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Abstract: This study firstly reports the characterization of a functional IκB homologue, FcCactus in Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcCactus consists of a 1359bp open reading frame (ORF) encoding a 453 amino acid protein with a predicted molecular weight (MW) of 48.46kDa and theoretical pI of 5.23. Phylogenetic analysis and multiple alignments revealed that the deduced amino acid sequence of FcCactus cDNA had high similarities to Cactus or IκB reported in seven other arthropods. Genomic DNA sequence of FcCactus was also obtained with a length of more than 17698bp and constituted of seven exons and six introns. Analysis on 5′-upstream regulatory region of its DNA sequence revealed that it contained the core promoter sequence with the TATA-box and transcription start site existing in it; furthermore, various transcription factor binding sites (HSF, Hb, BR-C Z, Dfd, CF2-II, Croc, Ttk, Dorsal, and c-Rel) were predicted. Spatial expression profiles showed that FcCactus mRNA had the highest expression level in muscle, hemocytes, heart and lymphoid organ. Gram-positive bacteria (Micrococcus lysodeikticus) and Gram-negative bacteria (Vibrio anguillarium) injection to shrimp caused the modulation of FcCactus at the transcription level. DsRNAi (double-strand RNA interference) approach was used to study the function of FcCactus and the data showed that FcCactus could regulate the expression of different antimicrobial peptides (AMPs) and antiviral factor (AV). The present data showed that FcCactus might play important roles in regulating the immune response of shrimp. [Copyright &y& Elsevier]

Peptides in shrimp hemolymph play an important role in the innate immune response. Analysis of hemolymph will help to detect and identify potential novel biomarkers of microbial infection. We used magnetic bead-based purification (ClinProt system) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to characterize shrimp hemolymph peptides. Shrimp serum and plasma were used as the source of samples for comparative analysis, and it was found that serum was more suitable for shrimp hemolymph peptidomic analysis. To screen potential specific biomarkers in serum of immune-challenged shrimps, we applied magnetic bead-based MALDI-TOF MS to serum samples from 10 immune-challenged and 10 healthy shrimps. The spectra were analyzed using FlexAnalysis 3.0 and ClinProTools 2.1 software. Thirteen peptide peaks significantly different between the two groups were selected as candidate biomarkers of lipopolysaccharide (LPS)-infection. The diagnostic model established by genetic algorithm using five of these peaks was able to discriminate LPS-challenged shrimps from healthy control shrimps with a sensitivity of 90% and a specificity of 100%. Our approach in MALDITOF MS-based peptidomics is a powerful tool for screening bioactive peptides or biomarkers derived from hemolymph, and will help to enable a better understanding of the innate immune response of shrimps. [ABSTRACT FROM AUTHOR]

Genetic linkage maps of Fenneropenaeus chinensis were constructed using a 'double pseudo-testcross' strategy with 200 single nucleotide polymorphisms ( SNPs) markers. This study represents the first SNP genetic linkage map for F. chinensis. The parents and F progeny of 100 individuals were used as mapping populations. 21 genetic linkage groups in the male and female maps were identified. The male linkage map was composed of 115 loci and spanned 879.7 cM, with an average intermarker spacing of 9.4 cM, while the female map was composed of 119 loci and spanned 876.2 cM, with an average intermarker spacing of 8.9 cM. The estimated coverage of the linkage maps was 51.94% for the male and 53.77% for the female, based on two estimates of genome length. The integrated map contains 180 markers distributed in 16 linkage groups, and spans 899.3 cM with an average marker interval of 5.2 cM. This SNP genetic map lays the foundation for future shrimp genomics and genetic breeding studies, especially the discovery of gene or regions for economically important traits in Chinese shrimp. [ABSTRACT FROM AUTHOR]

A C-type lectin-like protein was cloned and characterized from the Chinese shrimp Fenneropenaeus chinensis, and named as FcCTL. The results indicated that the full length cDNA of 859 bp had an open reading frame encoded a polypeptide of 220 amino acids with one carbohydrate-recognition domain, six conserved Cys and one key motif EPGD. The theoretical molecular weight and pI of mature protein was 25.3 kDa and 5.4. Sequence comparison of the deduced amino acid sequence of FcCTL showed varied identity of 26-34, 34, 31 and 30 % with those of F. chinensis, Portunus trituberculatus, Tetraodon nigroviridis, Penaeus monodon, respectively. qRT-PCR analysis indicated that FcCTL was expressed highest in hepatopancreas of normal shrimp, and it's expression was up-regulated in hepatopancreas and gills post white spot syndrome virus challenge. The purified recombinant FcCTL showed higher antimicrobial activity against Gram-positive bacteria than against Gram-negative bacteria and fungi. And the hemagglutinating activity of rFcCTL could be completely inhibited by GlcNAc (5 μg/ml), LPS (2.5 μg/ml), d-galactose (100 mM) and maltose (100 mM). These data suggested that FcCTL might play an important role in shrimp immune and would be helpful to better understand the innate immunity mechanism of shrimp. [ABSTRACT FROM AUTHOR]

Abstract: White spot syndrome virus (WSSV) is the most severe viral pathogen to the shrimp culture industry worldwide. The temporal and spatial dynamic changes of WSSV in Chinese shrimp Fenneropenaeus chinensis maintained at 22±1°C during infection via injection were studied through the quantitative real-time PCR method. The pleopods were selected for virus quantification at 0, 6, 12, 18, 24, 27, 30, 33, 36, 39, 42, 45 and 48hours post-injection (hpi). Besides the pleopod, different tissues including the hepatopancreas, nerve, heart, eyestalk, muscle, lymphoid organ, gut, stomach, gill and skin were also selected for viral load analysis at 0, 12, 27 and 30hpi as well as the moribund stage after 40hpi. The curve of viral load change in vivo assumed a very similar shape with the typical viral one-step growth curve. The infection process was classified into three stages: the light infection stage (0 to 18hpi) when the viral load in the pleopod was at the level of no more than 103/ng DNA and no mortality appeared; the moderate infection stage (18 to 45hpi), corresponding to the logarithmic phase of WSSV replication, when the viral load in the pleopod ranged from 103 to 105/ng DNA and a low mortality rate was observed; and the heavy infection stage when WSSV propagation went into the plateau phage and massive mortality happened. A turning point from chronic infection to acute infection was suggested from 27 to 30hpi when the viral load in most tissues increased substantially. The progress rate of WSSV in different tissues during the infection was different and the most favorite replication tissue of WSSV in early infection is the stomach, followed by the gill. The heaviest infected tissues are the skin, stomach and gill, explaining the symptoms of skin color changes, reduction of food consumption and gathering at the water surface due to dyspnea. The hepatopancreas, muscle and nerve were not the main replication sites in advanced infection. Also, the expression profiles of two anti-virus genes FcALF and FcAV were analyzed. This study might provide important data to study the virus infection mechanism. [Copyright &y& Elsevier]

