Lishi Yang

Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress.

ABSTRACT Chitinase is a multi‐gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase‐4 gene (PmChi‐4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi‐4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi‐4 protein was 67.7 kDa and shared 61%–88% identity with the type of Chi‐4s from other crustaceans. Quantitative real‐time (qRT‐PCR) analysis indicated that PmChi‐4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi‐4 was expressed throughout the whole larvae stages, and the highest level of PmChi‐4 transcripts was detected at Mysis3 stage, which indicated that PmChi‐4 may be involved in larval metamorphosis. In order to know whether PmChi‐4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi‐4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi‐4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi‐4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi‐4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon. HighlightsA chitinase‐4 gene was cloned from P. monodon by RACE method.The full length of PmChi‐4 cDNA was 2178 bp, including an 1815 bp ORF.qRT‐PCR was performed to study the PmChi‐4 expression pattern in different tissues and larvae stages.PmChi‐4 expression level increased after S. agalactiae and V. harveyi infection.PmChi‐4 expression level declined after ammonia nitrogen stress treatment.

Characterization of complement 1q binding protein of tiger shrimp, Penaeus monodon, and its C1q binding activity.

The receptor for the globular heads of C1q, C1qBP/gC1qR/p33, is a multicompartmental, multifunctional cellular protein with an important role in infection and in inflammation. In the present study, we identified and characterized the complement component 1q subcomponent binding protein (C1qBP) from the tiger shrimp Penaeus monodon (designated as PmC1qBP). The open reading frame of PmC1qBP encodes 262 amino acid residues with a conserved MAM33 domain, an arginine-glycine-aspartate cell adhesion motif, and a mitochondrial targeting sequence in the first 53 amino acids. PmC1qBP shares 32%-81% similarity with known C1qBPs and clusters with lobster gC1qR under phylogenetic analysis. The temporal PmC1qBP mRNA expression in the hepatopancreas was significantly enhanced at 9 h after Vibrio vulnificus challenge. The native PmC1qBP was expressed in the gills, hepatopancreas, ovaries, and intestines as a precursor (38 kDa) and the active peptide (35 kDa). The recombinant PmC1qBP protein was expressed in Escherichia coli BL21, and was purified using nickel-nitrilotriacetic acid agarose. A complement 1q binding assay indicated that the rC1qBP protein competitively binds to C1q in mouse serum. The data reveal that PmC1qBP is not only involved in shrimp immune responses to pathogenic infections, but also cross-binding to the mouse C1q.

Isolation and characterization of homologous TRBP cDNA for RNA interference in Penaeus monodon

The transactivation response RNA-binding protein (TRBP) interacts with Dicer and binds to double-stranded RNA as a critical component of the RNA-induced silencing complex, which is a key complex in the RNA interference pathway. The full-length cDNA of TRBP from the tiger prawn, Penaeus monodon, (PmTRBP; 1548 bp long with a 1029 bp coding region) was isolated. The encoded polypeptide of 343 amino acids had a predicted molecular mass of 36.8 kDa. Sequence homology and phylogenetic analysis indicated that PmTRBP was evolutionarily closest to TRBP1 from Litopenaeus vannamei, with the three double-stranded RNA-binding motifs that were typical of the TRBP family. Tissue expression profile analysis by quantitative real-time reverse transcription polymerase chain reaction showed that PmTRBP1 was constitutively expressed in all the examined tissues, with a predominant expression in the lymphatic organs and with the weakest expression in the ovaries. Significantly upregulated PmTRBP1 expression was elicited by systemic injections of Staphylococcus aureus, Vibrio vulnificus, and white spot syndrome virus, thereby revealing its pathogen inducibility. Furthermore, exogenous viral nucleoside analogs (high-molecular-weight poly(I:C) dsRNAs as well as R484 single-stranded RNA) were remarkably induced PmTRBP1 transcription at 48 h and 9 h post-injection, respectively, which suggested that PmTRBP1 might function in tiger prawn antibacterial and antiviral response.

