Apinunt Udomkit

Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference

One of the important peptide hormones that control reproduction in crustaceans is gonad‐inhibiting hormone (GIH). GIH is known to modulate gonad maturation by inhibiting synthesis of vitellogenin (Vg), the precursor of yolk proteins. In this study, a cDNA encoding a GIH (Pem‐GIH) from the eyestalk of Penaeus monodon was cloned using RT‐PCR and RACE techniques. Pem‐GIH cDNA is 861 bp in size with a single ORF of 288 bp. The deduced Pem‐GIH consists of a 17‐residue signal peptide and a mature peptide region of 79 amino acids with features typical of type II peptide hormones from the CHH family. Pem‐GIH transcript was detected in eyestalk, brain, thoracic and abdominal nerve cords of adult P. monodon. The gonad‐inhibiting activity of Pem‐GIH was investigated using the RNA interference technique. Double‐stranded RNA, corresponding to the mature Pem‐GIH sequence, can trigger a decrease in Pem‐GIH transcript levels both in eyestalk ganglia and abdominal nerve cord explant culture and in female P. monodon broodstock. The conspicuous increase in Vg transcript level in the ovary of GIH‐knockdown shrimp suggests a negative influence for Pem‐GIH on Vg gene expression, and thus implies its role as a gonad‐inhibiting hormone. This is the first report to demonstrate the use of double‐stranded RNA to elucidate the function of GIH in P. monodon.

Characterization of Argonaute cDNA from Penaeus monodon and implication of its role in RNA interference.

RNA interference (RNAi) has recently become a promising strategy for therapeutic of several viral diseases including those in the black tiger shrimp Penaeus monodon. However, the protein components that play role in RNAi in P. monodon have not yet been identified. Here, we report the cloning and functional characterization of a cDNA encoding Argonaute, a principal constituent of RNAi pathway in P. monodon. P. monodons Argonaute (Pem-AGO) exhibited the two signature domains, PAZ and PIWI. Substantial level of Pem-ago expression could be suppressed by double-stranded RNA (dsRNA) that targeted PAZ coding sequence in shrimp primary culture of Oka cells. The Pem-ago depleted cells showed impaired RNAi as the expression of an endogenous gene was rescued from the dsRNA-mediated silencing in these cells. Our results imply that Pem-ago is required for effective RNAi in P. monodon and thus identify the first protein constituent of RNAi machinery in penaeid shrimp.

Molecular cloning of a cDNA encoding a member of CHH/MIH/GIH family from Penaeus monodon and analysis of its gene structure

We report the nucleotide and deduced amino acid sequences of Pem-CMG peptide, a member of crustacean CHH/MIH/GIH peptide family, in black tiger prawn (Penaeus monodon). The 5′ and 3′ fragments of Pem-CMG cDNA were cloned by the method of rapid amplification of cDNA ends (RACE). The two fragments constitute a combined cDNA length of 593 bp with a 77 bp overlapping region. Sequence analysis reveals the presence of a 384 bp open reading frame which was subsequently cloned. The open reading frame encodes a precursor peptide that is comprised of 128 amino acids, with a putative processing site, KR. The mature peptide consists of 74 amino acid residues, the sequence of which is significantly homologous to those of the CHH/MIH/GIH family known from other crustaceans. Analysis of a genomic fragment of Pem-CMG reveals a single intron of 314 bp interrupting the coding sequence for the mature peptide. The presence of only one intron in Pem-CMG gene suggests that this gene is structurally different from the previously reported MIH gene of Charybdis feriatus and CHH-like gene of Metapenaeus ensis which possess two introns in their coding sequences.

