Shigui Jiang

Molecular cloning and mRNA expression of cathepsin C gene in black tiger shrimp (Penaeus monodon).

Cathepsin C (dipeptidyl-peptidase I, DPPI) is a lysosomal cysteine proteinase belonging to the papain superfamily, which is capable of removing dipeptides sequentially from the amino terminus of peptide and protein substrates. In the present study, the cDNA of a cathepsin C was cloned from black tiger shrimp Penaeus monodon (designated PmcathepsinC) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of PmcathepsinC consisted of 2051 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 1350 bp encoding a polypeptide of 449 amino acid residues with a predicted molecular weight of 50.0 kDa and theoretical isoelectric point of 5.65. The high identity of PmcathepsinC with Cathepsin C in other organisms indicated that PmcathepsinC should be a new member of the Cathepsin C family. By fluorescent quantitative real-time PCR, mRNA transcript of PmcathepsinC was detectable in all the examined tissues with higher level in ovary and heart. The temporal expression of PmcathepsinC mRNA in the hepatopancreas was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at 4 h post-stimulation, and then dropped back to the original level gradually. These results indicated that PmcathepsinC was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of P. monodon.

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.

Dietary supplementation of honeysuckle improves the growth, survival and immunity of Penaeus monodon

Two trials were conducted to determine the effects of honeysuckle on shrimp, Penaeus monodon, first on growth performance, secondly on the immune response of shrimp. In trial 1, shrimp (mean initial wet weight about 3.02 g) were fed with five diets containing 0% (basal diet), 0.1%, 0.2%, 0.4% and 0.8% honeysuckle in triplicate for 60 days. Growth performance (final body wet weight, FBW; weight gain, WG; biomass gain, BG) of shrimp fed honeysuckle diets were higher (P < 0.05) than that of shrimp fed the basal diet, shrimp fed 0.4% honeysuckle diet showed the highest value of growth performance. Shrimp fed 0.2% honeysuckle diet showed highest value of survival. The total antioxidant status (TAS) and glutathione peroxidase (GSH-Px) activity of shrimp fed 0.2%, 0.4% and 0.8% honeysuckle diets were higher (P < 0.05) than those of shrimp fed basal and 0.1% honeysuckle diets. Hepatopancreas malondialdehyde (MDA) of shrimp fed honeysuckle diets were lower (P < 0.05) than that of shrimp fed the basal diet. Total haemocyte count of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed honeysuckle diets. Haemolymph clotting time of shrimp had the opposite trend with the total haemocyte count of shrimp. In trial 2, the shrimp were exposed to air during a simulated live transportation for 36 h after the rearing trial. The antioxidant responses were characterized by lower TAS and higher antioxidant enzyme activities (superoxide dismutase: SOD, GSH-Px) and higher oxidative stress level (MDA) in the hepatopancreas compared to levels found in trial 1. No mortalities were observed in any diet groups after 36 h of simulated live transportation. The glutathione (GSH) content and TAS of shrimp fed 0.2%, 0.4% and 0.8% honeysuckle diets were higher (P < 0.05) than those of shrimp fed the basal and 0.1% honeysuckle diets. The SOD activity of shrimp fed the basal diet was higher (P < 0.05) than that of shrimp fed honeysuckle diets. The GSH-Px activity of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed 0.2%, 0.4% and 0.8% honeysuckle diets but without significant difference (P > 0.05) with shrimp fed 0.1% honeysuckle diet. Moreover, the oxidative stress level (MDA) recorded in the hepatopancreas with shrimp submitted to the honeysuckle diets were lower. In conclusion, results suggested that dietary intake containing honeysuckle could enhance the growth performance of P. monodon and improve its resistance to air exposure during simulated live transportation. Considering the effect of honeysuckle on both growth performance and survival of P. monodon, the level of honeysuckle supplemented in the diet should be between 0.2% and 0.4%.

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.

A preliminary study for identification of candidate AFLP markers under artificial selection for shell color in pearl oyster Pinctada fucata.

