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Siuming Chan - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression study of the lobster homarus americanus vitellogenin conservation in gene structure among decapods
General and Comparative Endocrinology, 2009Co-Authors: Shirley H K Tiu, Ho Lam Hui, Brian Tsukimura, Stephen S Tobe, Siuming ChanAbstract:This study reports the molecular characterization of the vitellogenin (Vg) of the lobster, Homarus americanus. Based on the annual collection of female lobsters, vitellogenesis commences in early March and continues through to September of each year. Using an antibody to vitellin of the lobster, H. americanus, several immunoreactive ovarian proteins were initially identified by Western blot analysis. The 80kDa protein contained the amino acid sequence APWGGNTPRC, identified subsequently by cDNA cloning to be identical to the lobster Vg. In common with the shrimp Metapenaeus Ensis and crab Charybdis feriatus, the lobster HaVg1 gene comprises 14 introns and 15 exons. The deduced HaVg1 precursor is most similar to the Vg of the crayfish Cherax quadricarinatus (57%), followed by M. Ensis (40-43% identity) and C. feriatus (38%). The results from genomic and RT-PCR cloning also confirmed the presence of multiple Vg genes in lobster. At early reproductive stages, the hepatopancreas HaVg1 transcript levels are low but increased to a maximum in animals with mature oocytes. The ovary, however, also expressed low levels of HaVg1. Using in vitro explant culture, treatment of hepatopancreas fragments with farnesoic acid or 20-hydroxyecdysone resulted in a significant stimulation in HaVg1 expression. From this study, it appears that Vg gene organization and expression pattern in decapods is highly conserved. Similar endocrine mechanisms may govern the process of vitellogenesis across the decapods.
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the use of recombinant protein and rna interference approaches to study the reproductive functions of a gonad stimulating hormone from the shrimp metapenaeus Ensis
FEBS Journal, 2007Co-Authors: Shirley H K Tiu, Siuming ChanAbstract:Although the crustacean crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone neuropeptides have been studied extensively in the last two decades and several neuropeptides from the shrimp Metapenaeus Ensis have been cloned, the functions of most of these neuropeptides remained putative. In this article, we describe the use of recombinant protein and an RNA interference approach to study the reproductive function of the previously reported molt-inhibiting hormone (MeMIH-B) in M. Ensis. When hepatopancreas and ovary explants were cultured in medium containing recombinant MeMIH-B, the vitellogenin gene (MeVg1) expression level was upregulated in a dose-dependent manner, reaching a maximum in explants treated with 0.3 nm recombinant MeMIH-B. Shrimp injected with recombinant MeMIH-B showed an increase in vitellogenin gene expression in the hepatopancreas. Moreover, a corresponding increase in the vitellogenin-like immunoreactive protein was detected in the hemolymph and ovary of these females. Injection of MeMIH-B dsRNA into the female shrimp caused a decrease in MeMIH-B transcript level in thoracic ganglion and eyestalk. These shrimp also showed reduction of vitellogenin gene expression in the hepatopancreas and ovary. Furthermore, the hemolymph vitellogenin level was also reduced in these animals. In summary, the results from recombinant protein and RNA interference experiments have demonstrated the gonad-stimulatory function of MeMIH-B in shrimp.
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vitellogenesis in the red crab charybdis feriatus hepatopancreas specific expression and farnesoic acid stimulation of vitellogenin gene expression
Molecular Reproduction and Development, 2005Co-Authors: Abby S C Mak, Shirley H K Tiu, Stephen S Tobe, Chi Lung Choi, Jerome H L Hui, Siuming ChanAbstract:Vitellogenesis in the mature female crab Charybdis feriatus occurs all year round during which active synthesis of the vitellogenin (Vg) precursor occurs. Several polypeptides from the ovaries were shown to be immuno-reactive to the shrimp vitellin (Vn) antibody. N-terminal amino acid sequence determination revealed that several ovarian polypeptides and one polypeptide secreted by the hepatopancreas were identical to part of the C. feriatus Vg (CfVg) precursor. The full-length cDNA sequence encoding a protein with high amino acid sequence similarity to the Vg of the shrimp Metapenaeus Ensis was cloned. In common with the shrimp M. Ensis MeVg2, the crab vitellogenin gene is expressed only in the hepatopancreas. The expression level of CfVg is undetectable in the non-reproductive females, increases to maximum at the middle stages of vitellogenesis and drops to a lower level in late vitellogenesis. Expression of CfVg also extended to females that are undergoing brooding of developing larvae. Although the 8 kb transcript for the full-length cDNA was detected, smaller transcripts specific to CfVg mRNA were also detected, suggesting the occurrence of alternative splicing/expression of the CgVg gene to produce the smaller transcripts. Using a short term in vitro hepatopancreas explant culture assay, we have demonstrated that low concentrations of farnesoic acid (FA) stimulate CfVg gene expression in the hepatopancreas. Although both methyl farnesoate (MF) and juvenile hormone III also caused up-regulation of the CfVg gene, their effects are only significant at much higher concentrations.
