Wilaiwan Chotigeat

The role of Pm-fortilin in protecting shrimp from white spot syndrome virus (WSSV) infection.

Crustacean fortilin or the product of the translationally controlled tumor protein (TCTP) gene isolated from Penaeus monodon, is well conserved and has a Ca(++) binding domain. Pm-fortilin has anti-apoptotic properties and is present at high levels during the onset of viral infections in P. monodon. The possibility of using rFortilin to protect against white spot syndrome virus (WSSV) infection was tested. Injection of shrimp with rFortilin, after infection with WSSV, resulted in 80-100% survival and detection of very low levels of WSSV by PCR, whereas in moribund samples WSSV levels were very high. This result implies that injection of recombinant rFortilin decreases viral infection by an unknown mechanism, but probably by inhibiting viral replication. Using a yeast two-hybrid screen for cellular protein partners to rFortilin we identified an unknown protein that bound to fortilin. This is a novel polypeptide of 93 amino acids with a number of XPPX signature sequences that are often reported to have a function in antiviral peptides.

Molecular Evolution of the Odorant and Gustatory Receptor Genes in Lepidopteran Insects: Implications for Their Adaptation and Speciation

Lepidoptera (comprised of butterflies and moths) is one of the largest groups of insects, including more than 160,000 described species. Chemoreception plays important roles in the adaptation of these species to a wide range of niches, e.g., plant hosts, egg-laying sites, and mates. This study investigated the molecular evolution of the lepidopteran odorant (Or) and gustatory receptor (Gr) genes using recently identified genes from Bombyx mori, Danaus plexippus, Heliconius melpomene, Plutella xylostella, Heliothis virescens, Manduca sexta, Cydia pomonella, and Spodoptera littoralis. A limited number of cases of large lineage-specific gene expansion are observed (except in the P. xylostella lineage), possibly due to selection against tandem gene duplication. There has been strong purifying selection during the evolution of both lepidopteran odorant and gustatory genes, as shown by the low ω values estimated through CodeML analysis, ranging from 0.0093 to 0.3926. However, purifying selection has been relaxed on some amino acid sites in these receptors, leading to sequence divergence, which is a precursor of positive selection on these sequences. Signatures of positive selection were detected only in a few loci from the lineage-specific analysis. Estimation of gene gains and losses suggests that the common ancestor of the Lepidoptera had fewer Or genes compared to extant species and an even more reduced number of Gr genes, particularly within the bitter receptor clade. Multiple gene gains and a few gene losses occurred during the evolution of Lepidoptera. Gene family expansion may be associated with the adaptation of lepidopteran species to plant hosts, especially after angiosperm radiation. Phylogenetic analysis of the moth sex pheromone receptor genes suggested that chromosomal translocations have occurred several times. New sex pheromone receptors have arisen through tandem gene duplication. Positive selection was detected at some amino acid sites predicted to be in the extracellular and transmembrane regions of the newly duplicated genes, which might be associated with the evolution of the new pheromone receptors.

Characterization and Small RNA Content of Extracellular Vesicles in Follicular Fluid of Developing Bovine Antral Follicles

Exosomes and microvesicles (i.e., extracellular vesicles: EVs) have been identified within ovarian follicular fluid and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, whether EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth remains unknown. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle.

Highly sensitive capacitive biosensor for detecting white spot syndrome virus in shrimp pond water.

Water is one major pathways by which the white spot syndrome virus (WSSV) pathogen enters aquaculture facilities. This paper describes the production and use of a capacitive biosensor for the quantitative detection of as little as 1copy/μl of WSSV in shrimp pond water. A glutathione-S-transferase tag for white spot binding protein (GST-WBP) was immobilized on a gold electrode through a self-assembled monolayer. Binding between WSSV and the immobilized GST-WBP was directly detected by a capacitance measurement. Under optimum conditions, the capacitive biosensor detected WSSV over a wide linear range of between 1 and 1 × 10(5)copies/μl. The system was highly selective for WSSV. One analysis cycle required only 20-25 min of analysis time and 25 min of regeneration time. The capacitive biosensor was applied to analyze WSSV concentration in eight shrimp pond water samples and the results were in good agreement with those obtained by a real time quantitative polymerase chain reaction (real-time PCR) method (P>0.05). The immobilized GST-WBP provided and could be reused for up to 39 analysis cycles for one electrode preparation with a relative standard deviation (RSD) of 2.4% and a good reproducibility of residual activity (95.8 ± 2.3%). The appealing performance of this biosensor indicated that it had great potential for an accurate very sensitive, quantitative, detection method for WSSV.

Molecular phylogenetic analysis of white prawns species and the existence of two clades in Penaeus merguiensis

Closely related species, Penaeus merguiensis and Penaeus silasi from Thai waters, were genetically examined using variation observed in 558 base pairs (bp) of sequence from cytochrome oxidase subunit I (COI) gene of mtDNA. The sequence divergences of COI between P. merguiensis and other Penaeus species were 5.76–6.15% (P. silasi), 13.17–13.97% (Penaeus indicus), 16.43% (Penaeus vannamei), 16.63% (Penaeus monodon), and 18.37% (Penaeus japonicus). From the alignment reported that there were four clades on phylogenetic tree, the distinction of the two monophyletic clades was referred as P. merguiensis, one monophyletic clade within P. silasi and P. indicus. These results point toward the possibility of P. merguiensis being a complex of two cryptic species or a single species with strong phylogeographic subdivision.

