Spawning behaviour of Penaeus (Fenneropenaeus) merguiensis de Man and the effect of light intensity on spawning

An experiment was conducted to investigate the effect of light intensity just prior to and during spawning of Penaeus (Fenneropenaeus) merguiensis de Man in the laboratory. Neither the presence of light nor light intensity (0, 10 and 1100 lux) had a significant effect on the spawning process or hatching rate in P. merguiensis. Difference in the percentage of spawners, percentage of incomplete spawning, fecundity and hatching rate were all insignificant (P > 0.05) among the three light treatments. However, spawning was delayed (up to 21 min on average) under 1100 lux with a high percentage of incomplete spawning, compared with the Control (0 lux). For the first time, spawning behaviour of P. merguiensis was observed and fully described. The spawning process can be divided into four phases: dormant, pre‐spawning, spawning and post‐spawning by distinct behaviour of prawn broodstock. The dormant phase was characterized by a quiescent and non‐feeding period, whereas movement of prawn broodstock was active during the pre‐spawning and post‐spawning phases. Depending on the individual, spawning was completed within 3–5 min. Prawns often stayed still on the water surface during spawning with occasional manoeuvres to change the position. After a short rest at the termination of the spawning phase, prawn broodstock re‐exhibited active movement as in the pre‐spawning phase, but showing conspicuous care to the deposited eggs. The role of light in controlling ovarian development and spawning, and the behaviour of prawn broodstock in relation to maximizing fertilization rate are discussed.     

Cold tolerance of the banana prawn Penaeus merguiensis de Man and its growth at different temperatures

Critical thermal minima (CTMin) were determined for subadults of Penaeus merguiensis acclimated at 15, 18, 21 and 24 °C for 3 weeks. The effect of cooling rate on CTMin was also investigated. The CTMin of prawns from these respective acclimation groups were 5.3, 6.0, 7.4, 9.0 and 4.7, 5.4, 6.0, 7.3 °C at the cooling rate of 1 °C h^?1 and 3 °C h^?1 respectively. Both acclimation temperature and cooling rate had a significant effect (P < 0.01) on the CTMin. Observation during the acclimation period showed that the growth rate of prawns acclimated at 24 °C was much higher than those acclimated at 21, 18 and 15 °C. The results indicate that P. merguiensis can overwinter in aquaculture ponds in south‐east Queensland, Australia, or other subtropical areas and attain good growth if simple overwintering facilities are available.     

Chilled storage of banana shrimp (Fenneropenaeus merguiensis) spermatophores with supplementation of moringa (Moringa oleifera Lam.) extract

This study was designed to optimize the chilled storage method for banana shrimp (Fenneropenaeus merguiensis) spermatophores and evaluate the potential of moringa (Moringa oleifera Lam.) extract on the reduction in bacterial contaminants during spermatophore preservation due to the uncertainty of the broodstock. Spermatophores were suspended in five extenders: mineral oil, Ringer's solution, phosphate buffer, calcium‐free saline and 0.8% NaCl and stored at 2–4°C. During a 28‐day storage, spermatophores stored in mineral oil showed the highest sperm viability (89.1%) and intact morphology with a slight formation of hardened adhesive matrices. The effect of moringa extract was investigated on chilled spermatophores. Spermatophores were suspended in mineral oil (the control) and mineral oil containing either penicillin–streptomycin (0.1%) or moringa extract (0.1 mg/ml) during a 28‐day storage. Supplementation of moringa extract resulted in a significant increase (p < .05) in sperm survival, compared to the control, and a complete elimination of culturable Vibrio (Vibrio alginolyticus, Vibrio mimicus and Vibrio furnissii), Staphylococcus kloosii, Bacillus macerans, Listeria ivanovii, Corynebacterium paurometabolum and Corynebacterium bovis, in chilled spermatophores. Chilled storage of spermatophores in mineral oil containing moringa extract was a promising technique due to the inhibition of shrimp and human pathogens without the spermicidal effect on banana shrimp sperm.     