Toll-receptor 9 gene in the black tiger shrimp (Penaeus monodon) induced the activation of the TLR–NF-κB signaling pathway

Toll receptors are important pathogen recognition receptors (PRRs) in shrimps, which play a vital role in defending against virus and bacterial challenge. In this paper, the characterization and functional analysis of a Toll9 receptor gene from Penaeus monodon was performed in HEK293T cells. Data showed that PmToll9 can activate the NF-κB promoter activities of TLR pathway, while ISRE and IFN-β promoter cannot be activated obviously in HEK293T cells using dual-luciferase reporter system. The downstream immune factors of IL-8, IκB-α, and TRAF6 were activated by PmToll9 and IL-8 showed the most significant up-regulation in expression levels, indicating the activities of NF-κB can be mediated by PmToll9. Six LRRs-deletion mutants were constructed and results showed these mutants had obvious declines in luciferase activities, among which the mutant pCMV-DeLRR4 showed the most significant decline. qPCR data indicated LRRs-deletion mutants efficiently impaired the activities of the downstream immune factors IL-8, IκB-α, and TRAF6. It demonstrates that LRRs-deletion mutants could result in the weaken abilities of PmToll9 in signaling transduction. Overexpression of PmToll9-GFP fusion protein in Hela cells revealed the primary cellular localization of PmToll9 is in the cytoplasm.

The comprehensive expression analysis of the G protein-coupled receptor from Penaeus monodon indicating it participates in innate immunity and anti-ammonia nitrogen stress

ABSTRACT The G protein‐coupled receptors (GPCRs) composed a superfamily that played an important role in physiological processes of crustaceans, with multiple functions such as growth and development, acting as a defense against stimulations from external factors. In this paper, one kind of GPCRs were identified from Penaeus monodon, called PmGPCR, included an open reading frame (ORF) of 1113 bp. Bioinformatic analysis showed that PmGPCR protein had the typical structure of seven transmembrane domains (7TM), especially the special Asp‐Arg‐Try motif (DRY motif) between the third transmembrane structures (TM3) and the second intracellular loops (IL‐2) which can prove that PmGPCR belongs to the rhodopsin‐like family. The analyses of phylogenetic tree indicated that the amino acid sequence of PmGPCR should be merged into Procambarus clarkiic with high identity (98%). Quantitative real‐time PCR (q RT‐PCR) revealed that PmGPCR mRNA was highly expressed in hepatopancreas, abdominal ganglia and lymph, in which it was significantly higher than that of other tissues (P < 0.05). In addition, the expression of PmGPCR was analyzed during three days post‐stimulation with the gram‐positive/negative bacteria, the mRNA expression level increased after challenged with gram ‐ positive bacteria in hepatopancreas, lymph and intestines. During the development stages, PmGPCR showed significantly higher expression in nauplius, zoea III, mysis III and post larvae stages than that in other development stages. Meanwhile, the highest transcripts expression of PmGPCR in abdominal ganglia, hepatopancreas, lymph and intestines respectively appeared at D0, D1, D2 and D3/D4 stages of molting. High or low concentration of ammonia nitrogen up‐regulated the expression level of PmGPCR at the initial stage in hepatopancreas and gill, and then down‐regulated at 48 h. These results indicated PmGPCR may mediate the pathways that involved in growth and development process, survival in the adversity, in addition, provided the useful data to research GPCR‐mediated physiological and biological process and explain the mechanisms to defense pathogens and anti‐stress in shrimp. HighlightsA GPCR gene belongs to the rhodopsin‐like subfamily was identified and characterized from Penaeus monodon.The transcripts of PmGPCR had a certain pattern in tissues during the whole growth and development stages.PmGPCR highly expressed in premolt stage in abdominal nerve, hepatopancreas, lymph and intestines.PmGPCR mRNA was up‐regulated after injected by gram‐positive bacteria.Ammonia‐N stress significantly influenced the mRNA levels of PmGPCR in the tissues.

