温度、盐度变化及鳗弧菌刺激对菲律宾蛤仔I型溶菌酶基因表达的影响

为研究在环境因素变化后,菲律宾蛤仔Ⅰ型溶菌酶参与免疫应答反应的变化模式,实验采用cDNA末端快速扩增技术,从菲律宾蛤仔体内克隆获得了一种Ⅰ型溶菌酶基因的全长cDNA序列(命名为RpiLYZ-2),该序列开放阅读框(ORF)为471 bp,编码156个氨基酸.RpiLYZ-2基因在所检测组织中均有表达,其中在外套膜中表达量最高,在肌肉组织中表达量最低.采用荧光定量PCR法,研究了不同温度[(29±1)、(21±1)和(13±1)℃]、盐度(32、22和12)及鳗弧菌刺激对菲律宾蛤仔Ⅰ型溶菌酶基因表达的影响.结果显示,在温度21℃和盐度22胁迫处理后,RpiLYZ-1基因的表达量呈现先上升后下降的趋势,而RpiLYZ-2基因的表达量则呈现先下降后上升的趋势.在高温29℃和低盐12胁迫条件下,RpiLYZ-1基因表达量总体呈下降趋势,而RpiLYZ-2基因表达量总体呈上升趋势;经鳗弧菌刺激后,RpiLYZ-1基因的表达量得到显著诱导,而RpiLYZ-2基因表达量则表现为先降低后上升的趋势.研究表明,环境因子变化和病原菌刺激能够显著影响菲律宾蛤仔两种Ⅰ型溶菌酶基因的表达量,且RpiLYZ-1和RpiLYZ-2可能存在着功能分化.     

三疣梭子蟹“中宁1号”的形态性状增长规律

以1~6月龄三疣梭子蟹"中宁1号"为材料,采用Logistic、Gompertz和von Bertalanffy3种模型分别拟合了体质量、体长、全甲宽、甲宽、体高、大螯长节长、大螯不动指长及第一步足长节长共8个形态性状的生长特征,旨在寻找各性状的最佳生长模型,并对其增长规律进行研究。结果表明:三疣梭子蟹体质量性状生长过程以Logistic生长模型(R2=0.999)的拟合效果最佳;除体质量外的其它7个性状则均以von Bertalanffy生长模型(R2为0.990~0.994)拟合效果最好;各性状模型经ANOVA检验后均具有统计学意义(P0.01)。根据各性状的最佳生长模型得出各性状的极限值分别为体质量231.44 g、体长84.45 mm、全甲宽164.44 mm、甲宽128.47 mm、体高43.69 mm、大螯长节长59.96 mm、大螯不动指长90.89 mm、第一步足长节长37.20 mm。体质量的快速生长区间及拐点分别为2.14~3.91月龄及3.02月龄;其它7个性状快速生长区间的始速点为0月龄,终速点为2.05~2.35月龄,拐点在1月龄左右。各性状间的生长速率、生长加速率、相对增长率与绝对增长率存在一定差异。总之,体质量性状生长过程符合"慢-快-慢"的特征,其它性状则表现为"快-慢"的特征。以上结果可为三疣梭子蟹"中宁1号"选择育种及养殖生产提供参考依据。     

贮藏在海藻酸钙胶丸中的荔枝种子活力

从健康果实中取出的荔枝种子分别放入加有0．1％代森锌的海藻酸钙溶液中，然后在2％氯化钙溶液中胶丸化，形成胶丸种子。胶丸种子和非胶丸种子在10℃下贮存80天，非胶丸种子在10℃中贮藏40天发芽率是11．7％，胶丸种子（4％海藻酸钙）在贮藏80天后仍有53．3％的发芽率。结果表明用海藻酸钙制成的胶丸可延长荔枝这样的“顽拗型”种子的贮藏寿命。     

三疣梭子蟹渔业生物学的初步调查

为探讨池塘养殖三疣梭子蟹的生长规律和雌雄差异,本研究对1~6月龄池塘养殖三疣梭子蟹各生长指标进行了比较分析,并采用Logistic模型分别拟合三疣梭子蟹的体重 (BW)、体长 (BL)、体高 (BH)、全甲宽 (FCW)、甲宽 (CW)、大螯长节长 (MLC)、大螯不动指长 (FFLC)、第一步足长节长 (MLFP)等8个形态性状的生长特征。结果显示,三疣梭子蟹雌性和雄性的生长存在差异,早期雄性生长较快,后期雌性生长较快,而雌雄混合分析介于二者之间。三疣梭子蟹各生长性状的Logistic模型拟合结果显示,除雌性MLC和MLFP以及雌雄混合分析的MLFP之外,其余性状R²均达到0.990以上;雌性和雄性体质量的极限生长值分别为290.27和195.91 g,快速生长区间分别为2.74~5.10月龄和2.33~4.14月龄,拐点分别为3.92和3.24月龄。研究表明,三疣梭子蟹的生长过程均符合“慢-快-慢”的特征,雄性比雌性更早进入快速生长期,但是快速生长期的持续时间不及雌性。本研究对三疣梭子蟹不同生长指标的规律特征,以及各指标在混合养殖条件下雌性和雄性的优势阶段进行了研究,为实现三疣梭子蟹精细化高效养殖提供了参考。     

