A chimeric recombinant crustacean hyperglycemic hormone from Litopenaeus schmitti (Burkenroad) obtained as C‐terminus fusion protein boost hemolymph glucose concentration in Litopenaeus vannamei (Boone)

To date, no hormonal treatments are available for control of shrimp reproduction and only eyestalk ablation is of practical use. The crustacean hyperglycemic hormone (CHH) is the most abundant neuropeptide of the eyestalk CHH family. It plays an important role in the regulation of hemolymph glucose levels, as its principal function, but it is also implicated in additional physiological processes such as moulting and reproduction. In the present study, the cDNA encoding Litopenaeus schmitti (Burkenroad) mature CHH was cloned into the Escherichia coli pTYB2 expression vector. Using this strategy we have obtained, for the first time, the recombinant CHH from L. schmitti with its C‐terminus fused to an intein tag. The expected fused protein of about 63 KDa was expressed in E. coli forming inclusion bodies. It was purified in a soluble form by electroelution following molecular size fractionation in sodium dodecil sulphate polyacrylamide gel electrophoresis. The ability of the chimeric CHH protein to elevate glucose levels in the hemolymph of the eyestalk‐ablated Litopenaeus vannamei (Boone) shrimps indicates that its biological activity as hyperglycemic protein is preserved. The results provide an alternative tool to obtain soluble recombinant proteins from the CHH family of neuropeptides to get a better understanding of shrimp endocrinology.     

Effect of recombinant crustacean hyperglycemic hormones rCHH‐B1 and rCHH‐B2 on lipid metabolism in the Pacific white shrimp Litopenaeus vannamei

The crustacean hyperglycemic hormones (CHH) are pleiotropic neuropeptides controlling diverse processes in the life cycle of crustaceans. In the Pacific white shrimp Litopenaeus vannamei, CHH‐B1 and CHH‐B2 are generated by alternative splicing of the same gene, and the recombinant versions of these peptides have shown differential effects on glucose metabolism and osmoregulation in this species. In this work, we explored the role of both recombinant variants, expressed in yeast Pichia pastoris, on lipid metabolism. The results showed that rCHH‐B1 and rCHH‐B2 injections into eyestalk‐ablated shrimp significantly increased the phospholipid and triglyceride levels in haemolymph and restored free fatty acid levels, suggesting that these CHH variants mobilize lipid reserves in shrimp. The effects on lipids were concomitant to its hyperglycemic activity, suggesting that both variants, in addition to their hyperglycemic activity, have hyperlipidemic functions.     

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.     

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.     

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.     

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.     

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。     

The Effect of Bilateral Eyestalk Ablation on Signal Transduction Pathways of Ion Regulation of Litopenaeus vannamei

A study was performed on the effects of bilateral eyestalk ablation on signal transduction pathways of ion regulation of Litopenaeus vannamei. The study included three treatments (starvation group, bilateral eyestalk ablation, and starvation and bilateral eyestalk ablation) in addition to a control group. The shrimp were sampled at 0, 12, 24, and 48 h. Results showed that the ablation of bilateral eyestalk had significant effects on the contents of three kinds of biogenic amines (BAs), cyclic guanosine monophosphate (cGMP), and the activities of three kinds of ion‐transport enzymes (P < 0.05). According to these results, bilateral eyestalk ablation had significant effects on the ion signal pathway of L. vannamei. The same changes were observed in 5‐hydroxytryptamine (5‐HT) contents, Na⁺‐K⁺‐ATPase, and HCO₃^?‐ATPase activities, suggesting that crustacean hyperglycemic hormone (CHH) regulated the changes in ion‐transport enzymes, mediated by BAs and cGMP. The specific pathways may be 5‐HT → cGMP → Na⁺‐K⁺‐ATPase, HCO₃^?‐ATPase, and BAs → cGMP → V‐ATPase. 5‐HT contents, Na⁺‐K⁺‐ATPase, and HCO₃^?‐ATPase activities in the starvation group were ultimately higher than those in the bilateral eyestalk ablation group, while the cyclic adenosine monophosphate (cAMP) contents increased slightly. Study results suggested that under the situation of bilateral eyestalk ablation, the shrimp could also use feed or its metabolites to increase 5‐HT or cAMP contents to regulate the Na⁺‐K⁺‐ATPase and HCO₃^?‐ATPase activities in gills.     

