Identification and characterization of Alix/AIP1 interacting proteins from the black tiger shrimp,Penaeus monodon

Apoptosis is proposed to be a major cause of death in shrimp viral infections. From our previous study, an apoptosis-related gene, Pm-Alix, was identified from the black tiger shrimp. Its expression was high in defence-related tissues including haemocytes and the lymphoid organ. To clarify its possible role in shrimp, we used Pm-Alix as bait in a yeast two-hybrid analysis to search for Alix interacting proteins in shrimp. Two cDNA sequences discovered had homology to a predicted ubiquitin C of the purple sea urchin, Strongylocentrotus purpuratus, and to a guanylyl cyclase of the red swamp crayfish, Procambarus clarkii. In vitro pull-down assays confirmed positive interaction between Pm-Alix and both proteins. Tissue distribution analysis revealed that Pm-Alix and the two binding partners were widely expressed in various tissues but more highly expressed in haemocytes. However, no significant positive or negative correlation was found in the expression of these genes as shrimp approached morbidity and death after challenge with white spot syndrome virus. Thus, the results suggested that Alix and its interacting partners did not play a direct role related to shrimp death.     

Delay of the egg activation process in the Black Tiger Shrimp Penaeus monodon by manipulation of magnesium levels in spawning water

The aim of this study was to determine whether magnesium (Mg²⁺) in seawater is required for egg activation of the black tiger shrimp Penaeus monodon and whether manipulation of Mg²⁺ levels can be used to delay the process and thereby synchronize egg activation. Female P. monodon broodstock were allowed to spawn in artificial seawater containing Mg²⁺ at varying levels with respect to the normal (100%) level: 100%, 50%, 20% and 0%. Egg activation occurred normally at 100% Mg²⁺, incompletely at 50% and 20% Mg²⁺ levels and did not occur at all with 0% Mg²⁺. The fertilization rate with 100% Mg²⁺ was observed to be 83%, but fertilization failed to take place in all the other groups. The fertilization rate was restored from 0% to 76% following the 20% Mg²⁺ level treatment when Mg²⁺ levels returned to normal (100%) as soon as spawning was completed. This study suggests that the level of Mg²⁺ in seawater plays a vital role in P. monodon egg activation, and that commencement of this process could be delayed by manipulation of the Mg²⁺ level during and immediately after spawning.     

Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre‐emptive treatment of a rearing system with antagonistic or non‐antagonistic bacterial probiotics

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non‐antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having sufficient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB 111 under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen. These inhibitory properties were further confirmed in the larval rearing systems of P. monodon. All these four probionts significantly improved larval survival in long‐term treatments as well as when challenged with a pathogenic strain of V. harveyi MCCB 111. We could demonstrate that Pseudomonas MCCB 102 and MCCB 103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems through active antagonism of vibrios, whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme‐supporting modes of action.     

Development of immunodot blot assay for the detection of white spot syndrome virus infection in shrimps (Penaeus monodon)

The VP 28 gene encoding a structural envelope protein of the white spot syndrome virus (WSSV) was cloned into a pET32a(+) expression vector for the production of the recombinant VP28 protein. A purified recombinant protein of 39.9 kDa size was used for polyclonal antibody production in rabbit. Specific immunoreactivity of the rabbit anti rVP28 antiserum to the viral antigen was confirmed by a Western blot. The specificity of this polyclonal anti‐rVP28 antiserum to detect the presence of the virus in WSSV‐infected Penaeus monodon was verified using a immunodot blot assay. Immunodot blot showed a positive reaction in infected shrimp tissues with prominent colour development using 3,3′,5,5′‐tetramethylbenzidine (TMB) as a chromogenic substrate when compared with 3–3′ diaminobenzidine tetrahydrochloride (DAB). Highest signal intensities of the immunodots were observed in infected shrimp pleopod extracts and haemolymph. On comparison with polymerase chain reaction (PCR), immunodot blot could detect 76% of PCR‐positive WSSV‐infected shrimp samples. Immunodot blot was found to be equivalent to first‐step PCR sensitivity to detect WSSV particles estimated to contain 1.0 × 10⁵ viral DNA copies.     

斑节对虾虾苗活力检测方法

该研究通过肉眼观察、镜检,进行干露、饥饿、盐度突降、福尔马林等抗性试验,并采用病毒检测等方法,以期建立评估斑节对虾（Penaeus monodon）虾苗活力和质量标准。结果表明,斑节对虾健康虾苗具有趋光性、集群性,体表光洁,肌肉透亮,肠胃食物充盈等特性。测试虾苗干露时间以15min为宜,健康虾苗干露后能立即恢复活力,而病弱虾苗多出现死亡、昏迷现象;虾苗的成活率随饥饿时间的延长而降低,随福尔马林浓度升高和时间延长而降低,随盐度突降幅度增加而降低。健康虾苗能忍受100～200μL·L^-1福尔马林溶液30min,成活率近100％;在盐度20～30下虾苗的成活情况较好,而其在淡水中仅能存活1h。对虾苗进行病毒检测,可以避免养殖中因虾苗携带病毒而可能导致的病毒性疾病的暴发。     

斑节对虾养殖池塘藻-菌关系初探

本研究通过对斑节对虾养殖系统中的异养细菌、浮游微藻进行为期3个月的监测，发现浮游微藻和异养细菌的总量都表现为养殖后期高于养殖前期，其中浮游微藻增加了2个数量级，异养细菌增加了1个数量级，施放有益芽孢杆菌群对池塘菌群和藻群的变动有明显的影响。施放有益芽孢杆菌群的池塘，异养细菌总数略低，弧菌数量维持在10^3 CFU／mL以下，浮游微藻平稳增长，蓝藻占20％以下；对照池异养细菌的总数略高，弧菌数量达到10^3 CFU／mL，浮游微藻数量波动，养殖后期蓝藻占60％，为绝对优势种群。表明有益芽孢杆菌群有促进浮游微藻平稳繁殖的作用，但对浮游蓝藻和弧菌的繁殖起抑制作用，浮游蓝藻与弧菌之间具有一定的繁殖相关性。     