Pearl oyster Pinctada fucata is widely cultured to produce seawater pearl in South China, and the quality of pearl is significantly affected by its shell color. Thus the Pearl Oyster Selective Breeding Program (POSBP) was carried out for the shell color and growth traits. The black (B), gold (G), red (R) and white (W) shell strains with fast growth trait were achieved after five successive generation selection. In this study, AFLP technique was used to scan genome of four strains with different shell colors to identify the candidate markers under artificial selection. Eight AFLP primer combinations were screened and yielded 688 loci, 676 (98.26%) of which were polymorphic. In black, gold, red and white strains, the percentage of polymorphic loci was 90.41%, 87.79%, 93.60% and 93.31%, respectively, Neis gene diversity was 0.3225, 0.2829, 0.3221 and 0.3292, Shannons information index was 0.4801, 0.4271, 0.4825 and 0.4923, and the value of FST was 0.1805. These results suggested that the four different shell color strains had high genetic diversity and great genetic differentiation among strains, which had been subjected to the continuous selective pressures during the artificial selective breeding. Furthermore, six outlier loci were considered as the candidate markers under artificial selection for shell color. This study provides a molecular evidence for the inheritance of shell color of P. fucata.

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.

Characterization of complete mitochondrial genome of fives tripe wrasse (Thalassoma quinquevittatum, Lay & Bennett, 1839) and phylogenetic analysis

To further supplement the genome-level features in related species, T. quinquevittatum complete mtDNA was firstly sequenced and de novo assembled by next-generation sequencing. The full-length mtDNA of T. quinquevittatum was a 16,896bp fragment, which was atypical of Labridae, with 2 ribosomal RNA (rRNA) genes, 13 protein-coding genes (PCGs), 23 transfer RNA (tRNA) genes, and a major non-coding control region (D-loop region). Additionally, the mtDNA of T. quinquevittatum exhibited characteristics of A (27.1%), T (29.3%), G (17.8%), and C (25.8%) with a high A+T content (56.4%). Furthermore, the analysis of the average Ka/Ks in the 13 PCGs of three Labridae species indicated a strong purifying selection within this group. Additionally, the phylogenetic analysis based on 13 concatenated PCGs nucleotide and amino acid datasets, showed high value support for the following sister clade among the four genera (T. quinquevittatum, Halichoeres trimaculatus, Halichoeres melanurus, Parajulis poecilepterus). The complete mtDNA of the T. quinquevittatum provided important information for the study in population genetics and evolutionary theory.

The complete mitochondrial genome of banana shrimp Fenneropenaeus merguiensis with phylogenetic consideration

Abstract The complete mitochondrial genome sequence of Fenneropenaeus merguiensis was determined by shotgun assembly method. The complete mitochondrial DNA sequence is a circular molecule with 16,023 bp in length including 13 protein-coding genes, 22 transfer RNA genes, 2 rRNA genes and a control region. The gene arrangements are consistent with the pan crustacean ground pattern. The molecular analyses provided robust evidence for the monophyly of Fenneropenaeus, but Litopenaeus was not monophyletic. Phylogenetic analyses robustly supported the fact that genus Penaeus s.l. contains the two lineages: Marsupenaeus and Penaeus s.s+ Fenneropenaeus + Litopenaeus + Farfantepenaeus.

Shotgun assembly of the mitochondrial genome from Fenneropenaeus penicillatus with phylogenetic consideration.

The complete mitochondrial genome is of great importance for better understanding of the genome-level characteristics and phylogenetic relationships among related species. In this study, Fenneropenaeus penicillatus mitochondrial genome sequence was determined by next-generation sequencing. The complete genome DNA was 16,040 bp in length and consisted of a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). The gene arrangement is identical to the pancrustacean pattern. The overall base composition of its mitochondrial genome is estimated to be 34.1% for A, 34.1% for T, 12.5% for G and 19.3% for C with a high A+T content (68.2%). The analysis of the average Ka/Ks in the 13 mitochondrial protein-coding genes of penaeid shrimps indicated a strong purifying selection within this group. The phylogenetic analysis based on mitochondrial sequences and 13 concatenated protein-coding genes showed strong statistic support for the following relationship among the five genera ((Penaeus s.s+Fenneropenaeus)+(Litopenaeus+Farfantepenaeus))+Marsupenaeus. The sequence data of F. penicillatus can provide useful information for the studies on molecular systematics, population structure, stock evaluation and conservation genetics.