Transcriptome reveals involvement of immune defense, oxidative imbalance, and apoptosis in ammonia-stress response of the black tiger shrimp (Penaeus monodon)

Abstract Ammonia is a major aquatic environmental pollutant that negatively impacts shrimp health and commercial productivity. However, we currently do not fully understand the underlying molecular mechanisms of ammonia stress in shrimp. We therefore performed transcriptomic analysis of hepatopancreas from black tiger shrimp (Penaeus monodon) treated with ammonia‐stress. We obtained 146,410,174 and 115,241,048 clean reads for the control and treatment groups, respectively. A total of 64,475 unigenes with an average length of 1275 bp and a N50 value of 2158 bp were assembled. A comparative transcriptome analysis identified 3462 differentially expressed genes, 177 of which are highly homologous with known proteins in aquatic species. Most of these genes showing the expression changes were related to immune function. Some significantly down‐regulated genes are involved in purine metabolism and other metabolic pathways, which suggests that purineolytic capacity is an ammonia detoxification process in P. monodon, and metabolic depression is a strategy to reduce shrimp exposure to ammonia. Additionally, ammonia stress altered the expression patterns of key apoptosis genes (Bcl‐xL, PERK, caspase 7, and caspase 10), confirmed that ammonia‐stress induce oxidative stress and eventually even apoptosis. We also found evidence for the involvement of antioxidant defense in response to oxidative imbalance, given the regulation of peroxiredoxin 1, SOD, and CAT under ammonia stress. In conclusion, our study clarifies shrimp defensive response to ammonia toxicity and should benefit efforts to breed more ammonia‐tolerant varieties. HighlightsTranscriptome analysis clarifies shrimp defensive response under ammonia stress.Ammonia detoxification occurs through conversion to purine via metabolic pathways.Metabolism is then decreased to minimize burden from excess purine.Ammonia treatment causes severe oxidative damage and triggers apoptosis.The antioxidant system then mitigates oxidative stress and associated cell death.

Influence of low dosage infection on WSSV in latent period and haemocyte of Penaeus monodon

The changes in mortalities of Penaeus monodon(with 1×105 copies/g WSSV),WSSV loads in muscle,THC and the percentage of different types of haemocyte were studied by injection with WSSV solution containing 3×103,6×102,2×102 copies/mL virus and PBS.Real-time PCR,and microscopical examination were used in this experiment.The results showed that the cumulative mortalities were 93.33%±2.89%,56.67%±5.77%,45.00%±5.00% respectively,when the concentration of WSSV solution were 3×103,6×102,2×102 copies/mL.No death was observed in the PBS group.When the concentration of WSSV solution was 3×103 copies/mL,the virus load in the muscle reduced to minimum(3.54×104 copies/g)at 30 min post-injection,and reached its maximum(3.12×108 copies/g)at 48 h post-injection,then decreased again.For the other two experiment groups,the minimal virus load(1.11×105,9.54×104 copies/g)in the muscle was observed at 1 h post-injection and the highest virus load(1.48×107,5.46×106 copies/g)occurred at 48 h post-injection,but a second highest virus load(1.58×105,1.11×105 copies/g)appeared at 6 h post-injection.No significant changes were found in the WSSV load of the control group throughout the experiment.The fluctuation range and the peak time of total haemocyte count(THC)were different between different dosage of WSSV solution.Especially THC reduced to minimum(3.48×106,4.05×106/mL)at 12 h and 48 h post-injection with 3×103 copies/mL solution.The variation of THC and WSSV load were negatively correlated except individual time points.Under all groups,the percentage of semi-granular cells showed upward trend obviously early post infection,but later remained at a high level although fluctuated;the percentage of granular and hyalinocyte cells showed downward trend early post-injection significantly,while increased slightly in the middle period,but the percentage of granular cells at the later stage of post-infection was significantly lower than that of early period,while the levels of hyaline cells between later and early stages of post-injection have no significant difference.