环介导恒温扩增技术检测血卵涡鞭虫

血卵涡鞭虫病是海水甲壳类的重要寄生虫病,其流行范围广、死亡率高、危害非常严重。本研究根据GenBank中已登录的血卵涡鞭虫(Hematodinium sp.)ITS1序列设计了1套引物,该引物可识别目标基因中6个不同区段。以此套引物建立了一种基于环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)的血卵涡鞕虫病诊断方法。特异性试验结果表明,该套引物对血卵涡鞕虫检测具有较高的特异性,能有效检出血卵涡鞕虫。敏感度试验结果表明,该LAMP技术的灵敏度比常规PCR技术高4个数量级。分别运用LAMP和常规PCR技术对25份临床疑似病例进行检测,LAMP方法共检出感染病例25份,常规PCR检出感染病例23份。该技术能在65℃恒温条件下45～60min完成目的DNA的扩增,可直接通过肉眼观察反应产物中是否产生白色沉淀或经SYBRGreenI染色后通过颜色变化、扩增产物的琼脂糖凝胶电泳来定性判断结果,这将为血卵涡鞕虫病的临床诊断提供一种更加简便、快速、实用的方法。     

海产蟹类血卵涡鞭虫间接荧光抗体快速检测技术

以寄生于三疣梭子蟹血淋巴中的血卵涡鞭虫虫体为抗原,制备了多克隆血清抗体.抗体经健康梭子蟹血淋巴吸附处理后,间接ELISA检测效价达7 680.应用该抗体建立了血卵涡鞭虫的间接荧光抗体检测技术(IFAT).采用常规显微镜检、PCR及IFAT 3种方法对采集的养殖青蟹、梭子蟹及海捕梭子蟹等18个样本进行了检测.检测结果显示:常规显微镜检阳性检出率为33.3%,而IFAT及PCR检测阳性率77.8%,符合率达100%;阳性虫体被染上黄绿色荧光,而正常梭子蟹血细胞则未被染色;可检测到血卵涡鞭虫不同生活阶段的营养体、腰鞭孢子及合孢体阶段.为血卵涡鞭虫的流行病学调查及生活史研究提供了简便实用的方法.     

海产甲壳类血卵涡鞭虫病研究进展

血卵涡鞭虫是导致海产甲壳类疾病的主要寄生虫病原之一,国内迄今尚未见相关报道。本文结合作者近年来对海水养殖甲壳类病害的研究结果,较系统地综述了国外甲壳类寄生血卵涡鞭虫的研究进展,包括分类地位、生活史、传播途径、流行病学、组织病理以及诊断方法,并提出了相应的防治措施,旨在为中国海水养殖蟹类血卵涡鞭虫病害的研究与防治提供参考。     

海产蟹类血卵涡鞭虫病间接荧光抗体快速检测技术

以寄生于三疣梭子蟹血淋巴中的血卵涡鞭虫虫体为抗原,制备了多克隆血清抗体。抗体经健康梭子蟹血淋巴吸附处理后,间接ELISA检测效价达7680。应用该抗体建立了血卵涡鞭虫的间接荧光抗体检测技术(IFAT)。采用常用显微镜检、PCR及 IFAT三种方法对采集的养殖青蟹、梭子蟹、及海捕梭子蟹等18个样本进行了检测。检测结果显示：常规显微镜检阳性检出率为33.3%,而IFAT及PCR检测阳性率77.8%,符合率达100%;阳性虫体被染上黄绿色荧光,而正常梭子蟹血细胞则未被染色;可检测到血卵涡鞭虫不同生活阶段的营养体、腰鞭孢子及合孢体阶段。本文为血卵涡鞭虫的流行病学调查及生活史研究提供了简便实用的方法。     

血卵涡鞭虫的发生及流行研究进展

血卵涡鞭虫(Hematodinium)是一类危害海水甲壳类动物的致病性寄生甲藻，自1931年以来感染超过40多种甲壳类动物，导致了一些经济类甲壳动物的大量死亡。为有助于加强对血卵涡鞭虫的防控，从季节、环境、宿主类型和传播模式方面，总结了血卵涡鞭虫流行和发生的原因。     

锯缘青蟹血蓝蛋白抑菌活性及其作用靶标

采用抑菌实验和抑菌抑制实验等方法探索锯缘青蟹(Scylla serrata)血蓝蛋白抑菌活性及其作用靶标.结果显示,锯缘青蟹血蓝蛋白对溶藻酸弧菌(Vibrio alginolyticus)、大肠杆菌K12(Escherichia coli K12)等细菌具有抑菌活性,其对大肠杆菌K12的抑菌活性可被大肠杆菌K12外膜蛋白(Outer membrane protein, Omp)所抑制,尤其是血蓝蛋白对大肠杆菌K12外膜蛋白敲除菌ΔOmpX、ΔOmpC、ΔOmpT、ΔTsx、ΔFadL和ΔOmpW的抑菌活性显著降低,与对照组相比,具有极显著性(P<0.01)和显著性差异(P<0.05).由此说明,锯缘青蟹血蓝蛋白具有抑菌活性,其抑菌作用靶标可能为多种外膜蛋白.     