Abstract: White spot syndrome virus (WSSV) caused a great economic loss in shrimp aquaculture. Although great efforts have been undertaken to characterize the virus disease during the last two decades, there are still lack of effective methods to prevent or cure it. In this study, we investigated the transcriptional expression profiles of 18 key immune-related genes in the Chinese shrimp Fenneropenaeus chinensis which was severely infected by WSSV. We found that the expression levels of 6 genes including chymotrypsin-like serine proteinase (CH-SPase), heat shock protein 70 cognate (HSP70), penaeidin (PEN), peroxinectin (PO), proliferating cell nuclear antigen (PCNA) and argonaute (AGO) changed significantly, while the expression of the other 12 genes had no significant changes compared to the control group. Among the 6 screened genes, CH-SPase showed significantly up-regulation, while the other 5 ones were significantly down-regulated. Knockdown of the expression of CH-SPase in WSSV-infected Chinese shrimp reduced the copy number of WSSV and delayed cumulative mortalities, suggesting that CH-SPase is important for WSSV infection. This study will be helpful to control the disease in shrimp caused by WSSV. [Copyright &y& Elsevier]

Trehalose is an important disaccharide and plays a key role in many organisms under different stress conditions. In the study, a gene ( FcTPS) encoded trehalose-6-phosphate synthase was reported from Chinese shrimp, Fenneropenaeus chinensis. The full-length cDNA of FcTPS is 3,281 bp including a poly A-tail of 20 bp, encoding a putative protein of 844 amino acids. The predicted protein contains a glycol_transf_20 domain and a trehalose_PPase domain. Genomic structure of FcTPS is composed of three exons with 192, 157 and 2,912 bp and two introns with 1,057 and 568 bp. In the second intron, four different SSRs are found. Transcripts of FcTPS gene are constitutively expressed in various tissues, with strongest level in hepatopancreas. After the shrimp were challenged with WSSV or Vibrio and the expression of FcTPS in hepatopancreas were analyzed using real-time PCR, the result showed that FcTPS transcript was down-regulated significantly in response to the challenge of Vibro at the early of 5 h post-challenge and then up-regulated significantly at 14 h. In addition, the expression of FcTPS showed the same result after the shrimp were challenged with WSSV. These results provide some new information about the tissue distribution, expression profiles and potential function of the trehalose-6-phosphate synthase in shrimp. [ABSTRACT FROM AUTHOR]

Abstract: An integrin β subunit (FcβInt) cDNA was cloned from hemocytes of Fenneropenaeus chinensis by rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of FcβInt consists of 2853bp and encodes a polypeptide of 783 amino acids with a predicted isoelectric point of 5.09. The deduced amino acid sequence of FcβInt possessed the classical domains of β integrin, including the βA domain and four epidermal-growth-factor (EGF)-like domains, which shared highest similarity with a β integrin of Pacifastacus leniusculus. The FcβInt mRNA was highly expressed in hemocytes, heart and gill, and moderately in gonad and intestine, whereas low expression level was found in muscle and hepatopancreas. FcβInt mRNA in gill was significantly up-regulated in 24h and then gradually decreased below the control level in response to WSSV challenge. The cDNA fragment encoding the extracellular region of FcβInt (FcβInt-ER) was expressed in Escherichia coli BL21 (DE3), and polyclonal antibodies against recombinant FcβInt-ER (rFcβInt-ER) were produced, which could specifically react to the hemocyte membrane by immunofluorescence assay and recognize a 120kDa protein in the hemocytes by western blotting. By ELISA and dot-blot binding assay, rFcβInt-ER exhibited specific WSSV binding activity. Neutralization assay in vivo further demonstrated that rFcβInt-ER could partially block WSSV infection to shrimp. These results suggested that FcβInt plays an important role in WSSV infection. [Copyright &y& Elsevier]

Abstract: Cathepsin C (Cath C) is a lysosomal cysteine protease that belongs to the papain superfamily. Cath C is capable of activating many chymotrypsin-like serine proteases and is reported to be a central coordinator for the activation of many serine proteinases in immune and inflammatory cells. In this study, Cath C cDNA was cloned from Fenneropenaeus chinensis (Fc). The complete cDNA of Fc-Cath C in Chinese white shrimp was found to be 1445-base pairs (bp) long. It contained an open reading frame (ORF) 1356-bp long and encoded a 451-amino acid residue protein, including a 17-amino acid residue signal peptide. Real-time PCR analysis results indicated that Fc-Cath C was present in all the tissues detected and exhibited high level of transcription in the hepatopancreas. In hemocytes, hepatopancreas, gills and intestine, Fc-Cath C was upregulated after stimulation by the Vibrio anguillarum and the white spot syndrome viruses (WSSVs). Replication of the WSSV increased after the injection of Fc-Cath C antiserum or knockdown Cath C by RNA interference. These results implied that Cath C might play a crucial role in the antiviral immune response of shrimp. [Copyright &y& Elsevier]

The transformer-2 gene, encoding a protein (Tra-2) which directs sex-specifically alternative splicing of doublesex (dsx) pre-mRNA in combination with the transformer (Tra) protein, has been proved to play important roles on sex differentiation and sex development in Drosophila melanogaster. In the present study, a tra-2 homologue (FcTra-2) was cloned and characterized in the Chinese shrimp, Fenneropenaeus chinensis. A FcTra-2 genomic DNA sequence with a length of 8,871 bp was obtained and verified to consist of 7 exons and 6 introns. Three alternatively spliced mRNA transcripts, designated as FcTra-2a, FcTra-2b and FcTra-2c, were isolated and characterized. Sequence analysis showed that FcTra-2 included a RNA recognition motif and a linker region, which shared high sequence identities with Tra-2 from other species and 2 arginine/serine rich regions. Further studies were performed on the isoform FcTra-2c, since it exhibited a significantly higher expression level in ovary than in other tissues. In early developmental stages of the shrimp, FcTra-2c was detected to suddenly increase its expression level at the mysis stage. In juvenile stage, FcTra-2c displayed a significantly higher expression level in female Chinese shrimp than in males. These data indicated that FcTra-2 might be involved in female sex determination in Chinese shrimp. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: ATP-binding cassette (ABC) transporters are integral membrane proteins which carry diverse substrates across biological membranes. In this study, we report the cloning of a full-length, 2122-bp cDNA sequence encoding a gene of the ABC transporter subfamily G (ABCG) from the shrimp Fenneropenaeus chinensis. The deduced protein, FcABCG, contains 633 amino acid residues with a theoretical molecular weight of 70.57kDa. Phylogenetic analysis showed that the FcABCG clusters with the shrimp Litopenaeus vannamei ABCG in the same clade and is more closely related to the insect ABC transporter/white proteins than to the ABCG2 of the fish Salmo salar. High-level mRNA expression of FcABCG was detected in the tissues of hepatopancreas and intestine using a real-time quantitative reverse transcriptase PCR. Furthermore, exposure to alkaline pH stress conditions and norfloxacin diets result in the activation of the FcABCG gene in F. chinensis. These results suggest that the ABCG transporter of F. chinensis might play a role in coping high pH stress and in exporting xenobiotics. [Copyright &y& Elsevier]

