溶藻弧菌肽聚糖对凡纳滨对虾白斑综合征病毒的抑制作用

从患病凡纳滨对虾(Litopenaeus vannamei)体内提取白斑综合征病毒(WSSV),PCR证实WSSV毒性,将病毒粗提液稀释10-2、10-3、10-4和10-5倍,攻毒实验表明,最佳攻毒剂量为10-4倍稀释液。对凡纳滨对虾(Litopenaeus vannamei)分别注射0.05、0.10、0.15 mg/m L的溶藻弧菌(Vibrio alginolyticus)肽聚糖,免疫24 h后感染WSSV,测定其免疫保护率和肝胰腺的免疫学指标,探讨溶藻弧菌肽聚糖对凡纳滨对虾抗WSSV的影响。结果表明:对虾的保护率随着肽聚糖的浓度增大而增大,与对照组相比,0.05、0.10、0.15 mg/m L组的保护率分别为36.67%、46.67%、56.67%;实验组凡纳滨对虾肝胰腺中的酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和一氧化氮合成酶(NOS)活力与对照组相比的差异有统计学意义(P0.01)。溶藻弧菌肽聚糖可有效增强凡纳滨对虾自身的非特殊性免疫能力,提高抗WSSV的能力。     

抗菌肽对凡纳滨对虾生长和机体免疫的影响

基础饲料(对照组)中添加10g/kg抗菌肽制剂喂养体重0.10±0.01g、体长2.17±0.11cm的凡纳滨对虾(Penaeus vannamei)30d,进行10d的水浴攻毒(鳗弧菌10~8/mL),探讨饲料中添加抗菌肽对凡纳滨虾生长、成活率、免疫保护率,以及肌肉中溶菌酶活性、超氧化物歧化酶活性、总抗氧化能力的影响。结果表明:饲料中添加抗菌肽可显著提高凡纳滨对虾相对增重率、成活率和对致病菌的免疫保护率(P<0.05),但对饲料系数无显著影响(P>0.05);未受外界微生物刺激时,饲料中添加抗菌肽对凡纳滨对虾总抗氧化能力有促进作用(P<0.05),受外界微生物刺激后,饲料中添加抗菌肽喂养凡纳滨对虾可提高对虾超氧化物歧化酶活性(P<0.05)和总抗氧化能力(P<0.01)。因此饲料中添加抗菌肽喂养凡纳滨对虾可提高其机体非特异性免疫。     

溶藻弧菌对凡纳滨对虾血清中一氧化氮及氧自由基的影响

对凡纳滨对虾注射浓度为1×105、1×106、1×107mL-1的溶藻弧菌悬液,在注射后4、12、24、48、72和120 h于腹血窦取血,测定其血清中一氧化氮(NO)、丙二醛(MDA)含量,以及一氧化氮合酶(NOS)、超氧化物岐化酶(SOD)活力。结果表明,注射溶藻弧菌后,凡纳滨对虾血清中NO含量及NOS活力显著高于对照组,且NOS活力最大值出现时间较NO早;而SOD活力先升高后下降,MDA含量在SOD活力开始下降后逐渐升高,说明一氧化氮系统对凡纳滨对虾感染的溶藻弧菌有清除作用,并能在一定程度上对损伤过程中生成的氧自由基产生作用。     

溶藻弧菌肽聚糖对凡纳滨对虾虾青素、免疫指标及保护率的影响

从溶藻弧菌中提取肽聚糖,以0.05、0.1、0.15 mg/mL 3种浓度为注射凡纳滨对虾后,测定血淋巴中的血细胞数目、酚氧化酶(PO)、超氧化物歧化酶(SOD)、一氧化氮合成酶(NOS)的活性和虾青素的含量,探讨溶藻弧菌肽聚糖对凡纳滨对虾虾青素含量、免疫指标、免疫保护率的影响。结果表明:注射不同质量浓度的溶藻弧菌肽聚糖都对血清中的血细胞数目、PO、SOD、NOS活性影响显著(p值﹤0.05),对虾青素的含量影响极显著(p值﹤0.01);虾青素的含量与血细胞数目、PO、NOS活性变化趋势无明显的相似性,与SOD活性变化有相似的变化趋势;0.05 mg/mL组的免疫保护率为40%,0.1 mg/mL组为53.33%,0.15 mg/mL组为66.67%。溶藻弧菌肽聚糖可有效地增强凡纳滨对虾非特异免疫力,提高对溶藻弧菌的抗病力,虾青素含量与SOD活性的高低变化趋势相似。     

