广东省阳江地区野生拟穴青蟹2种病毒感染情况调查

青蟹双顺反子病毒-1（mudcrabdicistrovirus-1,MCDV-1）和青蟹呼肠孤病毒（mudcrabreovirus,MCRV）是对养殖拟穴青蟹有较强致病性的2种病原,可导致高死亡率。文章采用双重巢式PCR检测法对2012年阳江地区5月-10月捕捞的野生拟穴青蟹（Scyllaparamamosain）病毒感染情况进行调查。结果显示,阳江地区2012年5月～10月的野生青蟹群体一直保持较高的病毒携带率（44．83％-100％）,其中MCDV-1检出率最高的月份为6月、8月和9月,MCRV检出率最高的月份为5月-6月和8月～9月;以单独感染类型为主的月份为5月、7月和10月（〉44．83％）,以双阳性感染类型为主的月份为6月、8月和9月（〉82．76％）。由此可见,2种病毒的流行月份为5月～6月和8月～9月;在流行月份期间2种病毒常常协同感染野生拟穴青蟹。     

盐度对中华绒螯蟹交配及产卵前后血淋巴中5种激素的影响研究

为探究盐度在中华绒螯蟹(Eriocheir sinensis)交配和产卵前后对血淋巴中5种激素的影响,本研究分别设置了0、2、4、6的交配盐度和3、6、9、12、15、18、21的产卵盐度,分析了不同交配和产卵盐度条件下,中华绒螯蟹血淋巴中双羟基孕酮(DHP)、促性腺激素(GTH)、前列腺素(PG)、雌二醇(E2)和睾酮的含量变化。结果显示,在淡水中,雌蟹无交配行为发生,当盐度<6时,雌蟹只交配不产卵。雌蟹在盐度为2～6中交配后,血淋巴中GTH、PG、E₂和睾酮与交配前相比均无明显变化,且交配后各盐度组相比无显著差异(P>0.05)。随着盐度的升高,雌蟹产卵前,血淋巴中DHP、PG、E₂和睾酮的含量均呈先下降再上升的趋势;产卵后血淋巴中DHP、PG、E₂则呈先上升后下降的趋势,其中,当盐度为18时,雌蟹产卵后,血淋巴中DHP、GTH、PG、E₂和睾酮含量均降到最低,且与产卵前相比均有显著差异(P<0.05)。当盐度为6时,雌蟹产卵前血淋巴中DHP、PG、E₂和...     

拟穴青蟹一种含CTLD结构域基因的克隆及表达分析

从拟穴青蟹(Scylla Paramamosain)中克隆得到一个含有CTLD(C-type lectin-like domain)结构域的C型凝集素基因,经分析未发现同源基因,将其命名为SpCTLD。该基因cDNA序列全长为1 237 bp,包含一个468 bp的开放阅读框,编码155个氨基酸,预测分子量为18.32 kDa,等电点为7.56。经一代测序,该基因在第390位(A390G)和第1 020位(T1020C)含有两个SNP位点。对SpCTLD基因在拟穴青蟹幼体不同时期和组织中的表达模式分析发现,SpCTLD在幼体的各个发育时期均有表达,其中在溞状幼体Ⅴ期表达量最高,仔蟹Ⅰ期次之,仔蟹Ⅱ期最低。对SpCTLD的组织分布分析表明,SpCTLD在鳃中表达量最高,其次为射精管,在血淋巴、肝胰腺等其他免疫组织中也有表达,其中在鳃和射精管的表达量远高于在其他组织中的表达量。副溶血弧菌胁迫实验表明,活菌组的肝胰腺、血淋巴、鳃中的SpCTLD表达量发生显著上调,且都在12 h达到最高。根据研究结果,推测SpCTLD是一种含CTLD结构域的新基因,它可能参与了拟穴青蟹抗细菌反应。该基因的发现丰富了拟穴青蟹C型凝集素相关基因的研究,有利于丰富甲壳动物先天免疫机制的研究。     