Abstract: Tetraspanins belong to the transmembrane 4 superfamily (TM4SF), which span the cell membrane 4 times and act as bridges or connectors. Increasing evidences have shown that tetraspanins play important role in virus infection. The large extracellular loop (LEL) of a tetraspanin is considered as a possible target of some virus. Tetraspanins are widely found in invertebrates, but the functional roles of most invertebrate tetraspanins have remained unknown. Recently, a tetraspanin, called FcTetraspanin-3, was cloned from the cDNA library of Chinese shrimp, Fenneropenaeus chinensis. The FcTetraspanin-3 constitutive expression in all examined tissues and the expression of the gene were highly induced in hepatopancreas, lymphoid organ and intestine by white spot syndrome virus (WSSV) challenge. In this study, we expressed and purified the recombinant peptide containing the LEL domain of FcTetraspanin-3, and produced the anti-LEL polyclone antibody. The expression of FcTetraspanin-3 was observed by real-time PCR and Western blot. Also, the localization of FcTetraspanin-3-positive cells in intestine and hepatopancreas were revealed by immunofluorescence. The results of anti-LEL antibody blocking experiments shown that the antibody can significantly reduce the mortality of shrimp challenged by WSSV. Additionally, dsRNA interference was utilized to examine the functional role of FcTetraspanin-3 in response to WSSV infection, and a sensible decrease of the viral copy number in the tetraspanin knockdown shrimp. These results suggested the blocking of LEL domain of FcTetraspanin-3 could inhibit the infection of WSSV. FcTetraspanin-3 might play an important role in response to WSSV infection, and the LEL domain of FcTetraspanin-3 might mediate the entry of WSSV. [Copyright &y& Elsevier]

This paper deals with the structure of mitochondrial DNA control region of Fenneropenaeus chinensis. The termination-associated sequence (TAS), cTAS, CSB-D-CSB-F, and CSB-1 are detected in the species. The results indicate that the structures of these parts are similar to those of most marine organisms. Two conserved regions and many stable conserved boxes are found in the extended TAS area, central sequences blocks, and conserved sequences blocks (CSBs). This is the special character of F. chinensis. All the mtDNA control region sequences do not have CSB2 and CSB3 blocks, which is quite different from most vertebrates. In addition, the complete mtDNA control region sequences are used to analyze the phylogenetic relationships of F. chinensis. The phylogenetic trees show a lack of genetic structure among populations, which is similar to many previous studies. [ABSTRACT FROM AUTHOR]

Hepatopancreatic parvovirus (HPV) of shrimp is distributed worldwide and the entire genome of Thailand and Indian strains ( PmDNV) and one Australian strain ( PmergDNV) have now been reported. The complete nucleotide sequence of a HPV strain isolated from the fleshy prawn Fenneropenaeus chinensis in Korea ( FcDNV) was determined and compared to previously reported sequences. The entire genome of FcDNV contains 6,336 nucleotides, with 40% G+C content, which is the biggest of the known HPV strains. The HPV genome has three open reading frames (ORFs) with a slight overlap between the first and second ORFs. The three ORFs encode the NS2 and NS1 proteins and VP that consist of 425, 578, and 820 amino acids, respectively. Among the three proteins, the NS1 protein shows the highest sequence similarity to the NS1 protein of other known HPV strains, followed by the NS2 protein and the VP protein. Phylogenetic analyses showed that HPV can be grouped into three genotypes, as previously reported, and FcDNV can be grouped as genotype I, with HPV strains isolated in Madagascar and Tanzania. The nucleotide sequences of the noncoding regions at the 5′- and 3′-ends of the plus-strand genome showed a Y-shaped hairpin structure and simple hairpin structure, respectively. [ABSTRACT FROM AUTHOR]

Abstract: Ras-related protein Rap GTPase has been implicated in cell adhesion, cell proliferation, and cell junction formation. The first shrimp Rap cDNA (FcRap) was recently identified from the Chinese white shrimp Fenneropenaeus chinensis. The full length of FcRap is 1013bp, with a 561bp open reading frame that encodes a 186 amino acid protein. FcRap has a calculated molecular mass of 20.90kDa and pI of 6.37. Phylogenetic analysis shows that FcRap and other Rap proteins are clustered into one group. Results from the quantitative real-time polymerase chain reaction show that FcRap could be detected mainly in the hemocytes, hepatopancreas, stomach, and gills, whereas a relatively lower expression level could be detected in the heart and intestines. FcRap in the hemocytes was upregulated 2h post Vibrio challenge, and it was upregulated 2h post white spot syndrome virus (WSSV) challenge, and peaked at 6h before it declined at 12h. No variation in the FcRap transcript was observed in the gills under the Vibrio challenge, but it was initially downregulated 2h post WSSV challenge, and then it was upregulated and peaked at 6h before it was eventually went down at 12h. The rFcRap protein was successfully expressed in Escherichia coli BL21DE3. The pull-down analysis showed that rFcRap protein could interact with VP28, an envelope protein of WSSV. The probable roles of Rap GTPase in shrimp innate immunity are presented for the first time. [Copyright &y& Elsevier]

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed. [ABSTRACT FROM AUTHOR]

Innate immunity is the first line of defense by a host against invading pathogens. Several signaling pathways participate in the immune response, one of which is the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Various evidences have been provided to suggest that the JAK/STAT pathway is involved in both antibacterial and antiviral immunities. In this study, the full-length cDNA and gene sequence of STAT (designated as FcSTAT) was cloned from the Chinese white shrimp, Fenneropenaeus chinensis. Phylogenetic analysis reveals that the FcSTAT is clustered with STAT5s and STAT6s from vertebrates and STATs from invertebrates. Quantitative real-time PCR exhibited that the FcSTAT had a wide distribution in all detected tissues and developmental stages. Time course analysis of the transcription level after WSSV challenge showed a noticeably early up-regulation of FcSTAT in hemocytes, hepatopancreas, and intestines. The expression levels of FcSTAT increased corresponding to Vibrio anguillarum stimulation in both hemocytes and hepatopancreas as well. All these imply that the JAK/STAT pathway participates in the immune response against bacteria and virus in F. chinensis. [ABSTRACT FROM AUTHOR]

Glutamine synthetase (GS) is considered to be one of the oldest existing functioning genes in evolution and plays a key role in two major biochemical pathways: in liver GS catalyzes ammonia detoxification, whereas in neural tissues it also functions in recycling of the neurotransmitter glutamate. In this study, a cytosolic glutamine synthetase was cloned and characterized from the Chinese Shrimp Fenneropenaeus chinensis, and named as FcGS. The results indicated that the full-length DNA of 1,756 bp had an open reading frame without intron, which encoded a polypeptide of 366 amino acids with several conserved active site residues. The calculated molecular mass of mature protein was 40.7 kDa and theoretical pI was 6.47. A phylogenetic analysis of GS sequences showed that FcGS clustered with the invertebrate group as expected. qRT-PCR analysis indicated that FcGS was expressed in intestines, hepatopancreas, muscles and gills, and its profile was up-regulated post-WSSV challenge in hepatopancreas and gills. Our results suggested that FcGS might participate in the regulation of shrimp immune response toward WSSV invasion. These data would be helpful to better understand the WSSV resistance mechanism of farming shrimps. [ABSTRACT FROM AUTHOR]