WSSV: VP26 binding protein and its biological activity.

White spot syndrome virus (WSSV) is one of the major causes of disease in the shrimp culture industry causing enormous economic losses. In this study, we displayed peptides from a cDNA library obtained from the hemolymph of shrimp infected with WSSV, on the surface of phage and screened for the peptides that interacted with the WSSV. One WSSV binding protein (WBP) gene was found to consist of 171 bp that had no matches in the NCBI database. This WBP was shown to bind to the VP26 protein of the WSSV by Western blotting. In addition, WBP reduced the binding of WSSV to shrimp haemocytes from 2.0 × 10(7)copies in the control to 6.0 × 10(2) after treatment with 80 μg of WBP. The survival rate of shrimp after WSSV were mixed with WBP at 80 μg, was 89% and the binding of WBP remained unchanged for at least 24h. Therefore, the results indicate that the WBP can bind to VP26 and inhibit the invasion of WSSV into host cells. This finding may introduce another future way to try to fight this disease in shrimp culture.

Production of a polyclonal antibody to the VP26 nucleocapsid protein of white spot syndrome virus (wssv) and its use as a biosensor

White spot syndrome virus (WSSV) is a major cause of high mortality in cultured shrimp all over the world. VP26 is one of the structural proteins of WSSV that is assumed to assist in recognizing its host and assists the viral nucleocapsid to move toward the nucleus of the host cell. The objective of this work was to produce a polyclonal antibody against VP26 and use it as a biosensor. The recombinant VP26 protein (rVP26) was produced in E. coli (BL21), purified and used for immunizing rabbits to obtain a polyclonal antibody. Western blot analysis confirmed that the antiserum had a specific immunoreactivity to the VP26 of WSSV. This VP26 antiserum was immobilized onto a gold electrode for use as the sensing surface to detect WSSV under a flow injection system. The impedance change in the presence of VP26 was monitored in real time. The sensitivity of the biosensor was in the linear range of 160–160000 copies of WSSV, indicating that it is good and sensitive for analysis of WSSV. The specificity of the biosensor was supported by the observation that no impedance change was detected even at high concentrations when using Yellow Head Virus (YHV). This biosensor may be applied to monitor the amount of WSSV in water during shrimp cultivation.

Cloning and characterization of pectate lyase from Hevea brasiliensis.

Latex from the commercial Hevea brasiliensis contains 30-50% (w/w) of natural rubber (cis-1,4-polyisoprene), the raw material for the many products of the rubber industry. We have constructed a cDNA library from the latex of H. brasiliensis to investigate the expressed genes and molecular events in the latex. We have isolated two cDNAs from this library, Hb-PEL-1 and Hb-PEL-2 that could encode for pectate lyase enzymes (EC4.2.2.2). From their sequence analysis Hb-PEL-1 and Hb-PEL-2 encode for proteins of 393 and 323 amino acids, respectively. Comparison of these deduced amino acid sequences with other pectate lyase enzymes showed they contained the conserved NADPH, Ca(2+) and substrate binding sites and had a 74% identity to Arabidopsis thaliana pectate lyase. Only the Hb-PEL-1 recombinant protein expressed from Escherichia coli had enzymic activity which was Ca(2+) dependent. Interestingly, Hb-PEL-1 contained an extra internal peptide between amino acid residue 38-108 when compared to Hb-PEL-2 and this peptide was also present in other pectate lyase enzymes. The transcript of pectate lyase (Hb-PEL) in the latex of rubber tree at various times after the first tapping was quantified by real-time PCR using 18s genes as internal standard. Most transcripts were detected on the first day after tapping and then decreased with time. This indicates that the pectate lyase may be involved in either the release of latex by breaking down the laticifer wall or in the development of laticifers.

Expression of a mammalian α2,6-sialyltransferase gene in Pichia pastoris

Abstract Terminal sialic acid on oligosaccharides of glycoproteins shows several biological functions of the glycoproteins. The yeast Pichia pastoris normally does not contain sialic acid on the oligosaccharides of glycoproteins. A sialyltransferase (ST) gene was transfected into P. pastoris to assess the possibility of using yeast cells as a host to produce sialoglycoproteins. The expression vectors pPIC3.5 and pPIC9 were used as carriers. The recombinant P. pastoris harbouring ST-pPIC3.5 and ST-pPIC9 had sialyltransferase activity of 1.1 and 10.2 mU l −1 respectively. The ability of the recombinant ST-pPIC3.5 and ST-pPIC9 to transfer the fluoresceinyl-NeuAc into the cell glycoproteins was 36.9 and 20.9 pmol mg −1 protein respectively.

Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein (PAP) gene.

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi (V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 μg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 μg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO (Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.