miR-34 regulates reproduction by inhibiting the expression of MIH, CHH, EcR, and FAMeT genes in mud crab Scylla paramamosain.

Mud crab Scylla paramamosain is a commercially important species widely cultured in China. It is well known that the eyestalk regulates reproductive activities in crustaceans. In our previous research, we found that the miR-34 expression level in male eyestalk was significantly higher than that in females. Thus, we assumed that it may play an important role in regulating reproduction. In this study, we used bioinformatic tools to identify the target genes of miR-34 in eyestalk. Six reproduction-related genes with an intact 3'-untranslated region (UTR), including molt-inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), vitellogenesis-inhibiting hormone, red pigment concentrating hormone, ecdysone receptor (EcR), and farnesoic acid methyltransferase (FAMeT) were identified. When the 3'-UTR plasmid vectors of the six genes were cotransfected with miR-34 mimics into 293FT cells, respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly decreased compared with that in the control group; on the contrary, when the six plasmid vectors were cotransfected with the miR-34 inhibitor respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly higher than that in the control group. When agomiR-34 and antagomiR-34 were injected into the eyestalk respectively in vivo, the expression levels of the MIH, CHH, EcR, and FAMeT genes were detected by a quantitative real-time polymerase chain reaction. The results showed that agomiR-34 suppressed the expression of the four genes, whereas antagomiR-34 enhanced their expression. These experimental results confirmed our hypothesis that miR-34 may indirectly regulate reproduction via binding to the 3'-UTRs of MIH, CHH, EcR, and FAMeT genes and suppressing their expression.

Identification and comparative analysis of the ovary and testis microRNAome of mud crab Scylla paramamosain.

The role of microRNA (miRNA) in reproductive regulation is attracting increasingly more attention. In this study, we obtained 9,643,114 and 15,498,999 raw reads from the ovary and testis library of important farmed mud crab Scylla paramamosain, respectively. After data mining, a total of 4,096,464 and 11,737,973 mappable small RNA sequences remained for analysis. By mapping to the reference genome and expressed sequence tag (EST) of Daphnia pulex and other crabs, a total of 1,417 miRNAs were identified. On the basis of 1,417 miRNAs, 514 (36.3%) unique miRNAs coexpressed in the gonad of female and male libraries, and 336 (23.7%) and 567 (40%) expressed preferentially in female and male libraries, respectively. Analysis of library sequencing data resulted in the identification of 108 miRNAs (out of 1,417; 7.6%) that showed significant differential expression between the two samples. Of these, 13 miRNAs were expressed only in the testis, two miRNAs were expressed only in the ovary, and 93 miRNAs were coexpressed: 57 (61.3%) were upregulated (ovary/testis) and 36 (38.7%) were downregulated (ovary/testis). To confirm the expression patterns of the predicted miRNAs, we randomly selected 14 candidate miRNAs from 108 differentially expressed miRNAs and performed stem-loop real time quantitative PCR (RT-qPCR) assays in five ovary developing stages. Five miRNAs showed similar expression patterns in almost every stage as those revealed by identification of differentially expressed genes (IDEG6) analysis. The above five miRNAs were predicted to match the 3'-untranslated region of the published S. paramamosain gene. Four out of five miRNA had a regulation effect on many genes, especially the genes related to gonadal development.

Molecular Characterization and Function of Bone Morphogenetic Protein 7 (BMP7) in the Pacific Abalone, Haliotis discus hannai.

