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.     

Parentage determination of the mud crab Scylla paramamosain using microsatellite markers

This study evaluated the properties of nine Scylla paramamosain microsatellite loci screened by us previously for inclusion in a parentage assignment marker suite. These nine highly polymorphic markers (mean He=0.847 and PIC=0.830) were determined as being suitable for parentage assignment. Simulations based on allele frequency data from 15 known maternal families (165 individuals) demonstrated that at least four loci were required to assign >95% of offspring to maternal parents with 95% confidence. In actual parentage assignments, all progenies were assigned to the maternal parents with six or more loci, which was similar to the simulation predictions. Our results suggest that this set of microsatellites provide a powerful and efficient tool for identifying pedigree information for selective breeding programmes of S. paramamosain.     

Overwintering behaviour affects distinctive flavour of mud crab Scylla paramamosain in commercial farms

In order to reveal the influence of overwintering behaviour on the flavour of Scylla paramamosain, this study selected crabs of before and after overwintering in SM (Sanmen County, Zhejiang Province), HP (Hepu County, Guangxi Province) and WN (Wanning city, Hainan Province). The contents of flavouring‐related substances in hepatopancreas, muscles and gonads were determined by high‐performance liquid chromatography (HPLC); taste activity value (TAV) and equivalent umami concentration (EUC) were calculated. The results showed that sweetish amino acids were mainly accumulated in muscles, umami amino acids were mainly accumulated in hepatopancreas, free nucleotides were mainly accumulated in gonads, and the EUC value in gonads was the highest. After overwintering, the content of sweetish, umami amino acids and free nucleotides in muscle and hepatopancreas decreased significantly, while the content of flavour‐tasting amino acids in gonads did not change significantly, but the content of umami nucleotides increased significantly. Therefore, overwintering behaviour decreased the flavour of muscle and hepatopancreas, slightly enhanced gonad and the overall flavour of S. paramamosain before overwintering was better than that after overwintering.     

拟穴青蟹保幼激素酯酶结合蛋白(JHEBP)基因的克隆与表达分析

甲基法尼酯(MF)是甲壳类的保幼激素(JH), MF的代谢可能主要由保幼激素酯酶(JHE)催化完成,保幼激素酯酶结合蛋白(JHEBP)能紧密结合JHE,在JHE的代谢中具有重要作用,研究JHEBP对于MF降解通路的探究具有一定促进作用。本研究获得了拟穴青蟹JHEBP基因的cDNA全序列,命名为Sp-JHEBP。Sp-JHEBP序列全长为1647 bp,包含一个891 bp的开放阅读框(ORF),可编码296个氨基酸,预测相对分子质量33.79 kD,等电点8.63。利用Target P预测Sp-JHEBP定位于线粒体, Mitoprot预测Sp-JHEBP的前31个氨基酸组成的肽段为线粒体定位信号肽。多重序列比对分析发现, Sp-JHEBP的氨基酸序列与多齿新米虾(Neocaridina denticulata)的一致度最高(67%),其次为黑脉金斑蝶(Danaus plexippus)(43%)。系统发育树分析JHEBP与物种分化并不一致,这表明JHEBP随着物种分化,功能上可能也出现了分化。Sp-JHEBP在所检测的9个组织中均有表达,在卵巢中的表达量远高于其它组织,其次是鳃、血淋巴、肝胰腺。在幼体发育过程中,Sp-JHEBP在受精卵时期最高,从Z1至Z5期逐渐升高,到Z5期达到最高,随后逐渐下降到C1期。体外处理MF和法尼酸(FA)显示Sp-JHEBP受高浓度MF上调,在0.1～5μmol/L之间随着FA浓度升高Sp-JHEBP表达量升高。综合分析我们认为Sp-JHEBP可能通过降解JHE来参与MF的代谢,该研究结果对青蟹MF降解分子网络的研究具有一定的推动作用。     

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.     

Effects of diets supplemented with either individual or combined probiotics,Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, on the immune response and disease resistance of the mud crab,Scylla paramamosain (Estampador)

Two trials, including firstly, diets incorporating individual or combined species of probiotics, Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, were fed to the mud crab, Scylla paramamosian, for 28 days to evaluate the growth, immunity and disease resistance, and secondly, the optimal level of B. subtilis E20 in the diet by tracking the growth, immunity and disease resistance after 28 days of feeding were analysed. No significant differences in growth, total haemocyte counts, respiratory bursts, superoxide dismutase, or glutathione peroxidase were found in the two trials. Crabs fed the B. subtilis E20‐containing diet (D20) had significantly increased phenoloxidase (PO) activity, phagocytic activity (PA) and disease resistance compared with crabs fed the control and the mixed probiotics diet (MD). The mortality of crabs fed the D20 was significantly lower than that of crabs fed the L. plantarum 7‐40‐containing diet. L. plantarum 7‐40 had a great inhibitory effect on the growth of B. subtilis E20, which may have led to the decreased probiotic effect of the MD. An analysis of the optimal level of B. subtilis E20 in the diet showed that crabs fed the B. subtilis E20‐containing diet at a level of 10^9–10 cfu kg^?1 had better immune response of PO and PA, and disease resistance in the second trial.     