血卵涡鞭虫单克隆抗体的制备与初步应用

用血卵涡鞭虫可溶性抗原免疫BALB/c小鼠,经常规融合、间接ELISA方法筛选,将所得阳性克隆再经3次亚克隆后,共获得3株针对血卵涡鞭虫的单克隆抗体(2B₂、3G₄、4G₇),单克隆抗体亚类鉴定表明,三者为IgG类抗体。用筛选的杂交瘤细胞株制备小鼠腹水抗体,其细胞上清及腹水效价分别为5.12×10^-4和8.00×10^-4。进一步利用单克隆抗体建立间接荧光抗体方法对单抗特异性进行鉴定,阳性虫体被染上黄绿色荧光,而正常梭子蟹血淋巴则未被染色。用单克隆抗体和多克隆兔抗血清以羊抗鼠HRP-IgG为酶标抗体,建立了检测血卵涡鞭虫的双抗体夹心ELISA方法,该方法对血卵涡鞭虫阳性标本检测符合率为100%。结果表明,制备的单克隆抗体效价高、特异性好,可用于血卵涡鞭虫的早期临床诊断。     

三疣梭子蟹 Portunus trituberculatus (Miers) 血卵涡鞭虫病诊断与综合防治措施

血卵涡鞭虫是导致海产甲壳类疾病的主要寄生虫病原之一，国内迄今尚未见相关报道。本文根据作者近年来对海水养殖梭子蟹血卵涡鞭虫病的研究结果．就该病在养殖梭子蟹当中的流行情况及诊断方法进行介绍，并提出了相应的综合防治措施，旨在为我国海水养殖接类血卵涡鞭虫病害的研究与防治提供参考。     

大黄鱼苗淀粉卵涡鞭虫病的诊断及防治方法

淀粉卵涡鞭虫是大黄鱼苗种培育危害最大的寄生虫之一.本文对大黄鱼苗淀粉卵涡鞭虫病发生的临床症状和防控方法进行了研究,除常规防治方法外,利用双氧水与高锰酸钾合用也可起到杀灭虫体,减少寄生数量的效果.同时,本文分析和比较了发生寄生虫病的育苗池水与正常池水的水质理化因子,为有效诊断和防治大黄鱼苗淀粉卵涡鞭虫病提供基础材料.     

“锯缘青蟹病害防控关键技术研究与示范”成果获肯定

根据中国水产科学研究院水科成发[2011]324号文件，由浙江省淡水水产研究所申报的《锯缘青蟹病害防控关键技术研究与示范》成果荣获2011年度中国水产科学研究院科技进步二等奖。     

锯缘青蟹苗种培育的科技进展

锯缘青蟹是我国南方沿海各省传统养殖的海水蟹类,在当前农业产业结构调整中被列为主要发展品种之一。鉴于苗种是限制青蟹养殖业发展的主要因素,本文根据目前的研究现状对青蟹的种质、性成熟机制,产卵、孵化条件,幼体培育过程中营养条件、盐度、温度、光照、疾病等,以及从大眼幼体到仔蟹的中间培育技术等作了简要归纳,并对研究现状和进入产业化急需解决的问题进行了探讨。     

锯缘青蟹促雄腺进行全浆分泌的证据

采用透射电镜技术，对锯缘青蟹(Scylla serrata)促雄腺细胞的分泌活动进行了观察。结果表明，腺细胞具有2种类型：A型细胞核内常染色质丰富，具有大量的扁平嵴的线粒体、粗面内质网与核糖体，A型细胞进行分泌时，细胞质大量排出，属于全浆分泌方式；B型细胞异染色质绕核内膜分布，仅见极少量的细胞器或细胞质完全消失。B型细胞为A型细胞完成分泌后的存在形式。本研究为甲壳动物促雄腺的全浆分泌活动提供了直接的证据。     

影响锯缘青蟹幼体成活的几个因素

本文对海捕和养殖的锯缘青蟹抱卵及胚胎发育情况进行了观察,并对幼体饵料营养强化、适口性、幼体的培育方式等进行了研究。结果表明：自然诲区捕获的亲蟹其抱卵率和幼体质量要好于人工养殖的亲蟹;胚胎发育的最适盐度为28～33,最适温度为26～30℃;幼体\期和大眼幼体时期摄食的饵料以边缘毛状的活体为佳,便于抱握;涵状幼体、期起应降低苗池中的直射光,投饵多餐少量．增加附着面积．可降低相互残杀率。     