The β-1,3- d-glucan binding protein (βGBP), one kind of the pattern recognition proteins (PRPs), was cloned and characterized from the Chinese Shrimp Fenneropenaeus chinensis, and named as FcβGBP-HDL. The results indicated that the full length cDNA of 6713 bp had an open reading frame encoded a polypeptide of 2139 amino acids with two glucanase-like motifs and one RGD motif, while without signal peptide. The calculated molecular mass of mature protein was 240.7 kDa and theoretical pI was 5.95. Sequence comparison of the deduced amino acid sequence of FcβGBP-HDL showed varied identity of 88, 54 and 53% with those of Litopenaeus vannamei βGBP-HDL, Pacifastacus leniusculus βGBP, and Pontastacus leptodactylus βGBP, respectively. qRT-PCR analysis indicated that FcβGBP-HDL was expressed in intestines, hepatopancreas, muscle, gill and hemocytes, and its profile was modified post WSSV challenge. The differential expressions of FcβGBP-HDL in different tissues post WSSV challenge suggested that FcβGBP-HDL might play an important role in shrimp immune and perform differently in different tissues. These data would be helpful to better understand the WSSV-resistance mechanism of farming shrimp. [ABSTRACT FROM AUTHOR]

This study introduced a kind of new SNP genotyping strategy in genetic analysis of Chinese shrimp ( Fenneropenaeus chinensis). Twenty SNP tetra-primer ARMA-PCR primer sets were validated and used to investigate the genetic structure of six selected families of marine shrimp F. chinensis. The effective number of alleles ranged from 1.127 to 1.993, with an average value of 1.600. The average values of expected and observed heterozygosities of the SNPs ranged from 0.505∼0.609 and 0.373∼0.487, respectively. Polymorphism information content ( PIC) values ranged from 0.145 to 0.373. Among 120 population-locus cases (six populations × twenty loci), only 8 (6.7%) showed significant deviation ( P < 0.05), while the other 112 (93.3%) were in Hardy-Weinberg equilibrium ( HWE) ( P > 0.05). The frequencies of minor alleles ranged from 0.378 to 0.497. For the six families, the wild genotype and its allele frequency were significantly higher than that of the mutant genotype and its allele frequency. Fifty five point forty two percent of the population-locus showed heterozygosity. The results indicated that tetra-primer ARMA-PCR is a simple, rapid and efficient method for SNP genotyping which make it useful in a broad aspects of F. chinensis genetic and breeding studies. [ABSTRACT FROM AUTHOR]

Caspases are a family of proteases, which play an important role in apoptosis.To evaluate the relationship between apoptosis and pH stress in crustaceans, a caspase gene (FcCasp) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length of FcCasp was 1329 bp with a 972 bp ORF, encoding a polypeptide of 323 amino acidswith a calculatedmolecular weight and pI of 36.0 kDa and 6.27 respectively. The deduced amino acid sequence of FcCasp contained a potential active site (QACRG pentapeptide) conserved in most caspases and two pro￠le hits (p20 and p10 domain pro￠le). Comparison of amino acid sequences revealed that FcCasp had an overall similarity of 76^83% with other penaeidshrimp caspases. The amino acid sequence of recombinant FcCasp protein expressed in Escherichia coli was identi￠ed by matrix-assisted laser desorption/ ionization-time of ￡ight-mass spectrometer analysis. High-level expression of FcCasp in six di!erent tissues was detected by real-time polymerase chain reaction after exposure to pH stress for 96 and148 h. TUNEL analysis indicated that apoptosis began to appear in F. chinensis hepatopancreas exposed to extreme pH for 12 h. The amount of apoptosis seems positively correlated with the length of exposure to the pH stressor. The results suggested that FcCaspwas involved in the response to environmental pH stress. [ABSTRACT FROM AUTHOR]

Abstract: The vasa gene, a member of the DEAD-box gene family, has been reported to be restricted exclusively to the germ cell lineage in most animals, and thus is the most reliable molecular marker for studies on germ cell development. In this study, the developmental expression pattern of the vasa-like gene in Fenneropenaeus chinensis (Fc-vasa-like) is presented. The origin and migration of the germ cell are revealed by whole mount in situ hybridization (ISH). Furthermore, gonadogenesis is observed by histological techniques and ISH. Fc-vasa-like mRNA is located in the cytoplasm of oocytes before and after spawning, and equally distributed in the cytoplasm of early embryos, from the 2-cell embryo to the blastula, indicating that Fc-vasa-like mRNA is maternally supplied. Later on, the expression of the Fc-vasa-like gene centralizes gradually, and is restricted to five cell clusters as precursor cells of primordial germ cells (PGCs) at the limb bud stage. The five cell clusters are located at the base of the antennules and antenna, as well as the cephalic lobe, implying an epigenetic mechanism of germ cell specification in this species. These cells migrate to a dorsolateral position in naupliar and zoeal stages, and gradually enter the genital ridge at the mysis 1 stage. The gonad is first detected histologically at the post-larval 1 stage (PL1, 1st day postlarva), and germ stem cells (GSCs) are distinguished in cluster in juveniles with the body length of 30mm. Our findings show that the developmental expression pattern of Fc-vasa-like is different from that of other Crustaceans, and suggest an epigenetic mechanism of germ cell development in Chinese shrimp. [Copyright &y& Elsevier]

Abstract: Hemocyte of shrimp as an important target cell for white spot syndrome virus (WSSV) was an optimal target for the investigation of interactions between WSSV structural proteins and host cells. In this study, hemocyte membrane of Fenneropenaeus chinensis (HmFc) was prepared and labeled with digoxigenin (DIG), then used as a probe to detect the HmFc-binding proteins in WSSV structural proteins by modified virus overlay protein binding assay (VOPBA) technique. A prominent immunoreactive protein band was identified in WSSV structural proteins, which was corresponding to VP 26. Subsequently, in order to investigate the VP26-binding protein in HmFc, VP26 was expressed, purified and labeled with fluorescein isothiocyanate (FITC). Detected by indirect immune fluorescence assay (IIFA), FITC-labeled recombinant VP26 (rVP26) showed binding ability to the shrimp hemocytes. Furthermore, result of VOPBA demonstrated that rVP26 could specifically recognize a protein in HmFc with an apparent molecular weight of 42kDa, which was identified to be β-actin by mass spectrometric analysis. These results indicate that the interaction between VP26 and β-actin might play an important role in WSSV infection. [Copyright &y& Elsevier]

Abstract: Protein disulfide isomerase (PDI) catalyzes formation and isomerization of disulfide bridges and has chaperone activity. Currently, increasing evidence suggests the significance of PDI in immune and stress responses. To clarify the role of PDIs in the innate immunity of shrimp, two PDI genes were isolated and identified from Fenneropenaeus chinensis (fleshy prawn). FcPDI1 is 1878bp in length and encodes a protein of 383 amino acids. It has 18-amino acid signal peptide, 3 thioredoxin domains with 3 active sites of CGHC, and KEDL retention signal at its C-end. FcPDI1 is an atypical PDI. The open reading frame of FcPDI2 encodes a 497-amino acid protein and shows the classical domain organization a-b-b′-a′. Phylogenic analysis and multiple alignments show that FcPDI1 is similar to PDI that contains 3 thioredoxin domains from other species including invertebrates and vertebrates. FcPDI2, LvPDI, and insect PDIs are grouped into one cluster and are similar to PDIs having a-b-b′-a′ domain organization. Tissue distribution shows that FcPDI1 and FcPDI2 were expressed in all detected tissues at the mRNA level. Changes in FcPDI1 and FcPDI2 expression at the mRNA level in hemocytes, hepatopancreas, gills, and ovaries upon Vibrio or white spot syndrome virus challenge were also analyzed. The results suggest that FcPDI1 and FcPDI2 might have roles in the innate immunity of shrimp. FcPDI1 was also successfully expressed in Escherichia coli and the recombinant FcPDI1 showed insulin reductase activity. Results show that FcPDI might play an important role in the innate immunity of shrimp. [Copyright &y& Elsevier]

Abstract: White spot syndrome virus (WSSV) is the most serious disease threatening the shrimp farming. Previous studies have elucidated the relationships between WSSV outbreak and environmental factors such as temperature, salinity and pH. But, these studies didn''t expatiate the relationships between the copy numbers of WSSV replication and the changes of environmental factors, even educed some contrary conclusions. Here, by the methods of Taguchi and real time PCR, the impact of three factors, temperature, salinity and pH on the proliferation of WSSV was studied together. The five levels selected for three factors were 15°C, 20°C, 25°C, 30°C and 35°C for temperature, 15‰, 20‰, 25‰, 30‰ and 35‰ for salinity and 6.5, 7.5, 8.0, 8.5 and 9.0 for pH. ANOVA analysis showed that significant differences existed between 15°C and 25°C (P=0.044), between 20°C and 25°C (P=0.047), and between 35°C and 25°C (P=0.044) for temperature. No significant differences were found among the levels of salinity and pH. This discovered that, of the three factors, the temperature was a crucial factor in determining WSSV proliferation. Hyperthermia (35°C) and hypothermia (15°C) could significantly decrease the WSSV proliferation while 25°C was the optimum temperature of WSSV replication. Moreover, low temperature was a more efficient factor to control WSSV replication and mortality of individuals infected WSSV, because the copy numbers of WSSV were only 0.626 per ng muscle at 15°C, while there were 5.94 copies at 35°C. Our study showed that the optimal WSSV proliferation condition was 30°C for temperature, 35‰ for salinity, and 8.0 for pH, and reversely, the worst WSSV proliferation condition was 15°C for temperature, 35‰ for salinity, and 9.0 for pH. [Copyright &y& Elsevier]

n analysis of a selection experiment was used to assess the impact of various animal model structures on REML estimates of variance components. The analyses were carried out based on 162 d body mass (BM) of 1 287 animals from 21 paternal half-sib groups of Fenneropenaeus chinensis. Estimated breeding values (EBV) of BM of all individuals were estimated using eight statistical models (A, AB, ABC, ABDC, ABMFC, ABMDC, ABFDC and ABMFDC) and BLUP (best linear unbiased prediction). These models were designed involving factors such as sex, spawn date as fixed effects, maternal genetic effects, full-sib family effects as random effects, mean BM of families at tagging and age at recording (covariate). The results demonstrate the importance of correct interpretation of effects in the data set, particularly those that can influence resemblance between relatives. The data structure and the particular model that was applied markedly influenced the magnitude of variance component estimates. Models based on few effects obtained upward biased estimates of additive genetic variance. The accuracy of genetic parameters and breeding value estimated by ABFDC model was higher than other models. The results imply that additive genetic direct value, full-sib family effects, and covariance effects besides sex and spawn date as fixed effects were very important for estimating genetic parameters and breeding value of body mass. This model had a heritability estimate of 162 d BM of 0.44. The comparison of the efficiency of selection based on breeding values or phenotypic value revealed great difference: average breeding value of the best 24 families selected by the 162 d BM breeding value and phenotype were 0.577 g and 0.366 g, respectively, representing a 36.57% higher efficiency in the former. In conclusion, selection based on breeding value was more effective than selection based on phenotypic value. Our results indicate that effects influencing the magnitude of estimates should be taken into account when estimating heritability and breeding values for BM. [ABSTRACT FROM AUTHOR]

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin ( FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp. [ABSTRACT FROM AUTHOR]

Two kinds of specific chicken egg yolk immunoglobulins (IgYs), IgY-WSSV and IgY-VP28, were, respectively, raised against the 2 mM binary ethylenimine (BEI)-inactivated white spot syndrome virus (WSSV) and a principal envelope protein VP28. The activity of purified specific IgYs was stable under the conditions of 20-70 °C, pH 3.0-10.0 and 0-700 g L sucrose solution. In the neutralization assay, these high-affinity IgY antibodies can specifically bind with the virus particles to protect shrimp ( Fenneropenaeus chinensis) against WSSV infection. After oral delivery for 20 days, the IgY-WSSV exerted a higher protection effect (RPS: 71.5%) than IgY-VP28 (RPS: 63.7%). Moreover, an increase in RPS (79.2%) was found on addition of IgY-WSSV:VP28 (0.1% IgY-VP28 plus 0.2% IgY-WSSV). This may indicate that neutralization of WSSV refers to the multiple-hit model. By time-course study of the levels of the specific IgYs in vivo, the data showed that the titre was enhanced to a relatively high level ( P/ N=8.35±0.45) at 3 days post administration, declined slightly ( P/ N=7.13±1.01) at 7 days post administration and then remained stable for further investigation. The stable antibody level potentially contributes towards blocking a large number of WSSV particles from entering and infecting on the major tissues at the early and late stages after challenge in shrimp. [ABSTRACT FROM AUTHOR]

The changes in the specific growth rate (SGR), feed intake (FI), feed conversion efficiency (FCE), and digestive enzyme activities (protease, amylase, and lipase in the hepatopancreas) of Fenneropenaeus chinensis juveniles (0.753 ± 0.041 g, wet weight) exposed to different periods of food deprivation were investigated during the period of refeeding in a controlled laboratory experiment. The starvation-refeeding cycles consisted of the following seven regimes of 24-day duration: satiate feeding (control, SC); no feeding for 4 (S4), 8 (S8), 12 (S12), 16 (S16), 20 (S20), and 24 (S24) days; then feeding to apparent satiation for the next 20, 16, 12, 8, 4, and 0 days, respectively. The various starvation-refeeding cycles have significant influences on the growth of the test shrimps. The SGR of the shrimps exposed to S4 and S8 treatments exceeded the controls, and no significant differences in SGR ( P > 0.05) were found between the SC and S12 or S16 treatments at the end of the refeeding. These changes may have resulted from the significant increase in FI and FCE. The protease activities of test shrimps exposed to different periods of food deprivation were significantly lower ( P < 0.05) than those of shrimps under SC treatment at the end of food deprivation then showed a tendency to rapidly increase during refeeding. However, nearly the exact opposite occurred for amylase activities and lipase activities. Lipid and carbohydrate reserves in the hepatopancreas are preferentially mobilized with protein-sparing effect at the beginning of food deprivation. Proteins are the main nutritional store during the refeeding. The findings of the research will be beneficial in the design of feeding regimes and will improve our knowledge on some aspects of the nutrition physiology of F. chinensis related to their biology. [ABSTRACT FROM AUTHOR]