Effects of different dietary lipid sources on tissue fatty acid composition,serum biochemical parameters and fatty acid synthase of juvenile mud crab Scylla paramamosain (Estampador 1949)

A 6‐week study was conducted to determine the effects of different lipid sources in pelleted diets on juvenile mud crab Scylla paramamosain. Five isonitrogenous and isolipidic diets containing 8% level of fish oil (FO), lard (LD), safflower oil (SO), perilla seed oil (PO) or mixture oil (MO; V_FO:V_SO:V_PO = 1:1:1), and a live food of marine bivalve Potamocorbula rubromuscula as the control diet (CF), were fed to groups of 25 juvenile crabs (average initial weight 7.4 g, carapace width 3.5 cm) in triplicate. The results showed that crabs fed MO had the highest survival (P < 0.05). The moisture content was significantly higher in crabs fed LD, SO and PO (P < 0.05). Crabs fed SO exhibited the lowest crude protein and lipid (P < 0.05). Ash contents were obviously lower in LD group (P < 0.05). Highest total lipid in the hepatopancreas and muscle was in LD and FO group respectively. Glucose, total cholesterol and low‐density lipoprotein were higher while high‐density lipoprotein was lower (P < 0.05) in LD group. Tissue fatty acid compositions were consistent with those in diets. FO and MO diets had the same depression effect like CF on fatty acid synthase activity and mRNA expression in the hepatopancreas. The results of this study indicated that FO and mixed oil are suitable for preparation of pelleted diets with better effects for juvenile S. paramamosain compared with live food, and the ratio of n‐6/n‐3 fatty acids in pelleted diets must be <1.     

Induction of spawning in Pacific white shrimp Litopenaeus vannamei (Boone, 1931) by injection of its molt inhibiting hormone isoform II produced in E. coli

The optimization of reproductive parameters in shrimp farming continues to be a challenge for most producing countries. Although the crustacean neuropeptides have been studied extensively in the last two decades, the functions of most of these neuropeptides remained putative. Among them, molt‐inhibiting hormone isoform II (MIH II) has shown an important role in vitellogenesis. In this study, the cDNA encoding mature MIH II peptide was isolated by RT‐PCR from the L. vannamei eyestalk. The cDNA was cloned into pET28a bacterial expression vector. Recombinant MIH II was obtained in the form of insoluble inclusion bodies and purified to ~88% purity. Two doses of rMIH II and a negative control group were assayed in vivo. The stages of ovarian maturation and spawning were recorded during 72 hr post‐injection. The results showed that ovarian maturation occurred approximately in 9% and 33% of females injected with rMIH II at the doses of 300 and 600 ng/gbw respectively. Neither maturation nor spawning was detected in the negative control group. Females injected with 600 ng/gbw, which showed vitellogenic stages III and IV, spawned. These preliminary results argue that the hormone rMIH II could be a promising candidate to induce spawning in L. vannamei shrimp.     

拟穴青蟹不同发育时期胚胎基因表达的内参基因的筛选

为获得适用于研究拟穴青蟹(Scylla paramamosain)不同发育时期胚胎基因表达的内参基因,本研究采用3个内参基因筛选软件geNorm、NormFinder及BestKeeper对微管蛋白α1A(Tuba1a)、核糖体蛋白L13(Rpl13)、泛素(UBQ)、核糖体蛋白S6激酶(Rps6)、精氨酸激酶(Ak)、3-磷酸甘油脱氢酶(gapdh)、28S核糖体RNA(28S)、18S核糖体RNA(18S)和延伸因子1A基因(EF-1α)等9个常用候选内参基因在拟穴青蟹不同发育时期胚胎的表达稳定性进行分析。geNorm分析表明, EF-1α和Rpl13的表达稳定性最高,并且采用2个内参基因同时校正目标基因的定量结果可以达到最优效果; NormFinder分析表明, gapdh和EF-1α是9个候选内参基因中表达最稳定的; BestKeeper分析表明,gapdh的标准偏差和变异系数是9个候选基因中最低的。综合3个内参基因筛选软件,可以选用EF-1α,或选用EF-1α和gapdh双内参同时校正拟穴青蟹不同胚胎发育时期各基因的表达情况。     