Expression of Biologically Active Crustacean Hyperglycemic Hormone (CHH) of Penaeus monodon in Pichia pastoris

Crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), and gonad-inhibiting hormone (GIH) are members of a major peptide family produced from the X-organ sinus gland complex in the eyestalk of crustaceans. This peptide family plays important roles in controlling several physiologic processes such as regulation of growth and reproduction. In this study the complementary DNA encoding a peptide related to the CHH/MIH/GIH family (so-called Pem-CMG) of the black tiger prawn Penaeus monodon was successfully expressed in the yeast Pichia pastoris under the control of the AOX1 promoter. The recombinant Pem-CMG was secreted into the culture medium using the α-factor signal sequence; of Saccharomyces cerevisiae without the Glu-Ala-Glu-Ala spacer peptide. The amino terminus of the recombinant Pem-CMG was correctly processed as evidenced by amino-terminal peptide sequencing. The recombinant Pem-CMG was purified by reverse-phase high-performance liquid chromotography and used in a biological assay for CHH activity. The final yield of the recombinant Pem-CMG after purification was 260 µg/L of the culture medium. Both crude and purified recombinant Pem-CMG produced from P. pastoris showed the ability to elevate the glucose level in the hemolymph of eyestalk-ablated P. monodon, which demonstrates that Pem-CMG peptide functions as hyperglycemic hormone in P. monodon.

Double-stranded RNA confers both preventive and therapeutic effects against Penaeus stylirostris densovirus (PstDNV) in Litopenaeus vannamei

Penaeus stylirostris densovirus (PstDNV) infection is found widespread in peneaid shrimp, especially in economically important species such as black tiger shrimp Penaeus monodon and Pacific white shrimp Litopenaeus vannamei. Although effective prevention method for viral diseases is not well established in shrimp, the treatment with viral specific double-stranded RNA (dsRNA) or siRNA has given promising results. In present study, dsRNAs corresponding to non-structural (ORF1 and ORF2 overlapping sequence) and structural (ORF3) genes of PstDNV were investigated for their potency to inhibit PstDNV replication in the shrimp. Periodically injection of either ORF1-2 dsRNA or ORF3 dsRNA at three days interval into L. vannamei resulted in substantial inhibition of PstDNV infection. In addition, a possibility for a therapeutic application of dsRNA in PstDNV-infected shrimp was demonstrated by the efficient suppression of PstDNV replication in L. vannamei when the ORF1-2 dsRNA was delivered into the shrimp within 24h post-PstDNV injection. Hence, our results established both the preventive and therapeutic potency of dsRNA to inhibit PstDNV in L. vannamei that could be applied as a potential treatment of PstDNV infection in shrimp.

Molecular cloning and functional characterization of Argonaute-3 gene from Penaeus monodon.

Argonaute (Ago) proteins play a crucial role in the shrimp RNA interference pathway. In this study, we identified and characterized a novel Ago gene from black tiger shrimp, Penaeus monodon. The complete open reading frame of P. monodon Ago3 (PmAgo3) consisted of 2559 nucleotides encoding a polypeptide of 852 amino acids with a predicted molecular weight of 97 kDa and an isoelectric point of 9.42. Analysis of the deduced amino acid sequence of PmAgo3 revealed the presence of two signature domains of the proteins in Argonaute family including PAZ and PIWI. Phylogenetic analysis indicated that PmAgo3 is classified into Ago subfamily and shared the highest amino acid sequence identity (83%) with Litopenaeus vannamei Ago2. Monitoring of the PmAgo3 expression by quantitative real-time PCR revealed that this gene was significantly up-regulated following dsRNA administration, while no significant difference in its expression was observed following yellow head virus (YHV) challenge. In contrast, inhibition of YHV mRNA expression was observed in PmAgo3-knockdown shrimp. These data imply that PmAgo3 is involved in the dsRNA-mediated gene silencing mechanism and plays an important role in YHV replication in the black tiger shrimp.

Potential roles of transglutaminase and thioredoxin in the release of gonad-stimulating factor in Penaeus monodon: implication from differential expression in the brain during ovarian maturation cycle.