青蟹呼肠孤病毒对拟穴青蟹的致病性研究

2008-2012年广西海洋研究所海水增养殖基地暂养的拟穴青蟹膏蟹出现“嗜睡”等典型的青蟹呼肠孤病毒病症状.采用套式PCR检测濒死膏蟹感染青蟹呼肠孤病毒阳性率为100％,经Blastn比对扩增序列与青蟹呼肠孤病毒基因(HQ414137.1)相似度达98％,属于青蟹呼肠孤病毒(JX213634).病毒粗提液人工回感后发现,攻毒1h,青蟹血淋巴中即可检测到青蟹呼肠孤病毒;攻毒后第4～6 d,出现死亡高峰,第10 d,累积死亡率达到100％.血清酶指标中碱性磷酸酶、谷丙转氨酶和谷草转氨酶活力明显高于对照组,而与免疫相关的过氧化物酶、超氧化物歧化酶活力低于对照组;肝胰腺、鳃和肌肉组织切片均呈现显著病理坏死损伤.青蟹呼肠孤病毒是此次拟穴青蟹膏蟹死亡的主要原因.     

白斑综合征病毒(WSSV)在拟穴青蟹体内增殖的研究

拟穴青蟹(Scylla paramamosain)因肉质鲜美、生长快速而成为我国主要的海水养殖经济蟹类,但是随着养殖规模的不断扩大,病害问题也越发严重。本研究通过流行病学调查和定量PCR跟踪检测的方法,研究了拟穴青蟹对白斑综合征病毒(WSSV)的易感性和WSSV在拟穴青蟹体内的增殖情况。结果表明,拟穴青蟹是WSSV的自然宿主,自然携带率为8.47%。WSSV可通过口服途径感染拟穴青蟹,并在拟穴青蟹体内快速增殖,注射感染5 d后,病毒量达到感染1d时的1.1×109倍,当病毒累积到一定量后即可致死拟穴青蟹。研究表明,拟穴青蟹是WSSV的天然宿主,在虾蟹混养过程中可以通过摄食WSSV感染虾而带毒或发病,并因此成为WSSV传播媒介,从而对虾蟹混养条件下WSSV的防治效果产生重要影响。     

盐度对中华绒螯蟹性早熟生理机制的影响

于2004年6月至11月在崇明岛中华绒螯蟹养殖基地进行1秋龄中华绒螯蟹(Eriocheir sinensis)的养殖试验,通过荧光免疫二步抗体检测法和原子吸收等方法,测定了1秋龄蟹血淋巴钙离子和雌二醇水平,研究了不同盐度水体中1秋龄中华绒螯蟹性早熟情况及盐度促进中华绒螯蟹性早熟的生理机制。结果表明:(1)随着水体盐度的升高,1秋龄中华绒螯蟹性早熟率上升,成活率则呈下降趋势;(2)水体盐度升高导致1秋龄未发育雌蟹血淋巴中Ca2 含量上升(P<0.05),但对1秋龄雄蟹作用不显著(P>0.05);(3)池塘养殖90 d之后,高盐度水体中1秋龄未发育雌蟹血淋巴中雌二醇水平显著高于低盐度水体1秋龄未成熟雌蟹(P<0.05);(4)1秋龄性早熟雌蟹血淋巴中雌二醇含量高达(1294.00±595.87)pg/mL,极显著地高于同期的1秋龄未成熟雌蟹(P<0.01)。研究表明:水体盐度升高引起的1龄雌蟹血淋巴中雌二醇水平和Ca2 含量上升是导致1秋龄雌性中华绒螯蟹性早熟的内在生理原因。     

2种饵料模式对稻田养殖中华绒螯蟹幼蟹养殖性能和生理代谢的影响

为优化稻田养殖中华绒螯蟹(Eriocheir sinensis)幼蟹的饵料模式,本研究分别采用配合饲料和传统饵料(豆粕、麸皮、玉米和小麦)投喂稻田养殖中华绒螯蟹幼蟹152 d,每种投喂组各3个重复,比较2种饵料模式对幼蟹养殖性能、蛋白质代谢、消化酶、抗氧化和免疫指标的影响。结果显示,配合饲料组的雌蟹和雄蟹体质量均高于传统饵料组,但差异不显著(P>0.05);就养殖性能而言,2种饵料模式下,配合饲料组正常幼蟹的平均体质量及产量、1龄早熟蟹平均体质量及其产量、总产量和成活率均略高于传统饵料组,饵料系数显著低于传统饵料组(P<0.05);就蛋白质代谢而言,配合饲料组雌蟹和雄蟹血淋巴中的总蛋白含量显著高于传统饵料组(P<0.05),而传统饵料组雄蟹血淋巴中的尿素氮含量和肝胰腺中的谷草转氨酶活力显著高于配合饲料组(P<0.05);配合饲料组雌蟹和雄蟹肝胰腺中的脂肪酶均显著高于传统饵料组(P<0.05),而雄蟹的α-淀粉酶活力显著低于传统饵料组;配合饲料组的雌蟹血淋巴中总抗氧化能力、碱性磷酸酶、酸性磷酸酶活力和肝胰腺中酸性磷酸酶活力均显著高于传统饵料组(P<0.05)。综上所述,投喂配合饲料可以提高稻田扣蟹的养殖性能、促进蛋白质沉积和脂类吸收、增强幼蟹抗氧化和免疫能力、提高蟹种质量。     