Abstract: The cDNAs encoding CathL and legumain from Chinese white shrimp Fenneropenaeus chinensis (FcCathL, FcLegu) were obtained. Both FcCathL and FcLegu mRNA were expressed mainly in the hepatopancreas of unchallenged shrimp. Time-course analysis of FcCathL showed that FcCathL was upregulated in the hepatopancreas of shrimp challenged with white spot syndrome virus (WSSV) at 12h. FcLegu mRNA in hepatopancreas was down-regulated by Vibrio. FcLegu transcript first declined from 2h to 6h and then recovered from 12h to 24h in hepatopancreas challenged with WSSV. FcCathL protein was detected in the hemocytes, hepatopancreas, gill, stomach, and intestine of unchallenged shrimp. Three bands of FcCathL protein detected in some tissues may represent preproenzyme, single chain and mature double chain form respectively. In hepatopancreas, FcLegu was detected in the proenzyme form. In other tissues, only active form could be detected. The protein of FcLegu was down-regulated by Vibrio or WSSV challenge in the stomach and gills. FcCathL and FcLegu were proposed to play a role in shrimp innate immunity for the first time. [ABSTRACT FROM AUTHOR]

Abstract: White spot syndrome virus (WSSV) infects all shrimp species and is the greatest detriment to shrimp culture. To better understand the mechanism of molecular responses to WSSV infection in Chinese white shrimp Fenneropenaeus chinensis, two dimensional electrophoresis (2-DE) was used. Differentially expressed proteins in the hepatopancreas of control and WSSV-injected Chinese white shrimp between (6, 12 and 24 h post-injection) were screened. Quantitative intensity analysis and mass spectrometry revealed that 54 spots of 47 proteins were significantly up-regulated, including some immune-related proteins, such as Toll receptor precursor, Leu-rich repeat protein, peroxinectin and serine proteinase-like protein. Fourteen spots of 13 proteins, such as heat shock protein, ATP synthase sub-unit beta and thrombospondin, were down-regulated in WSSV-infected shrimps. Protein expression patterns of the infected shrimp were drastically altered by WSSV infection. Some of the altered proteins were further investigated at the mRNA level using semi-quantitative reverse transcript PCR. These data may provide some information about shrimp proteins that participate in the WSSV infection process. [Copyright &y& Elsevier]

Abstract: To gain an insight into the function of shrimp lymphoid organ at protein level, we analyzed the proteome of lymphoid organ in healthy Chinese shrimp Fenneropenaeus chinensis (F. chinensis) through two-dimensional gel electrophoresis (2-DE) based proteomic approach. A total of 95 spots representing 75 protein entries were identified by liquid chromatography tandem mass spectrometry (LC–MS/MS) with both online and in-house database. According to Gene Ontology (GO) annotation of biological process, the identified proteins were classified into 13 categories. Among them, approximately 36% of proteins related to cytoskeleton are noticeable. Then, a comparative proteomic approach was employed to investigate the differentially expressed proteins in lymphoid organ of Vibrio anguillarum-challenged F. chinensis. At 24 h post-injection (hpi), 17 differentially expressed protein spots were successfully identified, including 4 up-regulated protein spots (represent 4 proteins: cathepsin L, protein similar to squid CG16901-PC, protein kinase C and protein similar to T-complex Chaperonin 5 CG8439-PA), and 13 down-regulated protein spots (represent 9 proteins: actin, beta-actin, cytoplasmic actin CyII, alpha tubulin, beta tubulin, protein similar to proteasome delta, vacuolar ATP synthase subunit B, elongation factor 2, carboxypeptidase B). These data may help us to understand the function of lymphoid organ and the molecular immune mechanism of shrimp responsive to pathogen infection. [Copyright &y& Elsevier]

Abstract: Rel/NFκB is a family of transcription factors. In the present study, a Rel/NFκB family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627bp, revealed a 1071bp open reading frame encoding 357 aa. The predicted molecular weight (MW) of the deduced amino acid sequence of FcDorsal was 39.78kDa, and its theoretical pI was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G+) bacteria (Micrococcus lysodeikticus) and Gram-negative (G−) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NFκB member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity. [Copyright &y& Elsevier]

Abstract: Ecdysone inducible gene, E75 is a primary target of ecdysone receptor (EcR), and is found to play a critical role in the molting process of arthropods. In this study, a cDNA encoding the E75 of Chinese shrimp Fenneropenaeus chinensis (FcE75) was cloned using RT-PCR and RACE techniques. FcE75 cDNA was 3611bp in length with an ORF of 2394bp. The deduced amino acid sequence of FcE75 had the highest sequence identity to E75 from a land crab Gecarcinus lateralis and E75 of the shrimp Metapenaeus ensis. Quantitative real-time PCR revealed a prominently high expression of FcE75 mRNA in the whole body RNA extract of late premolt period (D3) juvenile shrimp. The role of E75 in the process of shrimp molting was investigated using the RNA interference technique. Long double-stranded RNA corresponding to the FcE75 (dsE75) efficiently silenced the FcE75 transcript levels in juvenile F. chinensis. Further, injection with dsE75 completely arrested the molting process in experimental shrimp which eventually caused death. Setogenic analysis of the uropods from molt-arrested shrimp, showed defective epidermal retraction, poor development of setae and new cuticle. These results indicate that E75 might be related to the molting process and is essential for proper molting and survival of shrimp. This is the first report demonstrating the use of double stranded RNA to elucidate the possible role of E75 in the molting of decapod crustaceans. [Copyright &y& Elsevier]

Abstract: Two full-length cDNA sequences (Fc-CHH1, Fc-CHH2) encoding a crustacean hyperglycemic hormone (CHH) precursor homolog and their DNA sequences were cloned from Chinese shrimp Fenneropenaeus chinensis. The deduced amino acid sequences of them are predicted to contain a signal peptide and a mature peptide. The mature peptides of Fc-CHH1 and Fc-CHH2 shared 78% identity, but they showed low identities (less than 40%) to CHH peptides from other species. Both Fc-CHH1 and Fc-CHH2 proteins contain six highly conserved cysteine residues which are characteristic of the CHH family peptides. The transcripts of Fc-CHH1 and Fc-CHH2 were shown to be specifically present in the spermatophore sac of mature male Chinese shrimp through reverse transcription-polymerase chain reaction (RT-PCR) detection. The transcripts of Fc-CHH1 and Fc-CHH2 begin to appear at the immature stage (115days after the first post-larvae stage) when the spermatophore sac was first observed to be appeared. In situ hybridization analyses showed that Fc-CHH1 and Fc-CHH2 transcripts located at the epithelial cells in the internal wall of the spermatophore sac. In the cloned DNA sequences of Fc-CHH1 and Fc-CHH2, the predicted transcription factor binding sites in the 5′ flanking sequences are different from those previously reported for CHH family genes of crustacean. To our knowledge, these are novel CHH-like genes expressed specifically in male shrimp. Their function needs to be further investigated. [Copyright &y& Elsevier]

