Insulin-like androgenic gland hormone from the shrimp Fenneropenaeus merguiensis: Expression, gene organization and transcript variants.

Male sex differentiation in the crustacean is best known to be controlled by the insulin-like androgenic gland hormone (IAG). In this report, the cDNA and gene of the shrimp Fenneropenaeus merguiensis FmIAG were studied and characterized. FmIAG gene shares a high sequence identity in the coding region as well as the promoter region with that of F. chinensis. FmIAG gene is most likely consists of 5 exons and 4 introns. The cDNA reported here is the most abundant transcript that retained cryptic intron 4. The use of different splicing acceptor sites in exon 2 can produce a long-form FmIAG transcript variant with 6 additional amino acids inserted. Splicing of cryptic intron 4 would produce a transcript variant with a different C-terminal end. Therefore 4 different FmIAG transcripts can be produced from the FmIAG gene. During the molt cycle, the expression level of FmIAG was low in the early intermolt, increase steadily towards the late premolt and decreased rapidly in the early postmolt. In addition to the androgenic gland, FmIAG is also expressed in the hepatopancreas and ovary of adult females. Unilateral eyestalk ablation caused a significant increase in FmIAG transcript suggesting that the eyestalk consists of inhibiting factor(s) that suppressesFmIAGexpression. To explore the function of FmIAG in males, injection of FmIAG dsRNA knock-down the expression of FmIAG and up-regulated the expression of the vitellogenin gene in the testis and hepatopancreas. Interestingly a CHH-like gene identified in the androgenic gland was down-regulated. CHH-like gene knock-down resulted in altered expression of FmIAG in males suggesting that the CHH-like may be involved in FmIAG's regulation. RT-PCR with specific primers to the different transcript variant were used to determine if there is an association of different sizes of male and the type of IAG transcript. Results indicated that a high percentage of the large male shrimp expressed the long-form of FmIAG. The results suggested that FmIAG may be useful as a size marker for male shrimp aquaculture. In summary, the results of this study have expanded our knowledge of shrimp insulin-like androgenic gland hormone in male sex development and its potential role as a marker gene for growth regulation in shrimp.

Molecular characterization, RNA interference and recombinant protein approach to study the function of the putative Molt Inhibiting Hormone (FmMIH1) gene from the shrimp Fenneropenaeus merguiensis.

The Molt Inhibiting Hormone gene and cDNA of the banana shrimp Fenneropenaeus merguiensis (FmMIH1) has been cloned and characterized. FmMIH1 possesses most of the characteristics of the eyestalk CHH/MIH/GIH family subtype-II neuropeptides. FmMIH1 open reading frame consists of 315 bp encoding for 105 amino acid residues. The mature peptide of FmMIH1 consists of 76 amino acid residues, a glycine residue at position 11 of the mature peptide and 6 cysteine residues located in the conserved position. In addition to eyestalk, high levels of FmMIH1 transcript could also be detected in the intestine. FmMIH1 transcript level is low throughout the post-molt, early to mid-intermolt and premolt. However, a sharp increase could be observed in late intermolt (C3 stage). Both alignment and phylogenetic analysis reveal that FmMIH1 is most similar to the MIH1 of other shrimps. For functional assay, RNA interference results show that a significant 2.3 days (P < 0.05) reduction in molt cycle duration could be observed in shrimp receiving dsFmMIH1 injection. Surprisingly, injection of recombinant FmMIH1 could also cause a significant reduction of the molt cycle (average 1.9 days, P < 0.05). We hypothesize that the recombinant protein is biological inactive but it competes with the endogenous MIH for carrier protein binding and consequently reduces the amount of biological MIH that could reach the targets. In conclusion, the result of this study will provide us new insight in molting/growth control in crustacean.

Functional and evolutionary implications from the molecular characterization of five spermatophore CHH/MIH/GIH genes in the shrimp Fenneropenaeus merguiensis.