中国对虾亲虾反季节培育技术的初步研究

2001年11月18～28日自日照水产研究所等几处中国对虾越冬室筛选性腺有一定发育并已交尾的雌虾36尾，并采用光线控制、水温控制、饵料调节、性腺催熟等方法，促使对虾性腺快速发育。至2002年1月10日从性腺培育成熟的对虾亲虾中选取3尾，使用0．5m。塑料桶进行苗种培育。实验结果表明，中国对虾不经过常规的越冬管理，只要具备对虾亲虾发育的环境条件，在一定时间内性腺即可发育成熟．并能进行正常苗种培育。     

中国对虾卵黄蛋白原合成部位的研究

十足目甲壳类卵巢中卵黄的来源,在过去的几十年研究中一直存在争议,内源性合成和外源性合成都有报道。本文以雌性中国对虾为实验材料,根据组织学观察确定其发育阶段,试验虾可以分为:卵巢未发育期;卵黄发生前期;初级卵黄发生期和次级卵黄发生期4个时期。从处于不同卵巢发育期对虾的卵巢和肝胰腺中提取总RNA,用RT-PCR的方法初步探讨不同发育期的中国对虾卵巢和肝胰腺中卵黄蛋白原mRNA的表达,确定是否存在卵黄蛋白原的合成功能。结果发现,从卵巢未发育期到次级卵黄发生期的卵巢和肝胰腺中都有卵黄蛋白原mRNA的表达,说明二者为卵黄蛋白的合成部位。     

中国明对虾组织蛋白酶L 的原核重组表达及其组织分布

组织蛋白酶L属于半胱氨酸蛋白酶中的木瓜蛋白酶超家族,在哺乳动物中它与抗原提呈、肿瘤入侵和转移、骨质吸收、细胞凋亡等许多生命活动有关。但对其在无脊椎动物中的功能了解的不多。从中国明对虾(Fenneropenaeus chinensis)中克隆得到了组织蛋白酶L基因,为了进一步研究组织蛋白酶L的组织分布及其功能,对该基因进行了重组表达。将中国明对虾组织蛋白酶L基因的酶原片段亚克隆进原核表达载体pET30a中,转化大肠杆菌BL21(DE3),再进行诱导表达,表达产物经SDS-PAGE电泳检测,其分子量在40kD左右,与期望的中国明对虾组织蛋白酶L的分子量一致。表达产物形成包涵体,经过对包涵体变性和复性,并进行His-tag柱亲和纯化,得到了纯化的蛋白。利用重组蛋白制备了的兔抗血清。Western实验表明,组织蛋白酶L在中国明对虾的肝、胃、肠中有表达,而在卵巢中没有表达。     

磺胺甲基异噁唑在中国明对虾体内的残留和消除规律

在(20±2)℃水温条件下,研究磺胺甲基异唑在中国明对虾肌肉、血淋巴和肝胰脏3种组织中的残留及消除规律。药物在对虾肌肉、血淋巴、肝胰脏中的残留用二氯甲烷提取,反相高效液相色谱法检测,最低检测限可达0.01μg/mL,平均回收率为80%~90%。研究结果表明:磺胺甲基异唑在中国明对虾血淋巴中的残留量最小,在肝胰脏中的残留量最大且消除较慢,差异明显。建议把对虾的肝胰脏作为该药残留监控的靶组织,相应的休药期至少为20 d。     

The responsive expression of a caspase gene in Chinese shrimp Fenneropenaeus chinensis against pH stress

