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.     

Comparative study on the nutrition composition of adult male Chinese mitten crab (Eriocheir sinensis) with different coloured hepatopancreases

High lethality of white hepatopancreas syndrome has caused large economic loss of Chinese mitten crab (Eriocheir sinensis). Hepatopancreas colour of diseased crabs is gradually lighter, turning from golden yellow to yellow‐white to white. This study investigated composition of conventional nutrients in three edible parts of different coloured hepatopancreases of adult males, including normal yellow hepatopancreas group (Control), yellow‐white hepatopancreas group (YWH) and white hepatopancreas group (WH). No significant differences were observed in tissue indices and total edible yield among three crab types; however, condition factor level in crabs of YWH and WH was significantly lower. Dry matter content was significantly reduced in crabs of WH, especially crude fat level of hepatopancreas in WH males. Levels of all the amino acids of WH crabs in muscles and gonads were significantly lower than that of control. Proportion of saturated fatty acids and monounsaturated fatty acids were much lower in hepatopancreas of WH crabs than those of control, while proportion of polyunsaturated fatty acids and highly unsaturated fatty acids were significantly higher. Overall, YWH and WH do not affect growth. WH crabs showed poor nutritional quality, and nutritional value of YWH was in the middle as transition state between control and WH.     

Effect of lactic acid on enrofloxacin pharmacokinetics in Eriocheir sinensis (Chinese mitten crab)

As a representative acidifier, lactic acid (LA) is widely used in the diets of aquatic animals. LA is the main supporter of energy metabolism and may be associated with drug metabolism. This study was designed to explore the pharmacokinetics of enrofloxacin (ENR) in Eriocheir sinensis (Chinese mitten crab) fed diets containing 0.1%, 0.2% and 0.3% LA (designated groups LA1, LA2 and LA3 respectively). The concentrations of ENR in the haemolymph, hepatopancreas and muscle were determined by HPLC after a single oral dose of 10 mg/kg. Our results showed that LA had a significant effect on the peak ENR concentrations in all the tissues (p < 0.05) by one‐way ANOVA analysis. There was a trend that C_max (peak concentration) of ENR was elevated with LA levels increasing up to 0.3% in haemolymph, hepatopancreas, muscle and T_max (time‐point of the peak concentration of the drug), t_1/2β (elimination half‐life) and AUC_(0‐∞) of ENR were shortened. Taken together, 0.3% LA might be effective in improving ENR pharmacokinetics in E. sinensis. Furthermore, it can be speculated that the enhanced biotransformation of ENR in the hepatopancreas mediated by LA is responsible for the differences in the pharmacokinetics of ENR in E. sinensis.     

Screening and identification of microRNAs during larval metamorphic development of Chinese mitten crab Eriocheir sinensis

MicroRNAs (miRNAs) represent a class of endogenous small non‐coding RNAs with 18–25 nucleotides that are involved in diverse biological processes. In the present study, differentially expressed miRNAs in four different metamorphic stages of Eriocheir sinensis were comprehensively detected and validated from high‐throughput sequencing miRNA data sets. We identified and characterized 597 known miRNAs and 901 novel miRNAs from the four small RNA libraries. We also demonstrated the evolutionary conservation of miRNAs from Chinese mitten crab with those from other species. Analyses of miRNA expression profiles during brachyurization development of the Chinese mitten crab revealed that 86 mature miRNAs were dysregulated, 47 of which were significantly differentially expressed. The results of real‐time quantitative PCR analyses for a few selected miRNAs were similar to those of deep sequencing analyses. Intriguingly, the results of Gene Ontology annotation and KEGG pathway analyses indicated that miRNA gene clusters and families were consistently dysregulated at four different metamorphic stages, although they were enriched at various levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways) of these miRNAs. The results suggest that these miRNAs might play potential key roles in the regulation of gene expression in the brachyurization development of the Chinese mitten crab. Particularly, miR‐2a‐3p, miR‐305‐5p, miR‐263a‐5p and miR‐7‐5p may play major regulatory roles in the metamorphosis of E. sinensis.     