Bone morphogenetic proteins (BMPs) play important roles in a lot of biological processes, such as bone development, cell proliferation, cell differentiation, growth, etc. However, the functions of abalone BMP genes are still unknown. This study aimed to better understand the characterization and biological function of BMP7 of Haliotis discus hannai (hdh-BMP7) via cloning and sequencing analysis. The coding sequence (CDS) length of hdh-BMP7 is 1251 bp, which encodes 416 amino acids including a signal peptide (1-28 aa), a transforming growth factor-ß (TGF-ß) propeptide (38-272 aa), and a mature TGF-ß peptide (314-416 aa). The analysis of expression showed that hdh-BMP7 mRNA was widely expressed in all the examined tissues of H. discus hannai. Four SNPs were related to growth traits. The results of RNA interference (RNAi) showed that the mRNA expression levels of hdh-BMPR I, hdh-BMPR II, hdh-smad1, and hdh-MHC declined after hdh-BMP7 was silenced. After RNAi experiment for 30 days, the shell length, shell width, and total weight were found to be reduced in H. discus hannai (p < 0.05). The results of real-time quantitative reverse transcription PCR revealed that the hdh-BMP7 mRNA was lower in abalone of the S-DD-group than in the L-DD-group. Based on these data, we hypothesized that BMP7 gene has a positive role in the growth of H. discus hannai.

A Potential Negative Regulatory Function of Myostatin in the Growth of the Pacific Abalone, Haliotis discus hannai.

Myostatin, also known as GDF8, is a member of the transforming growth factor-ß (TGF-ß) superfamily. In vertebrates, myostatin negatively regulates the growth of skeletal muscle. In invertebrates, it has been reported to be closely related to animal growth. However, knowledge concerning the molecular mechanisms involved in the myostatin regulation of molluscan growth is limited. In this study, we found that the hdh-myostatin open reading frame (ORF) comprised 1470 base pairs that encoded 489 amino acids and contained structural characteristics typical of the TGF-ß superfamily, including a C-terminal signal peptide, a propeptide domain, and TGF-ß region. Gene expression analysis revealed that hdh-myostatin mRNA was widely expressed at different levels in all of the examined tissues of Haliotis discus hannai. Nine single nucleotide polymorphisms (SNPs) were associated with the growth traits. RNA interference (RNAi) against hdh-myostatin mRNA significantly downregulated hdh-myostatin at days 1, 15, and 30 post injection, and the pattern was correlated with downregulation of the genes TGF-ß receptor type-I (hdh-TßR I), activin receptor type-IIB (hdh-ActR IIB), and mothers against decapentaplegic 3 (hdh-Smad3). After one month of the RNAi experiment, the shell lengths and total weights increased in the abalone, Haliotis discus hannai. The results of qRT-PCR showed that the hdh-myostatin mRNA level was higher in the slow-growing group than in the fast-growing group. These results suggest that hdh-myostatin is involved in the regulation of growth, and that these SNPs would be informative for further studies on selective breeding in abalone.

Exploring the Intestinal Microbiota and Metabolome Profiles Associated With Feed Efficiency in Pacific Abalone (Haliotis discus hannai).

Feed efficiency (FE) is critical to the economic and environmental benefits of aquaculture. Both the intestines and intestinal microbiota play a key role in energy acquisition and influence FE. In the current research, intestinal microbiota, metabolome, and key digestive enzyme activities were compared between abalones with high [Residual feed intake (RFI) = -0.029] and low FE (RFI = 0.022). The FE of group A were significantly higher than these of group B. There were significant differences in intestinal microbiota structures between high- and low-FE groups, while higher microbiota diversity was observed in the high-FE group. Differences in FE were also strongly correlated to variations in intestinal digestive enzyme activity that may be caused by Pseudoalteromonas and Cobetia. In addition, Saprospira, Rhodanobacteraceae, Llumatobacteraceae, and Gaiellales may potentially be utilized as biomarkers to distinguish high- from low-FE abalones. Significantly different microorganisms (uncultured beta proteobacterium, BD1_7_clade, and Lautropia) were found to be highly correlated to significantly different metabolites [DL-methionine sulfoxide Arg-Gln, L-pyroglutamic acid, dopamine, tyramine, phosphatidyl cholines (PC) (16:0/16:0), and indoleacetic acid] in the high- and low-FE groups, and intestinal trypsin activity also significantly differed between the two groups. We propose that interactions occur among intestinal microbiota, intestinal metabolites, and enzyme activity, which improve abalone FE by enhancing amino acid metabolism, immune response, and signal transduction pathways. The present study not only elucidates mechanisms of variations in abalone FE, but it also provides important basic knowledge for improving abalone FE by modulating intestinal microbiota.