应用PCR方法检测患“黄水病”锯缘青蟹中的血卵涡鞭虫

"黄水病"是目前锯缘青蟹养殖过程中的主要疾病之一,病蟹主要症状表现为肌肉白浊,体液呈土黄色或浊白色牛奶状。因该病流行范围广、发病率和死亡率高,给青蟹养殖业主造成巨大经济损失,严重地影响了青蟹养殖的健康发展。本研究从病蟹体液中发现大量疑似血卵涡鞭虫的寄生原虫,应用已建立的梭子蟹血卵涡鞭虫病的PCR检测方法对患"黄水病"青蟹进行检测。结果从患病青蟹组织的DNA中扩增出产物大小为585bp的特异性目的片段,经序列分析比较,与三疣梭子蟹上发现的血卵涡鞭虫的序列同源,同源性达99.7%。综合病原流行病学调查、组织病理学、电镜观察等分析结果,初步确定血卵涡鞭虫是引起养殖青蟹"黄水病"的重要病原。     

三疣梭子蟹14-3-3基因的克隆及其在低盐和病原胁迫后的表达分析

本研究利用cDNA末端快速扩增技术(RACE)克隆了三疣梭子蟹(Portunus trituberculatus)14-3-3基因(Pt14-3-3)。Pt14-3-3基因cDNA序列全长为2510 bp,包含开放阅读框741 bp,编码由247个氨基酸组成的蛋白,分子量为27.98 kDa。序列比对结果显示,Pt14-3-3基因与中华绒螯蟹14-3-3基因同源性最高(100%)。系统进化树分析表明,Pt14-3-3氨基酸序列与中华绒螯蟹(Eriocheir sinensis)紧密聚为一支。组织表达分析显示,Pt14-3-3基因在肝胰腺、肌肉、鳃、心脏、眼柄、血淋巴中均有表达,其中,在肝胰腺中的表达量最高。低盐胁迫后,Pt14-3-3基因在鳃和肝胰腺中的表达量分别于48 h和12 h显著上调并达到最大值,分别为对照组的1.34倍(P<0.05)和7.54倍(P<0.05)。人工感染副溶血弧菌(Vibrio parahaemolyticus)和白斑综合征病毒(WSSV)后,Pt14-3-3基因在肝胰腺和血细胞中整体均呈显著上调表达,最高上调17.52倍(P<0.05)。本研究结果表明,Pt14-3-3基因可能在三疣梭子蟹低盐适应和免疫反应中发挥重要作用。     

中华绒螯蟹免疫因子-溶菌酶的初步研究

研究了在自然状态下中华绒螯蟹成蟹血淋巴和大眼幼体组织匀浆上清液中溶菌酶的活性。结果表明,在中华绒螯蟹成蟹血淋巴中均检测出溶菌酶的活性,为(0.0286±0.0081)活力单位(n=30),而在中华绒螯蟹大眼幼体组织匀浆上清液中亦检测到溶菌酶的活性,为(0.0318±0.0073)活力单位(n=4)。     

Innate immune response and gene expression of Scylla paramamosain under Vibrio parahaemolyticus infection

An epidemic of ‘milky disease’ in the mud crab (Scylla paramamosain) generally breaks out in the fall when the crab is near maturity, resulting in large economic losses in crab farming. Vibrio parahaemolyticus has been proven to be one of the major pathogens. In this study, the mud crabs were challenged with V. parahaemolyticus, and their innate immune responses were investigated in terms of total haemocyte counts (THCs), haemocytic enzyme activities and gene expression levels during a 114‐h period. The THCs of the mud crabs decreased significantly after 42 h of exposure. The activities of the haemocytic enzymes, including acid phosphatase‐alkaline phosphatase, phenoloxidase, superoxide dismutase (SOD), peroxidase and nitric oxidase synthethase, were significantly enhanced during the challenge course. The gene expression levels also significantly increased for all tested genes (proPO, Cu/Zn‐SOD, Prx, LYS, CRU and ALF) with the exception of CAT down‐regulated expression. The results may imply that the immune responses of the mud crab could be activated by the pathogens, and the data here will provide many clues for further systematic investigation of ‘milky disease’ caused by V. parahaemolyticus and the disease prevention in mud crab S. paramamosain.     