Effects of hypoxia on immune responses and carbohydrate metabolism in the Chinese mitten crab,Eriocheir sinensis

In aquaculture, hypoxia is an important stressor that can inhibit the healthy development of Chinese mitten crab, Eriocheir sinensis. The aim of this work was to research on how hypoxia (1 mg/L) followed by reoxygenation affects the immune response and carbohydrate metabolism in the Chinese mitten crab. The activities of immune‐related enzymes including lysozyme (LZM), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and phenoloxidase (PO) and concentration changes in haemolymph glucose, trehalose and hepatic glycogen were evaluated after 8 hr of hypoxia followed by 12 hr of reoxygenation. After 8 hr of hypoxia, the activities of LZM, PO, SOD and hepatic glycogen concentration significantly decreased to the minimum when compared to the control group, but the activities of ACP and AKP, and haemolymph glucose concentration significantly increased. After 12 hr of reoxygenation, the activities of LZM, ACP, AKP and SOD recovered to the normal level except SOD in plasma, while the PO, SOD in plasma and hepatic glycogen concentration were still significantly lower than the control level. No significant change was observed in trehalose concentration during hypoxia and reoxygenation periods. These results indicated that although the activities of ACP and AKP were likely increased to facilitate the hydrolysis of foreign matter, the overall strength of the immune response was decreased as LZM, PO and SOD activities were suppressed after exposure to hypoxic conditions. The energy stores of exposed crabs were reduced due to the expenditure of energy during exposure to hypoxic stress. In general, chronic hypoxia would impair the immunocompetence and increase the energy expenditure of Chinese mitten crab, further increased the risk of pathogen infection and feed coefficient, so the negative effects of hypoxia should not be ignored.     

Common specific indigenous bacteria reside in the intestinal tract of Chinese mitten crab (Eriocheir sinensis)

In our previous study, five bacterial phylotypes of Mollicutes group 1, Mollicutes group 2, Bacteroides, Meniscus and Marinifilum were found to be dominant (abundance > 0.5%) in the intestine of Chinese mitten crab (CMC, Eriocheir sinensis) farmed in Lake Tai, China. To shed light on whether these five bacterial lineages are common specific indigenous intestinal bacteria, samples of adult CMCs collected from eight geographically separated farms in China, juvenile crabs, farming water and crab feed are subjected to analysing by using 16S rRNA gene sequencing and phylotype‐specific, real‐time quantitative PCR (qPCR). Four phylotypes of the Mollicutes group 1, the Mollicutes group 2, Bacteroides and Meniscus are detected, with relatively higher number, in all crab samples, including adults and juveniles. The Mollicutes group 1 and 2 are more dominant, with the group 1 more abundant in midgut while the group 2 in hindgut. The Marinifilum phylotype is almost undetectable in crab samples but abundant in the farming water and feed. By contrast, phylotypes of the Mollicutes group 1, the Mollicutes group 2, Bacteroides and Meniscus are neither detectable in the water nor in feed. These four lineages appear to be the common specific indigenous intestinal bacteria in the entire CMC species, with the Mollicutes group 1 and 2 likely serving as the major symbiotic players in CMCs. Their beneficial contributions to CMC host await future deep investigation.     

Cottonseed meal protein hydrolysate stimulates feed intake and appetite in Chinese mitten crab,Eriocheir sinensis

One hundred and twenty crabs (initial weight: 51.32 ± 0.06 g) were fed three experimental diets with cottonseed meal protein hydrolysate (CPH) at 0, 6 and 12 g/kg for 60 days. The results showed that CPH0.6 diet significantly improved the 1‐hr feed intake, protease activity and weight gain of crabs. In thoracic ganglia, 4E‐binding protein (4E‐BP) expression level of crab fed the diets containing CPH was significantly higher than CPH0 group. In cerebral ganglia, protein kinase B and 4E‐BP expression levels of crab fed CPH0.6 diets were significantly lower than CPH0 group. In mid‐intestine, target of rapamycin (TOR) relative expression level of crabs fed the diets containing CPH was significantly lower than CPH0 group. 4E‐BP expression level showed an opposite pattern. In thoracic ganglia, cerebral ganglia and mid‐intestine, leptin receptor expression level of crabs fed CPH0.6 diet was significantly lower than CPH0 group. Neuropeptide Y and neuropeptide Y receptor expression level of crab fed CPH0.6 diet was significantly higher than CPH0 group. In conclusion, our results indicated that dietary supplementation of 6 g/kg CPH could stimulate appetite and quicken feeding rate via the TOR signalling pathway.     