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organization of the shrimp vitellogenin gene evidence of multiple genes and tissue specific expression by the ovary and hepatopancreas
Gene, 2003Co-Authors: Wingsze Tsang, Scott L Quackenbush, Billy K C Chow, Shirley H K Tiu, Siuming ChanAbstract:Abstract Vitellogenin is the major egg yolk protein synthesized in female shrimp during gonad maturation. Although there are several reports for the cloning of vitellogenin complementary DNA (cDNA) in different crustaceans, little is known of the gene organization of this protein. This study reports the first cloning and characterization of a full-length gene encoding the vitellogenin precursor from the shrimp Metapenaeus Ensis. By genomic DNA library screening, six different lambda clones were isolated using shrimp partial gene sequence as probe. Initial DNA sequence determination revealed that these clones are derived from different genes with coding sequence similar to other crustacean vitellogenins. Two of these clones were used for further analysis. One of the lambda clones (λ3.3) carries most of the coding sequence that correspond to the M. Ensis vitellogenin gene (MeVg1) and the other clone (λ8.3) carries a smaller portion of the coding sequence of a different vitellogenin gene (MeVg2). The λ3.3 clone was chosen for further characterization. To clone the remaining 5′ end upstream promoter region, 5′ untranslated region and the remaining coding sequence of MeVg1, a polymerase chain reaction (PCR)-based gene walking approach was used. Subsequently, a PCR clone with overlapping sequence identical to the genomic clone was obtained and the organization of MeVg1 gene was constructed. The MeVg1 gene consists of 15 exons and 14 introns spanning approximately 10 kb. Several potential cleavage sites were identified from the deduced vitellogenin precursor. Cleaving of the precursor in these sites would result in the production of several vitellogenin subunits. To clone the cDNA for the vitellogenin, 5′ and 3′ rapid amplification of cDNA ends was performed using ovary cDNA of the shrimp. A 4.4 kb 5′ cDNA clone and a 4 kb 3′ end cDNA clone were isolated. The size of the reconstructed cDNA for M. Ensis Vg is 7.97 kb and consists of the longest open reading frame of 7776 bp. Unlike the vitellogenin precursor of most insects and vertebrates, the deduced vitellogenin precursor lacks the polyserine domain important for receptor-mediated endocytosis. Phylogenetic analysis revealed a closer relationship of the MeVg1 with other crustacean vitellogenins but distantly related to other invertebrate and vertebrate vitellogenins. By reverse transcription-PCR, we have demonstrated that the shrimp MeVg1 gene is expressed only in the ovary and hepatopancreas while the MeVg2 gene is expressed exclusively in the hepatopancreas. In conclusion, the shrimp ovary also contribute significantly in the production of vitellogenin at transcription level and the gene organization of the shrimp protein may provide an insight in the evolution of this group of important proteins.
Ka Hou Chu - One of the best experts on this subject based on the ideXlab platform.