Caspases are a family of proteases, which play an important role in apoptosis. To evaluate the relationship between apoptosis and pH stress in crustaceans, a caspase gene (FcCasp) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length of FcCasp was 1329 bp with a 972 bp ORF, encoding a polypeptide of 323 amino acids with a calculated molecular weight and pI of 36.0 kDa and 6.27 respectively. The deduced amino acid sequence of FcCasp contained a potential active site (QACRG pentapeptide) conserved in most caspases and two profile hits (p20 and p10 domain profile). Comparison of amino acid sequences revealed that FcCasp had an overall similarity of 76–83% with other penaeid shrimp caspases. The amino acid sequence of recombinant FcCasp protein expressed in Escherichia coli was identified by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometer analysis. High‐level expression of FcCasp in six different tissues was detected by real‐time polymerase chain reaction after exposure to pH stress for 96 and 148 h. TUNEL analysis indicated that apoptosis began to appear in F. chinensis hepatopancreas exposed to extreme pH for 12 h. The amount of apoptosis seems positively correlated with the length of exposure to the pH stressor. The results suggested that FcCasp was involved in the response to environmental pH stress.     

塔玛亚历山大藻对中国明对虾鳃组织的氧化胁迫和对Caspase基因(FcCasp)表达的影响

选择在中国分离得到的一株能产生麻痹性贝毒(Paralytic Shellfish Poison, PSP)的赤潮甲藻塔玛亚历山大藻(Alexandrium tamarense) ATHK株, 研究其对中国明对虾(Fenneropenaeus chinensis)鳃组织氧化胁迫和Caspase基因(FcCasp)表达的影响。将中国明对虾分别暴露于200 cells/mL 和1 000 cells/mL塔玛亚历山大藻中, 于胁迫后3、6、12、24、48、72和96 h测定鳃的超氧化物歧化酶(SOD)活性、谷胱甘肽硫转移酶(GST)活性、丙二醛(MDA)含量和FcCasp基因相对表达量, 以不加藻的过滤海水作为对照。结果表明, 在200 cells/mL塔玛亚历山大藻胁迫下, SOD活性、GST活性、MDA含量和FcCasp相对表达量均随取样时间推移表现出先上升后下降的趋势。而在1 000 cells/mL塔玛亚历山大藻胁迫下, SOD活性随时间增加呈现先升高后降低的趋势, 在24~96 h 被显著抑制(P<0.05), GST活性除3和48 h外均被显著抑制(P<0.05), MDA含量和FcCasp相对表达量均整体表现上升的趋势, 呈现出一定的时间效应。相关性分析显示, 塔玛亚历山大藻胁迫下, 中国明对虾鳃的MDA含量和FcCasp相对表达量呈正相关(P<0.05)。实验结果表明, 塔玛亚历山大藻可破坏中国明对虾氧化−抗氧化系统的平衡, 引发对虾鳃组织的脂质过氧化(LPO), 造成其氧化损伤, 从而导致FcCasp表达的上调。本实验结果为将SOD活性、GST活性和MDA含量作为生物标志物, 用于评价塔玛亚历山大藻胁迫提供了依据, 同时也为研究塔玛亚历山大藻对中国明对虾的危害机制提供了参考。

     

中国明对虾serpin型丝氨酸蛋白酶抑制剂基因的重组表达及活性分析

Serpin家族成员作为主要的丝氨酸蛋白酶抑制剂,通过调节一系列丝氨酸蛋白酶和半胱氨酸蛋白酶的活性来调节蛋白酶级联反应,进而参与了包括血液凝结、补体激活、纤维蛋白溶解、炎症反应、肿瘤抑制和激素转运等大量的基本生物过程,在调节机体免疫及其它重要生理功能方面发挥着重要作用。我们在前期研究中,从中国明对虾血细胞中克隆得到一个serpin型丝氨酸蛋白酶抑制剂基因（Fc-serpin, GenBank注册号：DQ318857）,初步研究显示它在转录水平参与了病原菌和白斑杆状病毒（WSSV）引发的对虾先天免疫应答反应;本研究利用原核重组表达系统,成功获得了重组的对虾serpin型蛋白酶抑制剂（rFc-serpin）,纯化复性后得率为0.3g/L;活性分析显示,重组目的蛋白（rFc-serpin）对多种病原菌的生长均有一定抑制作用,研究结果进一步证实它参与病原微生物引发的对虾防御应答过程,为探讨甲壳动物先天免疫防御应答机制提供了有价值的参考。     

