Hematological Changes m White Spot Syndrome Virus-Infected Shrimp, Fenneropenaeus chinensis (Osbeck)

The pathological changes of hemocytes in the haemolymph and hepatopancreas were examined in experimentally and naturally WSSV (white spot syndrome virus) infected Fenneropenaeus chinensis. The results showed that the pathological manifesta- tions of hemocytes were similar among moribund shrimps infected via injection, feeding and by nature. Firstly, the total hemocyte counts (THCs) in WSSV-infected shrimp were significantly lower than those in healthy shrimp. Secondly, necrotic, broken and dis- integrated cells were often observed, and a typical hematolysis was present in the haemolymph smear of WSSV-infected shrimp. Thirdly, necrosis and typical apoptosis of hemocytes were detected with TEM in the peripheral haemolymph of WSSV-infected shrimp. Hyalinocytes and semi-granulocytes with masses of WSSVs in their nuclei often appeared, whereas no granular bemocytes with WSSV were found in the hepatopancreas of moribund infected shrimps. All our results supported that hemocytes were the main target cells of WSSV, and hyalinocytes and semigranular hemocytes seemed to be more favorable for WSSV infection in F. chinensis.     

Influence of white spot syndrome virus infection on hepatopancreas gene expression of ‘Huanghai No. 2’ shrimp (<Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>)

To elucidate the molecular response of shrimp hepatopancreas to white spot syndrome virus (WSSV) infection, microarray was applied to investigate the differentially expressed genes in the hepatopancreas of ‘Huanghai No. 2’ (Fenneropenaeus chinensis). A total of 59137 unigenes were designed onto a custom-made 60K Agilent chip. After infection, the gene expression profiles in the hepatopancreas of the shrimp with a lower viral load at early (48–96 h), peak (168–192 h) and late (264–288 h) infection phases were analyzed. Of 18704 differentially expressed genes, 6412 were annotated. In total, 5453 differentially expressed genes (1916 annotated) expressed at all three phases, and most of the annotated were either up- or down-regulated continuously. These genes function diversely in, for example, immune response, cytoskeletal system, signal transduction, stress resistance, protein synthesis and processing, metabolism among others. Some of the immune-related genes, including antilipopolysaccharide factor, Kazal-type proteinase inhibitor, C-type lectin and serine protease encoding genes, were up-regulated after WSSV infection. These genes have been reported to be involved in the anti-WSSV responses. The expression of genes related to the cytoskeletal system, including β-actin and myosin but without tubulin genes, were down-regulated after WSSV infection. Astakine was found for the first time in the WSSV-infected F. chinensis. To further confirm the expression of differentially expressed genes, quantitative real-time PCR was performed to test the expression of eight randomly selected genes and verified the reliability and accuracy of the microarray expression analysis. The data will provide valuable information to understanding the immune mechanism of shrimp’s response to WSSV.     

Detection of white spot syndrome virus (WSSV) ofPenaeus chinensis by in situ hybridization

White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I-digested fragments of the WSSV genome were cloned; three of these fragments were used as non-radioactive probes labeled with DIG-11-dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV-infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp. This work was supported by SRF for ROCS, SEM and the National 863 Project (Grant 819-Q-08) and Project under major State Basic Research Development Program (Grant G1999012002).     

Heat-shock protein 70 expression in shrimp Fenneropenaeus chinensis during thermal and immune-challenged stress

Using western immunoblotting we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimpFenneropenaeus chinensis during thermal and immune-challenged stresses. This is probably the first report of the effects of various stressors on the expression of HSP70 in shrimp. HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, when the shrimp were exposed to heat shock andVibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10°C above ambient temperature. Supported by the National Key Fundamental Research Program “Study of the Major Diseases and Resistance Mechanism of Mariculture Organisms”, NSFC, No. 30140017, and the international cooperative program “Immunaqua” by the European Commission, No. ICA4-CT-2001-10023.     

Chromosomal localization of 5S rDNA in Chinese shrimp (Fenneropenaeus chinensis): a chromosome-specific marker for chromosome identification

Chinese shrimp (Fenneropenaeus chinensis) is an economically important aquaculture species in China. However, cytogenetic and genomic data is limited in the organism partly because the chromosomes are difficult to isolate and analyze. In this study, fluorescence in-situ hybridization (FISH) was used to identify the chromosomes of F. chinensis. The 5S ribosomal RNA gene (rDNA) of F. chinensis was isolated, cloned and then used as a hybridization probe. The results show that the 5S rDNA was located on one pair of homologous chromosomes in F. chinensis. In addition, triploid shrimp were used to evaluate the feasibility of chromosome identification using FISH and to validate the method. It was confirmed that 5S rDNA can be used as a chromosome-specific probe for chromosome identification in F. chinensis. The successful application of FISH in F. chinensis shows that chromosome-specific probes can be developed and this finding will facilitate further research on the chromosomes of penaeid shrimps.     