盐碱水和海水养殖条件下的拟穴青蟹生长和营养成分比较分析

为探究不同水体条件下拟穴青蟹的生长和营养成分差异,本研究测定了黄河三角洲海水（DY）、盐碱水（ZD）及三门海水（SM）的水质离子组成,采用阿廖金分类法进行养殖水体分类;测量形态指标并计算肥满度,利用独立样本t-检验（t-Test）方法比较3种水体中养殖90和140日龄青蟹的生长;测定肝胰腺和肌肉氨基酸及脂肪酸含量,应用主成分分析和聚类分析方法分析各组青蟹氨基酸和脂肪酸的组成。结果显示,ZD组养殖水体碱度显著高于SM组（P<0.05）,与DY组无显著差异（P>0.05）;3个组90日龄青蟹肥满度在0.54~0.89之间,ZD、DY组青蟹肥满度显著高于SM组（P<0.05）,140日龄青蟹肥满度的组间差异不显著（P>0.05）;共测出20种氨基酸和26种脂肪酸,3个组青蟹的肝胰腺和肌肉总氨基酸含量（TAA）分别为3.94~6.87 g/100g和9.73~12.42 g/100g,SM组均为最高;3组青蟹的肝胰腺和肌肉总不饱和脂肪酸含量占总脂肪酸含量（TUFA/TFA）比值分别为59.68~68.80%和70.08~73.79%,其中,ZD组青蟹肝胰腺中C20:5n-3（EPA）含量为各组最高;主成分分析和聚类分析分别显示,ZD组的青蟹的氨基酸与脂肪酸含量与其他组区分较为明显,但差异不显著（P>0.05）,各组青蟹的肌肉、肝胰腺氨基酸和脂肪酸含量的聚类结果均相似。研究发现,与正常海水相比,盐碱水养殖青蟹的生长速度、肥满度及氨基酸和脂肪酸含量差异不大,部分指标占优,为保证养殖青蟹的生长速度和产量,可在盐碱水中适量补充Mg2+和K+。研究结果为盐碱水青蟹养殖和新品种培育提供参考。     

Characterization of a γ‐aminobutyrate type A receptor‐associated protein gene,which is involved in the response of Portunus trituberculatus to CO2‐induced ocean acidification

The γ‐aminobutyrate type A receptor‐associated protein (GABARAP) is a ubiquitin‐like modifier implicated in membrane trafficking and fusion events involving the γ‐aminobutyrate type A receptor, autophagy and apoptosis. In this study, the gene encoding GABARAP was cloned from swimming crab Portunus trituberculatus (PtGABARAP) based on the expression sequence tag (EST). The full‐length cDNA of 664 bp includes a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 223 bp with a poly(A) tail, and an open reading frame (ORF) of 354 bp encoding a polypeptide of 117 amino acids with a predicted molecular weight of 13.96 kDa. The deduced amino acid sequence shares high similarity (93%–100%) with GABARAPs from other species and includes a conserved Atg8 domain. In a phylogenetic analysis PtGABARAP clustered with GABARAPs from other species, and more widely with other GABARAP family proteins. The impact of elevated ocean acidification (OA) on P. trituberculatus behaviours was investigated, and real‐time RT‐PCR revealed that PtGABARAP expression was up‐regulated after OA exposure. Ocean acidification also caused crabs anxiety‐like behaviours, like the shoal average speed increase, preference for dark environment (scototaxis) and fast exploration. The results indicated that GABARAP might be involved in the interactions of GABA_A receptors and elevated‐CO₂ seawater.     