中国明对虾MKK3 基因cDNA 克隆及其在氨氮胁迫下的表达

采用RACE技术克隆获得中国明对虾(Fenneropenaeus chinensis)丝裂原活化蛋白激酶激酶3(MKK3)基因全长c DNA序列,并对该序列进行分析。结果表明,该基因全长为1434 bp,开放阅读框长1011 bp,5′非编码区长33 bp,3′非编码区长390 bp,将该基因命名为Fc MKK3。推测该基因编码336个氨基酸,分子量为37.89 k D,理论等电点为6.08。同源性和系统进化分析表明,Fc MKK3基因与丽蝇蛹集金小蜂(Nasonia vitripennis)和地中海实蝇(Ceratitis capitata)的相似性分别为69%和68%,与其他节肢动物MKK3聚为一类。荧光定量RT-PCR结果表明,Fc MKK3基因在肌肉中的相对表达量最高,其次为鳃。氨氮胁迫后该基因在中国明对虾肠、鳃、胃、心脏、肝胰腺、肌肉和血细胞中的表达量均显著增加,并有不同的时空表达趋势,表明Fc MKK3基因可能在中国明对虾应对非生物胁迫反应的过程中起着重要的作用。     

不同饵料搭配对中国明对虾生长和饵料转化效率的影响

采用沙丁鱼肌肉(FF)、鹰爪虾肌肉(SF)、菲律宾蛤仔足肌(CF)、沙蚕(PW)和配合饲料(FD)5种饵料,设计了CF PW、CF FD、PW FD、CF FD PW、FF FD PW FD、FF SF CF PW FD共6种饵料搭配投喂模式,研究了不同饵料搭配对中国明对虾的生长和饵料转化效率的影响。中国明对虾摄食混合饵料后的生长都比除PW处理外的4种单种饵料投喂处理快,CF PW处理的对虾在实验结束时的体重、增重率最大。混合投喂处理的饵料转化效率均高于除PW外的所有单种饵料投喂处理,所有混合投喂处理的实际饵料转化效率都比预测值高,并且除了PW FD和CF FD PW两个处理的实际值与预测值差异不显著外,其它混合处理的饵料转化效率实际值都显著高于预测值。文中还对不同饵料搭配的优势对对虾饵料选择性的影响及产生这些优势的原因进行了探讨。     

Comparison of white spot syndrome virus quantification of fleshy shrimp Fenneropenaeus chinensis in outdoor ponds between different growing seasons by TaqMan real‐time polymerase chain reaction

In the present study, we used TaqMan real‐time polymerase chain reaction to quantify and compare infection of white spot syndrome virus (WSSV) with shrimp production of Fenneropenaeus chinensis cultured in outdoor ponds along the west coast of the South Korea. In 2007, a total of 60 specimens in summer and 116 specimens in autumn were collected from 12 growing‐out ponds and 12 harvest ponds respectively. Pond harvest data were obtained from farmers. Of the summer samples, all specimens were WSSV positive, with a wide range of 12.4–7.0 × 10⁷ (mean 7.5 × 10⁶) copies ng^?1 DNA; shrimp production was 1.7 metric tonnes per hectare (mt ha^?1). Of the 116 autumn‐sample specimens, 81 (69.8%) were WSSV positive; WSSV infection had been decreased dramatically, to 0–7.2 (mean 3.5) copies ng^?1 DNA. Shrimp production of autumn ponds was 2.1 mt ha^?1. Statistical analysis indicated that the difference in WSSV infections detected in summer and autumn was highly significant (P<0.01). In summer, seven ponds (58.3%) with low‐WSSV infection loads (0–1000 WSSV copies ng^?1 DNA) had shrimp production of 2.7 mt ha^?1; the others had shrimp production of only 0.2 mt ha^?1. The mean shrimp production between the two infection levels showed a highly statistically significant difference (P<0.01).     