Ovarian maturation stages of the mud crab Scylla serrata

Ovarian maturation in adult wild‐sourced and pond‐grown Scylla serrata (Forsskål) was determined based on gross morphology and histological appearance. There were no significant differences noted in the histological features of both wild and pond‐reared S. serrata females. Ovarian maturation was classified into five stages: immature, early maturing, late maturing, fully mature and spent. The immature ovaries are thin and translucent to off white and contain oogonia, primary oocytes with large nuclei. The follicle cells were found around the periphery of the lobes and an area among groups of oogonia and oocytes. The follicle cells gradually enclosed the oocytes. The early‐maturing ovaries were yellow and small yolk globules started to appear in larger oocytes. In late‐maturing ovaries, the colour became light orange and lobules were apparent. Yolk globules occurred in the cytoplasm with larger globular inclusions towards the periphery, while follicle cells were hardly recognizable. Fully mature ovaries were orange to deep orange and had swollen lobules. Large yolk globules were apparent in the entire cytoplasm. Follicle cells were hardly seen. Spent ovaries were similar to the early‐maturing and late‐maturing stage in partially spawned females. The ovarian development was correlated closely to the gonadosomatic index, oocyte diameter, and ovarian histology. The classification of ovarian maturation provides baseline information for further studies on reproductive biology. Likewise, the information provides a guide for broodstock management in the hatchery.     

锯缘青蟹促雄腺发育的组织学研究

采用组织切片技术，以OlympusBH-2型显微镜对锯缘青蟹(Scylla serrata)促雄腺的发育进行观察。结果显示，促雄腺附着于射精管近末端，发育过程可分为4期：Ⅰ期促雄腺短小，腺细胞数量少；Ⅱ期促雄腺呈明显的索状；Ⅲ期促雄腺体积达到最大，一些部位膨大增生；Ⅳ期促雄腺不再生长，腺体急剧退化。腺细胞具有2种类型：A型细胞，核圆、异染色质少、胞质染色浅；B型细胞，核扁平、异染色质多，细胞染色深、界限不清晰，有时细胞质消失。促雄腺发育过程中，A、B型细胞数量、比例发生变化。B型细胞为A型细胞完成分泌后的存在形式，B型细胞比例反映了促雄腺分泌活动的状况。锯缘青蟹促雄腺发育和精巢成熟具有一定的相关性。     

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.     

利用AFLP技术筛选锯缘青蟹性别差异DNA片段

采用高盐和酚氯仿异戊醇 (PCI)结合法提取DNA ,利用AFLP技术 ,应用 5 2个引物组合 ,检测了锯缘青蟹 (Scyllaser rata)雌雄基因组DNA的多态性 ,筛选与锯缘青蟹性别相关的分子标记。实验中共扩增出 4 312条带 ,筛选出候选差异DNA片段 74 8条。这些差异DNA片段的获得 ,为研究锯缘青蟹性别的分子标记奠定了基础     

Ingestion of Brachionus plicatilis and Artemia salina nauplii by mud crab Scylla serrata larvae

Two feeding experiments were conducted to determine if Brachionus plicatilis and Artemia salina nauplii were ingested by mud crab Scylla serrata larvae. In the first experiment, larvae were fed with increasing densities of Artemia nauplii with or without Brachionus to determine consumption with increasing densities of Artemia and with increasing zoeal stage. This experiment also aimed to determine if the presence of Brachionus as an alternative prey influenced the intake of Artemia by the crab larvae. There was generally an increase in intake with increasing densities of Artemia and increased consumption of Artemia as the larvae grew. Consumption of Brachionus was consistently high in all zoeal stages. There was a significant reduction in the intake of Brachionus with increasing consumption of Artemia in the early zoeal stages (Z1, Z2, Z3), but at later stages (Z4, Z5) the intake of Artemia was no longer affected by the presence of Brachionus. In the second experiment, daily ingestion within instar of zoeal stages and megalopa were compared. There was an increased consumption of Artemia nauplii on the day before molting and increased ingestion of Brachionus on the day after larvae had molted, except at Z3. Megalopae exhibited a decline in Artemia nauplii intake on the days before metamorphosis to crablet.     

Acute toxicity of nitrite to mud crab Scylla serrata (Forsskål) larvae

Early larval stages of mud crab Scylla serrata were exposed to different concentrations of nitrite (40, 80 and 160 mg L⁻¹ and a control, without added nitrite) and three salinity levels (25, 30 and 35 g L⁻¹) using a static renewal method. No interactive effect of nitrite and salinity was detected. Estimated LT₅₀ in 96‐h toxicity tests decreased in all stages with increasing nitrite concentrations in all salinity levels. The 96‐h LC₅₀ values of nitrite‐N were 41.58, 63.04, 25.54, 29.98 and 69.93 mg L⁻¹ for zoea 1, 2, 3, 4 and 5 respectively. As the larvae grew, they showed a progressive increase in tolerance to nitrite. The toxicity of nitrite to larvae increased with exposure time. The median lethal concentration was not affected by salinity. The chloride component of salinity within 25–35 g L⁻¹ did not seem to be as effective in alleviating toxicity as has been reported in other crustacean species. Based on 96‐h LC₅₀ and an application factor of 0.1, the ‘safe level’ of rearing mud crab larvae was calculated to be 4.16, 6.30, 2.55, 2.99 and 6.99 mg L⁻¹ nitrite‐N for zoea 1, 2, 3, 4 and 5 respectively.