斑节对虾细胞周期蛋白B慢病毒载体的构建及对Hela细胞增殖影响

细胞周期蛋白B（Cyclin B）是影响卵母细胞发育成熟的重要基因。文章构建了PmCyB慢病毒表达载体,并和慢病毒包装复合物在293T细胞中包装成慢病毒颗粒。利用包装好的慢病毒颗粒,感染干扰了Cyclin B1基因的Hela细胞,研究了PmCyB对Hela细胞增殖的影响。结果表明,通过siRNA干扰细胞自身的Cyclin B1后,转染对照组病毒液的Hela细胞增殖减缓;同时,干扰后转入含有PmCyB重组慢病毒表达载体,Hela细胞增殖速度明显要高于转染对照组病毒液的Hela细胞的增殖速度。这一结果表明,PmCyB基因的过表达能补偿Cyclin B1缺失导致的细胞增殖减缓的现象,说明PmCyB基因具有细胞周期调控功能,是细胞增殖与分化的重要调控基因,且功能可能与人类的Cyclin B1相似。人类的Cyclin B1是卵母细胞发育成熟的重要基因。由此可以推断,PmCyB基因可能对斑节对虾（Penaeus monodon）卵巢发育成熟起着重要作用。     

池塘养殖斑节对虾生长、发育与性成熟

为了解不同养殖条件下斑节对虾的生长、外生殖器发育、性腺发育及性成熟之间的关系,对其养殖进行跟踪调查研究.结果显示:①斑节对虾雌雄外生殖器官发育和头胸甲长呈线性关系;②不同养殖环境条件下,斑节对虾性成熟生物学最小型个体无显著差异.雄性精荚出现的生物学最小型个体为头胸甲长3.1 cm,体长11.1 cm,体质量20.0 g;雄性性成熟个体的头胸甲长3.7 cm,体长13.0 cm,体质量37.0 g.池养雌性斑节对虾的性成熟生物学最小型个体以纳精囊的发育完全(可与雄虾交配)为标志,其最小性成熟个体的头胸甲长4.3 cm、体长15.1 cm、体质量53.0 g,雌性性成熟个体为头胸甲长5.0 cm,体长17.0 cm,体质量75.0 g以上;③池塘养殖斑节对虾性成熟与日龄和养殖环境相关.鱼塭雄虾精荚出现的最早时间为日龄120 d前后,其性成熟日龄约为160 d;池塘养殖雄虾精荚出现的最早时间为日龄150 d前后,其性成熟日龄约为260 d.鱼媪雌虾最早交配发生在日龄165 d前后,性成熟日龄205～236 d,池养雌虾最早交配发生在日龄240～ 280 d,性成熟日龄295～360 d以上.     

野生斑节对虾卵巢发育过程中不同组织的氨基酸组成

测定了海南岛南部海域野生斑节对虾（Penaeus mofbodon）亲虾在卵巢发育不同阶段的全虾、肝胰腺和卵巢的氨基酸组成。结果表明,在卵巢发育初级阶段（含I期和Ⅵ期）、中级阶段（含Ⅱ期、Ⅲ期和Ⅳ期）和高级阶段（V期）斑节对虾全虾样品中,牛磺酸（Tau）质量分数差异显著（P〈0．05）,由高到低为中级阶段〉高级阶段〉初级阶段;而其他的氨基酸质量分数、氨基酸总量和必需氨基酸总量略有变化,但差异不显著（P〉0．05）。肝胰腺和卵巢氨基酸总量随着性腺发育逐渐上升,临产时（V期）下降,但差异不显著（P〉0．05）。中级阶段卵巢中的天门冬氨酸（Asp）、谷氨酸（Glu）、脯氨酸（Pro）、酪氨酸（Tyr）和赖氨酸（Lys）等5种氨基酸质量分数处于峰值。     

亚硝态氮胁迫对斑节对虾(非洲群体)氧化应激、能量代谢和渗透调节的影响

为研究亚硝态氮(NO₂-N)对斑节对虾(非洲群体)氧化应激、能量代谢和渗透平衡的影响,实验选取体长(3.0±0.5) cm的斑节对虾,设置0 (对照组)、5、10和15 mg/L(3个胁迫组)的亚硝态氮浓度梯度,进行了为期72 h的急性胁迫。实验结果显示,肝胰腺氧化应激因子活性(或含量)随着胁迫时间发展而变化。超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)以及一氧化氮合酶(NOS)活性先升高后下降,24 h,SOD和GSH-Px、NOS活性达到最大值,10 mg/L胁迫组的SOD、GSH-Px活性和15 mg/L胁迫组的NOS活性显著高于其他3组;丙二醛(MDA)含量变化相反,24 h胁迫组的MDA含量最小,其中10 mg/L胁迫组显著低于其他3组;48 h,CAT活性达到最大值,10 mg/L胁迫组显著高于其他3组;一氧化氮(NO)含量随着胁迫时间的延长而逐渐升高,72 h,NO含量最高,15 mg/L胁迫组显著高于其他3组。血清能量代谢指标中脂肪酶(LPS)活性先上升后下降,48 h,LPS活性达到最大值,其中10 mg/L...