中国明对虾抗菌肽基因应答WSSV侵染的表达及其SNP分析

通过白斑综合征病毒(WSSV)感染实验,利用实时定量PCR技术研究了中国明对虾(Fenneropenaeus chinensis)应答病毒侵染后,已知的3种抗菌肽(对虾肽)在肝胰腺、肌肉、肠和鳃4种组织中的差异表达情况.结果显示,虽然3种抗菌肽表现出明显的组织表达特异性,即在不同组织中的表达趋势和表达丰富度存在明显的差异,但是就同一个组织而言,3种抗菌肽在1～120 h WSSV侵染区间内的表达趋势基本一致,在0 h(未侵染病毒)时,3种抗菌肽的表达量极低(为0);在6～24 h期间,检测到明显的表达量;48～120 h期间,3种抗菌肽的表达量总体呈现下降的趋势.这暗示3种抗菌肽在对虾机体内可能具有相似的生物学功能.在此基础上,本研究对各类型中国明对虾抗菌肽的SNP位点进行了筛选,进一步对不同SNP类型与抗WSSV或易感WSSV的关联程度进行了分析,结果显示3种抗菌肽基因的SNP位点很少,且在抗性和易感对虾群体内不存在明显的偏向分布.     

Genetic parameters of survival in four generations of Indian white shrimp (Fenneropeneaus indicus)

This study evaluated the genetic variance for survival at harvest of Indian white shrimp (Fenneropenaeus indicus) using a pedigreed synthetic population generated upon collecting broodstock from different fishery grounds in Egypt. The estimated heritabilities (h² ± SE) of survival from generations G₀ to G₃ ranged from 0.144 ± 0.026 to 0.008 ± 0.030 using a model that included the common environmental effect (c²). Low heritability (0.014 ± 0.015) was estimated across generations with the inclusion of common environmental effect (c²). However, the h² of survival from G₀ to G₁ ranged from 0.569 ± 0.071 to 0.410 ± 0.011 when c² was not included. The genetic correlation for survival between different ponds from G₀ to G₃ was moderate to high (0.79 ± 0.042 to 0.83 ± 0.012) but significantly different (P < 0.05). There were low correlation coefficients (?0.237 ± 0.089 to 0.316 ± 0.073) between body weight estimated breeding value (EBV) and survival EBV of families in four generations.     

中国明对虾天门冬氨酸转氨酶基因的克隆及氨氮胁迫对其时空表达的影响

利用RACE技术克隆获得中国明对虾(Fenneropenaeus chinensis)天门冬氨酸转氨酶GOT基因(FcGOT)。FcGOT 基因cDNA全长为1 910 bp, 其中, 开放阅读框1 284 bp, 编码427个氨基酸。同源性分析表明, 中国明对虾天门冬氨酸转氨酶GOT氨基酸序列与其他节肢动物高度保守, 与克氏原螯虾(Procambarus clarkii) 和桔粉蚧壳虫 (Planococcus citri) 的同源性分别为78%和73%。系统进化分析表明, FcGOT基因氨基酸序列与克氏原螯虾GOT聚为一支。组织表达分析发现FcGOT基因在肝胰腺、鳃、血细胞、肌肉、心脏、淋巴中均有表达, 其中肝胰腺中表达量最高。氨氮胁迫后, 荧光定量PCR分析结果表明, FcGOT基因在肝胰腺和鳃组织中的表达与对照组相比具有显著差异(P<0.05), 表明FcGOT基因在氨氮代谢方面具有重要的作用, 参与了中国明对虾机体的急性氨氮胁迫应答反应。

     

镉胁迫对凡纳滨对虾血细胞基因表达的影响

为探讨镉(Cd)胁迫下凡纳滨对虾(Litopenaeus vannamei)血细胞的分子响应,以不同浓度的Cd2+(0,0.5 mg/L和5 mg/L)进行胁迫,于不同胁迫时间取血淋巴,测定血细胞中Trx 2、Grx 2、Grx 3和MGST 3的基因表达量变化.结果显示,Trx 2、Grx 3和MGST 3表达量在胁...     