Identification of SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene and their association with precocity of the Chinese mitten crab Eriocheir sinensis

Moult‐inhibiting hormone (MIH), an important regulator of steroidogenesis, inhibits the synthesis of ecdysteroid in Y‐organ (YO) and plays a significant role in the regulation of moulting and post‐embryonic development of crustacean. Because unsuccessful moulting have been widely observed in precocious crabs, we investigated whether genetic variants in the 5′‐flanking region and 3′‐untranslated region (3′‐UTR) of the MIH gene are associated with precocity of the Chinese mitten crab. Thirty individual DNA samples were sequenced to search for SNPs in the 5′‐flanking region and 3′‐UTR of the MIH gene. Five SNPs (g.196 T>A, g.230 C>T, g.305 T>C, g.323 C>A and g.372 C>T) in the 5′‐flanking region and 6 SNPs (g.2677 C>T, g.2759 T>A, g.2807 T>C, g.3042 A>G, g.3088 T>G and g.3295 T>G) in the 3′‐UTR of the MIH gene were selected for the individual genotyping in a two‐stage association study. We found that a SNP g.3088 T>G in the 3′‐UTR of MIH gene was consistently associated with precocity of the Chinese mitten crab in stage 1 and stage 2, with a per‐allele OR (Odds Ratio) of 1.469 (95% CI: 1.169–1.844) after two stages combined (P = 0.001). However, no significant associations were observed between the other 10 SNPs and precocity of the Chinese mitten crab. To our best knowledge, this is the first association study between various SNP genotypes and phenotype attributes in Chinese mitten crab. Our findings suggest that the SNP g.3088 T>G may be a candidate marker for effective marker‐assisted selection to decrease the precocity of the Chinese mitten crab in future studies.     

“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.     

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。     

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.     

Lipids and fatty acids in wild and pond-reared mud crab Scylla serrata (Forsskål) during ovarian maturation and spawning

Wild‐caught and pond‐reared female mud crab Scylla serrata at different stages of ovarian maturation were collected from Samar and Capiz, Philippines. Crabs were categorized into five stages according to the external morphological and microscopic appearance of the most advanced oocytes. The ovaries, hepatopancreas, muscle and newly spawned eggs (NSE) were analysed for lipid class components and fatty acids. Total lipid was higher in pond‐reared than in wild‐caught crabs but increased with ovarian maturation in both groups. Ovarian lipid peaked at the fully mature stage, coinciding with a decline in hepatopancreatic and muscle lipids. Lipid levels declined significantly in spent females. The tissues contained elevated highly unsaturated fatty acids such as arachidonic (20:4n‐6), eicosapentaenoic (20:5n‐3) and docosahexaenoic (22:6n‐3) acids, but at higher levels in late maturing and fully mature ovaries and in NSE. The type of lipid class and fatty acid components in mature ovaries as well as in NSE are generally considered to be indicative of their importance in reproductive physiology and embryonic and larval development.     

饲料蛋白质水平对拟穴青蟹幼蟹生长性能、体成分和消化酶活性的影响

用蛋白质水平分别为31.45%,36.37%,41.55%,46.13%,51.72%和56.86%的6种等能饲料,对初始体重为(11.86?0.11)mg的拟穴青蟹(Scylla paramamosain)幼蟹进行为期3周的养殖实验,考察饲料蛋白质水平对拟穴青蟹幼蟹生长性能、体成分以及消化酶活性的影响,以期获得拟穴青蟹幼蟹饲料蛋白质的适宜添加量。结果表明,饲料蛋白质水平对拟穴青蟹幼蟹的成活率影响不显著(P0.05);幼蟹的增重率和特定生长率随饲料蛋白质水平的提高先显著升高(P0.05)后稍有下降,最大值出现在蛋白质水平为51.72%的实验组;随着饲料蛋白质水平的提高,幼蟹粗蛋白质含量显著升高(P0.05)后趋于稳定,粗脂肪含量显著降低(P0.05),灰分含量先显著升高(P0.05)后显著降低(P0.05),水分含量则没有显著变化(P0.05);随着饲料蛋白质水平的提高,幼蟹蛋白酶活性显著升高(P0.05),淀粉酶活性显著降低(P0.05),各组脂肪酶活性也有显著差异(P0.05),但没有明显的变化规律。本研究条件下,饲料蛋白质水平在41.55%~56.86%,拟穴青蟹幼蟹均表现出较好的生长率,增重率和蛋白质水平的回归分析表明,幼蟹饲料蛋白质适宜水平为49.03%。