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ABSTRACT: 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.
Marine Genomics 12/2011; 4(4):279-85. · 1.55 Impact Factor
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ABSTRACT: 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.
Virus Research 01/2011; 155(1):131-6. · 2.94 Impact Factor
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ABSTRACT: Ovarian maturation in crustacean is under the control of gonad-inhibiting hormone (GIH); a neuropeptide secreted from X-organ sinus gland complex in eyestalks. Unilateral eyestalk ablation that partially destroys GIH source is therefore a general practice in Penaeus monodon hatchery to induce ovarian maturation and spawning. Our previous report showed that silencing of GIH expression by GIH-specific double-stranded RNA (GIH-dsRNA) resulted in an increased expression level of vitellogenin in P. monodon, thus suggesting that GIH-dsRNA could be an alternative method to induce ovarian maturation in female P. monodon broodstock. In this study, we further demonstrated that a single injection of GIH-dsRNA into previtellogenic female P. monodon at the concentration of 3 µg GIH-dsRNA per gram body weight of shrimp was able to inhibit GIH expression for a minimum of 30 days. This dsRNA-mediated GIH silencing led to ovarian maturation and eventual spawning in both domesticated and wild female broodstock, particularly with a comparable effect to eyestalk ablation in wild shrimp. This is the first report that demonstrates a potential strategy to induce ovarian maturation in female P. monodon broodstock by GIH-dsRNA and thus provides a possible substitute for the cruel and detrimental eyestalk ablation practice.
Marine Biotechnology 03/2010; 13(2):163-9. · 3.43 Impact Factor
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ABSTRACT: 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.
FEBS Journal 04/2008; 275(5):970-80. · 3.79 Impact Factor
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ABSTRACT: 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. monodon's 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.
Biochemical and Biophysical Research Communications 04/2008; 367(4):768-74. · 2.48 Impact Factor
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ABSTRACT: Crustacean hyperglycemic hormone (CHH) plays a major role in controlling glucose level in the haemolymph and also triggers important events during molting and reproductive cycles. In Penaeus monodon, three types of CHH, namely Pem-CHH1, Pem-CHH2 and Pem-CHH3, have been previously characterized. In this study, mouse polyclonal antibody was raised against recombinant Pem-CHH1 that was expressed in Escherichia coli. The anti-Pem-CHH1 antibody recognized all three types of Pem-CHHs but did not cross-react with either related hormone, molt-inhibiting hormone of P. monodon, or unrelated human growth hormone. The hyperglycemic activity in the extract from the eyestalk neural tissues was significantly depleted after incubating with anti-Pem-CHH antibody. Direct injection of the antibody into shrimp caused about 30-50% reduction in the haemolymph glucose level. The result demonstrates the ability of anti-Pem-CHH1 antibody to deplete the activity of CHH in vivo, and thus provides a possibility of using anti-Pem-CHH1 antibody to inhibit the hormone activity as a strategy to modulate growth and reproduction in this species.
Journal of biochemistry and molecular biology 08/2006; 39(4):371-6. · 2.02 Impact Factor
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ABSTRACT: The Crustacean hyperglycemic hormone (CHH) has been shown to exist as multiple molecular forms in several crustacean species. In Penaeus monodon, a gene encoding CHH (so-called Pem-CHH1) was recently described. In this study, the molecular structures of two other CHH genes (Pem-CHH2 and Pem-CHH3) are reported. Both the Pem-CHH2 and Pem-CHH3 genes contain three exons that are separated by two introns that are similar to the structure of other genes in the same family. An analysis of the upstream nucleotide sequences of each Pem-CHH gene has identified the putative promoter element (TATA box) and putative binding sites for several transcription factors. The binding sites for CREB, Pit-1, and AP-1 were found upstream of all three Pem-CHH genes. A Southern blot analysis showed that at least one copy of each Pem-CHH gene was located within the same 10 kb genomic DNA fragment. These results suggest that the CHH genes are arranged in a cluster in the genome of P. monodon, and that their expression may be modulated by similar mechanisms.
