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.     

Effect of bioactive peptides (BPs) on the development of Pacific white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> Boone, 1931)

The present study was conducted to evaluate the feasibility of replacing fish meal (FM) with bioactive peptides (BPs) in diet of white shrimp (Litopenaeus vannamei). The changes in growth performance, body composition, non-specific immunity, and water quality were examined after the shrimp were fed four diets, in which 0% (control), 33.3%, 66.7% and 100% of FM was replaced by BPs, respectively. The groups were designated as Con, 1/3BPs, 2/3BPs, and 3/3BPs. A total of 720 shrimp with an initial body weight of 1.46 ± 0.78 g were fed the experimental diets for 56 days. The results revealed that: 1) the weight gain rate (WGR) in 1/3BPs, 2/3BPs, and 3/3BPs was significantly higher than that in Con (P < 0.05), while no significant difference was found on survival rate and feed conversion ratio (FCR); 2) the whole-body crude protein (CP) and crude lipids (CL) were significantly different among groups, while there was no significant difference between crude ash and phosphorus contents; 3) the levels of acid phosphatase (ACP), lysozyme (LZM), superoxide dismutase (SOD), phenol oxidase (PO) and bactericidal activity increased significantly with the inclusion of BPs; 4) in terms of water quality, no significant difference was found in pH and dissolved oxygen among diets during the whole experimental period. Moreover, even though nitrite and ammonium levels tended to increase with time, there was no significant difference among groups. The results indicated that BPs is an applicable alternative of protein source, which can substitute FM in the diets of L. vannamei; it is able to effectively promote growth performance and improve immunity. Moreover, BPs in the diets had no negative impact on water quality.     

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.     

Hematological changes in 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 manifestations 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 disintegrated 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 hemocytes 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.     

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.     

大黄对凡纳滨对虾生长和非特异性免疫指标的影响

在凡纳滨对虾饲料中分别添加大黄0、0.5、1.0、5.0、10.0和20.0g/kg,研究大黄对凡纳滨对虾(初始体重为0.34±0.004g)生长及非特异性免疫指标的影响。结果表明,大黄对凡纳滨对虾成活率、增重率、特定生长率、饲料系数、蛋白质效率和蛋白质累积率的影响不显著(P>0.05),对全虾和尾肌肉的灰分、脂肪和粗蛋白含量影响显著,全虾的粗蛋白和粗脂肪含量以1.0g/kg组最高(P<0.05),对凡纳滨对虾血清碱性磷酸酶、酸性磷酸酶、酚氧化酶、超氧化物歧化酶、溶菌酶以及血清总蛋白量等的影响显著,溶菌酶活性以1.0g/kg组最高(P<0.05);细菌感染实验中以1.0g/kg组存活率最高(P<0.05)。以非特异性免疫反应指标及感染实验存活率为指标,凡纳滨对虾饲料中大黄的适宜添加量为1.0g/kg。     

斑节对虾虾苗白斑综合症杆状病毒的检测和养殖跟踪

利用 2步 PCR检测技术对湛江和阳西地区的虾苗进行白斑综合病杆状病毒 ( WSSV)检测。在仔虾 2日龄最早检测到病毒 ,有 2 5%的虾苗带有 WSSV病毒。带病毒虾苗和不带病毒虾苗分别在不同养殖模式的养殖过程中跟踪。前者在湛江湖光镇普通虾塘跟踪养殖 ,它们在变化的环境中容易发病 ,p H、盐度和温度是重要的诱发因子 ,在养殖 50～ 60 d时发病死亡 ;后者在高位池和普通池养殖跟踪 ,它们对变化的环境有较大的适应性 ,养殖时间为 80～ 1 1 0 d,在相对优良的养殖技术条件下大部分可望养殖成功。环境中有 WSSV病原传入 ,不带病毒虾苗在养殖后期可以带有 WSSV病毒 ,出现白斑虾。跟踪的普通池有爆发病害 ,但是时间延后 ,跟踪的高位池没有爆发病害。     

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.     

Optimal content and ratio of lysine to arginine in the diet of Pacific white shrimp, Litopenaeus vannamei

The optimal quantity of dietary lysine (Lys) and arginine (Arg), and the optimal ratio of dietary Lys to Arg for Pacific white shrimp Litopenaeus vannamei were investigated. Coated Lys and Arg were added to a basal diet (37.99% crude protein and 7.28% crude lipid) to provide graded levels of Lys and Arg. The experimental diets contained three Lys levels (2.51%, 2.11%, and 1.70% of total diet), and three Arg levels (1.41%, 1.80%, and 2.21% of total diet) and all combinations of these levels were tested. Pacific white shrimp, with a mean weight of 3.62±0.1 g, were randomly distributed in 36 fiberglass tanks with 30 shrimp per tank and reared on the experimental diets for 50 days. After the feeding trial, the growth performance, survival, feed conversion rate (FCR), body composition and protease and lipase activities in the hepatopancreases of the experimental shrimps were determined. The results show that weight gain (WG), specific growth rate (SGR), FCR, body protein, body Lys and Arg content were significantly affected by dietary Lys and Arg (P <0.05) and improved when dietary Lys and Arg levels were 2.11% ～ 2.51% and 1.80%～2.21%, respectively. Protease and lipase activities in the hepatopancreases of the shrimps appeared higher when dietary Lys and Arg quantities were 2.11% ～2.51% and 1.80%～2.21%, although the difference was not statistically significant (P >0.05). Therefore, according to our results, the optimal Lys and Arg quantities in the diet of Pacific white shrimp, L. vannamei, were considered to be 2.11%–2.51% and 1.80%–2.21%, respectively, and the optimal ratio to be 1:0.88–1:1.05.     