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comparative proteomic profiling during ovarian development of the shrimp metapenaeus Ensis
Molecular Biology Reports, 2014Co-Authors: Ju Cui, Ka Hou ChuAbstract:Two-dimensional electrophoresis and mass spectrometry were used to identify proteins that are differentially expressed during ovarian maturation in Metapenaeus Ensis. 87 spots with consistently significant quantitative differences (≥1.5-fold for vol%) among stage I, III and V ovaries were chosen for MS/MS analysis. 45 spots were significantly matched to known proteins in the database (Mascot score >40). Half of them were down-regulated, in contrast to 9 out of 45 proteins that were up-regulated as ovarian maturation proceeded. Functionally, these identified proteins could be classified into five major groups, including cytoskeleton (11 %), metabolism (18 %), signal transduction (32 %), gene expression (14 %) and immune response (7 %). Among the differentially expressed reproduction-related proteins, the mRNA expression level of cellular retinoic acid/retinol binding protein in M. Ensis (MeCRABP) during ovarian maturation was further characterized by quantitative real-time PCR. It was down-regulated during ovarian maturation. In situ hybridization further revealed that MeCRABP transcript was localized in ooplasm of previtellogenic oocytes but not in vitellogenic oocytes. These results demonstrate the application of proteomic analysis for identification of proteins involved in shrimp ovarian maturation and they provide new insights into ovarian development.
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differential gene expression in hepatopancreas of the shrimp metapenaeus Ensis during ovarian maturation
Marine Biotechnology, 2008Co-Authors: Queenie W L Wong, Wai Yan Mak, Ka Hou ChuAbstract:Differentially expressed genes were identified in the hepatopancreas of Metapenaeus Ensis during ovarian maturation via differential display reverse transcription-polymerase chain reaction (DDRT-PCR). They are G-protein signaling modulator 2 (GPSM2), glutamate carboxypeptide II (GCPII), ligatin, C-type lectin, O-linked N-acetylglucosamine transferase (O-GlcNAc transferase), 1-acylglycerol-3-phosphate O-acyltransferase 4 (AGPAT4), vitellogenin (Vg), and hemocyanin. The hepatopancreas Vg gene identified in this study shows 92% and 49% amino acid sequence homology, respectively, to MeVg1 and MeVg2 previously isolated from this species, suggesting the identification of a new Vg gene in M. Ensis. Vg gene expression was highest when the ovary was actively developing. The two metabolic enzymes, O-GlcNAc transferase and AGPAT4, exhibited a similar trend of expression to Vg gene, suggesting their involvement in Vg synthesis. The signal transduction related genes (GPSM2, GCPII, ligatin, and C-type lectin) were highly expressed in the hepatopancreas in the initial phase of maturation. These genes may be important for the signaling in the hepatopancreas for synthesis and mobilization of vitellogenin and nutrients to the developing ovary. The present work provides candidate genes for further investigation on the role of hepatopancreas in shrimp reproduction.
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a change of heart cardiovascular development in the shrimp metapenaeus Ensis
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2002Co-Authors: Brian R Mcmahon, Kosuke Tanaka, J E Doyle, Ka Hou ChuAbstract:Abstract The larval development of penaeid shrimp is among the most complicated in crustaceans. In Metapenaeus Ensis, there are six naupliar, three protozoeal and three mysid larval instars, followed by postlarval development. Irregular heartbeat begins late in naupliar instar 6. Co-ordinated beating at 400–600 beats min−1 commences in the first protozoeal instar and continues throughout larval life. Initially, the contractile region is located more posteriorly in the cephalothorax and has a single pair of ostia, and the arterial distribution is limited to a single anterior vessel. In later mysid instars, a second cardiac pumping site develops posterior to, but connected with, the original site. This extension is more muscular, contains additional ostia and develops additional distribution vessels supplying the cephalothorax and abdominal areas. The original site is gradually merged into the new extension and only small refinements in the circulation occur in postlarval and juvenile life. Changes in physiological responses of the heart also occur throughout development. Responses to intra-pericardial microinjection of 5-hydroxytryptamine change drastically during development, as do cardiac responses to ambient hypoxia. Similarly, heartbeat of later juvenile instars is inhibited by injection of tetrodotoxin, while heartbeat of larval and early juvenile instars is not, suggesting that neurogenic regulation via the cardiac ganglion arises later in development. Our present studies attempt to integrate the anatomical and physiological changes in the development of the crustacean heart.
