Promotion of improved intestinal barrier health by soybean-derived bioactive peptides in Chinese mitten crab (Eriocheir sinensis) fed a low fishmeal diet.

To alleviate the growth inhibition, and intestinal damage of Chinese mitten crab (Eriocheir sinensis) induced by low fishmeal diets (LF), an 8-week feeding trial was conducted to evaluate the addition of dietary soybean-derived bioactive peptides (SBP) in LF diets on the regulation of growth, digestion and intestinal health. The crabs were fed isonitrogenous and isoenergetic conventional diet and LF diets (10 % fishmeal replaced by soybean meal, LF) supplemented with 0, 1 %, 2 %, 4 % and 6 % SBP, respectively. The results showed that LF diet inhibited growth while inclusion of SBP quadratically remitted the growth inhibition induced by LF. For digestive function, increasing addition level of SBP quadratically improved the α-amylase and trypsin activities. For antioxidant function, LF group significantly increased the malondialdehyde content, while SBP linearly decreased the malondialdehyde level and cubically increased the anti-superoxide anion activity and total antioxidant capacity level. For intestinal health, the peritrophic membrane (PM) almost completely separated from the inner wall of the intestinal lumen, the epithelial cells reduced, the muscularis became thinner and the apoptotic signals increased in LF group; with SBP addition, the intestinal morphology was improved, with the PM adhering to the inner wall of the intestinal lumen, an increase in the number of epithelial cells and an increase in the thickness of the muscularis. Additionally, there was a decrease in apoptotic signals. Dietary SBP also increased the expression of PT and Crustin1 quadratically and decreased the expression of ALF1 linearly, ALF3 and ILF2 quadratically.

Immune functions of the Dscam extracellular variable region in Chinese mitten crab.

In arthropods, there is only a single copy of Down Syndrome Cell Adhesion Molecule (Dscam) in the genome, but it can exist as numerous splice variants. There are three hypervariable exons in the extracellular domain and one hypervariable exon in the transmembrane domain. In Chinese mitten crab (Eriocheir sinensis), exons 4, 6 and 14 can produce 25, 34 and 18 alternative splice variants, respectively. In this study, through Illumina sequencing, we identified additional splice variants for exons 6 and 14, hence there may be > 50,000 Dscam protein variants. Sequencing of exons 4, 6 and 14 showed that alternative splicing was altered after bacterial stimulation. Therefore, we expressed and purified the extracellular variable region of Dscam (EsDscam-Ig1-Ig7). Exons 4.3, 6.46 and 14.18, three variable exons of the recombinant protein, were randomly selected. The functions of EsDscam-Ig1-Ig7 in immune defences of E. sinensis were subsequently explored. EsDscam-Ig1-Ig7 was discovered to bind to both Gram-positive Staphylococcus aureus and Gram-negative Vibrio parahaemolyticus, but it did not exhibit antibacterial activity. By promoting hemocyte phagocytosis and bacterial removal, EsDscam-Ig1-Ig7 can also shield the host from bacterial infection. The findings highlight the immunological activities of Dscam alternative splicing and reveal the potential for many more Dscam isoforms than were previously predicted in E. sinensis.

Dietary T-2 toxin induces transcriptomic changes in hepatopancreas of Chinese mitten crab (Eriocheir sinensis) via nutrition metabolism and apoptosis-related pathways.

Long-term feed route exposure to T-2 toxin was proved to elicit growth retarding effects and induction of oxidative stress and apoptosis in Chinese mitten crab (Eriocheir sinensis). However, no study with a holistic perspective has been conducted to date to further describe the in-depth toxicological mechanism of T-2 toxin in E.sinensis. In this study, an RNA-Sequencing (RNA-seq) was used in this study to investigate the effects of feed supplementation with 0 mg/kg and 4 mg/kg T-2 toxin on the hepatopancreas transcriptome of E.sinensis and establish a hepatopancreas transcriptome library of T-2 toxin chronically exposed crabs after five weeks, where 14 differentially expressed genes (DEGs) were screened out across antioxidant, apoptosis, autophagy, glucolipid metabolism and protein synthesis. The actual expression of all the DEGs (Caspase, ATG4, PERK, ACSL, CAT, BIRC2, HADHA, HADHB, ACOX, PFK, eEFe1, eIF4ɑ, RPL13Ae) was also analyzed by real-time quantitative PCR (RT-qPCR). It was demonstrated that long-term intake of large amounts of T-2 toxin could impair antioxidant enzyme activity, promote apoptosis and protective autophagy, disrupt lipid metabolism and inhibit protein synthesis in the hepatopancreas of E.sinensis. In conclusion, this study explored the toxicity mechanism of T-2 toxin on the hepatopancreas of E.sinensis at the mRNA level, which lays the foundation for further investigation of the molecular toxicity mechanism of T-2 toxin in aquatic crustaceans.