凡纳滨对虾和斑节对虾对WSSV敏感性的比较

用6.0×104拷贝、1.2×104拷贝和6.0×103拷贝3种剂量白斑综合症病毒(WSSV)对凡纳滨对虾和斑节对虾进行人工注射感染,比较了两种对虾对WSSV敏感性的差异。结果表明,凡纳滨对虾死亡时间随病毒剂量降低而延长,斑节对虾死亡时间没有明显差异;随病毒剂量的降低,凡纳滨对虾人工注射感染后病毒复制高峰时间显著延长,斑节对虾感染后病毒复制高峰时间相同,WSSV在凡纳滨对虾体内比在斑节对虾体内复制慢。对虾携带WSSV数量最低为3.3×107拷贝.g-1,最高为4.3×108拷贝.g-1。凡纳滨对虾比斑节对虾对WSSV的抵抗性更强。     

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。     

溶藻弧菌肽聚糖和vp28-siRNA对凡纳滨对虾白斑综合征病毒的抑制作用

对感染白斑综合征病毒(WSSV)的凡纳滨对虾(Litopenaeus vannamei)分别注射0.05和0.10μg/μL的vp28-si RNA,干扰效果实验表明,si RNA最佳浓度为0.10μg/μL。凡纳滨对虾感染WSSV 24 h时注射0.10μg/μL的vp28-si RNA,检测免疫后不同时间点的干扰效果。结果表明:在注射vp28-si RNA 6 h后,实验组与对照组WSSV病毒拷贝数差异有统计学意义(P0.05);在48 h时实验组病毒拷贝数急剧下降,对照组则保持较高水平,可较好干扰WSSV病毒复制,干扰效果持续120 h,对凡纳滨对虾的保护率为40%。凡纳滨对虾用0.10 mg/m L肽聚糖(Peptidoglycan)免疫24 h后感染WSSV,检测6、12、24 h注射vp28-si RNA的保护率。结果显示:6 h注射组的保护率为80%,12 h注射组为63.33%,24 h注射组为56.67%。凡纳滨对虾在肽聚糖和vp28-si RNA作用后,可增加其对WSSV抗感染作用,降低死亡率,干扰时间越早,对对虾的保护率越高。     

微囊藻毒素对凡纳滨对虾免疫相关酶活力的影响

通过毒性注射实验,探究微囊藻毒素(MC-LR)对凡纳滨对虾(Litopenaeus Vannamei)肝脏免疫相关酶活力的影响。结果表明,注射MC-LR后,凡纳滨对虾肝胰脏组织的超氧化歧化酶、谷胱甘肽过氧化酶、过氧化物酶、酸性磷酸酶、碱性磷酸酶和溶菌酶的酶活力呈先增大后减小的变化趋势。超氧化歧化酶、谷胱甘肽过氧化酶和过氧化物酶活力在对虾注射MC-LR 4 h后达到峰值,比对照组分别增加了15.62%、18.76%和32.86%,随后酶活力逐渐减小;而酸性磷酸酶和碱性磷酸酶酶活力在对虾注射MC-LR 2 h后即显著增加,比对照组分别增加了25.02%和37.99%,随后酶活力逐渐减小;溶菌酶在对虾注射MC-LR后8 h达到最大值,比对照组增加了33.57%,随后酶活力逐渐减小。MC-LR能显著影响凡纳滨对虾的免疫相关酶活力(P0.05),引起凡纳滨对虾的应激反应和抑制它们的免疫相关酶活力。     