拟穴青蟹不同发育时期胚胎基因表达的内参基因的筛选

为获得适用于研究拟穴青蟹(Scylla paramamosain)不同发育时期胚胎基因表达的内参基因,本研究采用3个内参基因筛选软件geNorm、NormFinder及BestKeeper对微管蛋白α1A(Tuba1a)、核糖体蛋白L13(Rpl13)、泛素(UBQ)、核糖体蛋白S6激酶(Rps6)、精氨酸激酶(Ak)、3-磷酸甘油脱氢酶(gapdh)、28S核糖体RNA(28S)、18S核糖体RNA(18S)和延伸因子1A基因(EF-1α)等9个常用候选内参基因在拟穴青蟹不同发育时期胚胎的表达稳定性进行分析。geNorm分析表明, EF-1α和Rpl13的表达稳定性最高,并且采用2个内参基因同时校正目标基因的定量结果可以达到最优效果; NormFinder分析表明, gapdh和EF-1α是9个候选内参基因中表达最稳定的; BestKeeper分析表明,gapdh的标准偏差和变异系数是9个候选基因中最低的。综合3个内参基因筛选软件,可以选用EF-1α,或选用EF-1α和gapdh双内参同时校正拟穴青蟹不同胚胎发育时期各基因的表达情况。     

A reovirus disease in cultured mud crab, Scylla serrata, in southern China

A reovirus, designated mud crab reovirus (MCRV), associated with large economic losses was recently isolated from marine cultured mud crab, Scylla serrata, in southern China. The complete viral particle is 70 nm in diameter, icosahedral and non-enveloped. The virus infects connective tissue cells of the hepatopancreas, gills and intestine in mud crab and develops in the cytoplasm. Hundred per cent mortality was observed in mud crab experimentally infected by intramuscular injection, bath inoculation and oral inoculation, while cohabitation infection caused 80% mortality. The viral genome consists of 13 linear dsRNA segments, with an electrophoretic pattern 1/5/7. The results of this study suggest that the virus is highly pathogenic and can be transmitted enterically as well as via the body surface of mud crab. Although the genomic organization of this virus is different from that of the other crab reoviruses, CcRV-W2 and DpPV, all three of these reoviruses have similar electrophoresis patterns. Therefore, MCRV may be a new member of the DpPV and CcRV-W2 group.     

Status of Biological Studies and Aquaculture Development of the Mud Crab, <Emphasis Type="Italic">Scylla serrata</Emphasis>, in China: An Experimental Ecological Studies

This is an initial paper in a series of overviews of biological research and aquaculture development of the mud crab, Scylla serrata, in China. Results of experimental ecological studies on mud crabs are reported here. As a result of these experimental studies, results that are important for mud crab culture were also discovered and these include, methods to condition and manage broodstock, determination of ecological conditions that are suitable for embryonic development, and the influence of temperature, salinity, diet and larval density on development and survival of larvae. Results of this work will be useful in establishing a good method for artificial mass culture of larvae.     

Studies on pathogenicity and prevalence of white spot syndrome virus in mud crab, Scylla serrata (Forskal), in Zhejiang Province, China

