拟穴青蟹STEAP4-like基因的克隆与表达分析

为了解拟穴青蟹(Scylla paramamosain)前列腺6跨膜上皮抗原4基因(six-transmembrane protein of prostate 4,STEAP4)在拟穴青蟹抗白斑综合征病毒(white spot syndrome virus, WSSV)免疫应答中的作用，表达与纯化GST-STEAP4重组蛋白，(1)通过聚合酶链式反应(PCR)克隆了STEAP4基因，利用生物信息学分析软件分析其序列特征；(2)通过实时荧光定量PCR(qRT-PCR)技术检测其组织分布及WSSV感染后的表达情况；(3)构建原核表达载体pGEX-6p-1-STEAP4-like,诱导表达与纯化重组蛋白GST-STEAP4-like。结果表明：(1)拟穴青蟹STEAP4-like基因ORF长1 443 bp,编码480个氨基酸，N端含有NAD结合位点和一个NADP氧化还原酶辅酶结构域，C端含有一个铁还原酶跨膜结构域；(2)氨基酸序列与三疣梭子蟹STEAP4-like相似性最高，与三疣梭子蟹和突眼蟹亲缘关系最接近；(3)STEAP4-like基因在被检测组织中均有表达；(4)WSSV刺激后ST...     

提高青蟹产量和商品档次的养殖技术试验

青蟹(Scylla serwata Forska)福建沿海俗称“蟳”。性成熟的雌性青蟹,因甲壳两侧充满“红膏”,俗称“红(膏)蟳”,它是海水养殖中经济价值较高,可出口创汇的高档水产品之一。近几年来,随着水产养殖结构的调整,虾蟹混养、鱼蟹混养及鱼虾蟹混养等多种养殖类型应运而生。笔者经过多年的养殖生产实践,感到只要在充分掌握青蟹生活习性的基础上,为其创造良好的生存环境,加上科学的饲养管理和积极的防病措施,就可提高养殖青蟹的产量和商品     

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.     

配合饲料在拟穴青蟹-脊尾白虾混养模式中的应用效果初探

为研究配合饲料在拟穴青蟹(Scylla paramamosain)-脊尾白虾(Exopalaemon carinicauda)混养模式中的应用效果,选取面积为5亩(15亩=1 hm2,下同)的青蟹-脊尾白虾混养池塘2口,分别投喂配合饲料+鲜杂饵料(试验塘)和鲜杂饵料(对照塘),开展为期70 d的养殖对比试验。结果显示:试验塘青蟹产量较对照塘增加33.5%,亩均效益提升28.3%,经济效益明显提高。试验塘青蟹的肌肉水分显著低于对照塘(P0.05),粗蛋白含量显著高于对照塘(P0.05);试验塘青蟹胃蛋白酶的活性相比于对照塘也有显著提升(P0.05)。试验表明,配合饲料在青蟹养殖实践中的应用具有可行性。     

黄河三角洲地区不同混养模式下拟穴青蟹生长与经济效益分析

为探讨黄河三角洲地区拟穴青蟹(Scylla paramamosain)养殖模式的结构优化，对其不同混养模式的生长与经济效益进行了研究。试验设置了4种混养模式，分别为海水池塘中的“拟穴青蟹-南美白对虾”模式(以下用CV表示)、“拟穴青蟹-日本对虾”模式(以下用CJ表示)、盐碱水池塘的“拟穴青蟹-脊尾白虾”模式(以下用CC表示)和盐碱水稻田中的“拟穴青蟹-耐盐碱水稻”模式(以下用CR表示),试验为期4个月。结果表明，CV模式中拟穴青蟹的体质量、全甲宽、甲宽、甲长、体高指标显著高于其他模式(P<0.05),CV模式中拟穴青蟹的存活率、质量增加率、特定生长率显著高于其他模式(P<0.05)。CV、CJ、CC和CR 4个模式的年利润分别为26.08万、87.37万、13.38万元/hm²和8.91万元/hm²。结果表明，海水池塘中经济效益最高的是CJ模式，盐碱水池塘或稻田经济效益最高的是CC模式，而CJ模式在所有模式中经济效益最高。     

