Xiaohan Chen

Transcriptome Analysis of Pacific White Shrimp (Litopenaeus vannamei) Hepatopancreas in Response to Taura Syndrome Virus (TSV) Experimental Infection

Background The Pacific white shrimp, Litopenaeus vannamei, is a worldwide cultured crustacean species with important commercial value. Over the last two decades, Taura syndrome virus (TSV) has seriously threatened the shrimp aquaculture industry in the Western Hemisphere. To better understand the interaction between shrimp immune and TSV, we performed a transcriptome analysis in the hepatopancreas of L. vannamei challenged with TSV, using the 454 pyrosequencing (Roche) technology. Methodology/Principal Findings We obtained 126919 and 102181 high-quality reads from TSV-infected and non-infected (control) L. vannamei cDNA libraries, respectively. The overall de novo assembly of cDNA sequence data generated 15004 unigenes, with an average length of 507 bp. Based on BLASTX search (E-value <10−5) against NR, Swissprot, GO, COG and KEGG databases, 10425 unigenes (69.50% of all unigenes) were annotated with gene descriptions, gene ontology terms, or metabolic pathways. In addition, we identified 770 microsatellites and designed 497 sets of primers. Comparative genomic analysis revealed that 1311 genes differentially expressed in the infected shrimp compared to the controls, including 559 up- and 752 down- regulated genes. Among the differentially expressed genes, several are involved in various animal immune functions, such as antiviral, antimicrobial, proteases, protease inhibitors, signal transduction, transcriptional control, cell death and cell adhesion. Conclusions/Significance This study provides valuable information on shrimp gene activities against TSV infection. Results can contribute to the in-depth study of candidate genes in shrimp immunity, and improves our current understanding of this host-virus interaction. In addition, the large amount of transcripts reported in this study provide a rich source for identification of novel genes in shrimp.

Transcriptome Analysis of Litopenaeus vannamei in Response to White Spot Syndrome Virus Infection

Pacific white shrimp (Litopenaeus vannamei) is the most extensively farmed crustacean species in the world. White spot syndrome virus (WSSV) is one of the major pathogens in the cultured shrimp. However, the molecular mechanisms of the host-virus interaction remain largely unknown. In this study, the impact of WSSV infection on host gene expression in the hepatopancreas of L. vannamei was investigated through the use of 454 pyrosequencing-based RNA-Seq of cDNA libraries developed from WSSV-challenged shrimp or normal controls. By comparing the two cDNA libraries, we show that 767 host genes are significantly up-regulated and 729 genes are significantly down-regulated by WSSV infection. KEGG analysis of the differentially expressed genes indicated that the distribution of gene pathways between the up- and down-regulated genes is quite different. Among the differentially expressed genes, several are found to be involved in various processes of animal defense against pathogens such as apoptosis, mitogen-activated protein kinase (MAPK) signaling, toll-like receptor (TLR) signaling, Wnt signaling and antigen processing and presentation pathways. The present study provides valuable information on differential expression of L. vannamei genes following WSSV infection and improves our current understanding of this host-virus interaction. In addition, the large number of transcripts obtained in this study provides a strong basis for future genomic research on shrimp.

Analysis of Hsp70 in Litopenaeus vannamei and detection of SNPS

Abstract The Hsp70 gene plays an important role in the animal immune response. In order to analyze the linkage between the genetic polymorphisms of Hsp70 and the virus-resistance trait of the shrimp, we detected SNPs in the coding region of Hsp70 by direct sequencing in 104 specimens of Litopenaeus vannamei from three shrimp populations (one TSV-resistance line and two TSV-susceptibility lines). Five SNPs were found including 661C/A, 712T/C, 782C/T, 892C/T, and 1090C/T in the fragment of Hsp70 (GenBank accession no. AY645906). There were significant difference of the allelic frequency of SNP 892C/T polymorphism between the TSV-resistance population and the two TSV-susceptibility populations (P < 0.001). A TSV-challenged test was performed to divide the shrimps into TSV-resistance group and TSV-susceptibility group. The 892C/T SNP genotypes of each shrimp sample were detected by pyro-sequencing. The allelic frequency of SNP 892C/T were significant difference between TSV-resistance group and TSV-susceptibility group (P < 0.001). The results show that the genetic polymorphisms of Hsp70 are likely associated with virus-resistance trait in populations of L. vannamei.