光合细菌对养殖水质及凡纳滨对虾抗病力的影响

在凡纳滨对虾养殖环境中人工引入不同浓度的光合细菌,监测氨氮、亚硝酸氮、化学需氧量的变化,15d后测定凡纳滨对虾的AKP、POD、PO、SOD、抗菌、溶菌活力,以及不同光合细菌浓度下凡纳滨对虾的成活率与体重增长率,研究光合细菌对水质和凡纳滨对虾抗病力的影响。结果表明,引入光合细菌可显著降低水体化学需氧量、氨氮含量,并抑制亚硝酸盐氮的产生,提高凡纳滨对虾的抗病力。当光合细菌投放浓度为3×103/mL时,水体中化学需氧量、氨态氮、亚硝酸盐氮含量的均值分别比对照组降低31.59%、43.42%、52.20%,差异显著(P<0.05);对虾的AKP、POD、PO、SOD、抗菌、溶菌活力分别比照组提高31.92%、72.29%、55.56%、31.61%、39.07%、69.51%,差异显著(P<0.05);成活率,体重增长率分别比对照组提高18.67%、34.03%,差异显著(P<0.05)。     

牟氏角毛藻对对虾育苗水质及抗逆性的影响

在凡纳滨对虾((Litopenaeus vannamei)育苗水体中引入不同浓度的牟氏角毛藻(Chaetoceros muelleri),检测主要水质因子,测定凡纳滨对虾对主要环境因子抗逆性,研究微藻对水质和凡纳滨对虾抗逆性的影响。结果表明,不同浓度牟氏角毛藻可明显改善对虾育苗水体的水质,提高幼虾的抗逆性(P<0.05)。牟氏角毛藻浓度为0.6×108～1×108/L时,实验组氨氮含量比对照组降低37.29%～46.32%,亚硝酸氮含量降低48.65%～62.70%,化学耗氧量(COD)值降低9.99%～12.19%;实验组育苗成活率达78.7%～84.0%,对照组育苗成活率46.0%;在氨氮、亚硝酸盐氮、硫化氢、甲醛抗逆性实验中,实验组幼虾成活率分别为75%～80%、75%、75%～85%、65%～70%,均显著高于对照组(P<0.05)。     

Diversity of bacteria isolated from crustacea larvae and their rearing water

1　Introduction　Blacktigershrimp (Penaeusmonodon)isthemajorshrimpspeciesculturedinIndonesia .Itsculturehasbecomemoreintensivebecauseofitshighdemandandeconomicvalue .Infact,shrimpexporthasyieldedthehighestIndonesianincomeinfisherysector .Itwasestimatedthattheproductionofculturedshrimpwas 80 0 0 0metrictonnesin 1995 .Consequently ,theneedforthefryforstockingpondculturesteadilyin creased .Thetotalnumberoffryrequiredwasabout 6millionperyear (FAO ,1995 )and 90 %oftherequiredanimalswereobtained…     

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.     

Glutamate dehydrogenase and Na+-K+ ATPase expression and growth response of Litopenaeus vannamei to different salinities and dietary protein levels

Improvement in the osmoregulation capacity via nutritional supplies is vitally important in shrimp aquaculture.The effects of dietary protein levels on the osmoregulation capacity of the Pacific white shrimp(L.vannamei) were investigated.This involved an examination of growth performance,glutamate dehydrogenase(GDH) and Na+-K+ ATPase mRNA expression,,and GDH activity in muscles and gills.Three experimental diets were formulated,containing 25%,40%,and 50% dietary protein,and fed to the shrimp at a salinity of 25.After 20 days,no significant difference was observed in weight gain,though GDH and Na+-K+ ATPase gene expression and GDH activity increased with higher dietary protein levels.Subsequently,shrimp fed diets with 25% and 50% dietary protein were transferred into tanks with salinities of 38 and 5,respectively,and sampled at weeks 1 and 2.Shrimp fed with 40% protein at 25 in salinity(optimal conditions) were used as a control.Regardless of the salinities,shrimp fed with 50% dietary protein had significantly higher growth performance than other diets;no significant differences were found in comparison with the control.Shrimp fed with 25% dietary protein and maintained at salinities of 38 and 5 had significantly lower weight gain values after 2 weeks.Ambient salinity change also stimulated the hepatosomatic index,which increased in the first week and then recovered to a relatively normal level,as in the control,after 2 weeks.These findings indicate that in white shrimp,the specific protein nutrient and energy demands related to ambient salinity change are associated with protein metabolism.Increased dietary protein level could improve the osmoregulation capacity of L.vannamei with more energy resources allocated to GDH activity and expression.     