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Purification and characterization of vitellin from the ovaries of the shrimp Metapenaeus Ensis (Crustacea: Decapoda: Penaeidae)
Invertebrate Reproduction & Development, 1997Co-Authors: Y. W. Qiu, Ka Hou ChuAbstract:Summary Vitellin from the ovaries of the shrimp Metapenaeus Ensis was isolated using gel filtration and anion exchange chromatography. Gel filtration revealed the presence of three peaks in an extract of mature ovaries. Two of these peaks were hardly noticeable in ovaries from immature shrimp. One of these was identified to be vitellin based on the results of Sudan black B and PAS staining for lipoprotein and glycoprotein, respectively. Purified vitellin with a molecular weight of 350 kD was composed of four subunits as revealed by SDS-PAGE. The two major subunits exhibited a molecular weight of 76 and 102 kD, respectively. Amino acid analysis of native vitellin revealed that glutamate/glutamine, alanine, valine and aspartate/asparagine were the major residues. A comparison with the amino acid compositions of vitellins reported for other penaeids shows that there are no great differences among penaeids. Vitellin from M. Ensis demonstrated an isoelectric point of 5.0 because of a high level of acidic amino...
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effects of copper on survival development and growth of metapenaeus Ensis larvae and postlarvae decapoda penaeidae
Marine Pollution Bulletin, 1995Co-Authors: Chris K C Wong, J K Y Cheung, Ka Hou ChuAbstract:Abstract Effects of copper on survival, development and growth of the early developmental stages of the penaeid shrimp, Metapenaeus Ensis, were studied in the laboratory. High variability in survival rates were observed among animals from different spawners. Larvae exposed to 0.06 and 0.10 mg Cu l−1 over a 10-day period had lower survival rates and exhibited slower development than control animals. No significant treatment effects were found at 0.04 and 0.08 mg Cu l−1. Effects of copper on survival and growth of postlarvae were studied over an 8-day period. Copper concentrations lower than 0.10 mg l−1 did not affect survival rates. Slower growth rates were observed in postlarvae exposed to 0.08 and 0.10 mg Cu l−1.
J M Jansen - One of the best experts on this subject based on the ideXlab platform.
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species delimitation and dna barcoding of atlantic Ensis bivalvia pharidae
Zoologica Scripta, 2014Co-Authors: Joaquín Vierna, Andres Martinezlage, J M Jansen, J Cuperus, Alejandra Perina, H M L Van Peltheerschap, Ana M GonzaleztizonAbstract:Ensis Schumacher, 1817 razor shells occur at both sides of the Atlantic and along the Pacific coasts of tropical west America, Peru, and Chile. Many of them are marketed in various regions. However, the absence of clear autapomorphies in the shell and the sympatric distributions of some species often prevent a correct identification of specimens. As a consequence, populations cannot be properly managed, and edible species are almost always mislabelled along the production chain. In this work, we studied whether the currently accepted Atlantic Ensis morphospecies are different evolutionary lineages, to clarify their taxonomic status and enable molecular identifications through DNA barcoding. For this, we studied 109 specimens sampled at 27 sites, which were identified as belonging to nine of those morphospecies. We analysed nucleotide variation at four nuclear (18S, 5.8S, ITS1, and ITS2) and two mitochondrial (COI and 16S) regions, although the 18S and 5.8S regions were not informative at the species level and were not further considered. The phylogenetic trees and networks obtained supported all morphospecies as separately evolving lineages. Phylogenetic trees recovered Ensis at each side of the Atlantic as reciprocally monophyletic. Remarkably, we confirm the co-occurrence of the morphologically similar E. minor (Chenu, 1843) and E. siliqua (Linne, 1758) along the NW Iberian coast, a fact that has been often overlooked. In South America, a relevant divergence between E. macha (Molina, 1792) individuals from Chile and Argentina was unveiled and suggests incipient speciation. We also confirm the occurrence of the North American species E. directus (Conrad, 1843) as far south as north-eastern Florida. Among the genomic regions analysed, we suggest COI as the most suitable DNA barcode for Atlantic Ensis. Our results will contribute to the conservation and management of Ensis populations and will enable reliable identifications of the edible species, even in the absence of the valves. The name Ensis coseli Vierna nom. nov. is proposed to replace E. minor Dall, 1899 non (Chenu, 1843).