Effect of dietary potassium on growth,nitrogen metabolism,osmoregulation and immunity of pacific white shrimp (Litopenaeus vannamei) reared in low salinity seawater

An 8 weeks feeding experiment was conducted to determine the effect of dietary potassium on the growth and physiological acclimation of Pacific white shrimp(Litopenaeus vannamei) reared in diluted seawater(salinity 4). Six semi-purified practical diets containing 0.59, 0.96, 1.26, 1.48, 1.74, and 2.17+ g potassium K per 100 g diet were formulated, respectively. The survival and feed conversion rate did not show significant difference among groups of shrimps given these diets(P0.05). The shrimps fed the diets containing 0.96-1.48 g K+ per 100 g diet gained the highest weight, specific growth rate, and protein efficiency ratio. Their ammonium-N excretion rate as well as hemolymph concentration of Na+ and Cl- were significantly lower than those of the control(P0.05), but a reverse trend was observed for their gill Na+/K+-ATPase. Moreover, the shrimps fed with 1.48 g K+ per 100 g diet were the highest in hemolymph urea level, and the phenoloxidase and lysozyme activities were significantly higher than those of the control(P0.05). The growth and physiological response of the test shrimps suggested that diet containing 1.48 g K+ per 100 g diet improved the growth of L. vannamei in low-salinity seawater, and enhanced the physiological acclimation of the organism.     

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.     

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…     

A Comparative study on the nonspecific immunity of juvenile Litopenaeus vannamei ever inhabiting freshwater and seawater

A study on the nonspecific immunity of Litopenaeus vannamei ever inhabiting freshwater and seawater was carried out at different molt stages by comparing their total hemocyte count(THC) and respiratory burst(RB) and activity of phenol oxidase(PO), nitric oxide synthase(NOS) and lysozyme(LY). Two-way ANOVA showed that salinity and molt stage independently affected THC and RB and the activity of PO, NOS and LY of juvenile L. vannamei significantly(P 0.05). The THC and RB and the activity of NOS gradually increased from the post-molt stages(A and B) to the pre-molt stages(D0–D3), which were common in shrimps inhabiting freshwater and seawater. The activity of PO peaked at the inter-molt stage(C), and touched the lowest at the post-molt stage in freshwater and pre-molt stage in seawater. The activity of LY was stable over the molt cycle. The RB and the activity of PO, NOS and LY of juvenile L. vannamei were significantly lower in freshwater than in seawater; whereas THC was significantly higher in freshwater than in seawater(P 0.05). It was concluded that the post-molt stage(especially stage A) was critical to shrimp culture, which should be intensively attended when L. vannamei was cultured in freshwater.     

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.     

Enhanced resistance of Portunus trituberculatus to Vibrio alginolyticus by selective breeding

We established a line (screened) of Portunus trituberculatus by selectively breeding individuals that survived from challenge with Vibrio alginolyticus, and compared the response of screened and unscreened (control) P. trituberculatus challenged with V. alginolyticus. We measured superoxide dismutase, catalase, acid phosphatase, alkaline phosphatase, and peroxidase activity and the content of hemocyanin in the plasma and phenoloxidase activity in serum. The cumulative survival rate after 24-h challenge with V. alginolyticus was significantly higher in the screened crabs than in the unscreened crabs (P<0.05). T-SOD and PO activity were significantly lower in the screened stock than in the unscreened stock (P<0.05). POD, CAT, and ACP activity and hemocyanin content were significantly higher in the screened stock than in the unscreened stock. Our results suggest that the screened stock was more resistant to infection. Furthermore, the indices we measured may be used to evaluate the health state of P. trituberculatus.     

Kinetic study of alkaline protease 894 for the hydrolysis of the pearl oyster Pinctada martensii

A new enzyme (alkaline protease 894) obtained from the marine extremophile Flavobacterium yellowsea (YS-80-122) has exhibited strong substrate-binding and catalytic activity, even at low temperature, but the characteristics of the hydrolysis with this enzyme are still unclear. The pearl oyster Pinctada martensii was used in this study as the raw material to illustrate the kinetic properties of protease 894. After investigating the intrinsic relationship between the degree of hydrolysis and several factors, including initial reaction pH, temperature, substrate concentration, enzyme concentration, and hydrolysis time, the kinetics model was established. This study showed that the optimal conditions for the enzymatic hydrolysis were an initial reaction pH of 5.0, temperature of 30°C, substrate concentration of 10% (w/v), enzyme concentration of 2 500 U/g, and hydrolysis time of 160 min. The kinetic characteristics of the protease for the hydrolysis of P. martensii were obtained. The inactivation constant was found to be 15.16/min, and the average relative error between the derived kinetics model and the actual measurement was only 3.04%, which indicated a high degree of fitness. Therefore, this study provides a basis for the investigation of the concrete kinetic characteristics of the new protease, which has potential applications in the food industry.     

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).