The synthesis of vitellogenin during ovarian maturation in crustacean is induced by gonad-stimulating factor(s) that are synthesized in the brain and thoracic ganglia. This process is negatively regulated by a gonad-inhibiting hormone (GIH) from the eyestalk. This study utilized differential-display RT-PCR technique to identify putative genes in brain and thoracic ganglia that may be involved in ovarian maturation of the black tiger shrimp, Penaeus monodon under the condition in which the expression of GIH was suppressed by GIH-specific dsRNA. After excluding redundant clones and subsequent verification by RT-PCR, 10 and 5 transcripts exhibited up-regulated and down-regulated expressions, respectively, in the GIH-dsRNA injected shrimp when compared with the Tris/NaCl injected shrimp. Among the up-regulated genes, a full sequence of thioredoxin cDNA was cloned, and nucleotide sequence analysis showed that it was highly similar to other crustacean thioredoxin. The thioredoxin gene as well as the other four genes including transglutaminase and three unknowns; U10-11, U10-15 and U13-11 that were up-regulated upon GIH-knockdown exhibited similar expression profile in the brain during ovarian maturation cycle. The highest expression level was detected in the brain of early-vitellogenic female shrimp suggesting that they are required for an initial stage of vitellogenesis. Our results posted for the first time a possible function of transglutaminase and thioredoxin in regulating the gonad-stimulating pathway in the brain of the shrimp.

Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei.

Protection of shrimp from yellow head virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA delivery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05μg dsYHV/g shrimp could give comparable level of YHV protection to the injection with 1.25μg naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25μg dsRNA/g shrimp showed only 50% mortality at 60days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encapsulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4days in the total of 960μg dsRab7/g shrimp could enhance YHV protection efficiency compared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems.

A novel gonad-specific Argonaute 4 serves as a defense against transposons in the black tiger shrimp Penaeus monodon

Argonaute is a key protein of the small-RNA guided gene regulation process. The Argonaute family is generally divided into two subfamilies; AGO and PIWI. In this study, a cDNA encoding a novel type of Argonaute (PmAgo4) in the black tiger shrimp Penaeus monodon was identified and characterized. PmAgo4 cDNA contained an open reading frame of 2433 nucleotides that can be translated into a deduced amino acid with the conserved PAZ and PIWI domains. PmAgo4 was phylogenetically clustered with the AGO subfamily while exhibited a gonad-specific expression pattern similar to that of proteins in the PIWI subfamily. The expression of PmAgo4 did not change significantly in response to either double-stranded RNA or yellow head virus injection suggesting that PmAgo4 may not be the main AGO proteins that play a role in dsRNA-mediated gene silencing or antiviral defense. Interestingly, PmAgo4 appeared to participate in the control of transposons since the activation of both DNA transposon and retrotransposon was detected in the testis of PmAgo4-knockdown shrimp. Our study thus provided the first evidence for an unusual type of the AGO proteins that was predominantly expressed in shrimp gonad and implication of its role in protecting the shrimp genome against an invasion of transposons.

Molecular characterization of a cDNA encoding red pigment-concentrating hormone in black tiger shrimp Penaeus monodon: Implication of its function in molt and osmoregulation.

Red pigment-concentrating hormone (RPCH) is a member of the AKH/RPCH peptide family present mainly in crustaceans and insects. Insect AKH is responsible for metabolic functions whereas RPCH plays a major role in the aggregation of red chromatophores in crustaceans. In this study, a full-length cDNA of RPCH of the black tiger shrimp, Penaeus monodon (PmRPCH) was cloned by Rapid Amplification of cDNA Ends strategies from the eyestalk RNA. A 770 bp full-length PmRPCH cDNA harbored 279 bp of an open reading frame encoding a signal peptide of 21 amino acid residues, an 8 amino acid mature RPCH peptide, followed by 61 amino acid residues of a RPCH precursor-related peptide. The highest levels of PmRPCH mRNA expression were detected in eyestalks while lower expression was found in other nervous tissues i.e. brain, thoracic ganglia and abdominal nerve cord. Expression of PmRPCH was transiently stimulated upon hypersalinity change within 12 h suggesting its osmoregulatory function. During the molting cycle, PmRPCH in the eyestalk was expressed at the lowest level in the early pre-molt stage (D0), then gradually increased over the pre-molt period and reached the highest level in the late pre-molt (D4) and post-molt (AB) stages. RPCH peptide at a dose of 100 pmol also increased gill Na(+)/K(+) ATPase activity in 36-48 h after injection. However, PmRPCH did not accelerate the duration of molting cycle. Our results provide the first evidence on the potential function of PmRPCH in molting, probably by mediating hemolymph osmolality and ion transport enzymes during the late pre-molt stage.