Journal of biochemistry and molecular biology 04/2004; 37(2):177-84. · 2.02 Impact Factor
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ABSTRACT: The CHH/MIH/GIH peptide family of black tiger prawn (Paneaus monodon) is important in shrimp reproduction and growth enhancement. In this study, the cDNA that encodes the complete peptide that is related to the CHH/MIH/GIH family (so-called, Pem-CMG) in the eyestalk of P. monodon was successfully expressed in a methylotrophic yeast Pichia pastoris under the control of an alcohol oxidase promoter. In order to obtain the secreted Pem-CMG, a secretion signal of either the Saccharomyces cerevisiae alpha-factor or Pem-CMG was employed. The results demonstrated that alphaPem-CMG, either with (alpha2EACMG) or without (alphaCMG) the Glu-Ala repeats, was secreted into the medium, while Pem-CMG with its own secretion signal failed to be secreted. The total protein amount that was secreted from the transformant that contained either alpha2EACMG or alphaMG was approximately 60 mg/l and 150 mg/l, respectively. The N-terminus of the Pem-CMG peptide of both alpha2EACMG and alphaCMG was correctly processed. This produced the mature Pem-CMG peptide.
Journal of biochemistry and molecular biology 10/2002; 35(5):476-81. · 2.02 Impact Factor
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ABSTRACT: 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.
Journal of Experimental Marine Biology and Ecology.
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ABSTRACT: 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 alpha-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 micro g/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.
Marine Biotechnology 5(4):373-9. · 3.43 Impact Factor
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ABSTRACT: Crustacean hyperglycemic hormone (CHH) is the most abundant peptide in the eyestalk of crustacean. This hormone not only plays its major role in controlling glucose level in the haemolymph, but is also significant to other processes such as ecdysteroid synthesis and ovarian maturation. Multiple forms of CHH have been reported in several species. In addition to the Pem-CHH1 of Penaeus monodon recently identified, here, we report the cloning and characterization of the cDNA encoding another two Pem-CHH peptides, so-called Pem-CHH2 and Pem-CHH3. Both cDNAs contained 381-bp open reading frame encoding 127 amino acids. The cleavage at the putative processing site of the signal peptide, KR, would generate a 74 amino acids mature hormone for both Pem-CHH2 and Pem-CHH3. Amino acid sequence analysis revealed that Pem-CHH2 and Pem-CHH3 shared 95% identity in their amino acid sequences to that of Pem-CHH1. Both recombinant Pem-CHH2 and recombinant Pem-CHH3 expressed as secreted proteins in Pichia pastoris exhibited hyperglycemic activity at the comparable level to that of Pem-CHH1. Furthermore, investigation of the transcripts of each Pem-CHH in several tissues by RT-PCR showed that expression of Pem-CHH1, Pem-CHH2 and Pem-CHH3 was not restricted only to the eyestalk but also detectable in the heart. In addition, the transcript of Pem-CHH1 was also present in the gill. CHHs from various origins may play different roles and thus contribute towards its pleiotropic nature.
Journal of Experimental Marine Biology and Ecology.
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ABSTRACT: The action of molt-inhibiting hormone (MIH) on the inhibition of ecdysone release from the Y-organ of decapod crustacean keeps the animal in the intermolt stage that dominates its molting cycle. MIH is thus one of the major keys in mediating growth and reproduction. This study has isolated cDNA encoding two types of MIH, Pem-MIH1 and Pem-MIH2, from the black tiger shrimp, Penaeus monodon on the basis of sequence homology to MIH from two other shrimp species. The full-length cDNA of Pem-MIH1 was characterized. Pem-MIH1 cDNA harbored 318 bp open reading frame that coded for a translated product containing 28 amino acids of the signal peptide and a putative mature Pem-MIH of 77 amino acids. The recombinant Pem-MIH1 was expressed in Pichia pastoris as a secreted protein. After purification by gel filtration, the purified Pem-MIH1 exhibited the ability to extend molting duration of P. monodon from 11.8 days to 16.3 days suggesting that Pem-MIH1 be responsible for molt-inhibiting function in the shrimp. The attempt to clone Pem-MIH1 and Pem-MIH2 genes was achieved by direct PCR amplification and PCR-based genome walking strategy, respectively. The structure of both Pem-MIH genes, containing three exons interrupted by two introns, was similar to each other and also to that of MIH genes of other crustaceans reported so far. Expression study of Pem-MIH1 and Pem-MIH2 in various tissues of P. monodon revealed the difference in expression patterns. Pem-MIH1 expressed in both the eyestalk and the thoracic ganglia whilst Pem-MIH2 expression was limited to the eyestalk. The expression of MIH in non-eyestalk tissue may suggest additional role of this hormone.
Journal of Experimental Marine Biology and Ecology.
