Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.     

凡纳滨对虾不同养殖密度高位池水体细菌群落动态

通过对养殖水体环境基因组DNA中细菌16S rRNA基因V4–V5区的高通量测序和生物信息学分析,研究了两种养殖密度的凡纳滨对虾(Litopenaeus vannamei)高位池水体中细菌群落在养殖过程中的动态。结果显示,养殖过程中各菌群相对丰度变化明显,细菌多样性随时间逐渐提高,优势菌群为变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes)。随着养殖时间增长,蓝藻丰度所占比例逐渐减少,而变形菌、拟杆菌和浮霉菌丰度逐渐增大,同时养殖前期高密度池浮霉菌丰度显著高于低密度池(P0.01),而其他菌群无显著差异。结果表明,养殖期前50 d不同养殖密度水体细菌群落差异较大,而后30 d内细菌群落的时间异质性大于空间异质性,这意味着高位池水体菌相被划分为两类,到养殖后期菌相快速转变,养殖密度所带来的影响被减弱。     

养殖曼氏无针乌贼白色卵膜营养成分分析及评价

测定分析并评价了养殖曼氏无针乌贼（ Sepiella maindroni）白色卵膜（WEM）的营养成分。结果表明,WEM （鲜样）中水分、粗灰分、粗蛋白和粗脂肪的质量分数分别为92.34％、1.76％、5.28％和0.34％。WEM（干样）中氨基酸（AA）总量为55.53％,其中必需氨基酸（EAA）总量为19.85％;必需氨基酸与非必需氨基酸（NEAA）的比值为65.6％,构成比例符合 FAO/ WHO 规定的优质蛋白质标准;鲜味氨基酸（ DTAA）总量为29.53％。WEM的第一限制性氨基酸为苯丙氨酸＋酪氨酸（Phe ＋ Tyr）,必需氨基酸指数（EAAI）为38.03。脂肪酸中多不饱和脂肪酸（PUFA）占19.21％。矿物质含量丰富,尤其以磷（P）质量分数最高（1423.2 mg·kg -1）。结果表明,WEM 营养组分较为全面,与野生黑色卵膜（BEM）的营养成分进行对比分析发现,在氨基酸组成、必需氨基酸和无机元素质量分数等方面存在差异,这种差异是否是导致白化受精卵孵化率降低的关键因素有待进一步研究。     

南美白对虾幼体和仔虾淀粉酶和脂肪酶活力的研究

以酶学分析方法测定了南美白对虾状幼体,糠虾和仔虾三个发育期淀粉酶和脂肪酶的活力。试验结果表明,糠虾期淀粉酶比活力显著高于状幼体和仔虾两期(P<0.05);脂肪酶比活力状幼体显著高于糠虾幼体(P<0.05)。在食性转化过程中,南美白对虾幼体消化酶活力与其食性相适应,不同发育阶段消化酶调节机制不同。     

白斑综合征病毒环介导等温扩增快速检测方法的建立

根据对虾白斑综合征病毒(WSSV)囊膜蛋白VP28基因保守序列,利用Primer Explorerv 4.0软件设计了4条引物,建立了白斑综合征病毒环介导等温扩增快速检测方法,对反应温度和反应时间等参数进行了优化,同时将建立的LAMP检测方法与巢式PCR进行了比较分析。结果表明,LAMP最适反应在64℃恒温条件60min内完成,凝胶电泳呈现梯型条带;反应体系中添加SYBR Green I荧光染料后,绿色的阳性结果明显区别于橙色阴性结果。LAMP方法的最低检出限为100拷贝/μL,灵敏度较巢式PCR高100倍,而且LAMP方法在1h内即可完成检测,操作简单,无需复杂仪器,肉眼可直接观察检测结果。用建立的LAMP方法对临床发病南美白对虾样品进行了检测,结果表明,LAMP方法适合对虾白斑综合征病毒的现场快速检测。     

