一株锯缘青蟹呼肠孤病毒的分离纯化及鉴定

在研究锯缘青蟹病害的过程中,从广东某养殖场分离到一株呼肠孤病毒(命名为SsRV)。病毒粒子为球状,直径约45 nm。病毒粒子基因组为12个节段的双链RNA;在1%琼脂糖电泳中SsRV基因组呈1/5/6带型,其分子量分别为3.8,2.5,2.4,2.0,1.9,1.8,1.3,1.2,1.1,1.1,1.0,1.0 kbp。根据病毒的宿主范围、基因组节段数及电泳型,此病毒很可能与地中海滨蟹呼肠孤病毒P和W 2共同构成呼肠孤病毒科一个新的属。     

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。     

Characterization of Aquimarina hainanensis isolated from diseased mud crab Scylla serrata larvae in a hatchery

Mass mortality due to necrosis signs occurred in hatchery-reared zoea stage larvae of the mud crab Scylla serrata in Okinawa, Japan, and a causative bacterium was isolated. In this study, we identified and characterized the bacterium by genome analysis, biochemical properties and pathogenicity. The bacterium was a Gram-negative, non-motile, long rod, forming yellow colonies on a marine agar plate. It grew at 20–33°C (not at 37°C) and degraded chitin and gelatin. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Aquimarina hainanensis. Genome sequence data obtained from Illumina MiSeq generated 29 contigs with 3.56 Mbp in total length and a G + C content of 32.5%. The predicted 16 chitinase genes, as putative virulence factors, had certain homologies with those of genus Aquimarina. Experimental infection with the bacterium conducted on larvae of four crustacean species, brine shrimp Artemia franciscana, freshwater shrimp Caridina multidentata, swimming crab Portunus trituberculatus and mud crab S. serrata, revealed that this bacterium was highly virulent to these species. The present study suggests that the bacterium caused mass mortality in mud crab seed production was A. hainanensis and can be widely pathogenic to crustaceans.     

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.     

Effect of selected feed meals and starches on diet digestibility in the mud crab,Scylla serrata

The present study examined the capacity of the mud crab, Scylla serrata to digest experimental diets that contained different animal and plant‐based feed meals or different levels or types of starch. The apparent dry matter digestibility (ADMD) coefficients for all feed meals tested in the first part of this study, except meat meal, were similar (78–88%). Crude protein digestibility (ACPD) coefficients for all feed meals were relatively high, with values ranging from 86% to 96%. Cotton seed meal, poultry meal, canola meal, fishmeal, soybean meal and lupin meal had similar gross energy digestibility (AGED) values (P>0.05) ranging from 84% to 89%. In the second part of this study, the impact of selected starches on the digestibility of fishmeal‐based formulated diets was assessed. The apparent starch digestibility (ASD) of wheat starch decreased significantly as the inclusion level was increased from 15% to 60%, however, there was no significant effect on ACPD values. At a 30% inclusion level, the ASD of diets containing different starches decreased in the order corn>wheat>potato=rice. Moreover, ACPD values were significantly higher (P<0.05) in the diets containing corn or rice starch than in those containing wheat or potato starches.     

锯缘青蟹血细胞的形态及分类

对锯缘青蟹（Scylla serrata）血细胞染色的抗凝剂、染色方法进行筛选。蟹样品体质量约250g。采用亚甲基蓝、瑞氏法染色后，在Olympus油镜下观察、记数、测量，再结合电镜超薄切片观察结果对锯缘青蟹血细胞进行分类。根据血细胞质中颗粒的有无、大小、折光性、染色特性及细胞的大小、核质比等，将锯缘青蟹血细胞分为4种：（1）无颗粒细胞，细胞质中无颗粒；（2）小颗粒细胞，细胞质中有深蓝色小颗粒；（3）中间型细胞，细胞质中既有深蓝色小颗粒，又有折光性红色大颗粒；（4）大颗粒细胞，细胞质中充满了具有折光性的红色大颗粒。4种血细胞的大小顺序从小到大依次为无颗粒细胞、小颗粒细胞、中间型颗粒细胞、大颗粒细胞；核质比则相反，分别为54．01％、37．13％、25．37％、17．49％；其数量百分比分别占20．92％、40．30％、19．39％、19．39％。根据伪足的多少。对4种血细胞在机体的免疫防御机制中所起的不同作用进行了简单探讨。     

Chemical constituents in the abdominal muscle of cultured mud crab Scylla serrata in relation to seasonal variation and maturation

ABSTRACT:     Seasonal changes in levels of chemical constituents in the abdominal muscle of male and female mud crab Scylla serrata were investigated. The gonadosomatic index in the female crab was higher in October and August, of which the muscle yield, glycogen, and total amount of adenosine 5'-triphosphate (ATP)-related compounds (ARC) in the October sample were the highest throughout the year, but the total amount of free amino acids (FAA) was the lowest. The total FAA and individual FAA such as glycine, alanine, and arginine increased in the August and November female samples, while that in the male specimens was higher in January, March, and August. The total ARC in both crabs was lower in the samples collected from winter than in other seasons whereas glycinebetaine was higher in winter and early spring. Glutamic acid, glycine, alanine, arginine, glycinebetaine, and adenosine monophosphate might be responsible for the taste of mud crab. Their level together was relatively higher in the August and January male samples, and in the August and November female specimens. However, the correlation between the taste component content and degree of gonad maturation was low.     