Molecular cloning, expression, promoter analysis and functional characterization of a new Crustin from Litopenaeus vannamei

ABSTRACT Antimicrobial peptides (AMPs) are the most important players in the innate immune system, providing a principal first‐line of defense against the invading pathogens. Crustin, a type of whey acidic protein (WAP) domain‐containing and cationic cysteine‐rich AMP, can function in a protease inhibition or an effector molecule manner. In the present study, a new Crustin was cloned and identified from Pacific white shrimp Litopenaeus vannamei and designated as LvCrustinA. The full‐length cDNA of LvCrustinA was 687 bp, with a 519 bp open reading frame (ORF) that encoded a peptide of 172 amino acids. Domain analysis indicated that LvCrustinA contained a Glycine‐rich region in the N‐terminal and a single WAP domain within eight cysteines in the C‐terminal. The 5′ upstream regulatory sequence of 1249 bp (promoter) was obtained using a genome walking method, and it contained several conserved transcription factors binding motifs including NF‐&kgr;B, AP‐1 and STAT (Signal transducers and activators of transcription). Dual‐reporter assay showed that NF‐&kgr;B transcription factors LvDorsal and LvRelish, and AP‐1 transcription factor Lvc‐Jun could up‐regulate the promoter activity of LvCrustinA, suggesting that NF‐&kgr;B and JNK‐c‐Jun pathways could be involved in regulating the expression of LvCrustinA. Moreover, LvCrustinA was abundantly expressed in immune related tissues such as gill, hemocyte and epithelium, and its expression was up‐regulated in response to Vibrio parahaemolyticus and White spot syndrome virus (WSSV) challenges in gill tissue, suggesting that LvCrustinA could be involved in the host defense against bacterial and viral infection. Additionally, RNAi mediated knockdown of LvCrustinA resulted in shrimps with the higher cumulative mortality during V. parahaemolyticus and WSSV infection. Taken together, these results provided some insight into the expression and transcriptional regulatory role of LvCrustinA, and its defensive role against pathogenic infection. HIGHLIGHTSThe new Crustin gene (LvCrustinA) is identified from pacific white shrimp Litopenaeus vannamei.LvCrustinA is transcriptionally regulated by both NF‐&kgr;B and AP‐1 pathways.LvCrustinA plays a protective role in Vibrio parahaemolyticus and WSSV infection.

Identification of cold responsive genes in Pacific white shrimp (Litopenaeus vannamei) by suppression subtractive hybridization.

The Pacific white shrimp (Litopenaeus vannamei) is one of the most widely cultured shrimp species in the world. Despite L. vannamei having tropical origins, it is being reared subtropically, with low temperature stress being one of the most severe threats to its growth, survival and distribution. To unravel the molecular basis of cold tolerance in L. vannamei, the suppression subtractive hybridization (SSH) platform was employed to identify cold responsive genes in the hepatopancreas of L. vannamei. Both forward and reverse cDNA libraries were constructed, followed by dot blot hybridization, cloning, sequence analysis and quantitative real-time PCR. These approaches identified 92 cold induced and 48 cold inhibited ESTs to give a total of 37 cold induced and 17 cold inhibited contigs. Some of the identified genes related to stress response or cell defense, such as tetraspanins (TSPANs), DEAD-box helicase, heat shock proteins (HSPs) and metallothionein (MT), which were more abundant in the forward SSH library than in the reverse SSH library. The most abundant Est was a tetraspanin-8 (TSPAN8) homolog dubbed LvTSPAN8. A multiple sequence alignment and transmembrane domain prediction was also performed for LvTSPAN8. LvTSPAN8 expression was also examined in the gills, muscle, heart and hepatopancreas following cold exposure and showed the highest expression levels in the hepatopancreas. Overall, this study was able to identify several known genes and novel genes via SSH that appear to be associated with cold stress and will help to provide further insights into the molecular mechanisms regulating cold tolerance in L. vannamei.

Cloning and analysis of full length cDNA of peritrophin in Litopenaeus vannamei

In order to study the peritrophin function of Litopenaeus vannamei related to IHHNV invasion,the full-length cDNA library from intestinal tissue of Litopenaeus vannamei was constructed by switching mechanism at 5′ end of the RNA transcript technique(SMART),a full-length cDNA of peritrophin was obtained by gene splicing and its characterization was analyzed.The capacity of constructed library was up to 1.05×106 pfu/mL,and its recombinant coefficient was 95%.1 600 clones selected randomly were sequenced,1 531 available sequences with inserted length ≥450 bp were obtained after removing vector.One cDNA of peritrophin gene sequence of 1 002 bp was obtained from the clones,its protein encodes 303 amino acids.The nucleotide sequences of Litopenaeus vannamei have an overall similarity of 80% to peritrophin 1,2 precursor and peritrophin 3 precursor of Penaeus monodon.Expression of peritrophin gene in different tissue was analyzed by semi-quantitative reverse transcription PCR(RT-PCR),the result showed that the expression of peritrophin transcript in myocardium and gastric was higher,the expression was lower in hepatopancreas,intestines and muscle,the minimal expression was in gills,but not in eyes;Expression was obviously weakened in heart of IHHNV injected Litopenaeus vannamei,expression was lower in hepatopancreas,eyestalk,muscle and gills tissues,there was almost no expression in intestines and stomach.These results suggest that peritrophin of Litopenaeus vannamei was involved in prevention of IHHNV invasion.