Integrated Analysis of Transcriptomics and Metabolomics Unveil the Novel Insight of One-Year-Old Precocious Mechanism in the Chinese Mitten Crab, Eriocheir sinensis.

Eriocheir sinensis is traditionally a native high-value crab that is widely distributed in eastern Asia, and the precocity is considered the bottleneck problem affecting the development of the industry. The precocious E. sinensis is defined as a crab that reaches complete sexual maturation during the first year of its lifespan rather than as normally in the second year. However, the exact regulatory mechanisms underlying the precocity are still unclear to date. This study is the first to explore the mechanism of precocity with transcriptome-metabolome association analysis between the precocious and normal sexually mature E. sinensis. Our results indicated that the phenylalanine metabolism (map00360) and neuroactive ligand-receptor interaction (map04080) pathways play an important role in the precocity in the ovary of E. sinensis. In map00360, the predicted aromatic-L-amino-acid decarboxylase and 4-hydroxyphenylpyruvate dioxygenase isoform X1 genes and the phenethylamine, phenylethyl alcohol, trans-2-hydroxycinnamate, and L-tyrosine metabolites were all down-regulated in the ovary of the precocious E. sinensis. The map04080 was the common KEGG pathway in the ovary and hepatopancreas between the precocious and normal crab. In the ovary, the predicted growth hormone secretagogue receptor type 1 gene was up-regulated, and the L-glutamate metabolite was down-regulated in the precocious E. sinensis. In the hepatopancreas, the predicted forkhead box protein I2 gene and taurine metabolite were up-regulated and the the L-glutamate metabolite was down-regulated in the precocious crab. There was no common pathway in the testis. Numerous common pathways in the hepatopancreas between male precocious and normal crab were identified. The specific amino acids, fatty acids and flavorful nucleotide (inosine monophosphate (MP), cytidine MP, adenosine MP, uridine MP, and guanosine MP) contents in the hepatopancreas and gonads further confirmed the above omics results. Our results suggest that the phenylalanine metabolism may affect the ovarian development by changing the contents of the neurotransmitter and tyrosine. The neuroactive ligand-receptor interaction pathway may affect the growth by changing the expressions of related genes and affect the umami taste of the gonads and hepatopancreas through the differences of L-glutamate metabolite in the precocious E. sinensis. The results provided valuable and novel insights on the precocious mechanism and may have a significant impact on the development of the E. sinensis aquaculture industry.

Effects of dietary glyceryl monolaurate supplementation on growth performance, non-specific immunity, antioxidant status and intestinal microflora of Chinese mitten crabs.

This study aims to investigate the effects of glycerol monolaurate (GML) on growth performance, non-specific immunity, antioxidant capacity and intestinal microflora in Chinese mitten crabs. The crabs were randomly arranged to three experimental diets groups containing 0 (control group), 1000 mg/kg GML (GML1000 group), and 2000 mg/kg GML (GML2000 group), respectively. After 8 weeks of breeding, results showed a better growth performance in GML2000 group, with a higher PWG, SGR and lower FCR (P < 0.05). Meanwhile, in GML2000 group the activities of phenoloxidase, alkaline phosphatase, acid phosphatase and lysozyme in hemolymph were increased (P < 0.05), also the activities of hemolymph and hepatopancreas superoxide dismutase (SOD) and glutathione peroxidase (GPx) were increased in hepatopancreas (P < 0.05). While malondialdehyde (MDA) concentrations were lower significantly (P < 0.05) both in GML1000 and GML2000 groups. Furthermore, the mRNA expression of TLR1, TLR2, which related to the Toll pathway were increased (P < 0.05). Supplementation of 2000 mg/kg GML up-regulated the expression of ALF and LZM (P < 0.05), and down-regulated the expression of caspase-3 (P < 0.05). The abundance of Firmicutes increased in GML2000 group (P < 0.05), and Shewanella was significantly increased (P < 0.05) in both GML1000 and GML2000 groups. In conclusion, dietary supplemented with GML enhanced the growth performance and antioxidant capacity, enhanced hemolymph immune enzymes activities and antimicrobial peptides expression through regulating the proPO system and Toll pathway, and improved gut microflora in Chinese mitten crabs.

Effects of insecticides on malacostraca when managing diamondback moth (Plutella xylostella) in combination planting-rearing fields.

The combination of crop planting and animal rearing in the same area is popular. However, if the methods of planting and rearing are not appropriate, it will result in losses and the disruption of pest management. The toxicities of 17 insecticides to Plutella xylostella, Eriocheir sinensis, and Procambarus clarkii were tested. The recommended maximum field doses were used in 2 d and 4 d bioassays, and the levels of resistance of P. xylostella to insecticides were determined. Of five insecticides that had relatively low toxicity to E. sinensis and P. clarkii, spinetoram and MbNPV showed the best control efficacy of P. xylostella, followed by tetrachlorantraniliprole, chlorantraniliprole, and avermectin. P. xylostella had relatively little resistance to spinetoram, MbNPV, chlorantraniliprole, and avermectin. Therefore, we concluded that the best insecticides suitable for combination planting and rearing fields (cauliflower-crab or cauliflower-crayfish) were spinetoram and MbNPV, followed by chlorantraniliprole and avermectin. Other insecticides, such as emamectin benzoate, indoxacarb, and chlorfenapyr were effective at controlling P. xylostella, but they were not suitable for use in combination planting and rearing fields because of their high toxicity to crabs and crayfish.

Comparative study of female Chinese mitten crabs based on their sizes and weights.

The Chinese mitten crab (Eriocheir sinensis) is an important aquaculture species in China. While the price of a large crab will generally be 2-5 times higher than that of smaller crabs, it remains unknown whether nutritional quality is affected by market price. To investigate the effect of size on nutritional composition, adult female crabs were collected and assigned to grades I-IV according to decreasing size. The results showed that meat yield and conditional factors were significantly (P < 0.05) reduced with the decreasing size. The different sizes did not significantly (P > 0.05) affect levels of moisture, crude protein (except for hepatopancreatic crude protein), and total lipid. Grade III crabs had the largest hepatopancreatic crude protein level, which was significantly (P < 0.05) increased compared with grade I crabs. A balanced amino acid composition was found in grade IV crabs, while crabs from grades II and IV had the highest essential amino acids score. Levels of highly unsaturated fatty acids, including C22:6n3, and the ratios of n-3 polyunsaturated fatty acids (PUFA)/n-6 PUFA and C22:6n3 (DHA)/C20:5n3 (EPA) in the hepatopancreas were significantly (P < 0.05) increased in grade III crabs compared with the other grades. In conclusion, among the four grades smaller female crabs (average weight: 93-112 g, grades III-IV) displayed an optimal nutritional quality.

Acute exposure to microcystin-LR induces hepatopancreas toxicity in the Chinese mitten crab (Eriocheir sinensis).

The Chinese mitten crab is an important economic species in the Chinese aquaculture industry due to its rich nutritional value and distinct flavor. The hepatopancreas is a popular edible part of the Chinese mitten crab, and therefore, hepatopancreatic health directly determines its quality. However, a large-scale outbreak of hepatopancreatic necrosis syndrome ("Shuibiezi" disease in Chinese), which is caused by abiotic agents correlated with cyanobacteria bloom outbreaks, adversely affects the Chinese mitten crab breeding industry. Cyanobacterial blooms that occur in high-density farming ponds can produce microcystin-LR (MC-LR), which is hepatotoxic in fish and mammals. Hepatopancreas toxicity of MC-LR (0, 25, 50 and 75 µg/kg) was investigated after 48 h of exposure. The MC-LR can cause hepatopancreatic injury by inducing hepatopancreatic structural damage, subcellular structural changes, and cell apoptosis, followed by enhanced lipid peroxidase, reactive oxygen species, and apoptosis-related enzyme (Caspase 3, 8, and 9) activities. These in turn promote gene and protein expression of apoptosis-associated proteases (Caspase 3, 7, and 8, Bcl-2, and Bax), and alter antioxidant system responses (superoxide dismutase, glutathione S-transferase, glutathione peroxidase, glutathione reductase activities, and glutathione content). The present study is the first report on MC-LR hepatotoxicity in the Chinese mitten crab and confirms hepatopancreas toxicity, providing a theoretical basis for enhancing MCs resistance and developing preventive and curative measures against hepatopancreatic disease in the Chinese mitten crab breeding industry.

Toxic effects of metal copper stress on immunity, metabolism and pathologic changes in Chinese mitten crab (Eriocheir japonica sinensis).

Copper (Cu2+), which represents a major physiological challenge for crab culture, is ubiquitous in the aquatic culture environment, and gills are the first organs that come into direct contact with the environment. However, the molecular basis of the response of crabs to Cu2+ stress remains unclear. Here, we conducted a transcriptome and differential expression analysis on the gills from Chinese mitten crab unexposed and exposed to Cu2+ for 24 h. The comparative transcriptome analysis identified 2486 differentially expressed genes (DEGs). GO functional analysis and KEGG pathway analysis revealed some DEGs, which were mostly related to immunity, metabolism, osmotic regulation, Cu2+ homeostasis regulation, antioxidant activity, and detoxification process. Some pathways related to humoral and cellular immunity, such as phagosome, peroxisome, lysosome, mTOR signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, and T cell receptor signaling pathway were enhanced under Cu2+ stress. In addition, Cu2+ stress altered the expression patterns of key phagocytosis and apoptosis genes (lectin, cathepsin L, Rab7, and HSP70), confirming that Cu2+ can induce oxidative stress and eventually even apoptosis. Histological analysis revealed that the copper can induce damage at the cellular level. This comparative transcriptome analysis provides valuable molecular information to aid future study of the immune mechanism of Chinese mitten crab in response to Cu2+ stress and provides a foundation for further understanding of the effects of metal toxicity.

Vitellogenin receptor expression in ovaries controls innate immunity in the Chinese mitten crab (Eriocheir sinensis) by regulating vitellogenin accumulation in the hemolymph.

The vitellogenin receptor (Vgr), which is specific for vitellogenin (Vtg), recognises and transports Vtg into the ovaries. Accumulating evidence suggests that Vtg also performs an immune defence function and plays critical roles in innate immunity in oviparous animals. However, whether Vgr is involved in innate immunity in the Chinese mitten crab (Eriocheir sinensis) is unknown. In this study, we obtained a 3009 nucleotide partial cDNA of the E. sinensis vitellogenin receptor gene (Es-vgr) encoding an open reading frame of 1003 amino acid residues. Bioinformatics analysis showed that the domains of Es-vgr were conserved during evolution. Quantitative real-time PCR and western blotting revealed that the highest Es-vgr expression levels occurred in the ovary, and expression was specific. Comparison of the expression levels of Es-vgr and the Vtg gene (Es-vtg1) at different ovary developmental stages suggested that there may be some regulatory relationship between them. Bacterial challenge induced high-level expression of antimicrobial peptide genes and reduced Es-vgr expression in ovaries, resulting in massive accumulation of Vtg in the hemolymph. The survival rate of crabs increased significantly after injection with recombinant Es-vtg1 protein following bacterial infection. Collectively, these results demonstrate that Es-vgr plays critical roles in antimicrobial function by regulating the accumulation of Vtg in the hemolymph.

Effects of graded levels of starch on the non-specific immune responses, antioxidant capacities and intestinal health in Chinese mitten crab, Eriocheir inensis.

A 9-week feeding trial was conducted to investigate the effects of graded levels of dietary starch (12%, 17%, 22%, 27% and 32%) on growth, non-specific immune responses, antioxidant capacities, immunity genes expression levels and pathogen resistance in Chinese mitten crab, Eriocheir inensis (initial body weight: 10.5 ± 0.5 g). Results showed that the highest weight gain rate of crabs was obtained in group containing 22% dietary starch. The highest activity of acid phosphatase, phenoloxidase and lysozyme in blood was found in crabs fed with 22-27% dietary starch. Additionally, 17%-27% dietary starch significantly increased the activities of superoxide dismutase and glutathione peroxidase, reduced malondinaldehyde content and then increased the total antioxidant capacities in hepatopancreas of crabs. The highest activity of alanine aminotransferase and aspartate aminotransferase was found in crabs fed with 32% dietary starch, indicating that excess starch had a negative effect on the liver function of crabs. With the dietary starch level increased, the transcription factors gene expression of the pro-inflammatory factors were significantly up-regulated, and the highest ILF2, IL-16, Relish and ADAM10 was found in crabs fed with dietary 32% starch, which may potentially promote the inflammatory response in intestines. Moreover, with the dietary starch increased, the activity of phenoloxidase and lysozyme, as well as the gene expression of crustin, were all increased in crabs after challenge against Citrobacter freundii, which demonstrated that additional dietary starch could provide immune-protection and help crabs improve their resistance against pathogens. In conclusion, these results suggest that adequate dietary starch can increase growth, enhance innate immune responses and promote disease resistance, reduce oxidative stress and inflammatory response in E. inensis. Taken together, 22-27% dietary starch (25.9-30.8% dietary carbohydrate) was suggested as a digestible energy source in crabs feed.

Bridging information domains to improve ecological understanding of biological invasions in a marine ecosystem.

We devised a practical method for integrating information on 2 marine invasive species using 3 different approaches: standardized ecological monitoring, online-reporting databases, and surveys of anglers and crabbers. Focusing on 2 recently introduced species with different characteristics, the Asian shore crab (Hemigrapsus sanguineus) and Chinese mitten crab (Eriocheir sinensis), in the Hudson-Raritan watershed of New York and New Jersey, we used sensitivity analyses to explore the relative contribution of each information source to knowledge of species abundance and distribution. All 3 information sources contributed something unique to understanding abundance and distribution of the introduced crabs. Online and survey data on Asian shore crabs significantly affected predictions of abundance, whereas monitoring data did not. When survey data were omitted, abundance estimates were unchanged over time, but when they were included, the model predicted an increased abundance in 2012. All 3 data sets for the Asian shore crab significantly affected estimates of species coverage; surveys had the biggest influence, increasing range size by 4097.25 km2 . For the catadromous Chinese mitten crab, ecological monitoring data collected in freshwater shortly after the original sighting significantly shaped model estimates for abundance and documented the establishment phase of the mitten crab in an area outside the spatial scope of the surveyed resource users. However, the survey data significantly enlarged mitten crab range-size estimates by 6498.01 km2 . By demonstrating that data integration produced an image of the invasion process that would not have emerged had we used any 1 method individually, model results provide evidence for the advantages of an interdisciplinary approach.

Acoplamiento entre los Dominios de Información para Mejorar el Entendimiento Ecológico de las Invasiones Biológicas en un Ecosistema Marino Resumen Diseñamos un método práctico para integrar la información sobre dos especies marinas invasoras usando tres estrategias diferentes: el monitoreo ecológico estandarizado, las bases de datos de reporte en línea y las encuestas realizadas a pescadores y recolectores de cangrejos. Con un enfoque sobre dos especies con características diferentes, Hemigraspus sanguineus y Eriocheir sinensis, introducidas recientemente en la vertiente Hudson-Raritan de Nueva York y Nueva Jersey, usamos varios análisis de sensibilidad para explorar la contribución relativa de cada fuente de información para el conocimiento sobre la abundancia y distribución de las especies. Las tres fuentes de información contribuyeron algo único al entendimiento de la abundancia y distribución de los cangrejos introducidos. Los datos obtenidos en línea y de las encuestas sobre la especie H. sanguineus afectaron significativamente las predicciones de su abundancia, mientras que los datos de monitoreo no lo hicieron. Cuando se omitieron los datos de las encuestas, las estimaciones de abundancia no sufrieron modificaciones con el tiempo, pero cuando incluimos esos datos, el modelo pronosticó un incremento en la abundancia en 2012. Los tres conjuntos de datos de la especie H. sanguineus afectaron significativamente las estimaciones de la cobertura de la especie; las encuestas tuvieron la mayor influencia incrementando el tamaño de la distribución por 4097.25 km2 . Para la especie catádroma E. sinensis, los datos del monitoreo ecológico recolectados en agua dulce poco tiempo después del avistamiento original moldearon significativamente las estimaciones del modelo para la abundancia y documentaron la fase de establecimiento de esta especie en un área fuera del alcance espacial de los usuarios del recurso encuestados. Sin embargo, los datos de las encuestas agrandaron considerablemente las estimaciones de la distribución de E. sinensis por 6498.01 km2 . Con la demostración de que la integración de datos produjo una imagen del proceso de invasión que no habría surgido de haber usado un método individualmente, los resultados del modelo proporcionan evidencias para las ventajas de una estrategia interdisciplinaria.

Effects of dietary yeast extract supplementation on growth, body composition, non-specific immunity, and antioxidant status of Chinese mitten crab (Eriocheir sinensis).

This study was conducted to evaluate the effects of dietary supplementation with yeast extract on growth, body composition, non-specific immunity, and antioxidant status of Chinese mitten crab (Eriocheir sinensis). A total of 432 crabs (initial average weight, 4.62 ±â€¯0.11 g) were randomly distributed into four treatment groups with six replicates of 18 crabs. The crabs were fed a basal diet or the same diet supplemented with 2.5, 5, and 10 g/kg yeast extract for 8 weeks. The results showed that dietary yeast extract inclusion enhanced the edible viscera index (linear, P < 0.001), edible viscera crude protein (CP) content (linear, P = 0.025) and serum phenoloxidase (ProPO) activity (quadratic, P = 0.023) at 56 day, increased the total superoxide dismutase (T-SOD) activity at 28 day (quadratic, P = 0.037) and catelase (CAT) activity at 56 day (quadratic, P = 0.034) of edible viscera, and muscular T-SOD activity (quadratic, P = 0.020) at 56 day in Chinese mitten crab. Compared with the control group, the inclusion of 5 g/kg yeast extract in the diet increased the edible viscera index, enhanced the CAT activity of edible viscera at 56 day in Chinese mitten crab (P < 0.05). Dietary 10 g/kg yeast extract inclusion enhanced the edible viscera index at 56 day in Chinese mitten crab than that of the control group (P < 0.05). These results implied that dietary yeast extract inclusion improved the edible viscera index and crude protein content of edible viscera, enhanced serum immunity, and increased the antioxidant status of edible viscera and muscle in Chinese mitten crab, especially when it is supplemented at 5 g/kg yeast extract in the diet.

Dietary fructooligosaccharide can mitigate the negative effects of immunity on Chinese mitten crab fed a high level of plant protein diet.

An 8-week feeding trial was carried out under controlled condition to evaluate the effect of dietary fructooligosaccharide (FOS) on growth performance, whole body composition, antioxidant status and immunity of crabs fed high levels of plant protein diets. Thus, six experimental diets were formulated (designated as F0P50, F0P60, F0P70, F0.2P50, F0.2P60 and F0.2P70), which contain two FOS levels (0 or 0.2%) and three plant protein levels (50, 60, or 70%) according to a 2 × 3 factorial design. The results showed that weight gain increased significantly as dietary plant protein level decreased from 70% to 50%. At 50% plant protein level, the addition of 0.2% FOS can significantly elevate weight gain (WG) (P < 0.05). The highest value in survival rate was observed in crabs fed F0.2P50 and F0.2P60 diet. Crabs fed F0.2P50 diet showed significantly higher crude protein content (P < 0.05) compared with those in other groups, but there were no significant differences in the contents of moisture, crude lipid and ash among all groups (P > 0.05). Catalase (CAT) activity in crabs fed F0.2P50 increased significantly (P < 0.05) compared with crabs fed F0P60, F0P70, F0.2P60 and F0.2P70, but malondialdehyde (MDA) concentrations decreased significantly (P < 0.05). Meanwhile, nitric oxide (NO) concentration, acid phosphatase (ACP) and alkaline phosphatase (AKP) activities of crabs fed 0.2% FOS diets increased significantly (P < 0.05) compared with crabs fed 0% FOS diets. The expressions of prophenoloxidase (propo) was significantly (P < 0.05) affected only by dietary plant protein levels with the highest values observed in 50% plant protein diet, whereas the opposite was true for Myeloid differentiation factor 88 (myd88). The mRNA expressions of mitochondrial manganese superoxide dismutase (mtmnsod), lipopolysaccharide-induced TNF-α factor (litaf) and toll like receptors (tlrs) were significantly affected (P < 0.05) by both FOS and plant protein levels. The cytosolic manganese superoxide dismutase (cytmnsod) mRNA expressions in F0.2P50 and F0.2P60 groups were significantly higher than those in F0P70 and F0.2P70 groups. The results in this study indicated that supplementation with 0.2% FOS can enhance growth performance in crabs fed lower plant protein diets and as well improve immunity in those fed with higher plant protein diets.

Effects of dietary supplementation with icariin on growth performance, antioxidant capacity and non-specific immunity of Chinese mitten crab (Eriocheir sinensis).

We investigated the effects of icariin (ICA) on growth performance, antioxidant capacity and non-specific immunity in Chinese mitten crab (Eriocheir sinensis). A total of 200 healthy crabs (average weight: 33.58 ±â€¯0.05 g) were randomly assigned to four treatments with five replicates, each with ten individuals per pool. There were four dietary treatments: the control group (fed with the basal diet), the ICA 50 group, the ICA100 group, and the ICA 200 group (fed with the basal diet supplemented with 50, 100, and 200 mg/kg ICA, respectively). These diets were provided for 8 weeks. Results indicated that ICA100 crabs had higher weight gain (WG), specific growth rate (SGR) and survival rate (SR) than the controls. Protein carbonyl content (PCC) and malondialdehyde (MDA) concentrations in the haemolymph and hepatopancreas of ICA100 crabs were significantly lower than in the control group, while the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly higher. The activities of PO, LZM, ACP and AKP were significantly enhanced with ICA supplementation at 50 and 100 mg/kg, yet decreased subsequently at 200 mg/kg. Furthermore, supplementation of 100 mg/kg ICA up-regulated the mRNA expression of prophenoloxidase (proPO), catalase (CAT), mitochondrial manganese superoxide dismutase (mtMnSOD), thioredoxin-1 (Trx1) and peroxiredoxin 6 (Prx6), while the mRNA expression of toll like receptors (TLRs), NF-κB-like transcription factor Relish and lipopolysaccharide-induced TNF-α factor (LITAF) were down-regulated in the hepatopancreas (P < 0.05). These findings indicate that dietary ICA supplementation at an optimum dose of 100 mg/kg may be effective in improving growth performance, antioxidant capability and non-specific immunity of Chinese mitten crab.

Arginine supplementation improves growth, antioxidant capacity, immunity and disease resistance of juvenile Chinese mitten crab, Eriocheir sinensis.

To investigate the effects of arginine (Arg) on the growth, antioxidant capacity, immunity and disease resistance of juvenile Chinese mitten crab, three diets containing Arg levels at 1.72% (control), 2.73% and 3.72% were formulated and fed to Chinese mitten crab (0.22 ±â€¯0.03 g) for eight weeks. The weight gain, ecdysterone and growth hormone in the serum, relative expression of insulin-like growth factor 2 in the hepatopancreas significantly increased in crabs fed the 2.73% and 3.72% Arg diets. The protein and lipid contents significantly increased in crabs fed the 3.72% Arg diet. The feed conversion ratios in crabs fed the diets with Arg additions were lower than in the control. Arg supplementation also enhanced the antioxidative capacity by increasing the activities of superoxide dismutase, catalase and the relative expression of Kelch-like ECH-associated protein 1 gene in the hepatopancreas, which subsequently decreased malondialdehyde content in the hepatopancreas. Besides, Arg also decreased nitric oxide content in the serum and the activity of nitric oxide synthetase in the hepatopancreas. The relative mRNA levels of crustin, relish, lysozyme and cryptocyanin genes were significantly upregulated by Arg supplementation. The activities of acid phosphatase and alkaline phosphatase in the serum significantly increased in crabs fed the 3.72% Arg diet than those in the control. Similarly, the relative mRNA levels of crustin, cryptocyanin and proPO genes were significantly upregulated in crabs fed the 2.73% Arg diet after lipopolysaccharide challenge, and in crabs fed the 3.72% Arg diet after the Poly (I:C) challenge. The crabs fed the 2.73% and 3.72% Arg diets had higher survival rate after bacterial infection than those fed the control diet. This study indicates that the addition of Arg to the diet at 2.7-3.7% can improve the growth, survival, antioxidant capacity, immunity and disease resistance in juvenile Chinese mitten crab.

Effect of single and combined immunostimulants on growth, anti-oxidation activity, non-specific immunity and resistance to Aeromonas hydrophila in Chinese mitten crab (Eriocheir sinensis).

This study evaluates the effect of dietary supplementation of immunostimulants on the Chinese mitten crab (Eriocheir sinensis) with a single administration of mannan oligosaccharide (MOS), or its combination with either ß-glucan or with inulin for 8 weeks. Four diets included an untreated control diet (C), MOS alone (3 g kg-1, M), MOS with ß-glucan (3 g kg -1 MOS + 1.5 g kg -1 ß-glucan, MB), and MOS with inulin (3 g kg -1 MOS + 10 g kg -1 inulin, MI). The weight gain and specific growth rate of the crabs fed M, MB, and MI diets were improved by lowing feed conversion ratio. The growth and feed utilization of the crabs fed the MB diet were improved compared with the other three groups. The crabs fed the M, MB and MI diets showed a higher intestinal trypsin activity than that in the M and control groups. The highest trypsin activity in the hepatopancreas was observed in the MB group. Crabs fed M, MB and MI diets increased antioxidant system-related enzyme activities, but reduced malondialdehyde. The highest activities of alkaline phosphatase, acid phosphatase, lysozyme and phenol oxidase in the gut and the respiratory burst of the crabs were found in the MB group. The MB diet promoted the mRNA expression of E. sinensis immune genes (ES-PT, ES-Relish, ES-LITAF, p38MAPK and Crustin) compared with the control. After 3 days of infection with Aeromonas hydrophila, the highest survival of crabs was also found in the MB group. This study indicates that the combination of MOS with ß-glucan or with inulin can improve growth, antioxidant capacity, non-specific immunity and disease resistance in E. sinensis.

Nutritional quality of different grades of adult male chinese mitten crab, Eriocheir sinensis.

This study mainly investigated the composition of adult male Chinese mitten crab (Eriocheir sinensis) from four grades/sizes (Grade I: 200-249 g; Grade II: 175-199 g; Grade III: 150-174 g; Grade IV: ≤ 150 g). The results showed that the grade III crabs had the largest gonadsomatic index (GSI), which was significantly higher than the grade I and grade II crabs, no significant difference was found with the grade IV crab. Significant differences in moisture and total lipid contents were observed among various edible parts from different grades of male Eriocheir sinensis. In particular, grade II crabs had the highest total lipid and dry matter content for hepatopancreas. A balanced amino acids composition and a high essential amino acids score (EAAS) were found in the muscle and gonads of grade III crabs. The levels of poly-unsaturated fatty acids (PUFA), n-3 PUFA, n-6 PUFA and docosahexaenoic acid (DHA) in the hepatopancreas, as well as the contents of PUFA, highly-unsaturated fatty acids (HUFA), n-3 PUFA, arachidonic acid (ARA), and eicosapentaenoic acid (EPA) in the gonads were significantly increased in the grade II crabs. Taken together, it can generally be concluded that adult male Eriocheir sinensis of 150-200 g (Grade II-III) weight have the highest nutritional quality even though they are not the largest crabs.

Identification and characterization of nascent polypeptide-associated complex alpha from Chinese mitten crab (Eriocheir sinensis): A novel stress and immune response gene in crustaceans.

Disease in aquatic animals is tightly linked to environmental challenges and their immune responses are greatly modified by their external environment. The chaperone protein nascent polypeptide-associated complex alpha (NACA) has been suggested to play important roles in the cellular response to stress and immune challenges, while the related biological functions remain largely unknown in invertebrates. In the present study we identified a NACA gene (termed EsNACA) from Chinese mitten crab Eriocheir sinensis and analyzed its expression changes in response to ambient (salinity and pH) stresses and immune challenges. The EsNACA protein is comprised of 209 amino acid residues with a conserved DNA binding domain, a C-Jun binding domain, a NAC domain and an ubiquitin-associated domain and shows the highest sequence identity (87%) with its counterpart in shrimp Penaeus monodon. EsNACA mRNA transcripts are presented in all tested normal tissues with predominant expression in hepatopancreas and lower expression in hemocytes. In addition, EsNACA expression was significantly altered in response to the ambient salinity (15‰ and 30‰ salinities) and pH (pH 6 and 8.5) stresses in gill, hepatopancreas, muscle, hemocytes and intestine tissues. Furthermore, EsNACA gene expression was substantially induced upon LPS and Poly(I:C) immune stimulations in E. sinensis hemocytes in vitro. Finally, EsNACA expression was up-regulated in E. sinensis hemocytes, gill, hepatopancreas, intestine and muscle tissues in response to Vibrio anguillarum challenges in vivo. Taken together, our findings for the first time show that EsNACA is an inducible gene involved in stress and immune response in crustaceans.

Single and multi-gene phylogeny of Hepatospora (Microsporidia) - a generalist pathogen of farmed and wild crustacean hosts.

Almost half of all known microsporidian taxa infect aquatic animals. Of these, many cause disease in arthropods. Hepatospora, a recently erected genus, infects epithelial cells of the hepatopancreas of wild and farmed decapod crustaceans. We isolated Hepatospora spp. from three different crustacean hosts, inhabiting different habitats and niches; marine edible crab (Cancer pagurus), estuarine and freshwater Chinese mitten crab (Eriocheir sinensis) and the marine mussel symbiont pea crab (Pinnotheres pisum). Isolates were initially compared using histology and electron microscopy revealing variation in size, polar filament arrangement and nuclear development. However, sequence analysis of the partial SSU rDNA gene could not distinguish between the isolates (~99% similarity). In an attempt to resolve the relationship between Hepatospora isolated from E. sinensis and C. pagurus, six additional gene sequences were mined from on-going unpublished genome projects (RNA polymerase, arginyl tRNA synthetase, prolyl tRNA synthetase, chitin synthase, beta tubulin and heat shock protein 70). Primers were designed based on the above gene sequences to analyse Hepatospora isolated from pea crab. Despite application of gene sequences to concatenated phylogenies, we were unable to discriminate Hepatospora isolates obtained from these hosts and concluded that they likely represent a single species or, at least subspecies thereof. In this instance, concatenated phylogenetic analysis supported the SSU-based phylogeny, and further, demonstrated that microsporidian taxonomies based upon morphology alone are unreliable, even at the level of the species. Our data, together with description of H. eriocheir in Asian crab farms, reveal a preponderance for microvariants of this parasite to infect the gut of a wide array of decapods crustacean hosts and the potential for Hepatospora to exist as a cline across wide geographies and habitats.