Mud crab, Scylla serrata (Forskal), is the most commercially important marine crab species in China. In recent years, serious diseases have occurred in major mud crab culture regions in SE China. PCR detection of white spot syndrome virus (WSSV) in diseased mud crabs collected from Zhejiang Province during 2006–2008 showed a prevalence of 34.82%. To study the pathogenicity of WSSV to mud crab, healthy mud crabs were injected intramuscularly with serial 10‐fold dilutions of a WSSV inoculum. The cumulative mortalities in groups challenged with 10^?1, 10^?2, 10^?3 and 10^?4 dilutions were 100%, 100%, 66.7% and 38.9% at 10 days post‐injection, respectively. All moribund and dead mud crabs except the control group were positive for WSSV by PCR. Based on the viral load of the WSSV inoculum by quantitative real‐time PCR, the median lethal dose (LD50) of WSSV in S. serrata was calculated as 1.10 × 10⁶ virus copies/crab, or 7.34 × 10³ virus copies g^?1 crab weight. The phenoloxidase, peroxidase and superoxide dismutase activities in haemolymph of WSSV‐infected moribund crabs, were significantly lower than the control group, whereas alkaline phosphatase, glutamate‐pyruvate transaminase and glutamic‐oxaloacetic transaminase were higher than in the control group. WSSV was mainly distributed in gills, subcuticular epithelia, heart, intestine and stomach as shown by immunohistochemical analysis with Mabs against WSSV. The epithelial cells of infected gill showed hypertrophied nuclei with basophilic inclusions. Numerous bacilliform virus particles were observed in nuclei of infected gill cells by transmission electron microscopy. It is concluded that WSSV is a major pathogen of mud crab with high pathogenicity.     

Bacterial septicaemia in prerecruit edible crabs,Cancer pagurus L.

Juvenile edible crabs, Cancer pagurus L., were surveyed from Mumbles Head and Oxwich Bay in South Wales, UK, and the number of heterotrophic bacteria and vibrios in the hemolymph was determined. The percentage of crabs with hemolymph containing bacteria was variable over the survey with higher numbers of animals affected in summer than in winter. Post‐moult crabs contained significantly higher numbers of heterotrophic bacteria in the hemolymph than pre‐ and intermoult animals. Crabs with cuticular damage to the gills also had significantly higher numbers of bacteria in the hemolymph. Crabs were found to have a high prevalence of infection by the dinoflagellate, Hematodinium. Such animals had significantly fewer bacteria in the blood in comparison with Hematodinium‐free animals. Of the 463 crabs surveyed, only 3 individuals had hemolymph containing 2000 + CFU mL^?1. Based on 16S rRNA gene sequences, two of these crabs contained a Vibrio pectenicida‐like isolate, while the other had a mixed assemblage of vibrios. Although 59% of the crabs surveyed had culturable bacteria in the hemolymph, the majority only had small numbers (<2000 CFU mL^?1), suggesting that such infections may be of limited importance to the sustainability of the crab fishery in this region.     

锯缘青蟹血清凝集素的特性和盐析提取研究

对锯缘青蟹(Scylla serrate)血清凝集素的凝集特性进行了研究。结果显示:锯缘青蟹血清凝集素对人、兔、鼠、鸽子、中华鳖、鲫红细胞均有凝集作用,其中对小鼠红细胞凝集活性最强,血凝活性可达29;凝集素活性最适pH 6.0~7.4;盐度对凝集活力有较大影响,NaC l浓度在0.4~0.5 mol/L时基本失活;未纯化血清凝集素对Ca2+、Mg2+未表现出依赖性;凝集素活性能被N-乙酰葡萄糖胺特异性抑制。对青蟹血清凝集素进行硫酸铵分级沉淀分离后,发现凝集素活性主要分布在25%饱和度硫酸铵沉淀区,该组分凝集比活比血清高27.44倍,SDS-PAGE结果表明主要蛋白带型为79 ku、75 ku、72 ku。     

基于计算机视觉和非参数估计的蟹苗数量估算方法研究

养殖池内蟹苗的数量估计在蟹苗养殖中有着重要意义。但现有的数量估计方法操作复杂且实用性不强,因此提出一种基于计算机视觉和非参数估计的蟹苗数量估算方法。首先在养殖池内分水层采集视频,通过背景建模得到前景图像,并使用分水岭算法和轮廓提取得到视频中的蟹苗数量,并作为样本数据,然后通过核密度估计得出概率密度函数,最后结合该函数和样本数据估算出池内蟹苗数量。结果表明,该方法对于容积约为1 000 L、蟹苗密度100~160只/L的小型蟹苗养殖池,估算蟹苗数量的平均正确率为82.14%。研究表明,采用该方法不仅可以解决采集视频过程的操作繁琐、幼苗转移的问题,而且能够避免图像处理过程中部分背景杂质的干扰。该方法还可以推广到虾苗和鱼苗等生物的幼苗估计,具有良好的通用性和可行性。