拟穴青蟹丝裂原活化蛋白激酶激酶（MAPKK）基因的克隆与表达分析

采用qRT-PCR、RACE等方法,获得了拟穴青蟹丝裂原活化蛋白激酶激酶(MAPKK)基因cDNA全长序列。该基因全长1 558 bp,开放阅读框长度为1 224 bp,编码407个氨基酸残基。同源分析显示,该基因编码的蛋白与昆虫的相似性高达70%,推测MAPKK基因在节肢动物具有较高的保守性。经荧光定量PCR检测,MAPKK基因在拟穴青蟹多个组织中有表达,且在脑神经节和卵巢中表达量较高。在拟穴青蟹卵巢发育过程中,MAPKK基因在卵巢发育期(Ⅲ期)表达量最高,发育期为卵母细胞快速生长期,推测MAPKK具有促进卵母细胞快速生长的作用。     

Innate immune response and gene expression of Scylla paramamosain under Vibrio parahaemolyticus infection

An epidemic of ‘milky disease’ in the mud crab (Scylla paramamosain) generally breaks out in the fall when the crab is near maturity, resulting in large economic losses in crab farming. Vibrio parahaemolyticus has been proven to be one of the major pathogens. In this study, the mud crabs were challenged with V. parahaemolyticus, and their innate immune responses were investigated in terms of total haemocyte counts (THCs), haemocytic enzyme activities and gene expression levels during a 114‐h period. The THCs of the mud crabs decreased significantly after 42 h of exposure. The activities of the haemocytic enzymes, including acid phosphatase‐alkaline phosphatase, phenoloxidase, superoxide dismutase (SOD), peroxidase and nitric oxidase synthethase, were significantly enhanced during the challenge course. The gene expression levels also significantly increased for all tested genes (proPO, Cu/Zn‐SOD, Prx, LYS, CRU and ALF) with the exception of CAT down‐regulated expression. The results may imply that the immune responses of the mud crab could be activated by the pathogens, and the data here will provide many clues for further systematic investigation of ‘milky disease’ caused by V. parahaemolyticus and the disease prevention in mud crab S. paramamosain.     

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。     

注射中华绒螯蟹及锯缘青蟹促雄性腺提取物对中华绒螯蟹雌蟹雄性化的影响

该论文首次报道了经由促雄性腺提取物的注射而在蟹类中引起的性逆转现象。此前关于蟹类促雄性腺活性研究的报道极少，而且蟹类的雄性化均是由促雄性腺的移植所产生。本实验中将锯缘青蟹和中华绒螯蟹的促雄性腺提取物分别注射到刚刚完成性别分化的中华绒螯蟹雌性幼蟹体内，此时幼蟹处于4至5期，壳宽为5～8 mm。注射之后，幼蟹经过大约1～2次蜕皮，此时在注射锯缘青蟹以及中华绒螯蟹的促雄性腺提取物的两组实验幼蟹中均能观察到雄性化现象，而注射生理盐水的对照组实验幼蟹中未能观察到相同现象。由此本实验可以证明促雄性腺确实是蟹类的一种雄性激素，注射促雄性腺提取物能引起雌性幼蟹发生性逆转；同时根据锯缘青蟹和中华绒螯蟹的促雄性腺提取物均能引起中华绒螯蟹雌性幼蟹发生性逆转的现象推测，锯缘青蟹和中华绒螯蟹两种间可能存在促雄性腺的交叉活性；不仅如此，性逆转还能在极低的注射剂量下获得，相当于中华绒螯蟹0.14促雄性腺当量和锯缘青蟹0.06促雄性腺当量。     

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.     

Effect of dietary cholesterol levels on growth performance,body composition and gene expression of juvenile mud crab Scylla paramamosain

A 56‐day feeding trial was conducted to investigate the effect of dietary cholesterol (CHOL) levels on growth performance, body composition and gene expression of juvenile mud crab (Scylla paramamosain). Four isonitrogenous and isoenergetic diets were formulated with 0.4%, 0.8%, 1.2% and 1.6% CHOL supplemented, and the final dietary CHOL concentrations were 0.72%, 1.11%, 1.49% and 1.83% respectively. Each dietary treatment was performed with three replicates (28 crabs per replicate, initial body weight 0.04 g). At the termination of the experiment, although the survival had no statistic difference in all treatments, the mud crabs fed the lowest CHOL diet had the lowest survival rate. The weight gain (WG) of mud crab significantly increased with dietary CHOL level up to 1.11% and then significantly decreased with dietary CHOL level further increased. The total‐cholesterol (T‐CHOL) content in whole body had an increasing trend with the dietary CHOL level increased. Besides, dietary CHOL supplement generally increased the hepatic superoxide dismutase (SOD) activity, and the mud crabs fed diets CHOL1.11 and CHOL1.49 showed significantly higher value than those fed other diets. The hepatic aspartate aminotransferase (AST) activity decreased slightly with dietary CHOL level up to 1.11% and then significantly increased with CHOL level further increased. The mRNA expression of ecdysone receptor (EcR) gene in the eyestalk obviously increased with dietary CHOL level up to 1.11% and then significantly decreased with dietary CHOL further increased. These results suggested that about 1.11% dietary CHOL seem fulfil to maintain good growth performance and healthy condition for juvenile S. paramamosain.     

Effect of different forms of Artemia biomass as a food source on survival,molting and growth rate of mud crab (Scylla paramamosain)

An experiment was conducted to evaluate the effect of different forms of Artemia biomass as a food source on survival, molting and growth rate of mud crab Scylla paramamosain. Instar 1 crablets with a mean weight of 0.0082 ± 0.0007 g were reared both individually and communally and fed with different diets consisting of fresh shrimp meat (control feed), live Artemia biomass, frozen Artemia biomass and a dried Artemia‐based formulated feed for 40 days. The highest survival was obtained for crablets receiving live Artemia (92.5% and 75.8%) followed by the groups fed with frozen biomass (90.0% and 47.5%), the control feed (72.5% and 24.2%) and the dried Artemia‐based diet (60.0% and 21.7%) for individual and communal cultures, respectively. The intermolt period, the total number of moltings and the growth rate, which were determined on individually reared crabs, showed the same pattern as for survival. The results suggest that crab performance decreased in the following order: live Artemia>frozen Artemia > fresh shrimp meat > dried Artemia‐based formulated feed. Live Artemia biomass proved an ideal feed for nursery of Scylla paramamosain crabs. Frozen Artemia biomass may be an alternative in times of shortage. Our findings illustrate the high potential for local utilization of Artemia biomass in Vietnam for reliable production of mud crab juveniles.     

Size at sexual maturity and body size composition of mud crabs <Emphasis Type="Italic">Scylla</Emphasis> spp. caught in Don Sak,Bandon Bay,Gulf of Thailand

Size at sexual maturity and body size composition of mud crabs Scylla spp. were examined as the basis for settling a minimum landing size as a fishing regulation in Don Sak, Bandon Bay, Gulf of Thailand, which has suffered serious mangrove habitat degradation. Mud crabs were caught using baited traps and gill nets. Hooked metal rods were also used to lever the crabs out of their burrows inside the mangroves. Two mud crab species, S. paramamosain and S. olivacea, occur in the bay; S. paramamosain is the dominant species, accounting for 87% of the samples. The size at which 50% of the S. paramamosain females reached maturity (SM₅₀) was estimated as an external carapace width (ECW) of 112.0 mm based on the morphology of the abdomen. Allometric changes in the crushing chelae height to ECW ratio suggested that the SM₅₀ of S. paramamosain males occurred at 106.4 mm ECW. The body size composition revealed that mainly immature mud crabs were exploited in Don Sak. To maintain a sustainable fishery for mud crabs, fishing regulations—including a minimum landing size based on the SM₅₀ estimates—are essential, as is habitat restoration.     

Molecular characterization of transformer‐2 gene in the mud crab Scylla paramamosain and its putative role in sexual development

Transformer‐2 (Tra‐2) is an mRNA splicing factor that acts in concert with Tra to regulate the female sexual development in Drosophila melanogaster. In decapods, as no Tra homologues have been identified, the role of Tra‐2 in sexual development is more often discussed accordingly. In the present study, the full‐length cDNA of Tra‐2 of the mud crab Scylla paramamosain (SpTra‐2) was cloned and characterized. The deduced amino acid sequence of SpTra‐2 contained an RNA‐recognition motif (RRM) flanking by two RS‐rich regions, which are the hallmarks of the Tra‐2 proteins. The SpTra‐2 mRNA was highly abundant in the hepatopancreas, muscles and androgenic glands of the male crabs, implying a potential role in male sexual development. The hypothesis was supported by the RNAi experiments, finding that the injection of SpTra‐2 dsRNA in vivo led to a decreased expression of SpIAG, a key gene in crustacean male sexual differentiation. The relationship between SpTra‐2 and SpIAG was further affirmed by eyestalk ablation (ESA) experiments, which caused SpTra‐2 expression earlier than SpIAG expression in both androgenic glands and hepatopancreas. In addition, treatment with SpTra‐2 dsRNA also reduced the mRNA level of SpSxl, but had no effects on SpDmrt expression, suggesting the sex determination system in S. paramamosain is different from that in D. melanogaster.     

“Butter Crab”: an environment‐induced phenotypic variation of Scylla paramamosain with special nutrition and flavour

An “abnormal” female Scylla paramamosain, named “Butter crab” (B_♀) is more popular than wild‐type mud crab (CK) with people. Histologically, “butter crab” ovaries are partially degraded, with full of yellow oily substances in body. Composition of the essential nutritional components is quite similar in “butter crab” and CK. In a comparison of fatty acids, B_♀ is richer. A comparison of flavour amino acids and nucleotide in muscle showed that sweet amino acids and bitter amino acids in B_♀ is lower than CK_♀, but umami amino acids are significantly higher in B_♀. However, the umami amino acid content in the gonads is significantly less in B_♀ than CK_♀. Moreover, umami nucleotides in B_♀ muscle is significantly higher. From the perspective of EUC evaluation, the umami taste of CK_ ♀ gonads is strongest, followed by B_ ♀ gonads. In the hepatopancreas, the umami taste ranks CK_♀> B_♀ >CK_♂, and there are no significant differences among muscles in the three groups. In conclusion, the characteristics of “butter crab” are more fragrant and nutritious, with sweet and umami taste. The article systematically scientifically analysed the nutritional composition and flavour characteristics of “butter crab”, providing a novel insight for development of S. paramamosain industry.     

Influence of highly unsaturated fatty acids in live food on larviculture of mud crab Scylla paramamosain (Estampador 1949)

Two experiments were carried out to investigate the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (ARA) levels in rotifers (Brachionus plicatilis) and Artemia on the survival, development and metamorphosis of mud crab Scylla paramamosain larvae. Five different lipid emulsions, varying in the level of total n‐3 and n‐6 highly unsaturated fatty acids (HUFA), DHA, EPA and ARA were used to manipulate the fatty acid profile of the live food. Fatty acid profiles of the live food and crab larvae at zoea one, three and five stages were analysed to study the HUFA uptake by the larvae. The fatty acid content of the live food affected the fatty acid profiles of the crab larvae. In both experiments, the survival rate in the zoeal stages was not statistically different among treatments. However, larval development rate and metamorphosis success were affected by the dietary treatments. In this respect, the DHA/EPA ratio in the live food seems to be a key factor. Enrichment emulsions with a very high (50%) total HUFA content but a low DHA/EPA ratio (0.6), or zero total HUFA content caused developmental retardation and/or metamorphosis failure. An emulsion with a moderate total HUFA (30%) and a high DHA/EPA ratio (4) was the best in terms of larval development during the zoeal stages and resulted in improved metamorphosis. Dietary ARA seemed to improve first metamorphosis, but its exact role needs further clarification. For the larval rearing of S. paramamosain, an enrichment medium containing about 30% total n‐3 HUFA with a minimum DHA/EPA ratio of 1 is recommended. Further investigation is needed on the total HUFA and optimum DHA/EPA ratio requirements for each crab larval stage.     

Immune Response of Mud Crab,Scylla Paramamosain,to Bacterial Lipopolysaccharide

Vibrio spp. is one of the main pathogens to cause high levels of mortality in the culture industry of mud crabs, Scylla paramamosain. Although considerable efforts have been devoted to understanding the immune response of S. paramamosain against pathogens, both immune‐response proteins and systematic responses to foreign agent invasions have yet to be investigated in detail. Hence, in this study, we challenged S. paramamosain with bacterial lipopolysaccharides (LPS) by injection. We examined a number of immune‐related indices in the crabs at 0, 2, 6, 12, 24, 36, 48, and 72 h postinjection, that is, the total hemocyte counts in hemolymph; the serum activities of phenoloxidase, superoxide dismutase, lysozyme, acid phosphatase, and alkaline phosphatase; and the mRNA levels of immune‐related genes encoding prophenoloxidase, serine protease inhibitors, antimicrobial peptide scygonadin, crustin, anti‐lipopolysaccharide factor, and catalase in hemocytes. In addition, the immune‐response proteins were identified in hemocytes at 24 h postinjection using two‐dimensional polyacrylamide gel electrophoresis. The results showed that the immune‐related enzymes and genes examined were induced in a time‐dependent manner post‐LPS challenge. Moreover, 18 altered proteins were present in the hemocytes and the main proteins to be altered were highly homologous to hemocyanin and protein kinase. Ultimately, this study provides useful information for understanding the immune response of S. paramamosain to bacterial infection.     