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.     

锯缘青蟹苗池微生态环境影响因素的初步探讨

通过对锯缘青蟹苗池微生态环境和水质环境的全过程检测,探讨了总菌数、弧菌数及弧菌数/总菌数的消长规律,并对苗种培育阶段涉及的各种外源因素及控制苗池微生态环境的相关手段的有效性作了初步的探讨。结果表明:苗池水体中总菌数在Z1~Z2少量添加水不换水阶段,呈相对稳定状态,进入Z3后总菌数逐渐增加,最高达3.5×106cfu/m l;弧菌数在Z1培育阶段出现2×102cfu/m l激增至5×104cfu/m l的峰值,第二峰值出现在Z5阶段,达6×105cfu/m l;弧菌数/总菌数比值在育苗起始时仅为0.4%,之后迅速升至30%~40%,在Z5阶段,由于弧菌数量急剧上升,达到50%的高比值;NH4-N与COD检测值在育苗过程中变动较大,两者与弧菌数/总菌数比值呈正相关关系。现行育苗方式中对苗池微生态环境产生影响的主要因素是苗种培育密度,海水、饵料的处理方式和抗生素;净化功能菌和蛭弧菌应用于育苗水体具有抑制弧菌繁殖的效果。     

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。     

Morphological deformities in mud crab Scylla serrata juveniles exposed to antibiotics during the larval stage

The effects of antibiotics on the external deformities, growth and survival of mud crab Scylla serrata larvae and juveniles were determined. Zoeae were exposed to oxytetracycline (OTC) (0, 3.0, 6.0, 9.0, 12 mg L^?1) and furazolidone (FZD) (0, 0.5, 1.0, 1.5, 2.0 mg L^?1) in the first and second experiments, respectively, until the late megalopa. The crab instars were grown in nursery tanks for 1 month. Larvae survived until megalopa only at 3.0 and 6.0 mg L^?1 OTC or 0.5 and 1.0 mg L^?1 FZD. These four concentrations were run simultaneously in another experiment. Morphological deformities in zoea 5 were bent dorsal, rostral and furcal spines. There was no significant difference (P > 0.05) on the deformities of zoea 5 in 3.0 and 6.0 mg L^?1 OTC and 0.5 and 1.0 mg L^?1 FZD. Significantly (P < 0.05) higher survival and faster growth were attained in 3.0 mg L^?1 OTC and 0.5 mg L^?1 FZD. Deformities observed in juveniles were fused frontal and lateral spines, asymmetrical and depressed tip of abdominal flap and gap between sternites. High percentage occurrence of deformities was observed in the 6.0 mg L^?1 OTC and 1.0 mg L^?1 FZD in the first and third experiments, respectively. There was no significant difference (P > 0.05) observed in the survival of juveniles in OTC and FZD treatments. However, growth was significantly (P < 0.05) faster in lower concentrations of the two antibiotics. The study shows the effects of OTC and FZD in the morphology of mud crab. Therefore, there is a need to eliminate the use of antibiotics and find alternatives.     

锯缘青蟹蜕皮抑制激素cDNA的分子克隆及其表达分析

根据近缘种类同源序列设计简并引物,采用逆转录聚合酶链式反应(RT—PCR)技术,首次扩增获得锯缘青蟹(Scylla serrata)眼柄组织中编码蜕皮抑制激素(MIH)成熟肽全长cDNA序列及部分信号肽序列。将该序列克隆到pUCm—T载体上进行序列测定。结果表明,编码锯缘青蟹MIH成熟肽的cDNA由234个碱基组成,由此推测MIH成熟肽含78个氨基酸残基。该序列不仅与其它短尾类甲壳动物的MIH氨基酸序列具有高度的同源性(79％～9l％),还与该激素同一家族的性腺抑制激素、大颚器抑制激素的氨基酸序列具有较高的相似性。RNA斑点杂交结果显示,MIH基因在眼柄神经节及脑组织中均有表达,而在肌肉、中肠腺中不表达。     

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.     

Ontogeny of feeding apparatus and foregut of mud crab Scylla serrata Forsskål larvae

The development of the feeding apparatus of the mud crab Scylla serrata larvae was studied using electron microscopy for mandibles and light microscopy for other paired mouthparts and the foregut. The six paired mouthparts, which consisted of the mandibles, maxillules, maxillae, first maxillipeds, second maxillipeds and third maxillipeds, were dissected from specimens representing each larval stage. The first five paired appendages were already present in newly hatched larvae while third maxillipeds appeared only at the megalopa stage. Mandibles displayed complex incisor and molar processes at early zoeal stages, which became simple in morphology at megalopa. Mandibular palp buds were observed at the zoea 5 stage and these became fully developed as three‐segmented mandibular palps at the megalopa stage. Endopods of other paired mouthparts exhibited increased number of setae and size as the individual metamorphosed from zoeal stages to megalopa and crab instar. The foregut appeared as a continuous cavity at zoea 1 where the cardiopyloric valve was indistinct while the filter gland was clearly identifiable. Zoea 2 and succeeding zoeal stages exhibited a setose foregut; the gastric mill and its lateral and median teeth were prominent at zoea 3 stage. The significance of these morphological changes is discussed in terms of its implication in larval feeding management.