基于图像识别技术研究不同海区三疣梭子蟹甲壳白色斑纹特征及蜕壳前后斑纹特征的变化

为进行三疣梭子蟹外观特征的研究,本研究通过计算机视觉技术建立三疣梭子蟹背部白色斑纹评估方法,经人工对比验证其准确性达100%。在此基础上,对来自海域的30只三疣梭子蟹[平均体质量(2.8±0.56)g]进行跟踪观察,发现蜕壳前后白色斑纹数量和位置保持不变,而白色斑纹面积和斑纹区域面积则会随着蜕壳后蟹壳面积的增加而扩大;蜕壳后,白色斑纹面积增加89.33%±8.61%,斑纹区域面积增加90.51%±7.95%,蟹壳面积增加94.66%±8.26%,均显著正相关。为进一步研究不同海区梭子蟹白色斑纹分布特征,对来自朝鲜、中国辽宁和浙江等不同海区的三疣梭子蟹进行白色斑纹个数(N)、白色斑纹面积(S1)、斑纹区域面积(S2)、白色斑纹面积百分比(R1)、斑纹区域面积百分比(R2)和密度(D)等6项参数分析,结果发现,不同海区三疣梭子蟹斑纹性状存在显著特点,朝鲜海区呈现斑纹小、分布面积少、占蟹壳总面积比例低的特点;辽宁海区呈现斑纹小、分布面积多、占蟹壳总面积比例高的特点;浙江海区呈现斑纹大、分布面积少、占蟹壳总面积比例低的特点。本研究将计算机视觉技术应用于水产养殖研究,通过建立评估参数阐明三疣梭子蟹白色斑纹的生长规律,揭示了不同群体的三疣梭子蟹白色斑纹具有地理多样性的特征。     

Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV.     

Effects of different dietary copper sources on the growth and intestinal microbial communities of Pacific white shrimp (Litopenaeus vannamei)

An 8‐week feeding trial was conducted to evaluate the effects of dietary copper sources on growth performance and intestinal microbial communities of juvenile Pacific white shrimp (Litopenaeus vannamei). Four isonitrogenous (420 g/kg crude protein) and isolipidic (70 g/kg crude lipid) experimental diets were formulated to contain different copper sources, Diet NSC (no‐supplemented copper), Diet CS (copper sulphate), Diet Availa (copper amino acid complex from Availa^®Cu100) and Diet M (1:1, copper sulphate + copper amino acid complex). A total of 360 Pacific white shrimp juveniles (initial weight: 1.86 ± 0.03 g) were randomly allocated into four groups, with three replicates per group and 30 shrimps each replicate. An Illumina‐based sequencing method was used to analyse the intestinal bacterial composition of shrimp juveniles. The results revealed that shrimps fed Diet M had significantly higher percent weight gain (PWG) than that fed Diet NSC and Diet CS, while there were no significant differences between Diet M and Diet Availa. Survival was not affected by the dietary copper sources. Proteobacteria and Bacteroidetes were dominant in the intestines of Pacific white shrimp. Shrimps fed Diet M and Diet Availa had higher bacterial richness and diversity than those fed Diet NSC and Diet CS with no significant differences among all treatments. Furthermore, Diet M and Diet Availa which contained copper amino acid complex reduced the relative abundance of Vibrionaceae in shrimp intestinal tract. This study indicated that dietary copper sources could affect the intestinal microbial communities and diets supplemented with Availa^®Cu100 could have a positive impact on the growth performance and decrease the potential risk of disease in Pacific white shrimp.     

日本对虾抗白斑病子三代的抗白斑综合征病毒感染能力及免疫特性

为了更好地进行日本对虾抗病良种的选育,本文用浓度为每毫升1×105个病毒粒子的对虾白斑综合征病毒(white spot syndrome virus,WSSV)粗提液注射感染抗白斑病日本对虾子三代和普通日本对虾,观察其死亡率,同时测定WSSV感染后0,4,24,48和96 h血淋巴酚氧化酶(PO)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)活力和蛋白含量.结果显示:注射WSSV 14 d内抗病子三代和普通对虾的平均死亡率分别为15%和85%,前者的抗病保护率达82.4%;感染前前者的各项免疫指标均高于后者,且PO,AKP和蛋白含量存在极显著差异(P＜0.01);感染后两者血淋巴检测的5项指标在96 h内的变化趋势基本相似,除ACP以外,抗病子三代的其他指标都比普通日本对虾的高.研究结果表明,所选育的抗白斑病日本对虾子三代能够有效地抵抗对虾白斑综合征病毒的侵染,其免疫指标也高于普通日本对虾.