中国明对虾C 型凝集素基因(Fclectin)的重组表达及活性分析

研究拟通过分析对虾C型凝集素的活性特点,探讨其在对虾先天免疫应答过程中的潜在功能以及在养殖生产实践中的应用。实验利用原核表达系统对中国明对虾C-型凝集素基因的两个串联的糖识别结构域(carbohydrate recognition domain,CRD)进行了重组表达,并通过纯化复性获得了重组目的蛋白(rFclectin-CRD1和rFclectin-CRD2)。活性分析结果显示,重组目的蛋白对多种病原菌有凝集和抑制生长的作用,并且具有Ca2+依赖活性;其凝集活性可被半乳糖、肽聚糖、脂多糖等多种病原相关分子模式所抑制,研究结果证实,Fclectin是一种典型的C-型凝集素,它可能作为中国明对虾先天免疫中重要的模式识别受体,在一定程度上参与了机体应答病原微生物的防御过程。     

Green fluorescent protein visualization of Vibrio parahaemolyticus infections in Indian white shrimp Fenneropenaeus indicus (H Milne Edwards)

The pathways by which pathogens invade Fenneropenaeus indicus, the potential colonization in various tissues and the disease transmission mechanisms are unclear. The aims of the present study were to visualize the colonization and pathogenesis of GFP‐tagged Vibrio parahaemolyticus, in various tissues of F. indicus to evaluate the pathogen interaction. Among the three strains isolated, a virulent strain VpDAHV2 was tagged with green fluorescent protein (GFP‐VpDAHV2) and validated for both its growth characteristics and its virulence as a genuine model for F. indicus infection. VpDAHV2 was positive for toxR and tlh genes and negative for tdh genes. CLSM images revealed that maximum colonization was observed in the haemolymph of the F. indicus challenged with GFP‐VpDAHV2. The haemolymph was the primary site for the colonization of GFP‐VpDAHV2 in F. indicus. The enteric localization occurred independently of the flagellum or motility of GFP‐VpDAHV2 through the intestinal route. The F. indicus infection model suggests that the haemolymph and the intestine represent the sites of infection by GFP‐VpDAHV2, and hence are the active sites of pathogen interactions. GFP tagging of V. parahaemolyticus is a new and systemic approach to determine the presence of bacteria in vivo for the confirmation of host pathogen interactions in aquaculture studies.     

中国明对虾放流对鱼类生态位的影响

为探究中国明对虾(Fenneropenaeus chinensis)增殖放流对水域内鱼类的影响及其表现形式,采用原位实验生态学方法,在对虾放流河道构建围隔,设置3种虾苗放流量(模拟莱州湾近岸放流5亿尾、7.5亿尾和10亿尾)和无放流处理,经过一个对虾生长季后,比较不同处理围隔中鱼类组成和生态位特征差异。结果显示,4种处理围隔中,回捕对虾数量随放流量增加而增加,但其生物量、平均个体大小和回捕率未随放流量增加而增加;回捕率在放流围隔间差异显著,以5亿尾模拟围隔的值最高;鱼的种类组成相似,可归于杂食性、浮游动物食性和鱼/虾食性。总鱼类和鱼/虾食性的鱼类产出量在围隔间差异不显著,杂食性鱼的产出量以放流围隔较高,浮游动物食性鱼的产出量随对虾放流量增加而减少。鱼种间高生态位重叠(>0.75)配对数随对虾生物量增多而减少。相似性分析显示,放流最多的和无放流围隔的鱼类生态位相异程度最高。综上认为,在实验水体中,放流对虾并不降低鱼类总量产出,但可能会影响不同食性鱼类的相对组成和生态位重叠度,且影响效应随放流量增加更为明显。