Temporal and spatial dynamics of white spot syndrome virus in the Chinese mitten crab,Eriocheir sinensis

White spot syndrome virus (WSSV) is the most severe viral pathogen to the crustacean aquaculture industry worldwide. Recently, serious WSSV outbreaks caused catastrophic losses in the Chinese mitten crab, Eriocheir sinensis in Jiangsu Province, Eastern China. However, to date, little is known about its infection mechanism in the new natural host. This study aimed to reveal the temporal and spatial dynamics of WSSV in E. sinensis. The slow viral growth in the early stage of infection was the light infection stage (from 0 to 24 hpi), and the exponential growth stage that followed was the logarithmic phase (from 24 to 72 hpi). The viral growth curve ended with the plateau phase (from 72 to 144 hpi) which demonstrated a consistent high level of viral load and accompanied heavy crab mortality. The viral load increased as time progressed with similar growth curves, however, at different degrees. The viral copy numbers of tissues at different time intervals, analysed using one‐way analysis of variance (anova ), showed significant differences between tissues at all time points (P < 0.05). Infection was detectable as early as 6 hpi in all the tissues screened. The severity of infection was found to be maximum in gill and pleopods, which could be recommended for diagnostic testing. This study might provide important data to analyse theoretically the interaction between WSSV and the host.     

Isolation and characterization of an additional crustacean hyperglycemic hormone from the greasyback shrimp <Emphasis Type="Italic">Metapenaeus ensis</Emphasis>

Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks. In this study, the most abundant CHH in the sinus gland of the greasyback shrimp Metapenaeus ensis was purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. Although two CHH molecules (Mee-CHH-A and Mee-CHH-B) have already been identified from M. ensis by cDNA cloning, this study revealed the presence of an additional CHH peptide based on differences in the N-terminal amino acid sequences of the CHH-A and CHH-B. Therefore, this novel CHH was designated as Mee-CHH-C. A cDNA encoding the Mee-CHH-C precursor was cloned by RT-PCR coupled with 5′- and 3′-RACE, and it was found that the mature Mee-CHH-C consisted of 72 amino acid residues containing 6 conserved cysteine residues and possessed an amidated C terminus. Mee-CHH-C had 62 and 68% identities with Mee-CHH-A and Mee-CHH-B, respectively, and was highly homologous to CHHs characterized from other penaeid shrimp species. The hyperglycemic activity of Mee-CHH-C was examined by an in vivo bioassay using the kuruma prawn Marsupenaeus japonicus. Injection of Mee-CHH-C increased hemolymph glucose levels significantly and dose-dependently. These results indicate that Mee-CHH-C is possibly one of the major molecules in M. ensis that regulate glucose levels in the hemolymph.     

Dietary astaxanthin supplementation for hatchery‐cultured red king crab,Paralithodes camtschaticus,juveniles

We conducted large‐scale production trials in Seward, Alaska, USA to investigate effects of dietary astaxanthin supplementation on survival, growth and shell colouration of recently settled juvenile (C1–C4) red king crabs (Paralithodes camtschaticus). We supplemented a control diet of commercial crustacean feeds with astaxanthin, and fed these diets to juvenile king crabs at densities of 2000 and 4000 crabs m^?2 for 56 days. We assessed survival and growth by counting crabs and individually measuring carapace width and weighing crabs at the start and end of the experiment, and quantified crab colour (hue, saturation, brightness) in digital photographs. Diets containing astaxanthin had higher survival, suggesting that astaxanthin may provide nutritional or immune system benefits. Crabs had lower hue, higher saturation and lower brightness values when fed diets containing astaxanthin, suggesting that red king crab colouration is plastic and responds to diet. Astaxanthin is likely an important dietary component for hatchery or laboratory reared red king crab juveniles, and should be considered for aquaculture and other rearing of this and possibly other crustacean species.     

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.     

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.     

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.     

Assessment of dietary lecithin and cholesterol requirements of mud crab, Scylla serrata, megalopa using semi-purified microbound diets

The effects of varying dietary lecithin and cholesterol levels on growth, development and survival of mud crab, Scylla serrata, megalopa were evaluated using six semi‐purified, microbound diets formulated to be iso‐energetic and containing three levels of supplemental lecithin (0, 20 and 40 g kg⁻¹ diet dry weight) and two levels of supplemental cholesterol (0 and 7 g kg⁻¹ diet dry weight). Fifteen megalopa were reared individually in each treatment and the nutritional value of diets was assessed on basis of mean dry weight and mean carapace width of newly settled first crab stage, as well as development time to the first crab stage and overall survival. A significant interaction between supplemental dietary lecithin and supplemental dietary cholesterol was found for final mean dry weight of newly settled crabs, and highest survival (60%) was recorded for megalopa fed diets containing the highest levels of dietary lecithin (39.7–44.1 g kg⁻¹) (diet 5 and 6) regardless of whether diets were supplemented with cholesterol; this rate of survival was identical to that of megalopa fed live Artemia nauplii. The results indicate that supplemental dietary cholesterol may not be essential for mud crab megalopa when fed diets containing sufficient levels of supplemental dietary phospholipids.     

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.