Isolation of Prawn(Exopalaemon carinicauda) 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)(Ec LGBP) was isolated.The full-length c DNA of Ec LGBP was 1338 bp,encoding a polypeptide of 366 amino acid residules.The deduced amino acid sequence of Ec LGBP shared high similarities with LGBP and BGBP from other crustaceans.Some conservative domains were predicted in Ec LGBP sequence.Ec LGBP 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 Ec LGBP 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.Ec LGBP expression after WSSV infection increased at 3 h,then significantly decreased in both hemocytes and hepatopancreas.The results indicated that Ec LGBP was involved in the immune defense against bacterial and viral infections.     

Effect of cadmium on the defense response of Pacific oyster Crassostrea gigas to Listonella anguillarum challenge

Heavy metal pollution can affect the immune capability of organisms.We evaluated the effect of cadmium(Cd) on the defense responses of the Pacific oyster Crassostrea gigas to Listonella anguillarum challenge.The activities of several important defensive enzymes,including superoxide dismutase(SOD),glutathione peroxidase(GPx),acid phosphatase(ACP),Na+,K+-ATPase in gills and hepatopancreas,and phenoloxidase-like(POL) enzyme in hemolymph were assayed.In addition,the expression levels of several genes,including heat shock protein 90(HSP90),metallothionein(MT),and bactericidal/permeability increasing(BPI) protein were quantified by fluorescent quantitative PCR.The enzyme activities of SOD,ACP,POL,and GPx in hepatopancreas,and the expression of HSP90 were down-regulated,whereas GPx activity in the gill,Na+,K+-ATPase activities in both tissues,and MT expression was increased in Cdexposed oysters post L.anguillarum challenge.However,BPI expression was not significantly altered by co-stress of L.anguillarum infection and cadmium exposure.Our results suggest that cadmium exposure alters the oysters’ immune responses and energy metabolism following vibrio infection.     

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 shrimp Fenneropenaeus 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 and Vibrio 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℃ above ambient temperature.     

Peritrophin-like protein from Litopenaeus vannamei(LvPT) involved in white spot syndrome virus(WSSV) infection in digestive tract challenged with reverse gavage

The peritrophic membrane plays an important role in the defense system of the arthropod gut. The digestive tract is considered one of the major tissues targeted by white spot syndrome virus(WSSV) in shrimp. In this study, the nucleotide sequence encoding peritrophin-like protein of L itopenaeus vannamei(Lv PT) was amplified from a yeast two-hybrid library of L. vannamei. The epitope peptide of Lv PT was predicted with the Gen Script Optimum Antigen? design tool. An anti-Lv PT polyclonal antibody was produced and shown to specifically bind a band at ~27 k Da, identified as Lv PT. The Lv PT protein was expressed and its concentration determined. L v PT ds RNA(4 μg per shrimp) was used to inhibit Lv PT expression in shrimp, and a WSSV challenge experiment was then performed with reverse gavage. The pleopods, stomachs, and guts were collected from the shrimp at 0, 24, 48, and 72 h post-infection(hpi). Viral load quantification showed that the levels of WSSV were significantly lower in the pleopods, stomachs, and guts of shrimp after L v PT ds RNA interference than in those of the controls at 48 and 72 hpi. Our results imply that Lv PT plays an important role during WSSV infection of the digestive tract.     

Proteomic analysis of acute responses to copper sulfate stress in larvae of the brine shrimp, Artemia sinica

Proteomics was used to reveal the differential protein expression profiles of acute responses to copper sulfate exposure in larvae of Artemia sinica.Fourteen differentially displayed protein spots were detected and seven of them were identified.Three spots were up-expressed and identified:actin, heat shock protein 70,and chaperone subunit 1;three down-regulated proteins were identified:arginine kinase,elongation factor-2,and glycine-rich protein;and a newly expressed protein was identified as peroxiredoxin.The study indicates the involvement of all the differentially expressed proteins in the early responses of protein expression,and in the survival of A.sinica in the presence of copper and other heavy metals;the findings improve understanding of the organism’s adaptive responses and resistance.     