White spot syndrome virus epizootic in cultured Pacific white shrimp Litopenaeus vannamei (Boone) in Taiwan

White spot syndrome virus (WSSV) has caused significant losses in shrimp farms worldwide. Between 2004 and 2006, Pacific white shrimp Litopenaeus vannamei (Boone) were collected from 220 farms in Taiwan to determine the prevalence and impact of WSSV infection on the shrimp farm industry. Polymerase chain reaction (PCR) analysis detected WSSV in shrimp from 26% of farms. Juvenile shrimp farms had the highest infection levels (38%; 19/50 farms) and brooder shrimp farms had the lowest (5%; one of 20 farms). The average extent of infection at each farm was as follows for WSSV‐positive farms: post‐larvae farms, 71%; juvenile farms, 61%; subadult farms, 62%; adult farms, 49%; and brooder farms, 40%. Characteristic white spots, hypertrophied nuclei and basophilic viral inclusion bodies were found in the epithelia of gills and tail fans, appendages, cephalothorax and hepatopancreas, and virions of WSSV were observed. Of shrimp that had WSSV lesions, 100% had lesions on the cephalothorax, 96% in gills and tail fans, 91% on appendages and 17% in the hepatopancreas. WSSV was also detected in copepoda and crustaceans from the shrimp farms. Sequence comparison using the pms146 gene fragment of WSSV showed that isolates from the farms had 99.7–100% nucleotide sequence identity with four strains in the GenBank database – China ( AF332093 ), Taiwan ( AF440570 and U50923 ) and Thailand ( AF369029 ). This is the first broad study of WSSV infection in L. vannamei in Taiwan.     

Dietary protease modulates nutrient retention efficiency and hepatopancreatic protease activity in juvenile Chinese mitten crab Eriocheir sinensis

An 8‐week growth trial was conducted with juvenile Chinese mitten crab fed a high fishmeal (FM) diet (PC), a low FM diet (NC) and three other diets (NC125, NC150 and NC175) supplemented with 125, 150 and 175 mg/kg of a dietary protease. In the NC diets, a portion of FM (from PC diet) was replaced by a combination of plant proteins. All diets were isoproteic (404 ± 3 g/kg) and isoenergetic (18.6 ± 0.2 kJ/g). No differences were observed among treatments in most parameters except protein (PRE, %) and lipid (LRE, %) retention efficiencies, where LRE was the highest (37.9%) in NC175 followed by the PC. A linear regression demonstrated significant effects of protease on hepatopancreatic trypsin activity (HPRO, p = .001), and PRE (p = .05) and LRE (p = .014) but not on energy retention efficiency (ERE). Dietary protease level was positively correlated with HPRO and nutrient retention efficiency (p < .05). On the other hand, the specific growth rate (SGR) was positively correlated with portein deposition (PD), lipid deposition (LP) and recovered energy (RE) and negatively correlated with survival (p < .05). This study is first to demonstrate that mainly nutrient retention efficiencies are modulated by specific dietary enzymes in animals.     

Experimental infection of European crustaceans with white spot syndrome virus (WSSV)

Eight European marine and freshwater crustaceans were experimentally infected with diluted shrimp haemolymph infected with white spot syndrome virus (WSSV). Clinical signs of infection and mortalities of the animals were routinely recorded. Diagnosis was by direct transmission electron microscopy (TEM), DNA hybridization (dot-blot and in situ hybridization) using WSSV probes and by PCR using WSSV specific primers. High mortality rates were noted between 7 to 21 days post-infection for Liocarcinus depurator , Liocarcinus puber , Cancer pagurus , Astacus leptodactylus , Orconectes limosus , Palaemon adspersus and Scyllarus arctus . Mortality reached 100%, 1 week post-infection in P. adspersus . When infection was successful, direct TEM observation of haemolymph revealed characteristic viral particles of WSSV, some observed as complete virions (enveloped), others as nucleocapsids associated with envelope debris. WSSV probes showed strong positive reactions in dot-blots and by in situ hybridization in sections and specific virus DNA fragments were amplified successfully with WSSV primers. White spot syndrome virus was pathogenic for the majority of the crustaceans tested. This underlines the epizootic potential of this virus in European crustaceans.     