DETECTION OF WHITE SPOT SYNDROME VIRUS(WSSV) OF PENAEUS CHINENSIS BY IN SITU HYBRIDIZATION

ＩＮＴＲＯＤＵＣＴＩＯＮＷｈｉｔｅＳｐｏｔＳｙｎｄｒｏｍｅＶｉｒｕｓ (ＷＳＳＶ)ｉｎＰｅｎａｅｕｓｃｈｉｎｅｎｓｉｓｉｓｔｈｅｃａｕｓａｔｉｖｅａｇｅｎｔｏｆａｖｅｒｙｓｅｖｅｒｅｅｐｉｚｏｏｔｉｃｄｉｓｅａｓｅｗｈｉｃｈ,ｓｔａｒｔｉｎｇｆｒｏｍ 1 993 ,ｈａｄｒｅｓｕｌｔｅｄｉｎｍｏｒｅｔｈａｎ 80 %ｍｏｒｔａｌｉｔｙｔｈｒｏｕｇｈｏｕｔｓｈｒｉｍｐｃｕｌｔｕｒｅｆａｒｍｓｉｎＣｈｉｎａ (Ｚｈａｎｅｔａｌ.,1 995;1 998) .Ｎｏｗ ,ｆｉｖｅｙｅａｒｓｌａｔｅｒ,ｔｈｅｖｉｒｕｓｄｉｓｅａｓｅｉｓｓｔｉ…     

Isolation of prawn (<Emphasis Type="Italic">Exopalaemon carinicauda</Emphasis>) lipopolysaccharide and β-1, 3-glucan binding protein gene and its expression in responding to bacterial and viral infections

The pattern recognition proteins (PRPs) play a major role in immune response of crustacean to resist pathogens. In the present study, as one of PRPs, lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) gene in the ridge tail white prawn (Exopalaemon carinicauda) (EcLGBP) was isolated. The full-length cDNA of EcLGBP was 1338 bp, encoding a polypeptide of 366 amino acid residules. The deduced amino acid sequence of EcLGBP shared high similarities with LGBP and BGBP from other crustaceans. Some conservative domains were predicted in EcLGBP sequence. EcLGBP constitutively expressed in most tissues at different levels, and the highest expression was observed in hepatopancreas. With infection time, the cumulative mortality increased gradually followed by the proliferation of Vibrio parahaemolyticus and white spot syndrome virus (WSSV). The expression of EcLGBP in response to V. parahaemolyticus infection was up-regulated in hemocytes and hepatopancreas, and the up-regulation in hepatopancreas was earlier than that in hemocytes. EcLGBP expression after WSSV infection increased at 3 h, then significantly decreased in both hemocytes and hepatopancreas. The results indicated that EcLGBP was involved in the immune defense against bacterial and viral infections.     

Distribution of Bacteria Injected in Body of Giant Black Shrimp,Penaeus Monodon

Distribution of injected Vibrio anguillarum in body of P,,naeus monodon was studied with immunohistochemical method. Bacteria could be detected throughout the experiment in some individuals; however in lymphoid tissue, gill, heart and haemolymph of all vibrio injected shrimp, the bacteria could be observed only 5 min after injection. The bacteria density in haemolymph, haemolymph of the hepatopancreas and gills decreased with time. In the lymphoid organ and heart, the bacteria density was the highest 48 h after injection, then decreased. Nodules could be formed in the heart, lymphoid organ and injection site.     

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK’s (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.     

Screening white spot syndrome virus (WSSV)-resistant molecular markers from <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>

White spot syndrome virus (WSSV)-resistant molecular markers were screened from the selectively bred new variety ‘Huanghai No. 2’ of Fenneropenaeus chinensis using unlabeled-probe high-resolution melting (HRM) technique. After the artificial infection with WSSV, the first 96 dead shrimps and the last 96 surviving shrimps were collected, representing WSSV-susceptible and -resistant populations, respectively. The genotypes at well-developed 39 single nucleotide polymorphisms (SNPs) loci were obtained. As revealed in the Chi-square test, 3 SNPs, genotype A/A of contig C364-89AT, genotype A/A of C2635-527CA and genotype C/T of contig C12355-592CT, were positively correlated with disease-resistance traits. Other 2 SNPs, genotype G/G of contig C283-145AG and genotype C/C of contig C12355-592CT, were negatively correlated. Moreover, analysis with BlastX program for disease-resistant SNPs indicated that 3 contigs, Contig283, Contig364 and Contig12355, matched to the functional genes of effector caspase of Penaeus monodon, peptide transporter family 1-like protein, and 40S ribosomal protein S2 of Perca flavescens with high sequence similarity. The results will be helpful to provide theoretical and technical supports for molecular marker-assisted selective breeding of F. chinensis.     