中国东南沿海青蟹线粒体COI基因部分序列分析

对我国东南沿海5个地区的72个青蟹个体的线粒体细胞色素氧化酶亚基I (COI)部分序列序列进行了测定和分析。获得的72个细胞色素氧化酶亚基I (COI)序列可分为12个单倍型，与GenBank中已知的Scylla paramamosain COI序列的相似性达到98%以上，与其它3种 青蟹的差异为7.36%～15.54%。这些序列与S. paramamosain的遗传距离仅为0.00783，但是与S. serrata、S. olivacea和S. tranquebarica〗的遗传距离却分别达到0.11659、0.17812和0.08423。序列特征、遗传距离和系统进化等分析结果都表明本文研究的青蟹为S. paramamosain。结果提示，在进行青蟹属相关研究应当仔细鉴别采集样本的种类。     

Improved techniques for rearing mud crab Scylla paramamosain (Estampador 1949) larvae

A series of rearing trials in small 1 L cones and large tanks of 30–100 L were carried out to develop optimal rearing techniques for mud crab (Scylla paramamosain) larvae. Using water exchange (discontinuous partial water renewal or continuous treatment through biofiltration) and micro‐algae (Chlorella or Chaetoceros) supplementation (daily supplementation at 0.1–0.2 million cells mL⁻¹ or maintenance at 1–2 millions cells mL⁻¹), six different types of rearing systems were tried. The combination of a green‐water batch system for early stages and a recirculating system with micro‐algae supplementation for later stages resulted in the best overall performance of the crab larvae. No clear effects of crab stocking density (50–200 larvae L⁻¹) and rotifer (30–60 rotifers mL⁻¹) and Artemia density (10–20 L⁻¹) were observed. A stocking density of 100–150 zoea 1 (Z1) L⁻¹, combined with rotifer of 30–45 mL⁻¹ for early stages and Artemia feeding at 10–15 nauplii mL⁻¹ for Z3–Z5 seemed to produce the best performance of S. paramamosain larvae. Optimal rations for crab larvae should, however, be adjusted depending on the species, larval stage, larval status, prey size, rearing system and techniques. A practical feeding schedule could be to increase live food density from 30 to 45 rotifers mL⁻¹ from Z1 to Z2 and increase the number of Artemia nauplii mL⁻¹ from 10 to 15 from Z3 to Z5. Bacterial disease remains one of the key factors underlying the high mortality in the zoea stages. Further research to develop safe prophylactic treatments is therefore warranted. Combined with proper live food enrichment techniques, application of these findings has sustained a survival rate from Z1 to crab 1–2 stages in large rearing tanks of 10–15% (maximum 30%).     

Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development

Treated with combined bilateral eyestalk ablation and maintenance of water temperature at 22.5±1.5 °C, mud crab Scylla paramamosain females with mature ovaries were induced to produce eggs outside the natural spawning season in subtropical southern China. Newly extruded eggs from a crab were incubated in vitro at 10, 15, 20, 25, 27, 30, 35 °C, respectively, and the embryonic development was closely monitored. Abnormal cell division was observed at temperatures 10 and 35 °C. At 15 °C, development remained at the gastrula stage by day 32 post‐spawn, at which time the experiment was terminated. Hatching of in vitro incubated eggs occurred between 20 and 30 °C. An increase in incubation temperature from 20 to 25 °C reduced the incubation duration by 14 days, 2.6 times of that measured for a similar 5 °C increase from 25 to 30 °C. Embryonic development of S. paramamosain was divided into stage 0–10, and the duration of each stage was recorded for each incubation temperature. The information obtained allows accurate prediction of hatching time of female crabs incubated under variable temperatures. Larvae hatched from in vitro incubated eggs were reared to reach first juvenile crab stage and their dry weights were similar to those of larvae hatched naturally.     

Tissue distribution of hepatopancreatic parvo‐like virus of shrimp in freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of hepatopancreatic parvo‐like virus (HPV) of shrimp in different organs of freshwater rice‐field crab Paratelphusa hydrodomous (Herbst) using bioassay, PCR, RT‐PCR, ELISA, Western blot and q‐PCR analyses. Another attempt was made to use this crab as an alternative to penaeid shrimp for the large‐scale production of HPV. This crab was found to be highly susceptible to HPV by intramuscular injection. The systemic HPV infection was confirmed by PCR and Western blot analyses in freshwater crab. The expression of capsid protein gene in different organs of infected crab was revealed by RT‐PCR analysis. Indirect ELISA was used to quantify the capsid protein in different organs of the crab. The copy number of HPV in different organs of the infected crab was quantified by q‐PCR. The results revealed a steady decrease in C_T values in different organs of the infected crab during the course of infection. The viral inoculum that was prepared from different organs of the infected crab caused significant mortality in post‐larvae of tiger prawn, Penaeus monodon (Fabricius). The results revealed that this rice‐field crab could be used as an alternative host for HPV replication and also for large‐scale production of HPV.