White spot syndrome virus inactivation study by using gamma irradiation

The present study was conducted to investigate the effect of gamma irradiation on white spot syndrome virus(WSSV). White spot syndrome virus is a pathogen of major economic importance in cultured penaeid shrimp industries. White spot disease can cause mortalities reaching 100% within 3–10 days of gross signs appearing. During the period of culture, immunostimulant agents and vaccines may provide potential methods to protect shrimps from opportunistic and pathogenic microrganisms. In this study, firstly, WSSV was isolated from infected shrimp and then multiplied in crayfish. WSSV was purified from the infected crayfish haemolymph by sucrose gradient and confirmed by transmission electron microscopy. In vivo virus titration was performed in shrimp, Penaeus semisulcatus. The LD50 of live virus stock was calculated 10 5.4 /mL. Shrimp post-larvae(1–2 g) were treated with gamma-irradiated(different doses) WSSV(10 0 to 10-4 dilutions) for a period of 10 days. The dose/survival curve for irradiated and un-irradiated WSSV was drawn; the optimum dose range for inactivation of WSSV and unaltered antigenicity was obtained 14–15 kGy. This preliminary information suggests that shrimp appear to benefit from treatment with gammairradiated WSSV especially at 14–15 KGy.     

Antioxidant Response of Ridgetail White Prawn Exopalaemon carinicauda to Harmful Dinoflagellate Prorocentrum minimum 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.     

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.     

Transcriptome Analysis of Crassostrea sikamea (♀) × Crassostrea gigas (♂) Hybrids Under and After Thermal Stress

Crossbreeding is an effective approach to manage the genetic decline in aquaculture.One-way hybrids of Crassostrea sikamea(♀)and Crassostrea gigas(♂)have advantages in growth traits and adaptation to high temperature.Here,we used high-throughput sequencing to analyze the molecular processes in the hybrids under and after thermal stress.The hybrids were cultured in the seawater with an increasing temperature from 25℃to 40℃during 10 hours,which is regarded as the thermal stress stage.Then the temperature decreased from 40℃to 25℃within 2 h,which is regarded as the recovery stage.In this study,1293 significant diffe-rentially expressed genes(DEGs)were obtained under thermal stress,of which 576 were upregulated and 717 were downregulated,and 740 significant differentially expressed genes(DEGs)were obtained in the recovery stage,of which the number of upregulated and downregulated genes was 409 and 331,respectively.The antigen processing and presentation,NOD-like,and NF-kappa B path-ways were significantly enriched during the thermal stress stage.The MAPK and PPAR signaling pathways were significantly enrich-ed during the recovery stage.The HSP70,HSP90,and CANX genes were strongly and rapidly upregulated in the control/thermal stress groups but were slightly less upregulated in the thermal stress/recovery group.These results indicate that the innate immune system or nonspecific immunity was deployed to protect interior tissues from thermal stress.In addition,85%of the mutual DEGs were involved in bidirectional regulation(up/down or down/up)when the oysters were removed from the thermal stress to recover.This study provides preliminary insight into the molecular response of C.sikamea(♀)and C.gigas(♂)hybrids to thermal stress and provides a basis for future studies on temperature-adaptation and the possible expansion of hybrid breeding.     

Peptidomic analysis of Chinese shrimp (Fenneropenaeus chinensis) hemolymph by magnetic bead-based MALDI-TOF MS

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.     

Development and application of antibody microarray for white spot syndrome virus detection in shrimp

Detecting white spot syndrome virus (WSSV) in shrimp in high efficiency and veracity is important for disease prevention in aquaculture. Antibody-based microarray is a novel proteomic technology that can meet the requirements. In this study, we developed an antibody microarray for WSSV-detection in a specific and parallel way at multiple samples. First, seven slides each with different modifications were characterized by atomic force microscope, and were compared in the efficiency of immobilizing proteins. Of the seven, 3-dimensional structured agarose gel-modified slides were chosen appropriate for the microarray for having higher signal value and superior spot size. A purified rabbit anti-WSSV antibody was arrayed as the capture antibody of the microarray on the agarose gel-modified slides, and then the microarray slides were incubated in the tissue homogenate of sampled shrimp and the antibody-antigen complex was detected by Cy3-conjugated anti-WSSV monoclonal antibody. The results were measured by a laser chipscanner and analyzed with software. To obtain satisfied fluorescence signal intensity, optimal conditions were searched. The detection limit of the antibody microarray for WSSV is 0.62 μg/mL, with a proven long shelf life for 6 months at 4°C or 8 months at -20°C. Furthermore, concordance between antibody microarray and traditional indirect ELISA reached 100% for WSSV detection. These results suggest that the antibody microarray could be served as an effective tool for diagnostic and epidemiological studies of WSSV.