Abstract: The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. [Copyright &y& Elsevier]

Abstract: Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences (Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473bp and 574bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro. [Copyright &y& Elsevier]

A muscle cDNA library of Chinese shrimp ( Fenneropenaeus chinensis) was constructed with the SMART™ cDNA Library Construction Kit. The titer of optimal primary library was 7.7×105 pfu mL−1 and that of the amplified library was 3.0×109 pfu mL−1. The percentages of the recombinant clones of primary and amplified libraries were over 98%. The insert sizes were longer than 400 bp with an average of 1000 bp. A positive clone containing a 794 bp insert was sequenced and identified encoding fast skeletal troponin I gene. This library provided a useful resource for the functional genomic research of F. chinensis. [ABSTRACT FROM AUTHOR]

The ion-transport enzyme activities were studied in the nauplii 1–2, zoea 1–3, mysis 1–3, and postlarva 1–7 of the shrimps Marsupenaeus japonicus and Fenneropenaeus chinensis. The results showed that total ATPase, Na+-K+-ATPase, and V-ATPase (V-type H+-ATPase) activities increased during the early development for both the species, from zero in nauplii to stable levels after zoea stage. The enzyme activities of the latter species were significantly higher than those of the former species after zoea stage ( F> F0.05). The contributions of Na+-K+-ATPase, V-ATPase and HCO3 −-ATPase to the total ATPase activity of the two species varied in different developmental stages and accounted for 40%–70%, 22%–46% and 2%–13% in M. japonicus from zoea to postlarva stage, whilst the shares of them were 42%–69%, 28%–44% and 2.5%–22%, respectively in F.chinensis. These findings suggest the possible culture of the two species with varying water quality, especially the salinity and pH. [ABSTRACT FROM AUTHOR]

Abstract: The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp. [Copyright &y& Elsevier]

Three members of the tetraspanin/TM4SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM4SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene ( FcCD9) was specifically expressed in the hepatopancreas. While for the CD63 gene ( FcCD63), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63, the tetraspanin-3 gene ( FcTetraspanin-3) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9, FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM4SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection. [ABSTRACT FROM AUTHOR]

Abstract: In invertebrates, the Toll signaling pathway is important for activation of antimicrobial peptides in the innate immune system. Activation of the Toll pathway requires binding of Toll with its ligand Spätzle. Here we described a Spätzle-like protein, designated as Fc-Spz, from hemocytes of Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequence of Fc-Spz shares 54% identity with Spätzle-like protein of salmon louse (Lepeophtheirus salmonis). Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), quantitative real time PCR, and Western blot analyses were carried out to analyze the expression pattern and distribution profile of Fc-Spz in shrimp after challenged with bacteria and virus. The results showed that Fc-Spz mRNA was up-regulated in all the tissues tested in shrimp injected with Vibrio anguillarum and white spot syndrome virus (WSSV). The C-terminal active Fc-Spz domain (114 residues) was expressed in Escherichia coli and purified by affinity chromatography. The recombinant Fc-Spz C-114 was injected into crayfish (Procambarus clarkii) to determine the expression levels of several antimicrobial peptide genes. The results showed that recombinant Fc-Spz C-114 could up-regulate crustin 2 expression in crayfish. These results suggest that Fc-Spz may play a role in the innate immune defence of Chinese shrimp and crayfish. [Copyright &y& Elsevier]

Abstract: The whey major component, whey acidic protein (WAP), has one or more WAP domains characterized by a four-disulfide core (4-DSC) structure. These kinds of proteins are involved in multiple functions, including proteinase inhibitor activity, antimicrobial activity, ATPase inhibitor activity, and regulatory function in cell proliferation. Recent research indicates that WAP domain-containing proteins play an important role in the innate immunity of crustaceans. In this study, a novel double WAP domain (DWD)-containing protein named Fc-DWD was found for the first time in Chinese white shrimp, Fenneropenaeus chinensis. The open reading frame of Fc-DWD encodes a protein of 117 amino acids, including a signal peptide of 16 amino acids and two WAP domains. The predicted molecular mass of the mature protein is 12.78kDa with an estimated pI of 8.49. The first WAP domain, named WAP 1, composed of 49 amino acids locates in the amino-terminal of Fc-DWD, and the second WAP domain, named WAP 2, composed of 45 amino acids locates in the carboxy-terminal. Fc-DWD mRNA was upregulated in hemocytes, hepatopancreas, gills, and stomach of bacteria- and virus-challenged shrimp. Results of the binding assay showed that rFc-DWD could bind to both Gram-negative bacteria and Gram-positive bacteria. rWAP 1 could only bind to Gram-positive bacteria, but rWAP 2 could bind to both Gram-negative and positive bacteria. Moreover, rFc-DWD exhibited proteinase inhibitory activity against the secretory proteinase(s) from Bacillu s subtilis and Pseudomonas aeruginosa. All of these findings suggest that Fc-DWD may play an important role in enabling the host defense to execute its proteinase inhibitory activity against pathogens. [Copyright &y& Elsevier]

Abstract: Penaeid shrimp, as an invertebrate, relies on the innate immunity to oppose the microbial invaders. Antimicrobial peptides (AMP) are an integral component of the innate immune system in most organisms and function as an early first line of defense against pathogens, but the knowledge about the pathways to regulate the shrimp AMP gene expression is still absent up to date. In the current study, a Relish homolog (FcRelish) was cloned from Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcRelish consists of 2157bp, including 1512bp open reading frame, encoding 504 amino acids. The predicted molecular weight of FcRelish is 57kDa, and the theoretical PI is 7.00. Spatial expression profiles showed that FcRelish had the highest expression levels in the hemocytes and lymphoid organ. Both Vibrio anguillarium and Micrococcus lysodeikticus stimulation to shrimp can affect the transcription profile of FcRelish. Silencing of FcRelish through DsRNA interference can greatly change the transcription profile of AMP. Therefore, we suggest that FcRelish identified in the present study is closely related to the transcription of AMP, and then we inferred that Imd pathway might exist in shrimp. [Copyright &y& Elsevier]