盐度对尼罗罗非鱼血清甲状腺素浓度和成活率的影响

在尼罗罗非鱼(Oreochromis niloticus)由淡水逐渐向不同盐度海水(5,10,15,20,25)适应过程中,运用放射免疫测定法研究血清中三碘甲腺原氨酸(T3)浓度的变化,并分析盐度变化对尼罗罗非鱼成活率的影响。结果表明,盐度对尼罗罗非鱼血清中T3水平和成活率有着显著的影响。在盐度5~20的范围内变化后,血清T3的浓度在6 d内随着盐度上升而增大,第7天达到高峰;随后,逐渐降低并趋于稳定,在第14天时,各盐度组尼罗罗非鱼血清中T3开始处于稳定状态;稳定后,各实验组血清中T3的浓度均比对照组升高,并表现出随盐度的升高而升高的趋势。在盐度20时,实验第17天,尼罗罗非鱼的成活率为80%。而在盐度为25时,实验第11天,尼罗罗非鱼的成活率仅为54%。对不同盐度下尼罗罗非鱼死亡原因及血清甲状腺激素在盐度变化下的生理意义和对死亡率的影响作了讨论。     

植酸酶对凡纳滨对虾生长性能和体成分的影响

在凡纳滨对虾饲料中添加不同量微生物植酸酶(0,500,1000,2000 U/kg),观察虾的存活率、增重率、饲料系数及虾体和虾壳成分,研究饲料中添加植酸酶对凡纳滨对虾生长性能的影响。结果表明:饲养8周后,饲料中添加500~2000 U/kg植酸酶对凡纳滨对虾增重率、存活率和饲料利用无显著影响(P>0.05);植酸酶添加各组与对照组虾体的水分、粗蛋白、脂肪、灰分、总钙和总磷含量差异不显著;虾壳中粗灰分和钙含量在500和1000 U/kg组显著高于对照组和2000 U/kg组(P<0.05),虾壳磷含量在各饲料组间差异不显著。血清磷浓度在对照组和2000 U/kg组显著高于500 U/kg组(P<0.05),血清钙浓度在组间差异不显著,血清碱性磷酸酶活性以对照组最高,显著高于500和1000 U/kg组。结果说明,在特定试验条件下,饲料中添加植酸酶对凡纳滨对虾幼虾的虾壳和血清成分有显著影响(P<0.05),对幼虾生长性能影响不显著(P>0.05)。     

Effects of Na^＋/K^＋ Ratio of Groundwaters on the Gill Ion-Transport Enzyme Activity, Plasma Osmolality and Growth of Cynoglossus semilaevis Juveniles

The effects of environmental Na /K ratio on the gill ion-transport enzyme activity,plasma osmolality and growth ofCynoglossus semi/aevis juveniles were investigated.The results showed that,plasma osmolality was similar among flounder adaptedto different Na /K ratios of saline groundwaters (P>0.05),while the growth,gill Na ,K -ATPase and HCO,3'--ATPase activities wereaffected by Na /K ratio significantly (P<0.05).The gill Na ,K -ATPase activity reached its maximum on day 3,then decreasedgradually from day 3 to day 9 and remained constant from day 9 to day 15.The peak values of gill Na ,K -ATPase activity weredetected on day 3 for all Na /K ratios of saline groundwaters,then the enzyme activities descended,and on day 9 the enzyme activi-ties achieved steady state,and the gill HCO,3--ATPase activity increased rapidly and achieved steady state after one day.Duringsteady state,the gill Na ,K -ATPase and HCO,3--ATPase activity of Na /K ratios 20 and 40 treatments were significantly higherthan those in the control group (Na /K ratio 27.5),while there were no significant differences between the Na /K ratio 30 treatmentand the control group; the gill Na ,K -ATPase activity of Na /K ratio 20 and 40 treatments were significantly higher than that forratio 30 treatment,but there were no significant differences of gill HCO3-ATPase activity among these treatments.At the end of the15-day experiment,the weight gain (%) and specific growth rate (SGR) of flounders maintained in seawater were significantly higherthan those in groundwaters; significant differences also occurred among the treatments; Na /K ratio 30 treatment had the highestvalues (33.7% and 1.94 respectively),which were significantly higher than those under Na /K ratios 20 and 40 treatments.There-fore,for the saline groundwater used in this experiment,it is suggested that the Na /K ratio be adjusted to approximately 30,I.e.,asclose to that of natural seawater as possible in the culture of flounder.     