The expression regulation of Cyclins and CDKs in ovary via miR-9c and miR-263a of Scylla paramamosain.

Scylla paramamosain is an economically important cultured crab species in China. Cyclins and cyclin-dependent kinases (CDKs) play important roles in regulations of cell cycle and ovarian development. MiRNAs can negatively regulate gene expression at the post-transcriptional level through base-complementary pairing with the 3'-untranslated region (3-UTR) of the target gene. In this study, bioinformatics prediction showed that miR-9c and miR-263a identified from our group's gonad miRNAome of S. paramamosain may bind to the 3' UTR region of cyclin A, cyclin B, cyclin E, cyclin H, CDK1, and CDK2. Furthermore, the results of double luciferase reporter gene assay showed that the luciferase activities of HEK293T cells co-transfected with miR-9c mimics/miR-9c inhibitor and the 3'-UTR plasmid vectors of the five genes (cyclin A, cyclin B, cyclin H, CDK1, and CDK2) were significantly decreased/increased compared with those in the NC (negative control) and BC (blank control) groups. The results in miR-263a were similar to miR-9c, but all of the six genes could be regulated by miR-263a. In in vivo experiments, agomiR-9c (miR-9c enhancer) injection resulted in decreases of cyclin A and CDK1 expression level, and reverse effects were observed by injecting antagomiR-9c. AgomiR-263a decreased the expression of cyclin A, cyclin B, cyclin H, CDK1, and CDK2, but antagomiR-263a increased their expression. Both the in vitro and in vivo experiments confirmed functions of miR-9c and miR-263a in cell cycle progress of ovarian development by expression regulation of cyclin A, cyclin B, cyclin E, cyclin H, CDK1, and CDK2. The findings provide new insights into the reproductive regulation mechanism in mud crab and further enrich the knowledge of cell cycle and ovarian development regulation in invertebrates.

miR-9 and miR-263 Regulate the Key Genes of the ERK Pathway in the Ovary of Mud Crab Scylla paramamosain.

Mud crab Scylla paramamosain is one of the most important economic crabs in China. The molecular regulatory mechanism of ovarian development has received considerable attention in recent years. Some studies found that ERK (extracellular signal-regulated protein kinase) signaling pathway plays an important role in ovarian development and is negatively regulated by microRNAs (miRNAs). However, the study about the regulation of miRNA on the ERK pathway in crustacean's ovary remains unknown. In this study, the target genes of the ERK signaling pathway regulated by selected miRNAs identified from the ovary of mud crab in our previous research were predicted by using bioinformatics tools. The results showed that the ERK2 might be a target gene of miR-9c, miR-263a, and miR-263b; MEK2 may be a target gene of miR-263a; and Rap-1b may be a target gene of miR-9, miR-9c, and miR-263a. Results of in vitro dual-luciferase reporter assay showed that the relative luciferase activities were significantly lower in HEK293T cells co-transfected with the combination of miRNA mimics and pmir-RB-REPORTTM-target gene-3'UTR than those with the combination of mimics NC and pmir-RB-REPORTTM-target gene-3'UTR. In contrast, the relative luciferase activities were significantly higher in HEK293T cells co-transfected with miRNA inhibitor than those with inhibitor NC. To further validate in vitro results, the miRNA reagents were injected into the living female mud crabs, and the expression levels of miRNAs and target genes after the injection were analyzed by quantitative real-time PCR. The in vivo experimental results showed that miRNAs (miR-9c/miR-263a) agomir (enhancers)/antagomir (inhibitors) can enhance/decrease the expression of two miRNAs, respectively, and the expression of target genes in the ovary was declined/increased after injection of agomir/antagomir reagent. In conclusion, miR-9/miR-263 can negatively regulate the expression of the ERK pathway genes (ERK2, MEK2, and Rap-1b) in the ovary of mud crab.