Abstract: C-type lectins play important roles in innate immunity of invertebrates. In the present study, we report a novel C-type lectin, named FcLec4, from the Chinese white shrimp Fenneropenaeus chinensis. FcLec4 contains a single carbohydrate recognition domain (CRD) with a putative signal peptide. Phylogenetic analysis indicated that FcLec4 was distant from most reported C-type lectins from shrimps. The expression of FcLec4 increased at both mRNA and protein level after stimulation of Vibrio anguillarum. Recombinant FcLec4 could agglutinate both Gram-positive and -negative bacteria in the presence of calcium. The recombinant protein could bind to peptidoglycan and selectively bind to microorganisms. Interestingly, the tight binding of recombinant FcLec4 to V. anguillarum might facilitate the subsequent clearance of the bacteria in vivo. To the best of our knowledge, this might be the first report that a C-type lectin was found to be directly involved in the anti-V. anguillarum response in shrimps. [Copyright &y& Elsevier]

Growth traits in 150 Chinese shrimps Fenneropenaeus chinensis were analyzed by the sequence characterized amplified regions (SCAR) technique in two populations of sixth-generation cultured shrimps with different ranges of body lengths (CP-a and CP-b) and in the wild-type population (WP). First, 240 random primers were used to screen polymorphic fragments by random amplified polymorphic DNA (RAPD). Population analysis revealed nine RAPD markers correlated with growth traits, including seven positively and two negatively correlated ones. The sequences of the RAPD markers were then used to design longer primers for SCAR. Six primer pairs were obtained, and two of these produced polymorphic fragments among the three groups. One amplified 39, 26 and 27 polymorphic fragments, with band frequencies of 78, 52 and 54% in the CP-a, CP-b and WP groups, respectively. The distributions of polymorphic fragments in the three groups were significantly different ( P < 0.05) according to a χ2 test, indicating that they may be candidate markers linked to growth traits. Another primer pair amplified three alleles, resulting in six combinations of genotypes among three groups. Since allele A1 was only found in the population of shorter shrimps, it may be a negative growth marker. [ABSTRACT FROM AUTHOR]

This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an initial wet body weight of 1.115 g±0.012 g. The salinity of the control treatment (represented by D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The range of salinity fluctuation was 4. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8 d, respectively; each original salinity was decreased abruptly to salinity 24, which first lasted for another 2 d and was then raised to its initial value 28. This constituted a salinity fluctuation cycle and afterwards the cycle repeated. It was found that: 1) The weight gain of shrimp in treatment D4 was 202.9% and significantly higher than that in treatment D0 ( P<0.05). The molting rate in treatment D2 was the lowest, while that in treatment D6 was the highest and significantly higher than those in treatments D0 and D2 ( P<0.05). 2) The hemolymph 20-HE concentration of shrimps in all treatments was at low levels and increased first gradually during the inter-molt period and then increased sharply. It reached a peak value during the pre-molt stage, and then decreased abruptly during the post-molt stage when the lowest level occurred. 3) During the post-molt stage, the hemolymph 20-HE concentration in treatment D2 was 7.47 pg μL−1 and significantly higher than that in treatment D0 ( P<0.05). There were, however, no significant differences in hemolymph 20-HE concentration among all five treatments during the inter-molt stage ( P>0.05). During the pre-molt stage, the hemolymph 20-HE concentration in treatment D6 was significantly higher than that in treatment D0 ( P<0.05), whereas the hemolymph 20-HE concentration in treatment D2 was significantly lower than that in other treatments ( P<0.05). Adequate salinity fluctuation promoted the molting rate through increasing hemolymph 20-HE concentration at the pre-molt stage. [ABSTRACT FROM AUTHOR]

Abstract: Serine proteinase inhibitor plays an essential role in arthropods by restraining the activities of endogenic or exogenic serine proteinases. Four Kazal-type serine proteinase inhibitors, Fcspi-1–4, from the hepatopancreas of Chinese white shrimp, Fenneropenaeus chinensis, were cloned and identified. The open reading frames (ORFs) of Fcspis are 1389, 1236, 1080, and 939 base pairs, encode the pre-proteins of 462, 411, 359, and 312 amino acids and form the 9, 8, 7, and 6 typical Kazal domains, respectively. When analyzing the amino acid sequences of the four inhibitors, it was found that they might have been derived from the same transcript, which was subjected to alternative splicing, and none of the Kazal domains were identical within each inhibitor. Multiple alignments showed that the Kazal inhibitors were homologous with a conserved motif of Cx3Cx6VCGSDGxTYx3CxLx5Cx5ITx6GC. The results from RT-PCR indicated that the expression of Fcspis as a whole was upregulated by bacterial challenge, no obvious change was noticed after viral challenge, and Fcspi-1 had a similar expression pattern with that of Fcspis. Recombinant FcSPIs were successfully expressed in bacteria and purified for further study. Recombinant FcSPI-1 was sensitive to DTT and had thermal stability. The inhibitory kinetics assay suggested that rFcSPI-1 was a mixed-type fast tight binding inhibitor with inhibitory activities against subtilisin A at a molar ratio of 1:1, 1:2 against proteinase K, and 2:1 against elastase. It can firmly bound to two Gram-positive and one Gram-negative bacteria but without anti-bacterial ability. In addition, it inhibited the activities of both bacterial-secreted proteinases and natural chymotrypsin of Chinese white shrimp, suggesting that FcSPI-1 may participate in the immune defence response by inhibition of bacterial pathogen proteinases and possibly be involved in the regulation of shrimp proteinase activity. [Copyright &y& Elsevier]

Abstract: Retinoid X receptor (RXR)/ultraspiracle (USP) is the heterodimeric partner of ecdysteroid receptor and is required for the molting process of arthropods. To better understand the molecular aspects governing the process of molting in shrimp, the full-length cDNA of two RXRs, named as FcRXR-1 and FcRXR-2 were obtained from Chinese shrimp Fenneropenaeus chinensis which were of 1715 and 1700 bp long, revealed a 1315 and 1300 bp open reading frame (ORF) respectively. Quantitative Real time PCR analysis showed a marked tissue-specific difference in the expression of FcRXR transcript, which revealed that the expression of FcRXR could be regulated in a tissue-specific manner. Moreover, high expression of FcRXR mRNAs was observed in late pre-molt period (D3) and post molt stages (A–B) of shrimp. Among the two isoforms, FcRXR-2 appeared in a considerably high level in all the stages compared to the FcRXR-1. In addition, we examined the temporal expression of two chitinase genes: FcChitinase (FcChi) and FcChitinase-1 (FcChi-1) during the molt cycle of F. chinensis. Both the FcChi and FcChi-1 transcripts were detected in all stages of molting, although considerable fluctuations observed through the molt cycle. Injection of double stranded RXR (dsRXR) into juvenile shrimp resulted in a maximum silencing effect at 48 h post injection. We analyzed the expression levels of FcChi, FcChi-1 and the ecdysone inducible gene E75 (FcE75) in samples of dsRXR injected shrimp. Significant reduction in levels of both FcE75, FcChi and FcChi-1 transcripts occurred in the silenced shrimp. This correlation suggested that RXR might involve in the downstream regulation of E75 and chitinase gene transcription in the ecdysone signaling pathway of decapod crustaceans. [Copyright &y& Elsevier]