斑节对虾、青蟹、缢蛏半咸水土塘生态混养模式研究

正半咸水土塘养殖是广西北海市沿海一带最具代表性的养殖方式,养殖面积近10万亩,南美白对虾、拟穴青蟹为最主要的两种养殖品种,养殖模式以单养为主,虾、蟹混养模式零星可见。该传统养殖模式易为村民掌握,投资少,但收益不乐观,养殖户信心也越来越差。为改变北海市海水养殖模式,提升当地水产养殖效益,广西区海洋研究所承担实施了广西科学院基本科研业务费资助项目"拟穴青蟹养殖系统优化及营养物质收支研究"课题,试验总面积80亩,2018年进行了拟穴青蟹、斑节对虾、缢蛏的不同放苗密度和不同搭配比例的试验,相比于传统单养,获得了较高的混养产量和经济效益。现将该项目技术试验情况总结如下。     

红树林滩涂瓦缸养殖锯缘青蟹关键技术

锯缘青蟹是广西防城港市主要养殖品种之一,养殖方式有池塘单养、池塘虾蟹混养、池塘蟹鱼混养等传统养殖方式。但由于青蟹互相残食、捕捞困难等问题的困扰,青蟹养殖成活率无法提高,效益不高。2008年至2010年,笔直针对这些问题,在红树林滩涂上利用瓦缸1缸1蟹放养青蟹,提高了青蟹的养殖成活率,     

致急性肝胰腺坏死病副溶血弧菌(VpAHPND)自然感染三疣梭子蟹

近年来包括急性肝胰腺坏死病(AHPND)在内的多种新发疫病的流行,使我国甲壳类养殖业遭受了严重的经济损失。为了筛查导致山东潍坊某养殖场中一虾蟹混养池塘内患病三疣梭子蟹感染的可能病原,本研究采用分子生物学检测方法,对三疣梭子蟹样品进行了白斑综合征病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、虾血细胞虹彩病毒(SHIV)、致急性肝胰腺坏死病副溶血孤菌(Vp_(AHPND))、虾肝肠胞虫(EHP)、偷死野田村病毒(CMNV)、黄头病毒(YHV)和肝胰腺细小病毒(HPV)等8种病原的检测,并对样品进行了组织病理和原位杂交分析。分子生物学检测结果显示,患病三疣梭子蟹样品呈Vp_(AHPND)阳性,而呈现WSSV、IHHNV、SHIV、EHP、CMNV、YHV和HPV阴性。对样品进行Vp_(AHPND)套式PCR第二轮扩增产物的序列测定、比对和进化树分析,结果显示,扩增产物序列与致病副溶血弧菌质粒上pirA~(vp)毒力基因片段具有99%的同源性,该序列与已报道的多个致病副溶血弧菌PirA聚在进化树的同一主分支上。组织病理学分析显示,患病三疣梭子蟹的肝胰腺小管上皮细胞坏死,心肌纤维呈溶解样病变,鳃丝上皮柱突细胞明显坏死,胸神经节的神经细胞损伤严重,并且这些组织中还可见大量的细胞核固缩现象;原位杂交结果显示,肝胰腺、心肌、鳃组织及胸神经节中的病变部位均存在Vp_(AHPND)探针的蓝紫色杂交信号。以上表明,虾蟹混养池塘中三疣梭子蟹在自然状态下感染了Vp_(AHPND),并导致肝胰腺、心肌、鳃和胸神经节发生了严重病理损伤。本研究首次在养殖三疣梭子蟹中检测到Vp_(AHPND)感染并揭示了感染所致的病理变化,相关结果为揭示Vp_(AHPND)自然宿主种类和养殖三疣梭子蟹病害防控提供了基础信息。     

