despite this, the developmental dynamics of myogenesis in bivalves remains largely unknown, particularly at the early developmental stages. In the present study, we investigate the characteristics of muscle development of Yesso scallop Patinopecten yessoensis by phalloidin staining, light, electron and confocal microscopy. Myoblasts containing chaotic myofilaments are initially found in the blastocoel of trochophore, and become more organized during the transformation from trochophore into veliger. This is followed by a well-structured musculature including an anterior adductor, velum retractors and ventral retractors at the early veliger stage. With development into late veliger, larval muscle system is composed of the branched velum retractors and ventral retractors, anterior and posterior adductors. The most striking change for pediveliger is the development of foot retractor and mantle related muscles at this stage. During metamorphosis, the retractor muscles and anterior adductor undergo the irreversible shrink until vanishing completely towards the end of larval life, which coincide with the growth of foot retractor and mantle margin. All retractor muscles are found to be composed of striated fibres, whereas the adductor muscles have both smooth and striated components. The present study provides new evidences on the dynamic pattern of myogenesis during embryonic and larval development in scallops, which will greatly improve our understanding of scallop myogenesis and provide the basis for comparative analysis of muscle development in bivalves.

Ferritin has an important immune function in the ark shell Scapharca broughtonii.

Ferritin, the principle cytosolic iron storage protein in the majority of living organisms, has important roles during immune process in invertebrates. Detailed information about ferritin in the ark shell Scapharca broughtonii, however, has been very limited. In this study, full-length ferritin (termed SbFer) was cloned by the rapid amplication of cDNA ends (RACE) method based upon the sequence from the transcriptome library. The cDNA contained a 182 bp 5'-untranslated region, a 519 bp open reading frame encoding a polypeptide of 172 amino acids, a 229 bp 3'-untranslated region, and three introns (902, 373 and 402 bp) embedded in four exons. There was an iron response element (IRE) in the 5'-untranslated region. The deduced amino acid sequence of SbFer possessed many characteristics of vertebrate H type ferritin, shared 63%-91% identity with mollusks and greater identity with vertebrate H type ferritin compared to the L type. The SbFer gene expression pattern examined by quantitative real-time PCR showed ferritin mRNA was expressed in all ark shell tissues examined. The highest levels of expression were found in hemocytes with decreasing levels of expression in foot, mantle, gill, adductor muscle and hepatopancreas. A challenge with Vibrio anguillarum resulted in time-dependent significant upregulation of SbFer mRNA, indicating SbFer participated actively in the bacterial defense process. Further analysis of the antibacterial activity indicated recombinant SbFer could function as an immune antibacterial agent to both Gram-positive and Gram-negative bacteria. Taken together, these results suggested strongly that ferritin of the ark shell is involved in immune defense against microbial infection and it is a constitutive and inducible acute-phase protein.

Complete mitochondrial genome of Ark shell Scapharca subcrenata.

Complete mitochondrial genome of Scapharca subcrenata was determined in this report. It is 48,161 bp in length, being the largest mitochondrial genome among reported shellfish at present. The entire mitochondrial genome consists of 57 genes including 12 protein-coding genes, 2 ribosomal RNAs and 41 transfer RNAs.