Transcriptome responses of RNAi-mediated ETH knockdown in Scylla paramamosain at different premolt substages.

Ecdysis triggering hormone (ETH) plays an important role in molting, reproduction, and courtship behavior in insects. To investigate the potential downstream pathways and genes of ETH in Scylla paramamosain, RNA interference (RNAi) was conducted on crabs at early (D0) and late (D2) premolt substages, and the transcriptome profiles of each group were compared by RNA sequencing. Real-time quantitative polymerase chain reaction (RT-qPCR) and semiquantitative polymerase chain reaction (RT-PCR) results showed a significant knockdown of ETH at D0 stage, whereas a significant increase was shown conversely in crabs at D2 substage after the injection of dsETH. A total of 242,979 transcripts were assembled, and 44,012 unigenes were identified. Transcriptomic comparison between crabs at D2 and D0 substages showed 2,683 differentially expressed genes (DEGs); these genes were enriched in ribosome and pathways related to transcription factor complex and cell part. Twenty DEGs were identified between dsETH-injected and dsGFP-injected crabs at D0 substage; these DEGs were involved in carbohydrate metabolism, one carbon pool by folate, and chitin binding. Twenty-six DEGs were identified between dsETH-injected and dsGFP-injected crabs at D2 substage; these DEGs were involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction. RT-qPCR verified the differential expression of the selected genes. In conclusion, crabs at D0 substage are more active in preparing the macromolecular complex that is needed for molting. Moreover, ETH has potential roles in carbohydrate metabolism, one carbon pool by folate, and chitin binding for crabs at D0 substage, while the role of ETH turns to be involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction at D2 substage to facilitate the occurrence of molting. The selected DEGs provide valuable insight into the role of ETH in the regulation of crustacean molting.

Characterization of a molt-related myostatin gene (FmMstn) from the banana shrimp Fenneropenaeus merguiensis.

Myostatin is an important member of the transforming growth factor (TGF) family that functions to regulate muscle growth in animals. In this study, the myostatin gene (FmMstn) and two slightly different (short and long forms) cDNAs of the banana shrimp Fenneropenaeus merguiensis were cloned and characterized. Similar to Mstn gene of the scallop, fish and mammal, FmMstn gene consists of 3 exons and 2 introns. The 2kb upstream promoter region of the FmMstn gene consists of putative response elements for myocyte enhancing factor (MEF2) and E-box factors. The longest open reading frame of the short Mstn consists of 1260bp encoding for a protein with 420 amino acid residues. The long FmMstn is almost identical to the short FmMstn with the exception of 8 amino acid insertions. FmMstn is most similar to the Mstn of Litopenaeus vannamei and Penaeus monodon sharing >92-98% amino acid sequence identity. Multiple sequence alignment results revealed high degree of amino acid conservation of the cysteine residues and mature peptide of the FmMstn with Mstn from other animals. FmMstn transcript was detected in the heart, muscle, optic nerve and thoracic ganglion. FmMstn transcript level in muscle is higher in early postmolt, decreases in intermolt and increases again towards ecdysis. Higher expression level of FmMstn is also observed in smaller shrimp of the same age. Knock-down of FmMstn gene by RNAi can cause a significant increase in molt cycle duration and failure of some shrimp to undergo ecdysis. Direct DNA sequencing results revealed that FmMstn gene is highly polymorphic and several potential SNPs have been identified. Some SNPs are associated with the size difference of the shrimp. In summary, the result of this study indicates that shrimp FmMstn gene is molt/growth-related and the presence of SNP suggests that it could be a candidate gene for shrimp genetic improvement research.

Identification of mud crab reovirus VP12 and its interaction with the voltage-dependent anion-selective channel protein of mud crab Scylla paramamosain.