三疣梭子蟹受精卵离体孵化技术

为了探究三疣梭子蟹受精卵的离体孵化技术及效果,本研究先后开展了受精卵块最适分离液种类及作用条件的筛选、分离液处理不同发育期受精卵离体孵化的差异、分离液处理对受精卵卵膜的结构影响,及分离液处理受精卵对孵化后幼体活力的影响实验。结果显示,木瓜蛋白酶是一种较为理想的分离液,在浓度为0.09 g/mL,分离时间30 min时,分离率可达到99%以上;经过分离液处理后的各期受精卵均能孵化出幼体,卵内溞状幼体期离体胚胎孵化率最高,为89.0%±3.3%,未经处理的对照组为70.0%±4.8%;卵裂期离体胚胎孵化率最低,为58.0%±3.9%,对照组为31.0%±2.3%,与对照组相比,处理组的孵化率明显提高。透射电镜结果显示,经过分离液处理的受精卵卵膜结构疏松,且厚度降低,符合处理组孵化率增加这一现象,干露、福尔马林溶液胁迫和行为学测试对不同处理组的幼体进行质量评价的结果显示,处理组和对照组幼体活力无显著差异。研究表明,实验所获得的分离液可以有效提高三疣梭子蟹受精卵的分离率和孵化率,且不影响幼体质量,可为三疣梭子蟹及其他甲壳动物受精卵的离体孵化提供参考。本研究可为三疣梭子蟹的苗种繁育提供新的技术手段,也将为基因编辑辅助育种等技术的实施奠定基础。     

Long‐term low‐salinity stress affects growth,survival and osmotic related gamma‐aminobutyric acid regulation in the swimming crab Portunus trituberculatus

A 30‐day experiment was conducted to investigate the effects of long‐term low‐salinity stress on the growth performance and osmotic related chlorine ion channel ligand regulation: gamma‐aminobutyric acid (GABA) content and GABA_A receptor‐associated protein (GABARAP) expression in Portunus trituberculatus. The salinity levels of both the control group and the experimental group were 30 and 12 psu respectively. After rearing for 30 days, the specific growth rate and survival rate were compared between the two groups, and salinity 6 psu was used to test the salinity tolerance. The results were as follows: (a) Both the specific growth rate and survival rate were significant lower in the experimental group (p < 0.05) after 30 days; (b) After challenge with salinity 6 psu for 72 hr, the crabs of experimental group had a 100% survival rate, whereas the crabs of the control group were all dead within 48 hr; (c) The content of GABA and the gene expression level of GABARAP in experimental group were significant different from control group (p < 0.05) after challenge via salinity 6 psu. In the control group, the GABA content increased rapidly from 9.96 ± 2.09 to 42.00 ± 5.94 µg/g; however, in the experimental group, it only increased to 27.82 ± 2.55 µg/g; the gene expression of GABARAP in the experimental group increased to the maximum at 24 hr, then decreased and stabilized at 48 hr, suggesting that GABA and GABARAP were trigged during the early stage of low‐salinity stress resistance.     

Peroxiredoxin 6 from cuttlefish ( Sepiella maindroni ): molecular characterisation during development and the response to thermal stress

The aim of this work was to investigate the function of the peroxiredoxin family member Peroxiredoxin 6 in Sepiella maindroni (Smprx6) during embryonic development and the response to thermal stress. The 1064 bp full-length coding sequence of Smprx6 includes an open reading frame of 660 bp encoding a polypeptide of 219 amino acids with a predicted molecular mass of 24.4 kDa. The deduced protein sequence shares significant homology with other reported Prx6 enzymes. Phylogenetic analysis classified SmPrx6 into the 1-Cys-Prx cluster, and quantitative real-time PCR (qPCR) demonstrated constitutive mRNA expression in all tested tissues and during five different embryonic developmental stages, as previously documented in other cephalopods. Thermal stress induced Smprx6 expression, which peaked at 6 h then returned to control levels. Recombinant SmPrx6 was active at different concentrations and was thiol-dependant. The results suggest that SmPrx6 plays a major role in the development of Sepiella maindroni and its response to oxidative and thermal stress.     

三疣梭子蟹“科甬1号”生长速率、形态特征和对溶藻弧菌的耐受性

为探究三疣梭子蟹“科甬1号”新品种(简称“科甬1号”)和普通海捕野生三疣梭子蟹子1代(简称“普通蟹”)在生长速率、形态特征和对溶藻弧菌耐受性上的差异,通过对相同生长条件下 “科甬1号”和普通蟹的30、60、90、120和150日龄5个时间点进行体质量和形态指标测量,并在60、90、120和150日龄4个时间点,用浓度为1.14×10⁷ cfu/mL溶藻弧菌按10 μL/g(体质量)剂量进行体腔注射攻毒并统计死亡率。对数据进行ANOVA检验表明,在全部5个测量时间点,“科甬1号”体质量均极显著大于普通蟹体质量;“科甬1号”和普通蟹各形态特征差异均不显著;在60和90日龄2个攻毒时间点,“科甬1号”溶藻弧菌耐受性均极显著强于普通蟹,在120和150日龄2个攻毒时间点,“科甬1号”溶藻弧菌耐受性均显著强于普通蟹。LSD检验分析表明,在60～150日龄期间“科甬1号”对溶藻弧菌的耐受性较稳定,不随生长时间不同而存在显著差异,此外也表明相同浓度溶藻弧菌对相同品种60～150日龄的三疣梭子蟹根据体质量按10 μL/g剂量进行体腔注射攻毒具有相对稳定的致死率。