Immune response of Pseudosciaena crocea to the injection of Vibrio alginolyticus

For the investigation of anti-infection immune response of Pseudosciaena crocea, 160 healthy fish samples were categorized into infected and control groups. Each individual fish in the infected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension of Vibrio alginolyticus in density of 2×107 CFU/ml, while each individual in the control group was injected i.p. with 0.2 ml sterile saline solution (0.85%). It was observed that the artificial injection of V. alginolyticus significantly inc...     

Effects of different enzymatic hydrolysis methods on the bioactivity of peptidoglycan in Litopenaeus vannamei

The effects of different hydrolysis methods on peptidoglycan(PG) were assessed in terms of their impact on the innate immunity and disease resistance of Pacific white shrimp,Litopenaeus vannamei.PG derived from Bifidobacterium thermophilum was prepared in the laboratory and processed with lysozyme and protease under varying conditions to produce several different PG preparations.A standard shrimp feed was mixed with 0.05% PG preparations to produce a number of experimental diets for shrimp.The composition,concentration,and molecular weight ranges of the soluble PG were analyzed.Serum phenoloxidase and acid phosphatase activity in the shrimp were determined on Days 6-31 of the experiment.The protective activity of the PG preparations was evaluated by exposing shrimp to white spot syndrome virus(WSSV).Data on the composition of the PG preparations indicated that preparations hydrolyzed with lysozyme for 72 h had more low-molecular-weight PG than those treated for 24 h,and hydrolysis by protease enhanced efficiency of hydrolysis compared to lysozyme.SDS-PAGE showed changes in the molecular weight of the soluble PG produced by the different hydrolysis methods.Measurements of serum phenoloxidase and acid phosphatase activity levels in the shrimp indicated that the PG preparations processed with enzymes were superior to the preparation which had not undergone hydrolysis in enhancing the activity of the two serum enzymes.In addition,the preparation containing more low-molecular-weight PG enhanced the resistance of the shrimp to WSSV,whereas no increased resistance was observed for preparations containing less low-molecular-weight PG.These findings suggest that the immunity-enhancing activity of PG is related to its molecular weight and that increasing the quantity of low-molecular-weight PG can fortify the effect of immunity enhancement.     

波吉卵囊藻对弧菌生长的影响

研究波吉卵囊藻及藻―菌体系对创伤弧菌(Vibrio vulnificus)、副溶血弧菌(Vibrio parahaemolyticus)和溶藻弧菌(Vibrio alginolyticus)生长的影响,结果显示:无菌波吉卵囊藻对创伤弧菌、副溶血弧菌的生长均有显著促进作用(P<0.05),对溶藻弧菌的生长无显著影响(P>0.05);波吉卵囊藻―沼泽红假单胞菌体系对溶藻弧菌有明显抑制作用(P<0.05);无菌波吉卵囊藻―枯草芽孢杆菌体系对溶藻弧菌的生长有显著促进作用(P<0.05),对创伤弧菌、副溶血弧菌的生长无显著影响(P>0.05);带菌波吉卵囊藻对三株弧菌均有明显抑制作用(P<0.05);带菌波吉卵囊藻共栖细菌对三株弧菌均无明显抑制作用(P>0.05);在无菌波吉卵囊藻中回加带菌藻共栖细菌,分析实验10～18d的数据发现的数据发现藻―菌体系对创伤弧菌、副溶血弧菌的生长有显著影响(P<0.05),藻―菌体系逐渐恢复对三株弧菌的抑制作用。