Quality assessment of newly hatched mud crab,Scylla serrata,larvae

Starvation and exposure to formalin were investigated as possible stress tests for evaluating the quality of mud crab, Scylla serrata, larvae. For the starvation stress test, newly hatched zoeae stocked in 150‐ml containers were either starved or fed rotifers. Similarly, newly hatched zoeae were stocked in containers with seawater of 0 (control), 20, 30 and 40 mg/L formalin for the formalin stress test. The zoeae from the same batches were used for seed production to monitor their performance and validate the results of stress tests. Starvation was found to be unsuitable for larval quality evaluation. However, the impact of initial food deprivation on the newly hatched larvae indicates that feeding immediately after hatching is necessary for mud crab larvae. Exposure of larvae to 40 mg/L formalin for 3 hr appeared to be a reliable and practical method for larval quality assessment as the survival of larvae in the mass production tanks validated the classification of good and poor quality batches in the stress tests. On this basis, a hatchery operator can decide which batch should be cultured further. Finally, there appears to be a link between the quality of larvae and the performance at the megalopa and early juvenile crabs.     

锯缘青蟹精氨酸激酶基因的克隆与表达

通过分子生物学方法,克隆得到锯缘青蟹（Scylla serrata）精氨酸激酶（Arginine Kinase,AK）开放阅读框基因序列.测序结果显示：该开放阅读框基因的序列长度为1 071 bp,编码356个氨基酸残基;该序列登录Genbank（GQ：851626）,序列比对结果显示,锯缘青蟹精氨酸激酶与凡纳滨对虾（Litopenaeus vannamei）精氨酸激酶的氨基酸序列同源性高达94%,与岸蟹（Carcinus maenas）、中华绒鳌蟹（Eriocheir sinensis）等甲壳类动物的精氨酸激酶也有较高的同源性,均高于90%;将克隆得到的锯缘青蟹精氨酸激酶基因与pGEX-4T-3载体连接,转化大肠杆菌BL21,经IPTG诱导得到分子质量为65 ku的融合表达蛋白（GST-AK）,经兔抗锯缘青蟹精氨酸激酶多克隆抗体的免疫印迹分析证实,GST-AK具有抗原性.     

Comparative study on the embryonic development of three mud crabs Scylla spp.

Morphological changes in the embryos, egg size and development, incubation period and morphological structures of newly hatched zoea of three mud crab Scylla species were determined. The three species exhibited similar embryonic development composed of 10 stages. The mean egg diameter of Scylla serrata was significantly larger (P<0.05) at the prehatch stage. The mean egg diameters of Scylla tranquebarica and Scylla olivacea were similar (P>0.05). The incubation period was the longest in S. serrata and the shortest in S. olivacea. There was a positive relationship between egg size and larval size, as S. serrata exhibited the largest egg size and first zoea. However, no correlation was detected between egg size at prehatch and lengths of the morphological structures of the newly hatched zoea. The three species exhibited similar lengths of cephalic structures, but S. olivacea had significantly shorter (P<0.05) abdominal structures. The duration of spawning from ablation was the shortest in S. tranquebarica and the longest in S. olivacea. The study is relevant to aquaculture and fisheries management of Scylla species.     

青蟹野生与养殖群体遗传多样性的微卫星分析

利用微卫星分子标记对青蟹东海三门湾野生群体和浙江三门养殖群体的遗传多样性进行分析。6个微卫星位点在2个青蟹群体中共检测到66个等位基因,多态信息含量为0.746 ～ 0.895,均表现为高度多态性,可有效应用于青蟹遗传多样性的研究。青蟹野生群体与养殖群体的等位基因数分别为4 ～ 18,6 ～ 14,平均杂合度分别为0.577,0.561,平均多态信息含量分别为0.779,0.828;群体间遗传分化指数FST值为0.0317,遗传相似性指数为0.7930,遗传距离为0.2319。结果表明,青蟹野生与养殖群体的遗传多样性均较丰富,群体间存在一定的遗传分化。     

拟穴青蟹卵泡抑素相关蛋白基因的克隆和表达分析

卵泡抑素相关蛋白(follistatin related protein,FRP)是一种细胞外基质糖蛋白,该蛋白参与细胞增殖、迁移、组织重塑、胚胎发育、细胞间相互作用等多种生理过程。迄今甲壳动物FRP的研究尚未见诸报道。实验采用RACE技术首次克隆了拟穴青蟹FRP基因部分cDNA序列。该序列长度1 948 bp,FRP肽段含有484个氨基酸残基,包括KAZAL-FS结构域、EFh结构域和两个Ig结构域。系统进化树显示拟穴青蟹FRP基因的分子进化地位与其生物学分类地位一致。半定量PCR及荧光定量PCR结果表明,FRP基因在拟穴青蟹的胸神经团、脑和卵巢中表达。FRP基因在卵巢发育各期的表达量各不相同,卵巢未发育期最高,据此我们推测FRP具有抑制卵巢发育的作用。     