Effect of dietary lipid source and vitamin E on growth,non‐specific immune response and resistance to Aeromonas hydrophila challenge of Chinese mitten crab Eriocheir sinensis

Six purified diets were formulated to contain three lipid sources, fish oil (FO), linseed oil (LO) and soybean oil (SO), at 6% diet lipid crossing two levels of vitamin E (100 and 300 mg α‐tocopheryl acetate/kg diet) for each lipid source (FO100, FO300, LO100, LO300, SO100, SO300). The juvenile Chinese mitten crab, Eriocheir sinensis, respectively, fed on these diets with four replicates for 6 weeks. The crab weight gain (WG) and specific growth rate (SGR) were significantly affected by dietary lipid sources. No difference was found between the crabs fed two levels of vitamin E, but the WG and SGR were numerically higher in crab fed 300 mg/kg vitamin E than those fed the other level of vitamin E. The lipid source and vitamin E level could affect fatty acid composition in the hepatopancreas. The contents of saturated fatty acids (SAFA) and n‐3HUFA were significantly higher in the crab‐fed fish oil. The highest contents of n‐6PUFA and n‐3PUFA were found in the crab‐fed soybean oil and linseed oil respectively. The contents of SAFA, n‐3HUFA and n‐3PUFA were higher in the 300 mg/kg vitamin E treatment. A lower malondialdehyde (MDA) content and higher phenoloxidase (PO) activity were observed in the crab fed 300 mg/kg vitamin E. The results of this study indicate that the Chinese mitten crab fed the diet with 6% fish oil and 300 mg/kg vitamin E showed better growth, antioxidant capacity and resistance to Aeromonas hydrophila.     

Label‐free quantification proteomics reveals the effects of dietary fish oil and soybean oil on the immune response of Chinese mitten crab,Eriocheir sinensis

The replacement of fish oil (FO) in Eriocheir sinensis can significantly reduce the cost of E. sinensis cultivation, while several studies have indicated that replacing FO with soybean oil (SO) could significantly reduce the resistance of E. sinensis to disease. However, the molecular mechanisms underlying these effects remain poorly understood. In this study, crabs were fed two diets containing FO or SO, following which a label‐free quantification proteomic analysis was employed. And the activity of enzymes involved in the nonspecific immune response was also measured. Growth performance was undifferentiated between the crabs fed with FO and SO. A total of 519 proteins were identified, and 70 proteins were significantly altered between the crabs fed the two different diets. Five proteins related to the immune response were identified to be differently expressed. C‐type lectin, haemocyanin subunit 6 and cryptocyanin were significantly downregulated, while fatty acid‐binding protein and catalase were highly expressed in the crabs fed SO. The activities of acid phosphatase, alkaline phosphatase, superoxide dismutase and phenoloxidase were all significantly changed in crab fed with different diets. These findings will provide novel insight into the molecular mechanism regarding the replacement of FO on the immune response of E. sinensis and provide evidences for the relationship between nutrition and immunity in E. sinensis.     

Effects of dietary supplementation of Haematococcus pluvialis powder on gonadal development,coloration and antioxidant capacity of adult male Chinese mitten crab (Eriocheir sinensis)

The green algae Haematococcus pluvialis is an important source of natural astaxanthin as feed additive. This study was conducted to investigate the effects of dietary supplementation of H. pluvialis powder on gonadal development, coloration and antioxidant capacity of adult male Chinese mitten crab Eriocheir sinensis, and four experimental diets were formulated to contain 0, 0.2%, 0.4% and 0.6% of H. pluvialis powder. There were four treatments (defined as D1~D4) in this study and each treatment had three replicates. Dietary H. pluvialis contents had no significant effects on survival, body weight gain rate and gonadal development of male E. sinensis. For colour parameters, the total carotenoids content in carapace and hepatopancreas as well as hepatopancreatic lightness (L*) and carapace redness (a*) increased significantly with increasing dietary H. pluvialis (p < 0.05). For the antioxidant indices in the serum, D4 had the lowest activities of superoxide dismutase (SOD) and peroxidase (POD), but the highest glutathione peroxidase (GSH‐Px) and lactic dehydrogenase (LDH), while the malondialdehyde (MDA) in serum and hepatopancreas decreased significantly with the rising content of dietary H. pluvialis (p < 0.05); D1 had the highest levels of SOD, POD and GSH‐Px in hepatopancreas. For the non‐specific immune indices, the highest activities of acid phosphatase (ACP) and γ‐glutamyl transpeptidase (γ‐GT) were found on the serum of D3 and D4 (p < 0.05). D1 had the highest levels of ACP and alkaline phosphatase (ALP) in hepatopancreas, while D2 and D3 had the lowest levels of ALP and ACP respectively. These results suggested the optimal dietary natural astaxanthin level was around 40 mg/kg diets.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

