Project description:Acute hepatopancreatic necrosis disease (AHPND) is a shrimp farming disease, caused by a pathogenic Vibrio parahaemolyticus carrying a plasmid encoding Vp_PirAB-like toxin (VpAHPND). Whiteleg shrimp, Litopenaeus vannamei were fed food pellets containing formalin-killed VpAHPND (FKC-VpAHPND) to select for toxin resistance. To identify genes associated with Vp_PirAB-like toxin resistance, total RNA was sequenced to identify differentially expressed genes (DEGs) in the stomach and hepatopancreas among surviving shrimp (sur-FKC), AHPND-infected shrimp (Vp-inf) and normal shrimp (control). From a total of 79,591 genes, 194 and 224 DEGs were identified in the stomach and hepatopancreas transcriptomes, respectfully. The expressions of DEGs were validated by qPCR of ten genes. Only one gene, a gene homologous to L vannamei anti-lipopolysaccharide factor AV-R isoform (LvALF AV-R), was expressed significantly more strongly in sur-FKC than in the other groups. The association of LvALF AV-R expression and toxin resistance was affirmed from the surviving shrimp in a second-trial of FKC-VpAHPND feeding. These results suggest that LvALF AV-R may be involved in shrimp defense mechanisms against Vp_PirAB-like toxin virulence.
Project description:The transcriptomic response of two strains of the Pacific whiteleg shrimp, different in their resistance to Taura Syndrome Virus (TSV), in response to infection with TSV and Yellow Head Virus (YHV). Changes in gene expression in the shrimp’s hepatopancreas were assessed using a cDNA microarray containing 2,469 putative unigenes. The patterns of gene expression between the shrimp strains were considerably similar, except for the more advanced stages of Taura Syndrome. Between the different treatments approximately 250 genes were differently expressed. The most advanced stages of YHV infection showed the highest number of differently expressed genes. During infection there were profound changes in the expression of genes related to lipid and protein metabolism, cellular trafficking, immune defense and stress response. Keywords: Disease state analysis, disease resistance There were 5 biological replicates for each of the groups in this experiment. Also, two strains of Litopenaeus vannamei were used: a strain resistant to TSV and a strain susceptible to TSV (Kona line). The treatments consisted of injecting both strains with 60mL of a shrimp extract made from shrimp previously injected with either a SPF shrimp extract (1x10-4), Taura Syndrome Virus (1x10-5) or Yellow Head Virus (1x10-4). The 2 initial control groups were composed of hepatopancreas samples from both strains prior the injections. Samples were also collected from at days 1 and 2 from both strains from the 3 different treatments (control, TSV and YHV).
Project description:Here we used microarrays to characterize changes in global gene expression in the hepatopancreas of Pacific white shrimp, Litopenaeus vannamei, exposed to short term (4 h) hypoxia (H) or hypercapnic hypoxia (HH) or long term (24 h) H or HH, compared to animals in air-saturated water (normoxia). The transcriptomes of crustaceans exposed to low O2 and high CO2 contained both shared and treatment-specific signature genes (q M-bM-^IM-$ 0.01, FC M-bM-^IM-% 1.5), with shifts characteristic of metabolic depression rather than anaerobic metabolism. Down-regulated signature genes dominated the transcript profile in three of the four treatments (H 4 h, H 24 h, 4 h HH); many of these genes were involved in amino acid or RNA metabolism or in translation, including several tRNA synthetases. Unique patterns of gene expression such as increased lipid metabolism and hemocyanin synthesis (H 24 h) and initiation of apoptosis (24 h HH) were tied to specific treatments. This work contributes insight to the effects that human perturbations might have on estuarine organisms, and the importance of examining the impacts of environmentally relevant combinations of hypoxia and hypercapnia on estuarine populations. L. vannamei were exposed for 4 or 24 hours to one of the following conditions: normoxia, hypoxia or hypercapnic hypoxia. Hepatopancreas tissue from individual animals was dissected, total RNA extracted, labelled and hybridized to oligonucleotide microarrays with probes for 21,864 L. vannamei unigenes. Treatments were repeated until a total of 7 biological replicates was obtained for each time:treatment combination, except for the 24 h normoxia group, represented by 6 replicates.
Project description:Here we used microarrays to characterize changes in global gene expression in the hepatopancreas of Pacific white shrimp, Litopenaeus vannamei, exposed to short term (4 h) hypoxia (H) or hypercapnic hypoxia (HH) or long term (24 h) H or HH, compared to animals in air-saturated water (normoxia). The transcriptomes of crustaceans exposed to low O2 and high CO2 contained both shared and treatment-specific signature genes (q ≤ 0.01, FC ≥ 1.5), with shifts characteristic of metabolic depression rather than anaerobic metabolism. Down-regulated signature genes dominated the transcript profile in three of the four treatments (H 4 h, H 24 h, 4 h HH); many of these genes were involved in amino acid or RNA metabolism or in translation, including several tRNA synthetases. Unique patterns of gene expression such as increased lipid metabolism and hemocyanin synthesis (H 24 h) and initiation of apoptosis (24 h HH) were tied to specific treatments. This work contributes insight to the effects that human perturbations might have on estuarine organisms, and the importance of examining the impacts of environmentally relevant combinations of hypoxia and hypercapnia on estuarine populations.
Project description:The functional diversity of crustacean hemocyanins is broad, encompassing O2 delivery, innate immune response, metabolite storage, and osmolyte balance, all in a heterogeneous protein structure. As such, the sequence diversity of this class of proteins and its subunit composition are the focus of many studies on crustacean adaptation to environmental challenges. Recent transcriptomic and genomic sequencing on the Pacific whiteleg shrimp Litopenaeus vannamei has identified unique isoforms of hemocyanin including an ancestral β-type subunit thought to be lost in penaeid shrimp. However, it is unknown the degree to which these isoforms are translated as proteins, and whether they differ in function. The present study uses proteomic approaches to characterize the protein-level abundance and organization of these hemocyanin isoforms within their native oligomeric structures. Fractions of each hemocyanin oligomeric form were purified by size-exclusion high performance liquid chromatography for identification of subunit isoforms using tandem mass spectrometry at <1% protein false discovery rate. Relative abundances of hemocyanin oligomers and monomeric subunits from hemolymph and fractions were also quantified by polyacrylamide gel electrophoresis with and without denaturation for comparison of relative abundance. Ten hemocyanin isoforms were identified by tandem mass spectrometry in both hemocyanin oligomer fractions including a single small subunit, eight large subunits, and the first protein-level evidence of a β-type subunit in penaeid shrimp. Hemocyanin subunits were organized primarily as hexamers (95-99% relative abundance) as opposed to dodecamers. Hexamers utilized a significantly higher ratio (2.05:1) of small subunit to large subunit compared to dodecamers (1.04:1), and the relative abundances of the large subunit isoforms was dominated by HcL1 in both fractions. The ability to distinguish and quantify hemocyanin isoforms within oligomeric structures will aid future studies linking hemocyanin genes to function and physiology as well as offer insight into the evolutionary history of crustaceans.