饲料糖脂比对拟穴青蟹仔蟹生长性能、体组成和消化酶活性的影响

本试验旨在研究饲料糖脂比对拟穴青蟹仔蟹生长性能、体组成和消化酶活性的影响。以初始体重为(41.4±0.3)mg的拟穴青蟹仔蟹为试验对象,分别投喂糖脂比为0.54、0.88、1.39、2.08和3.50的等氮(约44%)等能(约19.5 MJ/kg)饲料3周。结果表明:1)饲料糖脂比对拟穴青蟹仔蟹的终末平均体重(FABW)、成活率(SR)、增重率(WGR)以及特定生长率(SGR)均有显著影响(P0.05)。随着饲料糖脂比的增大,拟穴青蟹仔蟹的FABW、SR、WGR以及SGR均呈先升高后降低的趋势,且均是糖脂比1.39试验组最高,显著高于糖脂比0.54和3.50试验组(P0.05)。2)饲料糖脂比对拟穴青蟹仔蟹的水分、粗蛋白质和粗灰分含量没有显著影响(P0.05),而对粗脂肪含量的影响显著(P0.05),糖脂比0.54试验组的脂肪含量最高,显著高于糖脂比2.08和3.50试验组(P0.05)。3)饲料糖脂比对拟穴青蟹仔蟹的淀粉酶活性没有显著影响(P0.05),但显著影响蛋白酶和脂肪酶活性(P0.05)。随着饲料糖脂比的增大,蛋白酶活性呈先增大后减小的趋势,脂肪酶活性呈降低的趋势,糖脂比1.39试验组的蛋白酶活性显著高于其他试验组(P0.05),糖脂比0.54和0.88试验组的脂肪酶活性显著高于糖脂比2.08和3.50试验组(P0.05)。以增长率为评价指标,经回归分析得出拟穴青蟹仔蟹饲料的适宜糖脂比为2.07。     

饲料脂肪水平对拟穴青蟹仔蟹生长性能、体组成及消化酶活性的影响

本试验以初始体重为(0.042±0.002)g的拟穴青蟹仔蟹为研究对象,以鱼油和大豆油(1∶1)为脂肪源,配制脂肪水平分别为1.93%、3.95%、6.35%、8.14%、10.54%、12.30%、14.22%(实测值)的7种等氮等能试验饲料,养殖期为3周,用以研究饲料脂肪水平对拟穴青蟹仔蟹生长性能、体组成及消化酶活性的影响。每100只拟穴青蟹仔蟹为1个重复,每3个重复饲喂1种试验饲料。结果表明:1)饲料脂肪水平对拟穴青蟹仔蟹的增重率和特定生长率有显著影响(P0.05)。随着饲料脂肪水平的升高,增重率和特定生长率均呈先增后降的趋势,在饲料脂肪水平为8.14%时,增重率和特定生长率达到最大值。二次曲线回归分析确定当增重率达到最大值时,饲料脂肪水平为7.52%。拟穴青蟹仔蟹的成活率在饲料脂肪水平为14.22%的组最低,显著低于其余各组(P0.05)。2)饲料脂肪水平对全蟹中粗蛋白质和粗脂肪含量有影响显著(P0.05)。全蟹粗蛋白质含量随着饲料脂肪水平的升高先升后降,在饲料脂肪水平为8.14%的组全蟹粗蛋白质含量达到最高;全蟹粗脂肪含量则随着饲料脂肪水平的升高而持续上升,在饲料脂肪水平为14.22%的组全蟹粗脂肪含量达到最高。饲料脂肪水平对全蟹中水分和粗灰分含量没有显著影响(P0.05)。3)随着饲料脂肪水平的升高,拟穴青蟹仔蟹蛋白酶、淀粉酶和脂肪酶活性均呈先增后降的趋势,3种酶活性的最大值均出现在饲料脂肪水平为8.14%的组。由此得出,以增重率为评价指标,拟穴青蟹仔蟹饲料中最适脂肪水平为7.52%。     

拟穴青蟹GRP78基因的克隆与应激表达

葡萄糖调节蛋白78(glucose regulated protein 78 ku,GRP78)是热休克蛋白70家族成员之一,在调节蛋白质折叠和维持内质网稳态过程中起着分子伴 侣作用。采用RT-PCR、RACE等技术,首次从拟穴青蟹获得了GRP78的cDNA全长序列。该序列全长2 284 bp,开放阅读框(ORF)为1 962 bp,编码653个氨基酸残基。 同源分析显示,该基因编码的蛋白含有HSP70家族的签名序列,C末端为内质网蛋白滞留信号KDEL,与其他物种具有很高相似性。实时荧光定量PCR结果表明,GRP78 基因在拟穴青蟹多个组织中均有表达。第一期仔蟹在不同的温度和盐度下暴露12 h后,GRP78基因表达量随环境温度升高而增加;在高盐(30)条件下GRP78表达量 较高,进而推测拟穴青蟹GRP78参与蛋白质折叠和环境胁迫的应答。     

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.