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mesheften Ensis directus strandschelpen spisula subtruncata kokkels cerastoderma edule mosselen mytilus edulis en otterschelpen lutraria lutraria in de nederlandse kustwateren in 2009
IMARES Wageningen Report, 2009Co-Authors: P C Goudswaard, J J Kesteloo, K J Perdon, J G Jol, C Van Zweeden, J M JansenAbstract:Het doel van deze inventarisaties is het in kaart brengen van de bestanden van commercieel interessante soorten en het weergeven van de fluctuaties in de tijd, ten behoeve van het visserijbeleid. Het onderzoek is in eerste instantie gericht op de Amerikaanse zwaardschede (mesheften) (Ensis directus), de halfgeknotte strandschelp (Spisula subtruncata) en de kokkel (Cerastoderma edule). Daarnaast bleek er een voortgezette toename van een voorheen minder algemene soort, de gewone otterschelp (Lutraria lutraria), tot een niveau dat commerciele exploitatie wellicht mogelijk maakt. In dat perspectief is de otterschelp in de berekening van de bestanden meegenomen.
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mesheften Ensis directus halfgeknotte strandschelpen spisula subtruncata kokkels cerastoderma edule en otterschelpen lutraria lutraria in de nederlandse kustwateren in 2008
2008Co-Authors: P C Goudswaard, J J Kesteloo, K J Perdon, J M JansenAbstract:Ten behoeve van het beleid voor de visserij op Amerikaanse zwaardschedes (mesheften) (Ensis directus), halfgeknotte strandschelpen (Spisula subtruncata) en kokkels (Cerastoderma edule) heeft Wageningen IMARES, in opdracht van LNV het bestand in de Nederlandse kustwateren geinventariseerd. Deze inventarisatie is uitgevoerd in het voorjaar van 2008 en is daarmee de veertiende opeenvolgende survey die op deze manier sinds 1995 wordt uitgevoerd.
Josefina Méndez - One of the best experts on this subject based on the ideXlab platform.
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Identification of razor clams Ensis arcuatus and Ensis siliqua by PCR-RFLP analysis of ITS1 region
Fisheries Science, 2008Co-Authors: Ruth Freire, Juan Fernández-tajes, Josefina MéndezAbstract:Ensis arcuatus and Ensis siliqua are the most economically important species of razor clams in the European Union. Due to similarities between their shell morphology, and the differing retail value, these species are often misidentified. Therefore, it is necessary to develop an appropriate protocol to allow accurate differentiation between these species of razor clam in order to protect consumer rights, avoid commercial fraud (whether intentional or unintentional), and to enforce labeling and safety regulations. With the aim of developing a rapid and reliable method of differentiation, individuals of E. arcuatus and of E. siliqua were examined by polymerase chain reaction restriction fragment polymorphism (PCR-RFLP) using the internal transcribed spacer region 1 (ITS1). A species-specific restriction endonuclease pattern was found with the enzyme Kspl for both species, allowing their exact identification. Thus, this work provides a simple, reliable and rapid protocol for accurate discrimination between E. arcuatus and E. siliqua, which proves useful for traceability and enabling the enforcement of labeling regulations.
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identification of the razor clam species Ensis arcuatus e siliqua e directus e macha and solen marginatus using pcr rflp analysis of the 5s rdna region
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Juan Fernandeztajes, Josefina MéndezAbstract:Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the 5S ribosomal DNA region has been applied to the establishment of DNA-based molecular markers for the identification of five razor clam species: Ensis arcuatus, E. siliqua, E. directus, E. macha, and Solen marginatus. PCR amplifications were carried out using a pair of universal primers from the coding region of 5S rDNA. S. marginatus was simply distinguished by the different size of the amplicons obtained. Species-specific restriction endonuclease patterns were found with the enzymes Hae III for E. arcuatus, E. siliqua, and E. directus, and Acs I for E. macha, and when two enzymes were combined, the four species were also identified. Thus, this work provides a simple, reliable, and rapid protocol for the accurate identification of Ensis and Solen species in fresh and canned products, which is very useful for traceability and to enforce labeling regulations. Keywords: Species identification; razor clam; Ens...
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identification of the razor clam species Ensis arcuatus e siliqua e directus e macha and solen marginatus using pcr rflp analysis of the 5s rdna region
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Juan Fernandeztajes, Josefina MéndezAbstract:Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis of the 5S ribosomal DNA region has been applied to the establishment of DNA-based molecular markers for the identification of five razor clam species: Ensis arcuatus, E. siliqua, E. directus, E. macha, and Solen marginatus. PCR amplifications were carried out using a pair of universal primers from the coding region of 5S rDNA. S. marginatus was simply distinguished by the different size of the amplicons obtained. Species-specific restriction endonuclease patterns were found with the enzymes Hae III for E. arcuatus, E. siliqua, and E. directus, and Acs I for E. macha, and when two enzymes were combined, the four species were also identified. Thus, this work provides a simple, reliable, and rapid protocol for the accurate identification of Ensis and Solen species in fresh and canned products, which is very useful for traceability and to enforce labeling regulations.
Shirley H K Tiu - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression study of the lobster homarus americanus vitellogenin conservation in gene structure among decapods
General and Comparative Endocrinology, 2009Co-Authors: Shirley H K Tiu, Ho Lam Hui, Brian Tsukimura, Stephen S Tobe, Siuming ChanAbstract:This study reports the molecular characterization of the vitellogenin (Vg) of the lobster, Homarus americanus. Based on the annual collection of female lobsters, vitellogenesis commences in early March and continues through to September of each year. Using an antibody to vitellin of the lobster, H. americanus, several immunoreactive ovarian proteins were initially identified by Western blot analysis. The 80kDa protein contained the amino acid sequence APWGGNTPRC, identified subsequently by cDNA cloning to be identical to the lobster Vg. In common with the shrimp Metapenaeus Ensis and crab Charybdis feriatus, the lobster HaVg1 gene comprises 14 introns and 15 exons. The deduced HaVg1 precursor is most similar to the Vg of the crayfish Cherax quadricarinatus (57%), followed by M. Ensis (40-43% identity) and C. feriatus (38%). The results from genomic and RT-PCR cloning also confirmed the presence of multiple Vg genes in lobster. At early reproductive stages, the hepatopancreas HaVg1 transcript levels are low but increased to a maximum in animals with mature oocytes. The ovary, however, also expressed low levels of HaVg1. Using in vitro explant culture, treatment of hepatopancreas fragments with farnesoic acid or 20-hydroxyecdysone resulted in a significant stimulation in HaVg1 expression. From this study, it appears that Vg gene organization and expression pattern in decapods is highly conserved. Similar endocrine mechanisms may govern the process of vitellogenesis across the decapods.
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the use of recombinant protein and rna interference approaches to study the reproductive functions of a gonad stimulating hormone from the shrimp metapenaeus Ensis
FEBS Journal, 2007Co-Authors: Shirley H K Tiu, Siuming ChanAbstract:Although the crustacean crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone neuropeptides have been studied extensively in the last two decades and several neuropeptides from the shrimp Metapenaeus Ensis have been cloned, the functions of most of these neuropeptides remained putative. In this article, we describe the use of recombinant protein and an RNA interference approach to study the reproductive function of the previously reported molt-inhibiting hormone (MeMIH-B) in M. Ensis. When hepatopancreas and ovary explants were cultured in medium containing recombinant MeMIH-B, the vitellogenin gene (MeVg1) expression level was upregulated in a dose-dependent manner, reaching a maximum in explants treated with 0.3 nm recombinant MeMIH-B. Shrimp injected with recombinant MeMIH-B showed an increase in vitellogenin gene expression in the hepatopancreas. Moreover, a corresponding increase in the vitellogenin-like immunoreactive protein was detected in the hemolymph and ovary of these females. Injection of MeMIH-B dsRNA into the female shrimp caused a decrease in MeMIH-B transcript level in thoracic ganglion and eyestalk. These shrimp also showed reduction of vitellogenin gene expression in the hepatopancreas and ovary. Furthermore, the hemolymph vitellogenin level was also reduced in these animals. In summary, the results from recombinant protein and RNA interference experiments have demonstrated the gonad-stimulatory function of MeMIH-B in shrimp.
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vitellogenesis in the red crab charybdis feriatus hepatopancreas specific expression and farnesoic acid stimulation of vitellogenin gene expression
Molecular Reproduction and Development, 2005Co-Authors: Abby S C Mak, Shirley H K Tiu, Stephen S Tobe, Chi Lung Choi, Jerome H L Hui, Siuming ChanAbstract:Vitellogenesis in the mature female crab Charybdis feriatus occurs all year round during which active synthesis of the vitellogenin (Vg) precursor occurs. Several polypeptides from the ovaries were shown to be immuno-reactive to the shrimp vitellin (Vn) antibody. N-terminal amino acid sequence determination revealed that several ovarian polypeptides and one polypeptide secreted by the hepatopancreas were identical to part of the C. feriatus Vg (CfVg) precursor. The full-length cDNA sequence encoding a protein with high amino acid sequence similarity to the Vg of the shrimp Metapenaeus Ensis was cloned. In common with the shrimp M. Ensis MeVg2, the crab vitellogenin gene is expressed only in the hepatopancreas. The expression level of CfVg is undetectable in the non-reproductive females, increases to maximum at the middle stages of vitellogenesis and drops to a lower level in late vitellogenesis. Expression of CfVg also extended to females that are undergoing brooding of developing larvae. Although the 8 kb transcript for the full-length cDNA was detected, smaller transcripts specific to CfVg mRNA were also detected, suggesting the occurrence of alternative splicing/expression of the CgVg gene to produce the smaller transcripts. Using a short term in vitro hepatopancreas explant culture assay, we have demonstrated that low concentrations of farnesoic acid (FA) stimulate CfVg gene expression in the hepatopancreas. Although both methyl farnesoate (MF) and juvenile hormone III also caused up-regulation of the CfVg gene, their effects are only significant at much higher concentrations.
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organization of the shrimp vitellogenin gene evidence of multiple genes and tissue specific expression by the ovary and hepatopancreas
Gene, 2003Co-Authors: Wingsze Tsang, Scott L Quackenbush, Billy K C Chow, Shirley H K Tiu, Siuming ChanAbstract:Abstract Vitellogenin is the major egg yolk protein synthesized in female shrimp during gonad maturation. Although there are several reports for the cloning of vitellogenin complementary DNA (cDNA) in different crustaceans, little is known of the gene organization of this protein. This study reports the first cloning and characterization of a full-length gene encoding the vitellogenin precursor from the shrimp Metapenaeus Ensis. By genomic DNA library screening, six different lambda clones were isolated using shrimp partial gene sequence as probe. Initial DNA sequence determination revealed that these clones are derived from different genes with coding sequence similar to other crustacean vitellogenins. Two of these clones were used for further analysis. One of the lambda clones (λ3.3) carries most of the coding sequence that correspond to the M. Ensis vitellogenin gene (MeVg1) and the other clone (λ8.3) carries a smaller portion of the coding sequence of a different vitellogenin gene (MeVg2). The λ3.3 clone was chosen for further characterization. To clone the remaining 5′ end upstream promoter region, 5′ untranslated region and the remaining coding sequence of MeVg1, a polymerase chain reaction (PCR)-based gene walking approach was used. Subsequently, a PCR clone with overlapping sequence identical to the genomic clone was obtained and the organization of MeVg1 gene was constructed. The MeVg1 gene consists of 15 exons and 14 introns spanning approximately 10 kb. Several potential cleavage sites were identified from the deduced vitellogenin precursor. Cleaving of the precursor in these sites would result in the production of several vitellogenin subunits. To clone the cDNA for the vitellogenin, 5′ and 3′ rapid amplification of cDNA ends was performed using ovary cDNA of the shrimp. A 4.4 kb 5′ cDNA clone and a 4 kb 3′ end cDNA clone were isolated. The size of the reconstructed cDNA for M. Ensis Vg is 7.97 kb and consists of the longest open reading frame of 7776 bp. Unlike the vitellogenin precursor of most insects and vertebrates, the deduced vitellogenin precursor lacks the polyserine domain important for receptor-mediated endocytosis. Phylogenetic analysis revealed a closer relationship of the MeVg1 with other crustacean vitellogenins but distantly related to other invertebrate and vertebrate vitellogenins. By reverse transcription-PCR, we have demonstrated that the shrimp MeVg1 gene is expressed only in the ovary and hepatopancreas while the MeVg2 gene is expressed exclusively in the hepatopancreas. In conclusion, the shrimp ovary also contribute significantly in the production of vitellogenin at transcription level and the gene organization of the shrimp protein may provide an insight in the evolution of this group of important proteins.