Distribution of Bacteria Injected in Body of Giant Black Shrimp, Penaeus Monodon

1　INTRODUCTIONThepenaeidshrimpcultureindustriesintheworlddevelopedfromtheirexperimentalbeginningslessthanthirtyyearsagointoamajoraquaculturein dustrytodaythatprovideshundredsofthousandsofjobs,billionsofU .S.dollarsinrevenue,andaug mentstheglobalfoodsupplywithahighvaluecrop(LighterandRedmam ,1 998) .Upto 1 995,farmedshrimpatvolumeof71 2 0 0 0MT ,almostallconsistingofPenaeidspecies,accountedfor 2 7%ofworldshrimpproduction.Inthatyear,asoneofthemostimportantculturedshrimp,theproductionofP…     

Effects of dietary vitamin C on the immune function of shrimps, Penaeus chinensis

Prepared in this experiment were six groups of diets, i.e. VC0, VC1, VC2, VC3, VC4 and VC5 with the contents of vitamin C (VCmg(100 g)⁻¹ diet) of 0, 100, 200, 400, 800, and 1200 respectively. It was found that vitamin C increased the concentrations of immunoglobulin-like (IgG-like, IgA-like and IgM-like) substances in the serum of Penaeus chinensis after a feeding period of 3 weeks. The differences among groups were significant (P<0.01), but there was no difference in the contents of complement3-like and complement4-like substances in the serum (P>0.05). Phenoloxidase (PO) activity in the serum of VC3 group shrimps was higher than that of VC0 and other groups, but no significant difference was observed between VC0 group and other groups. Furthermore, bactericidal activity of the serum to Vibrio parahaemolyticus in shrimps fed with the VC1 diet was higher than that in the other groups (P<0.01), while no difference was demonstrated among all groups for the bactericidal activity to Vibrio alginolyticus (P>0.05). It is, therefore, suggested that vitamin C (100–400 mg(100 g)⁻¹ diets) could be used as an immunostimulant of P. chinensis.     

Antioxidant response of ridgetail white prawn <Emphasis Type="Italic">Exopalaemon carinicauda</Emphasis> to harmful dinoflagellate <Emphasis Type="Italic">Prorocentrum minimum</Emphasis> exposure and its histological change

The dinoflagellate Prorocentrum minimum, one of the most widespread red tide causing species, affects marine aquaculture and ecosystems worldwide. In this study, ridgetail white prawn Exopalaemon carinicauda were exposed to P. minimum cells (5 × 10⁴ cells mL^?1) to investigate its harmful effects on the shrimp. Antioxidant activities and histological changes were used as indicators of health status of the shrimp. In 72 hours, the mortality of E. carinicauda was not affected, but its antioxidant response and histology were statistically different from those of control. Elevated superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities and depressed catalase (CAT) activity were observed in gill; while increased SOD, glutathione S-transferase (GST), CAT activities and modulated GPX activity were observed in hepatopancreas. Thus, antioxidant activities in gill and hepatopancreas seem to respond differentially to harmful alga exposure. Increased malondialdehyde (MDA) content in early a few hours indicates the damage of the antioxidant defense system. Although MDA content recovered to a low level thereafter, a series of histological abnormalities including accumulation or infiltration of hemocytes, tissue lesions and necrosis were discovered in gill and hepatopancreas. Exposure to P. minimum induced sublethal effects on E. carinicauda, including temporary oxidative damage and histological injury.     

Impact of Vibrio parahaemolyticus and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp Litopenaeus vannamei

White spot syndrome virus(WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect L itopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus(WSSV+Vp), or we fi rst infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection(Vp+WSSV). The effect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of V ibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction(PCR) method. L v My D88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the different mortality rates. Our results show that(1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection;(2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed signifi cant increases in the numbers of Vibrio( P 0.05);(3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10 5 to 10 7 /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.     

Impact of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect Litopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus (WSSV+Vp), or we first infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection (Vp+WSSV). The eff ect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of Vibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction (PCR) method. LvMyD88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the diff erent mortality rates. Our results show that (1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection; (2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed significant increases in the numbers of Vibrio (P<0.05); (3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10⁵ to 10⁷ /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.