Mud crab reovirus (MCRV) is the causative agent of a severe disease in cultured mud crab (Scylla paramamosain), which has caused huge economic losses in China. MCRV is a double-stranded RNA virus with 12 genomic segments. In this paper, SDS-PAGE, mass spectrometry and Western blot analyses revealed that the VP12 protein encoded by S12 gene is a structural protein of MCRV. Immune electron microscopy assay indicated that MCRV VP12 is a component of MCRV outer shell capsid. Yeast two hybrid cDNA library of mud crab was constructed and mud crab voltage-dependent anion-selective channel (mcVDAC) was obtained by MCRV VP12 screening. The full length of mcVDAC was 1180 bp with an open reading frame (ORF) of 849 bp encoding a 282 amino acid protein. The mcVDAC had a constitutive expression pattern in different tissues of mud crab. The interaction between MCRV VP12 and mcVDAC was determined by co-immunoprecipitation assay. The results of this study have provided an insight on the mechanisms of MCRV infection and the interactions between the virus and mud crab.

Characterization of the shrimp neuroparsin (MeNPLP): RNAi silencing resulted in inhibition of vitellogenesis.

The full-length Metapenaeus ensis neuroparsin (MeNPLP) cDNA was cloned which encodes a shrimp protein homologous to the insect neuroparsin and vertebrate insulin-like growth factor binding protein (IGFBP). MeNPLP cDNA is 1389 bp in length and the longest open reading frame is 303 bp in length. The first 27 aa are predicted to be the signal peptide and aa 28-101 is the mature peptide with an estimated molecular weight of 7.83 kDa and pI of 5. It shows high amino acid sequence similarity (42-68%) to the neuroparsin of insects and N-terminal end of the IGFBP of vertebrates. The cysteine residues in MeNPLP responsible for disulfide bond formation are conserved as in other neuroparsin-like proteins. The expression level of MeNPLP is the highest in the hepatopancreas, followed by the nerve cord, brain, heart, ovary, and muscle. However, it was not expressed in the testis. Using an insect neuroparsin antibody, MeNPLP could only be detected in the hepatopancreatic tubules, suggesting that MeNPLP may be a secretary product. Although MeNPLP expression was stimulated in the ovary, it was inhibited in the hepatopancreas after treatment with neurotransmitter serotonin (5-HT). In vivo gene silencing of MeNPLP could cause a significant decrease of vitellogenin transcript level in the hepatopancreas and ovary. As a result, a corresponding decrease in vitellogenin protein level was observed in the hemolymph and ovary. In conclusion, this study has provided the first evidence that MeNPLP is involved in the initial stage of ovary maturation in shrimp.

The shrimp heat shock cognate 70 functions as a negative regulator in vitellogenin gene expression.

Within the 2.6-kb 5' flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2), several clusters of putative heat shock factor (HSF) response elements were identified. Deletion of these response elements has caused significant increases in MeVg2 promoter activity, suggesting that the HSF and Hsc70 complex may regulate vitellogenin gene expression in a negative manner. To confirm the role of Hsc70 in the regulation of vitellogenin gene expression, the ovary cDNA for Hsc70 was cloned and characterized. The Hsc70 transcript level was high in the ovary and hepatopancreas of females at the early vitellogenic stage but dropped during ovarian maturation. In addition, Western blot analysis revealed the presence of Hsc70 in the nuclear but not in the cytoplasmic fraction during the early stage of ovary maturation. Electrophoretic mobility shift assay (EMSA) results showed that ovary nuclear extract contained a factor that binds to the HSF response element. Since the addition of ATP caused a decrease in the binding of Hsc70, Hsc70 may form a repressor complex with HSF to inhibit MeVg2 expression. An in vitro RNA interference technique was used to study the gene function of Hsc70. Hsc70 gene knockdown resulted in an increased MeVg2 mRNA level in the ovary (54%) and hepatopancreas (62%). In summary, this report describes the first study of vitellogenin gene regulation at the transcription level in crustaceans and provides strong evidence that Hsc70 acts as a molecular chaperone to negatively regulate MeVg2 gene expression in shrimp.