Changes in the gut microbiome of the Chinese mitten crab (Eriocheir sinensis) in response to White spot syndrome virus (WSSV) infection

Intestinal microorganisms play important roles in maintaining host health, but their functions in aquatic animal hosts have yet to be fully elucidated. The Chinese mitten crab, Eriocheir sinensis, is one such example. We attempted to identify the shift of gut microbiota that occurred in response to infection of white spot syndrome virus (WSSV), an emerging viral pathogen in the crab aquaculture industry. The microbiota may exert some control over aspects of the viral pathogenesis. We investigated the changes in composition and structure of the crab gut microbiome during various WSSV infection stages of 6 h post‐infection (hpi) and 48 hpi, using a 16S rRNA approach on the MiSeq Illumina sequencing platform. Four phyla (Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes) were most dominant in the gut of E. sinensis regardless of the WSSV infection stages. However, further analysis revealed that over 12 bacterial phyla, 44 orders and 68 families were significantly different in abundance at various states of WSSV infection. Several intriguing aspects of E. sinensis gut bacteria that had not been previously reported were also uncovered, such as class Mollicutes was dominant here, but absent in crabs from Yangtze River estuary and Chongming Islands. Overall, this study provided the first evidence that changes in gut microbiome were closely associated with the severity of WSSV infection and that indicator taxa could be used to evaluate the crab health status.     

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.     

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.     

正常与性早熟中华绒螯蟹的Y-器官转录组分析

性早熟是中华绒螯蟹养殖过程中普遍存在的一个问题。为发掘中华绒螯蟹性早熟相关的重要功能基因,实验采用Illumina Hiseq 2000高通量测序技术获得了正常与性早熟雌蟹的Y-器官转录组数据,并进行比较分析。结果显示,测序分别获得44 619 538和43 052 958个clean reads,比对发现2655个差异表达基因,Gene Ontology(GO)功能分类分析将文库中的差异表达基因归类到3大功能(生物过程、细胞组分和分子功能)的42个类别中。KEGG富集分析将文库中的差异表达基因富集到134条特定的KEGG代谢途径,其中4条是显著性富集,包括酮体合成和降解、丁酸甲酯代谢、神经营养因子信号通路和碱基切除修复通路。通过RNA-seq技术获得了丰富的中华绒螯蟹Y-器官转录组信息,为中华绒螯蟹新基因克隆、性早熟成因与预防和Y-器官的研究提供有价值的数据。     

miR‐122‐5p as a plasma biomarker of liver injury in fish exposed to microcystin‐LR

Recent studies have shown the presence of large amounts of microRNAs (miRNAs; miRs) from damaged cells in the peripheral blood. In this study, we investigated the levels of miRNAs circulating in the blood plasma of whitefish (Coregonus lavaretus) after exposure to microcystin‐LR. We used real‐time PCR to examine the relative expression of plasma levels of 4 miRNAs (miR‐122‐5p and let‐7c‐5p, the liver‐enriched microRNAs, miR‐148a‐3p which promotes the hapatospecific phenotype in mammals, and miR‐92a‐3p, a cell proliferation and angiogenesis promoter, potentially hepatocarcinogenic) during the first 48 h after exposure to MC‐LR. We observed a rapid increase of miR‐122‐5p levels 8 h after exposure (P < 0.05), which continued to the end of the experiment. Our results demonstrated that the plasma miR‐122‐5p was indicative of MC‐LR‐induced liver injury, exhibiting areas under the curve close to 1 in ROC analysis (AUC = 0.976, P < 0.001). Although plasma levels of miR‐148a‐3p and miR‐92a‐3p were significantly elevated by the end of the experiment, their discriminative power was lower than reported for the miR‐122‐5p. Based on these results and reports on miRNA‐based diagnosis of liver injuries in mammals, plasma miR‐122‐5p could be considered as a robust, new generation diagnostic biomarker in fish, helpful for the non‐invasive diagnosis of liver damage.     

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.     

Examination of a practical method for copper enrichment of euryhaline rotifers (Brachionus plicatilis) as diet of Eriocheir sinensis zoea larvae

In this study, an effective method to enrich the rotifer Brachionus plicatilis with copper was developed as a feed for the Chinese mitten crab Eriocheir sinensis zoea larvae. Changes in the concentrations of other minerals in rotifers were also examined when copper was added for rotifer enrichment. The ability of Chlorella to absorb waterborne copper is much higher than that of rotifers, and hence, copper was preaccumulated in Chlorella before its ingestion by rotifers. The copper content in rotifers was comparable to the dietary copper requirement of the crab larvae when the rotifers were enriched with 0.1 mg Cu g⁻¹ Chlorella for 12 h. Further enrichment in rotifers with Cu‐enriched Chlorella and lipid emulsions did not significantly change the profile of major fatty acids and mineral composition in the rotifers. Evidence shows the feasibility of copper enrichment in rotifers using microalgae that can accumulate copper. This study indicates that copper in rotifers can be enriched by feeding copper‐enriched algae at a concentration of 0.1–0.2 mg Cu g⁻¹ Chlorella. The developmental rates of E. sinensis can be improved by feeding zoea larvae with copper‐enriched rotifers, but survival rates were not affected by dietary copper enrichment.     

Effects of short‐term starvation on the rhythmic expression of microRNAs in skeletal muscle of goldfish (Carassius auratus)

Molecular oscillators exist in peripheral tissues such as in skeletal muscles and diet is a dominant factor to affect Zeitgeber for peripheral clocks. MicroRNAs (miRNAs) play an important role in muscle cell proliferation and differentiation. However, the knowledge of starvation effects on miRNA rhythmic expression remains limited in teleost. In this study, the circadian expression pattern of miRNAs was investigated in goldfish muscle upon restricting feeding treatment. The data showed that 15 miRNAs exhibited a daily rhythmicity among the 70 miRNAs assayed in muscles of normally fed goldfish. While after 7‐day and 15‐day fasting treatment, 23 and 18 miRNAs showed circadian rhythmicity in goldfish muscles respectively. Only 4 miRNAs (miR‐23a, miR‐29, miR‐199a‐3p and miR‐455) exhibited a daily rhythmicity in all three groups of different nutrient treatments. Correlation analysis of the circadian‐miRNAs indicates different feeding stimulation could alter the circadian‐miRNA components and their expression profile in skeletal muscle upon short‐term feed deprivation. These miRNAs may play important roles in regulating circadian expression of genes involved in muscle cell differentiation and growth by nutritional alteration, thus having a potential application in fish aquaculture.     

Food sources and trophic relationships of three decapod crustaceans: insights from gut contents and stable isotope analyses

Chinese mitten crab (Eriocheir sinensis), freshwater shrimp (Macrobrachium nipponensis) and red swamp crayfish (Procambarus clarkii) are important culture species in China and have world‐wide introduced distributions. The trophic role of these three decapods has not been compared within a system although they consume plants, animals, and detritus and often co‐occur in freshwater ponds. A combination of stable isotope measurements and gut content analysis was used to determine the main food sources and trophic niche of three benthic decapods, in commercial crab ponds around Lake Gucheng, China. Observation of the gut contents reflected a variety of prey items ingested by three decapods. Macrophytes made up the highest contribution to the volume of their gut contents, followed by forage fish, corn and mollusks. Stable isotope analysis supported a stronger relationship between crayfish and vegetable matter than with animal matter, while both crab and shrimp were the opposite. An isotopic mixing model indicated that about 60% of crab and shrimp production originated from animal matter, while only 40% of crayfish production was ascribed to consumption of animal matter. Although results from the mixing model corroborate the gut content findings in most cases, stable isotope results showed that three decapods obtained more energy for growth from animal matter than what would be estimated if gut analysis was used alone. Estimates of niche overlap indices indicated a high degree of dietary overlap among the three decapods examined, suggesting that shrimp and crayfish density should be controlled if considering economic benefits of pond aquaculture.     

Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir,a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real‐time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue‐specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.     

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.     

Growth and immune response of Chinese mitten crab (Eriocheir sinensis) fed diets containing different lipid sources

A 10‐week feeding trial was conducted to evaluate the effects of dietary lipid sources on the growth and immune responses of Chinese mitten crab Eriocheir sinensis. Six isonitrogenous and isoenergetic diets were formulated with fish oil (FO), linseed oil (LO), soybean oil (SO), rapeseed oil (RO), coconut oil (CO) and beef tallow (BT) as the sources of lipid with five replicates each. Thirty crabs (2.35 ± 0.14 g) were stocked into each tank and fed twice daily. Weight gain and specific growth rate of crab fed the FO diet were significantly lower than those fed other diets (P < 0.05), except for crabs fed LO diet (P < 0.05). Crab fed the SO diet weighed more than those fed FO diets (P < 0.05). Serum superoxide dismutase and malondialdehyde of crab fed the FO diet were significantly higher than in other groups (P < 0.05). Crab fed the FO diet had the highest activities of serum phenoloxidase, acid phosphatase, alkaline phosphatase and lysozyme (P < 0.05). The fatty acid composition in the liver of crab reflected the change in test diets. Our results indicate that the use of dietary vegetable or animal oils can achieve similar growth performance to the use of dietary FO in Chinese mitten crab, but non‐FOs may impair crab immunity. Soybean oil is recommended as a suitable replacer for FO in Chinese mitten crab diet.     

Isolation and partial characterization of a new reovirus in the Chinese mitten crab, Eriocheir sinensis H Milne Edwards

A second type of freshwater crab reovirus has been isolated from Chinese mitten crab, Eriocheir sinensis H. Milne Edwards, in China; we named it E. sinensis reovirus (EsRV816). The negatively stained virion is a non‐enveloped icosahedral particle, 60 ± 5 nm in diameter. Its genome is composed of 10 dsRNA linear pieces exhibiting an electrophoretic pattern of 5/3/2. The largest segment (RNA‐1) was cloned and sequenced. The deduced amino acid sequence, corresponding to the RdRp of the virus, showed 26% identity with the RdRp of Operophtera brumata (L.) cypovirus 19 in the genus Cypovirus and 24% identity with RdRp of Nilaparvata lugens (Stal) reovirus in the genus Fijivirus. On the basis of its ultrastructure and physicochemical properties, this virus is quite different from other crab reoviruses, and particularly with another freshwater crab reovirus EsRV905, recently classified in a new genus Cardoreovirus. This virus (EsRV816) possesses all the characters of the members of the reoviridae family and could represent a new genus.     

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。     

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.     

Effects of dietary protein to energy ratios on growth,body composition and digestive enzyme activities in Chinese mitten‐handed crab,Eriocheir sinensis

This study aimed to determine the optimal protein to energy ratio (P/E ratio) and evaluate the effect of dietary protein and lipid levels on growth performance, body composition and digestive enzymes activities in Chinese mitten‐handed crab, Eriocheir sinensis. Nine practical diets containing three levels both for protein (DP 30%, 35% and 40%) and lipid (DL 2%, 7% and 12%) with P/E ratios ranging from 13.69 to 19.79 mg KJ^?1 were fed to four replicates of crabs (3.39 ± 0.10 g) for 10 weeks. Weight gain increased significantly with the increase in DP level at each DL level. Moreover, weight gain increased in crabs fed with diets containing DL level from 2% to 12% and DP level from 30% to 35%. However, the diet containing 40% DP and 12% DL levels significantly decreased the growth performance and protein efficiency of the crabs. The whole crab and hepatopancreas lipid contents also increased as dietary lipid increased, but not dietary protein. The total protease activity increased significantly with the increase in dietary protein at each lipid level. The lipase activity was statistically comparable among different DL levels at each DP level. Taken together, the crab fed the diet containing 35% protein and 12% lipid levels with P/E 15.77 mg KJ^?1 revealed optimal growth, feed utilization efficiency and digestive enzymes activities. Moreover, our study indicated that the higher dietary lipid level at a relatively lower dietary protein level could provide protein sparing effect in Eriocheir sinensis.