Identification of microRNAs involved in cold adaptation of Litopenaeus vannamei by high-throughput sequencing

The Litopenaeus vannamei (L. vannamei) is one of the most widely cultured shrimp species in the world, with low temperature being one of the most serious threats to its growth and survival. To examine the potential regulatory mechanism of cold adaptation, we conducted a microRNAs (miRNAs) analysis on the hepatopancreas of L. vannamei under normal temperature 28 °C (M28), cold acclimation 16 °C for 6 days (M16), and recovered under normal temperature (MR). In total 14,754,823, 14,945,246 and 15,880,093 raw reads representing 10,690,259, 8,587,144, and 11,512,941 unique sequences of 18-32 nt length were obtained from the M28, M16 and MR libraries, respectively. After comparing the miRNA sequences with the miRBase database, 68 known mature miRNAs and 47 novel miRNAs were identified. Expression analysis showed that 34 miRNAs were significantly differential expressed in response to cold adaptation. Compared to the M28 library, 21 miRNAs were upregulated and 13 miRNAs were downregulated significantly in the M16 library. After recovery to normal temperature, there are 16 miRNAs upregulated and 15 miRNAs downregulated significantly compared to M28 library. Then, five significantly differential expressed miRNAs under cold acclimation including three known miRNAs (mja-miR-6491, mja-miR-6494, and Bta-miR-2478) and two newly-identified miRNAs (novel_68 and novel_5) were selected for validation by RT-qPCR in the hepatopancreas and muscle tissues of cold treated shrimps. The expression trend of most the miRNAs from RT-qPCR were consistent with the next-generation sequencing data. Further, the Gene Ontology (GO) annotation showed that the metabolic process GO term was significantly enriched with target genes of the differentially expressed miRNAs. Additionally, KEGG pathway analysis suggested that the fatty acid degradation and glycerolipid metabolism pathways etc. are significantly enriched with the target genes. These findings may contribute to a better understanding of the molecular mechanisms governing the responses to low temperature in L. vannamei.

Proteomic responses under cold stress and recovery reveal unique cold tolerant mechanism in the Pacific white shrimp (Litopenaeus vannamei)

The Pacific white shrimp (Litopenaeus vannamei), one of the most widely cultured shrimp species in the world, often suffers from cold stress. To understand the molecular mechanism of cold tolerance in Pacific white shrimp, we conducted a proteomic analysis on two contrasting shrimp cultivars, namely, cold-tolerant Guihai2 (GH2) and cold-sensitive Guihai1 (GH1), under normal temperature (28°C), under cold stress (16°C), and during recovery to 28°C. In total, 3,349 proteins were identified, among which 2,736 proteins were quantified. Based on gene ontology annotations, differentially expressed proteins largely belonged to biological processes, cellular components, and molecular functions. KEGG pathway annotations indicated that the main changes were observed in the lysosome, ribosomes, and oxidative phosphorylation. Subcellular localization analysis showed a significant increase in proteins present in cytosol, extracellular regions, and mitochondria. Combining enrichment-based clustering analysis and qRT-PCR analysis, we found that glutathione S-transferase, zinc proteinase, m7GpppX diphosphatase, AP2 transcription complex, and zinc-finger transcription factors played a major role in the cold stress response in Pacific white shrimp. Moreover, structure proteins, including different types of lectin and DAPPUDRAFT, were indispensable for cold stress tolerance of the Pacific white shrimp. Results indicate the molecular mechanisms of the Pacific white shrimp in response to cold stress and provide new insight into breeding new cultivars with increased cold tolerance.

Effect of low temperature on globin expression, respiratory metabolic enzyme activities, and gill structure of Litopenaeus vannamei

Low temperature frequently influences growth, development, and even survival of aquatic animals. In the present study, physiological and molecular responses to low temperature in Litopenaeus vannamei were investigated. The cDNA sequences of two oxygen-carrying proteins, cytoglobin (Cygb) and neuroglobin (Ngb), were isolated. Protein structure analysis revealed that both proteins share a globin superfamily domain. Real-time PCR analysis indicated that Cygb and Ngb mRNA levels gradually increased during decrease in temperatures from 25 to 15°C and then decreased at 10°C in muscle, brain, stomach, and heart, except for a continuing increase in gills, whereas they showed a different expression trend in the hepatopancreas. Hemocyanin concentration gradually reduced as the temperature decreased. Moreover, the activities of respiratory metabolic enzymes including lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were measured, and it was found that LDH activity gradually increased while SDH activity decreased after low-temperature treatment. Finally, damage to gill structure at low temperature was also observed, and this intensified with further decrease in temperature. Taken together, these results show that low temperature has an adverse influence in L. vannamei, which contributes to systematic understanding of the adaptation mechanisms of shrimp at low temperature.