Effects of xylan and cottonseed meal on growth performance,digestion, intestinal morphology and the expression of two peritrophin genes of Chinese mitten crab,Eriocheir sinensis

Xylan is one of the main non‐starch polysaccharides (NSPs) in cottonseed meal. This study aimed to evaluate the anti‐nutrition effect of xylan and to better understand cottonseed meal as a feed ingredient for the Chinese mitten crab, Eriocheir sinensis. A diet containing 280 g/kg fishmeal without cottonseed meal or xylan was formulated as a control (FM diet). A second diet (XYL diet) was supplemented with 53.3 g/kg xylan according to its amount in the third diet, the cottonseed meal diet (CSM diet), which was formulated to include 400 g/kg cottonseed meal. The last diet (CSM + XYLase diet) included 400 g/kg xylanase hydrolysed cottonseed meal, in which most xylan was hydrolysed. Diets were isonitrogenous (370 g/kg crude protein) and isoenergetic (17.58 KJ/g) and were randomly fed to 12 tanks of crabs for 8 weeks. Results showed that no difference was detected among groups in growth performance, serum biochemical index, or digestive and metabolism enzyme activity (p > 0.05). The apparent digestibility coefficient (ADC) of dry matter was FM > XYL > CSM + XYLase > CSM (p < 0.05). The ADC of protein in crab fed diet FM and XYL was significantly higher than CSM and CSM + XYLase (p < 0.05). Cottonseed meal decreased the fold height of the hindgut, upregulated the expression of peritrophin genes of Es‐PL44 and Es‐PP1 (p < 0.05), while xylan decreased the fold height of the hindgut and upregulated the expression of Es‐PP1 (p < 0.05). These results indicated that the negative effect of cottonseed meal and xylan on digestibility partly compensated by enhanced absorption of the hindgut innerly and by supplementation of xylanase externally, and cottonseed meal is an attractive alternative protein source in general for Chinese mitten crab.     

对虾白斑综合征病毒在螯虾动物模型的感染特性

应用克氏原螯虾作为动物模型研究对虾白斑综合征病毒（ＷＳＳＶ）的感染增殖特性，涉及感染温度，感染途径，继发感染，半数致死量（ＬＤ５０），免疫保护及保存期等，结果显示，２２－２５℃时，接种ＷＳＳＶ的螯虾一般于２－７d内死亡，温度升高对病毒增殖影响不显著，３０－３２℃时，平均死亡时间２－６d，温度降低对病毒增殖影响较显著，１５－１９℃时，接种螯虾平均２－１０d内死亡，８－１０℃时，平均死亡时间３－６d，感染途径分别用腹节肌肉，腹节皮下洲射及口服，均能使螯虾感染发病，而浸泡方式不能使螯虾发病，细菌分离和细菌定量结果表明，寄考于螯虾心脏，肝胰腺内的阴沟肠杆菌在感染后期大量增殖，菌量分别是正常螯虾的２５和３０倍，形成继发感染，用螯虾心脏，肝胰腺内的阴肠杆菌在感染后期大量增殖，菌量分别是政党螯虾的２５和３０倍，形成继发梁。用螯虾测定ＷＳＳＶ的ＬＤ５０为１０^-6.5.mL^-1种毒液，将病毒５６℃，３０min灭活后免疫螯虾，不能使螯虾形成免疫保护，ＷＳＳＶ匀浆液－３０℃冻存１年后失活，而－３０℃冻存于螯虾体内ＷＳＳＶ保存１年后仍有活力。     

Purification of white spot syndrome virus by iodixanol density gradient centrifugation

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60 000  g ) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80 000  g ) through a discontinuous iodixanol gradient (phosphate‐buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1 000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures.