Shrimp Genetic Improvement Center

Surat Thani, Thailand

Shrimp Genetic Improvement Center

Surat Thani, Thailand
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Wimuttisuk W.,National Science and Technology Development Agency | Tobwor P.,National Science and Technology Development Agency | Deenarn P.,National Science and Technology Development Agency | Intaraudom C.,National Science and Technology Development Agency | And 6 more authors.
Biochemical and Biophysical Research Communications | Year: 2017

Dietary polyunsaturated fatty acids (PUFAs) are critical to the success of ovarian development in marine crustaceans, especially for domesticated species such as the black tiger shrimp Penaeus monodon. These fatty acids are stored in a midgut gland called the hepatopancreas and subsequently serve as an energy source or are incorporated in yolk during ovarian development. PUFAs are known precursors of hydroxy fatty acids, including hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid (HEPE), which are catalyzed by lipoxygenases (LOX). In previous studies, 8-HEPE has been shown to regulate female reproduction and adipogenesis in marine crustaceans. However, whether the biosynthesis of 8-HEPE in these species is the result of LOX activity has yet to be investigated. In this study, 8-HEPE was identified exclusively in P. monodon hepatopancreases using liquid chromatography-mass spectrometry. Treatment with nordihydroguaiaretic acid resulted in the reduction of 8-HEPE, suggesting the enzyme-dependent catalysis of 8-HEPE in hepatopancreases. Additionally, a full-length P. monodon LOX (PmLOX) was amplified from shrimp ovary cDNA. Sequence analysis revealed that the putative PmLOX contains domains and catalytic residues required for LOX catalytic function. Furthermore, PmLOX expression increased steadily as shrimp ovary maturation progressed, while PmLOX expression and the amount of 8-HEPE decreased in shrimp hepatopancreases. These findings not only suggest differential requirements for hydroxy fatty acid biosynthesis in shrimp ovaries and hepatopancreases during the P. monodon ovarian development, but also provide insights into the LOX pathway in marine crustaceans. © 2017 Elsevier Inc.

Utari H.B.,Mahidol University | Soowannayan C.,Mahidol University | Soowannayan C.,National Science and Technology Development Agency | Flegel T.W.,Mahidol University | And 4 more authors.
Developmental and Comparative Immunology | Year: 2017

The viral accommodation hypothesis proposes that endogenous viral elements (EVE) from both RNA and DNA viruses are being continually integrated into the shrimp genome by natural host processes and that they can result in tolerance to viral infection by fortuitous production of antisense, immunospecific RNA (imRNA). Thus, we hypothesized that previously reported microarray results for the presence of white spot syndrome virus (WSSV) open reading frames (ORFs) formerly called 151, 366 and 427 in a domesticated giant tiger shrimp (Penaeus monodon) breeding stock might have represented expression from EVE, since the stock had shown uninterrupted freedom from white spot disease (WSD) for many generations. To test this hypothesis, 128 specimens from a current stock generation were confirmed for freedom from WSSV infection using two nested PCR detection methods. Subsequent nested-PCR testing revealed 33/128 specimens (26%) positive for at least one of the ORF at very high sequence identity (95–99%) to extant WSSV. Positive results for ORF 366 (now known to be a fragment of the WSSV capsid protein gene) dominated (28/33 = 84.8%), so 9 arbitrarily selected 366-positive specimens were tested by strand-specific, nested RT-PCR using DNase-treated RNA templates. This revealed variable RNA expression in individual shrimp including no RNA transcripts (n = 1), sense transcripts only (n = 1), antisense transcripts only (n = 2) or transcripts of both sense (n = 5). The latter 7 expression products indicated specimens producing putative imRNA. The variable types and numbers of the EVE and the variable RNA expression (including potential imRNA) support predictions of the viral accommodation hypothesis that EVE are randomly produced and expressed. Positive nested PCR test results for EVE of ORF 366 using DNA templates derived from shrimp sperm (germ cells), indicated that they were heritable. © 2017 Elsevier Ltd

Kiatmetha P.,Prince of Songkla University | Kiatmetha P.,Shrimp Genetic Improvement Center | Siangdang W.,King Mongkut's University of Technology Thonburi | Bunnag B.,King Mongkut's University of Technology Thonburi | And 2 more authors.
Aquaculture International | Year: 2011

The purpose of this study was to compare the performance of two species of diatoms, Thalassiosira weissflogii and Chaetoceros gracilis, in the larviculture of the black tiger shrimp Penaeus monodon. Shrimp larvae were fed with either C. gracilis, T. weissflogii, or a combination of the two species of diatoms. The larvae fed solely with T. weissflogii or a combination of the two types of diatom had significantly higher survival rates and faster metamorphosis than those fed solely with C. gracilis. The numbers of diatom cells consumed by larvae during 3-h periods were determined, revealing that larvae consumed significantly higher numbers of C. gracilis than T. weissflogii. However, when the protein, total fatty acid, and polyunsaturated fatty acid content of the two species of diatom are compared, significantly higher amounts of each are found in T. weissflogii. Converting the number of diatom cells consumed into equivalent protein, total fatty acid, eicosopentaenoic acid, and decosahexaenoic acid reveals that larvae fed with T. weissflogii received significantly higher amounts of all the nutrients, compared to those consuming C. gracilis. The results showed an advantage of feeding T. weissflogii to C. gracilis in enhancing survival and metamorphosis in P. monodon larvae. © 2010 Springer Science+Business Media B.V.

Pongtippatee P.,Aquatic Animal Biotechnology Research Center | Laburee K.,Aquatic Animal Biotechnology Research Center | Thaweethamsewee P.,Prince of Songkla University | Hiranphan R.,Prince of Songkla University | And 5 more authors.
Aquaculture | Year: 2012

This study seeks to determine the best method for preventing the second body (PBII) extrusion as a means to induce triploidy in the black tiger shrimp Penaeus monodon. Chemical (cytochalasine-B and 6-dimethylaminopurine) and temperature (heat and cold) shocks were applied to newly fertilized eggs. Cold shock that was administered at 8 °C, for 10. min at 8. min post-spawning, was the best method for PBII triploidy induction, as evidenced by the highest percentage of forming three pronuclei in syngamy. Therefore, cold shock induction was employed to further explore the advantage of triploid over diploid shrimp. After hatching of the cold-shocked eggs, the larvae were allowed to reach juvenile and adult levels of development. The number and amount of chromosomes were determined in juveniles and adult stages, using Fluorescence Activating Cell Sorting methods, by which the shrimp were divided into diploid and triploid groups. At day 150 in culture, the average body weight of the triploid females (35.2 ± 3.2. g) and triploid males (31.5 ± 3.5. g) was significantly higher (P< 0.0001) than that of their diploid counterparts (24.5 ± 0.5. g for females and 23.1 ± 3.8. g for males), having a ratio of 2 females:1 male for triploid shrimp, and 2 females:3 males for diploid shrimp. These results reveal the advantages of growing triploid over diploid P. monodon, and its feasibility for commercial production. © 2012 Elsevier B.V.

Sangsuriya P.,Mahidol University | Huang J.-Y.,National Taiwan University | Chu Y.-F.,National Taiwan University | Phiwsaiya K.,Mahidol University | And 13 more authors.
Molecular and Cellular Proteomics | Year: 2014

White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSVWSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein-protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture. Molecular & Cellular Proteomics 13: 10.1074/mcp.M113.029199, 269-282, 2014. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Kanjanasopa D.,Prince of Songkla University | Pongtippatee P.,Prince of Songkla University | Vanichviriyakit R.,Mahidol University | Wongtripop S.,Shrimp Genetic Improvement Center | And 3 more authors.
Aquaculture | Year: 2015

Monodon baculovirus (MBV) causes slow growth of the shrimp being due to its infection in the hepatopancreas, the organ producing digestive enzymes and for nutrient storage. It has long been documented that the virus is transmitted from broodstock to offspring via contamination in rearing water by feces of the infected broodstock that contained sloughing-off damaged hepatopancreatic cells containing MBV. As a management practice, the washing of eggs and nauplii with disinfectant has been recommended to eliminate MBV that are attached to their surface and thus prevent the infection at the later development stage of the shrimp. However, we detected the MBV infection in postlarvae and juveniles of the black tiger shrimp Penaeus monodon, even after our repeated attempts to eliminate the virus by washing the eggs and nauplii with povidone iodine as disinfectant. Therefore we used MBV-infected broodstock to identify the cause of this problem by tracing the presence of MBV in hepatopancreas and ovary of the broodstock, eggs, nauplii and postlarvae, using histology with hematoxylin-eosin staining, polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) specific for polyhedrin, a protein produced by MBV. We found all the ovaries collected from the broodstock, which were detected MBV-positive in the hepatopancreas by histology, PCR and ISH, were also PCR-positive. By ISH, positive signals were detected in the cell membrane, cytoplasm and nuclear membrane of the oocytes. The eggs and nauplii from the MBV-positive broodstock were also positive by PCR, in both "wash" and "no-wash" specimens. By ISH, positive signals were detected in ooplasm and subcuticular region of nauplii, as well as inside its body. Using IHC, positive signals were detected inside the body and appendage of the nauplius. Taken all these together, it is most likely that MBV could be vertically transmitted through trans-ovarian route. Hence, simply washing the eggs and nauplii with disinfectant may not be an adequate procedure to eliminate the MBV infection in offspring from MBV-infected broodstock. © 2015 Elsevier B.V.

Tangprasittipap A.,National Center for Genetic Engineering and Biotechnology | Tangprasittipap A.,Mahidol University | Chouwdee S.,National Center for Genetic Engineering and Biotechnology | Chouwdee S.,Mahidol University | And 6 more authors.
General and Comparative Endocrinology | Year: 2012

Although many crustacean neuroendocrine hormones have been reported, the enzymes responsible for post-translational modification of neuroendocrine hormones have rarely been characterized. A prohormone convertase 2 (PC2)-like enzyme has been isolated from the optic lobe of the giant tiger shrimp, Penaeus monodon and referred as PmPC2. The full length cDNA sequence of PmPC2 has been identified and found to resemble evolutionarily conserved PC2 enzymes of vertebrates and invertebrates. PmPC2 was expressed in all larval developmental stages and in neuroendrocrine cells in the adult optic lobe. Its expression was found to be negatively related with shrimp body weight by qPCR (P< 0.05). Immunohistochemistry results using an anti-rPmPC2 antibody with adult shrimp revealed high staining intensity in specific neurosecretory cells including the sinus gland, the organ of Hanström (also referred to as the medullar terminalis X-organ) and the organ of Bellonci (also referred to as the sensory or X-organ). By using the yeast two hybrid technique, PmPC2 was found to bind with P. monodon hyperglycemic hormone (Pem-CHH1) that plays an important role in glucose metabolism. Since PmPC2 is a subtilisin-like serine proteinase, it is expected to cleave the synthetic substrate, pyr-RTKR-MCA, but the expressed recombinant catalytic domain of PmPC2 (rPmPC2-cat) showed no enzymatic activity as expected. In vivo injection of dsRNA-PmPC2 resulted in reduced transcripts for both PmPC2 and Pem-CHH1 on day 3 post injection, but there was no accompanying reduction of glucose level in the hemolymph. Taken together, PmPC2 localization, expression and activity suggest that it has a function(s) in the shrimp neuroendrocrine system and that it may not only activate Pem-CHH1 but also affect its expression. However, there is no obvious explanation for the negative correlation between PmPC2 expression level and shrimp body weight. © 2012 Elsevier Inc.

Tangprasittipap A.,Mahidol University | Tiensuwan M.,Mahidol University | Withyachumnarnkul B.,Mahidol University | Withyachumnarnkul B.,National Science and Technology Development Agency | Withyachumnarnkul B.,Shrimp Genetic Improvement Center
Aquaculture | Year: 2010

The optic lobe of the female-shrimp eyestalk was selected as the target tissue for suppressive subtractive hybridization (SSH) because it is a place for synthesis of many hormones and peptides/enzymes involved in molting and growth. SSH was performed to screen genes differentially expressed in the optic lobe between large female (LF; body weight>90 percentile of weight distribution curve) and small female (SF; body weight<10 percentile). A total of 426 recombinant clones were obtained from the two directions. After sequencing and analysis less than 30% of the resulting expressed sequence tag (EST) exhibited high homology to known records at GenBank (BlastX with E-value<10-4) indicating little available molecular information on the optic lobe. Among the EST with relatively high homology to GenBank records, 5 candidate ESTs had homology to records for genes involved in cell differentiation/proliferation, cell cycle and hormone processing. These included genes resembling cyclophilin, cyclophilin A, fibrillarin, SPARC and PC2. These candidate ESTs were selected to confirm the reproducibility of the SSH data using semi-quantitative RT-PCR normalized with EF-1α. Pearson's correlation analysis confirmed that the index of relative cyclophilin, SPARC and fibrillarin-like expression was negatively correlated with body weight (p<0.05) and this supported the SSH data indicating their low relative expression in LF shrimp when compared to SF shrimp. The index of relative cyclophilin-like expression showed the highest correlation coefficient with body weight (r=-0.678). The relationship could be expressed by inverse fitted equation: body weight (g)=38.243/(index of relative cyclophilin-like expression). This fitted inverse model could predict the body weight of female shrimp with 91.6 % coefficient of determination (R2) based on the index of relative cyclophilin-like expression in the optic lobe of female P. monodon. © 2010 Elsevier B.V.

Dong H.T.,Chulalongkorn University | Nguyen V.V.,Chulalongkorn University | Phiwsaiya K.,Mahidol University | Phiwsaiya K.,National Science and Technology Development Agency | And 7 more authors.
Aquaculture | Year: 2015

Flavobacterium columnare and Edwardsiella ictaluri are two major bacterial pathogens threatening catfish aquaculture globally. Earlier studies have focused on the characterization of single bacterial infection. In reality, multiple bacterial pathogens are present in aquaculture systems and are probably responsible for disease outbreaks and considerably outweigh single infection. The objectives of this study, therefore, were to investigate whether single or concurrent bacterial pathogens were involved in naturally diseased striped catfish (Pangasianodon hypophthalmus) and subsequently investigate the pathogenicity of single- and dual-infection through experimental challenges. The investigation revealed coinfections of F. columnare and E. ictaluri found in naturally diseased Thai striped catfish exhibiting columnaris and edwardsiellosis diseases. Bacterial identification was confirmed by phenotypic tests, species-specific PCR and 16S rDNA sequence analysis. Molecular data analysis also identified that the infected fish species was P. hypophthalmus. Experimental challenges of striped catfish juveniles with single and dual bacterial species using both immersion (i.m) and injection (i.p) approaches were performed. Injection of two different doses of combined bacteria caused markedly high mortality of 86.7-100%, indicating high virulence of the bacterial isolates. Immersion (i.m.) coinfection of E. ictaluri (2.6×106CFUmL-1) and F. columnare (1.0×104CFUmL-1) caused significantly high cumulative mortality (96.7±5.8%) compared to i.m. of single infection of E. ictaluri (80.0±20%) or F. columnare (3.3±5.7%) with the same dose of bacteria. Both coinfection challenge routes i.p. and i.m. successfully mimicked typical signs and histopathological manifestations of natural coinfection. This study had fulfilled Koch's postulates through single- or dual-challenged tests to mimic the natural disease case in striped catfish. Statement of relevance: The authors strongly believe that our manuscript would provide significant knowledge to fish aquaculture especially to that of the striped catfish P. hypophthalmus. © 2015 Elsevier B.V.

PubMed | Mahidol University and Shrimp Genetic Improvement Center
Type: Journal Article | Journal: Molecular reproduction and development | Year: 2016

Protein and lipid composition of sperm plasma membrane are modified as these gametes continue to mature during their transit along the spermatic tract. Our previous study revealed that during its journey through the spermatic duct of the black tiger prawn, Penaeus monodon, sperm cholesterol content decreases through the action of lipid-binding proteins within the luminal environment. In this study, the full cDNA sequence of epididymal secretory protein E1 (HE1), or Niemann-Pick C2 (NPC2), was cloned from P. monodon (termed Pmnpc2), and its conserved cholesterol/lipid-binding domain was characterized. The putative tertiary structure of PmNPC2 showed high similarity with the structure of Bos taurus NPC2. Pmnpc2 is expressed in many tissues, including the spermatic tract (i.e., testis, vas deferens, terminal ampoule) and the female thelycum. In situ hybridization revealed the presence of Pmnpc2 transcripts in the vas deferens, terminal ampoule, and thelycum epithelia, suggesting that PmNPC2 could be secreted into the lumen of the spermatic duct. A recombinant hexahistidine-tagged PmNPC2 (rPmNPC2-6His) was able to bind cholesterol and sperm lipid extracts, while co-incubation of sperm from the vas deferens with rPmNPC2-6His resulted in the depletion of cholesterol from these gametes. Together, these results suggest that PmNPC2 participates in sperm cholesterol efflux during the sperm maturation process in P. monodon. Mol. Reprod. Dev. 83: 259-270, 2016. 2016 Wiley Periodicals, Inc.

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