几种锯缘青蟹健康养殖技术

广西沿海许多养殖户因地制宜,采取滩涂生态单养、鱼蟹或虾蟹混养、轮养的新型养殖方式进行健康养殖,经济效益十分显著,青蟹养殖面积和产量不断增长,成为海水养殖发展的新亮点。一、红树林滩涂生态养殖青蟹利用红树林空隙的滩涂设置养殖坛(罐)进行青蟹生态养殖,可充分利用自然生态系统的自身调节作用,又可利用红树林枝条上附着的小牡蛎(藤壶)等作为青蟹辅助饵料,减轻小牡蛎等对红树林的伤害,减少饵料的投喂,同时青蟹的排泄物等有机物又可促进红树林的生长,避免残饵对养殖环境的污染,使蟹、     

聚合酶链反应（PCR）检测养殖对虾的白斑症病毒（WSSV）感染

对虾WSSV病是亚洲对虾养殖业中的一个棘手问题。本研究采用Kimura引物 ,用PCR技术对不同生长期的中国对虾 (Penaeuschinensis)进行了WSSV的检测 ,同时也检测了对虾发病时养殖池中多见的野生厚蟹 (Helicesp .)和矛尾刺虎鱼 (Acanthogobiushasta)。检测结果表明 :分别在检测的 5尾亲虾中的 1尾 ,6尾仔虾中的 1尾 ,5尾稚虾中的 3尾及所检测的 5尾病虾和 2只厚蟹中获得到 982bp的PCR扩增产物 ,说明为WSSV感染阳性。在检测的 2尾矛尾刺虎鱼中均未获得PCR扩增产物 ,说明为WSSV感染阴性。在亲虾、虾苗以及虾池内的野生厚蟹中检测到WSSV感染的阳性结果表明 :WSSV感染的亲虾有可能是病毒的储主 ,WSSV感染的野生厚蟹有可能是病毒中间宿主或病毒的携带者 ,它们在对虾WSSV病的感染、传播中起了重要的作用     

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.     

乳山对虾养殖场白斑综合征病毒(WSSV)调查研究

2002年采用PCR-核酸探针斑点杂交法检测了乳山对虾养殖场1000余份样品携带白斑综合征病毒(WSSV)的情况。结果显示,639例对虾样品中阳性检出率为26·6%;77例蟹类样品中阳性检出率为18·2%;266例浮游动物样品中阳性检出率为38·3%,3~9月份浮游动物阳性率呈下降趋势,消毒后水体中浮游动物的阳性率仍很高;30例贝类样品检测均为阴性;204例底泥样品中,阳性检出率为17·6%,22例抽滤海水样品检测均为阴性。结果表明,虾、蟹类在传播WSSV中起着重要作用,贝类、海水传播WSSV的可能性很小,浮游动物、底泥在传播WSSV中的作用和机制应引起高度重视。     

Experimental host range and histopathology of white spot syndrome virus (WSSV) infection in shrimp, prawns, crabs and lobsters from India

Experimental studies were conducted by injecting or feeding white spot syndrome virus (WSSV) derived from infected shrimp, Penaeus monodon (Fabricius), collected from the south-east coast of India, to five species of shrimp, two species of freshwater prawns, four species of crabs and three species of lobsters. All species examined were susceptible to the virus. Experimental infections in the shrimp had the same clinical symptoms and histopathological characteristics as in naturally infected P. monodon . A cumulative mortality of 100% was observed within 5–7 days in shrimp injected with WSSV and 7–9 days in shrimp fed with infected tissue. Two species of mud crab, Scylla sp., survived the infection for 30 days without any clinical symptoms. All three species of lobsters, Panulirus sp., and the freshwater prawn, Macrobrachium rosenbergii (De Man), survived the infection for 70 days without clinical symptoms. However, bioassay and histology using healthy P. monodon revealed that crabs, prawns and lobsters may act as asymptomatic carriers/reservoir hosts of WSSV. This is the first report to suggest the carrier/reservoir capacity of these hosts through histological and bioassay evidences. Ultrastructural details of the virus in experimentally infected shrimp, P. vannamei , (Boone), were also studied.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

A Profound Effect of Hyperthermia on Survival of Litopenaeus vannamei Juveniles Infected with White Spot Syndrome Virus