The recent use of RNA-Seq to study the transcriptomes of different species has helped identify a large number of new genes from different non-model organisms. In this study, five distinctive transcripts encoding for neuropeptide members of the CHH/MIH/GIH family have been identified from the spermatophore transcriptome of the shrimp Fenneropenaeus merguiensis. The size of these transcripts ranged from 531 bp to 1771 bp. Four transcripts encoded different CHH-family subtype I members, and one transcript encoded a subtype II member. RT-PCR and RACE approaches have confirmed the expression of these genes in males. The low degree of amino acid sequence identity among these neuropeptides suggests that they may have different specific function(s). Results from a phylogenetic tree analysis indicated that these neuropeptides were likely derived from a common ancestor gene resulting from mutation and gene duplication. These CHH-family members could be grouped into distinct clusters, indicating a strong structural/functional relationship among these neuropeptides. Eyestalk removal caused a significant increase in the expression of transcript 32710 but decreases in expression for transcript 28020. These findings suggest the possible regulation of these genes by eyestalk factor(s). In summary, the results of this study would justify a re-evaluation of the more generalized and pleiotropic functions of these neuropeptides. This study also represents the first report on the cloning/identification of five CHH family neuropeptides in a non-neuronal tissue from a single crustacean species.

Effects of artificial infection of Litopenaeus vannamei by Micrococcus lysodeikticus and WSSV on the activity of immunity related enzymes.

In this study, the activities of 5 immunity related enzymes namely acid phosphatase (ACP), alkaline phosphatase (AKP), phenoloxidase (PO), peroxidase (POD) and lysozyme phosphatase (LZM)) of Litopenaeus vannamei after they have been injected with different concentrations of Micrococcus lysodeikticus and the white spot syndrome virus (WSSV) were examined. The cumulative mortality at 0, 24, 48, 72, 96 h was obtained. Copy numbers of WSSV in L. vannamei after a single infection, secondary infection and concurrent infection were measured. Hemolymph samples of M. lysodeikticus and WSSV injected shrimp were collected at 0, 6, 12 24, 48, 72, 78, 84, 96 and 120 h. The results were: (i) The cumulative mortality of L. vannamei increased as the shrimp were infected with higher concentration of the bacteria; (ii) The most sensitive changes of ACP, AKP and LZM were in the 6.2 × 10(5), 6.2 × 10(6), 6.2 × 10(7) cfu/mL M. lysodeikticus group; (iii) ACP but LZM were more sensitive to M. lysodeikticus than WSSV, and AKP, PO and POD is more sensitive to WSSV; (iv) The copies of WSSV in the co-injected group were higher than WSSV-single infection and WSSV-bacteria-secondary infection group at 48 h. The amount of WSSV in L. vannamei of concurrent infection and WSSV-bacteria-secondary infection groups were higher than that of the WSSV-single infection group.

PmPPAF is a pro-phenoloxidase activating factor involved in innate immunity response of the shrimp Penaeus monodon.

One of the major steps in the innate immune response of shrimp includes the activation of serine proteinases of the pro-phenoloxidase pathway by the prophenoloxidase activation enzyme (PPAF). In this study, the cDNA encoding a serine proteinase homologue (SPH) with prophenoloxidase activating activity of Penaeus monodon (PmPPAF) was cloned and characterized. PmPPAF cDNA consists of 1444 nucleotides encoding a protein with 394 amino acid residues. The estimated molecular weight of PmPPAF is 43.5 kDa with an isoelectric point of 5.19. PmPPAF consists of a signal peptide, a CLIP domain and a carboxyl-terminal trypsin-like serine protease domain. It is highly similar to the masquerade-like protein 2A (61% similarity) of the crayfish Pacifastacus leniusculus, other serine proteases (42.9-67% identity) of P. monodon, and the PPAF of the crab (61% similarity). Unlike other SPH of P. monodon, which express mainly in the hemocytes, PmPPAF transcripts were detected in the hemocytes, eyestalk, hypodermis, gill, swimming leg and brain. Similar to the crab PPAF, PmPPAF transcript level is high in shrimp at the premolt stages and PmPPAF expression is up-regulated in shrimp infected with white spot syndrome virus (WSSV). Gene silencing of PmPPAF decreased expression of a prophenoloxidase-like gene and injection of Anti-PmPPAF antibody causes a decrease in PO activity. Taken together, these results provided evidence that PmPPAF is a serine proteinase homologue, and is involved in the pro-PO activation pathway of the shrimp innate immune system.