This study was conducted to examine the effect of increasing seawater temperature on White Spot Syndrome Virus (WSSV) infection in juvenile Pacific White shrimp ( Litopenaeus vannamei ). Infection by WSSV was achieved using two methods, intramuscular injection and per os (oral) administration. Forty injected and 20 per os infected animals were kept in heated tanks at 32.3 ± 0.8 C, and the same number of WSSV infected animals were maintained in tanks at ambient temperature (25.8 ± 0.7 C). Despite the route of exposure, there were no survivors among the animals kept at ambient temperature; whereas, in heated tanks the survival of the WSSV infected juvenile shrimp was always above 80%, suggesting the existence of a beneficial effect from hyperthermia that mitigated the progression of WSSV disease. Moreover, this beneficial effect was not attributable to viral inactivation. Infected animals kept at 32 C had histologically detectable lymphoid organ spheroids suggestive of a chronic viral infection but were PCR negative (hemolymph) for WSSV. These findings might be related to low viral replication in WSSV-infected shrimp held at the higher environmental temperature. When the WSSV-infected shrimp were transferred from 32 C to ambient temperature, the mortality from WSSV ensued and was always 100%. Although the mechanism related to the beneficial effect of heating was not determined, our results indicate that increasing the water temperature modifies dramatically the natural history of the WSSV disease and the survival curves of WSSV-infected juvenile Pacific White shrimp.     

利用微卫星标记比较分析拟穴青蟹不同家系的遗传多样性

拟穴青蟹(Scylla paramamosain)是我国重要的海水养殖蟹类,随着市场需求量的增加及养殖规模的不断扩大,有必要培育高产、抗逆的优质新品种.家系选育是选择育种的有效途径,目前已应用于拟穴青蟹人工育种工作中.本研究利用8个微卫星标记对拟穴青蟹6个家系共275 ind个体进行了遗传多样性分析,共检测到50个等位...     

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。     

拟穴青蟹两种养殖模式的比较

以拟穴青蟹为主养品种,比较半封闭式养殖模式与传统开放式中水体理化生因子、放养品种的存活率和产量,以探索汕头牛田洋的适宜青蟹养殖模式。经过10个月的养殖试验,半封闭式养殖模式水体理化指标、污染物指标、菌体指标等总体优于开放式养殖模式。半封闭式养殖模式青蟹存活率高出开放式养殖模式66.22%,其他养殖品种的存活率和产量也均高于开放式养殖模式。研究结果表明,与传统的开放式养殖模式相比,半封闭养殖模式能有效降低水体污染指标,提高养殖产量,较为适合在汕头牛田洋青蟹养殖区进行推广。     

自然养殖水体投喂CpG ODN和表面展示VP28的解脂耶罗维亚酵母对凡纳滨对虾(Litopenaeus vannamei)抗WSSV的免疫保护作用

用添加CpG寡聚核苷酸(CpG ODN)和表面展示VP28的解脂耶罗维亚酵母(VP28-yl)的饵料投喂凡纳滨对虾,进行田间中试实验。投喂30 d后进行WSSV感染实验,评估其对凡纳滨对虾的免疫保护作用。投喂实验结束后,CpG ODN投喂组对虾的相对增重率达到(65.8±7.8)% (P<0.05),这暗示CpG ODN可能具有促生长作用。WSSV攻毒后,CpG ODN和VP28-yl投喂组对虾中WSSV拷贝数与对照组相比均显著降低(P<0.05),相对免疫保护率分别可达到26.7%和36.7%。在投喂结束和WSSV刺激后,CpG ODN组对虾中的呼吸爆发水平均显著升高(P<0.05)。而在VP28-yl投喂组,WSSV引起的细胞凋亡则显著受到抑制(P<0.05)。此外,WSSV刺激后,STAT基因在CpG ODN组和VP28-yl组对虾中的表达水平均显著上调(P<0.05),分别在第5天和第3天达到最大值,而对照组中则显著下调。研究结果表明,CpG ODN和VP28-yl增强了凡纳滨对虾抗病毒免疫力,对养殖对虾病毒性疫病的防控具有显著作用,可以作为免疫增强剂添加在饵料中,具有在养殖生产中推广使用的前景。