Temporal and Spatial Signatures of Scylla paramamosain Transcriptome Reveal Mechanistic Insights into Endogenous Ovarian Maturation under Risk of Starvation.

Variability in food availability leads to condition-dependent investments in reproduction. This study is aimed at understanding the metabolic response and regulatory mechanism of female Scylla paramamosain in response to starvation in a temporal- and tissue-specific manner. The mud crabs were starved for 7 (control), 14, 28, and 40 days for histological and biochemical analysis in the hepatopancreas, ovary, and serum, as well as for RNA sequencing on the hepatopancreas and ovary. We further highlighted candidate gene modules highly linked to physiological traits. Collectively, our observations suggested that starvation triggered endogenous ovarian maturation at the expense of hepatopancreas mass, with both metabolic adjustments to optimize energy and fatty acid supply from hepatopancreas to ovary in the early phase, followed by the activation of autophagy-related pathways in both organs over prolonged starvation. These specific adaptive responses might be considered efficient strategies to stimulate ovarian maturation of Scylla paramamosain under fasting stress, which improves the nutritional value of female mud crabs and other economically important crustaceans.

Glyphosate-based herbicide causes spermatogenesis disorder and spermatozoa damage of the Chinese mitten crab (Eriocheir sinensis) by affecting testes characteristic enzymes, antioxidant capacities and inducing apoptosis.

Glyphosate-based herbicide (GBH) is a popular herbicide, which may contaminate the water environment and affect aquatic animals. In this study, testes morphology, physiology function, apoptosis pathway, and spermatozoa quality of Chinese mitten crab (Eriocheir sinensis) were evaluated after 7 days of GBH exposure (48.945 mg/l,1/2 of the 96 h LC50 value of GBH). Results showed that GBH induced spermatogenesis disorder by H.E. staining. The obvious vacuolar degenerations and fewer spermatids of the testes accompanied by decreased primary spermatocytes-type seminiferous tubules (PSc-STs) were observed. The extensive apoptosis of spermatids by TUNEL staining was visible. Meanwhile, testes'' characteristic enzyme activities associated with spermatogenesis, including lactate dehydrogenase (LDH) and acid phosphatase (ACP) were significantly decreased. Testes suffered oxidative damage as reflected by the significant decrease in superoxide dismutase (SOD) activities, the significant increase in malondialdehyde (MDA) contents, and heat shock proteins (HSP-70) mRNA expression. Further studies demonstrated that GBH induced apoptosis of testes through the mitochondrial apoptotic pathway by upregulating the relative mRNA expression of cysteinyl aspartate specific proteinase 3 (Caspase-3), Bcl-2-associated X protein (Bax), and downregulating B-cell lymphoma 2 (Bcl-2). Oxidative damage may be one of the causes of GBH-induced apoptosis in testes. After GBH exposure, the morphology of spermatophores was changed. The survival and the acrosome reaction (AR) ratio of spermatozoa was significantly decreased. Altogether, these results demonstrated that GBH affects spermatogenesis, spermatophore and spermatozoa quality of E.sinensis, which provides novel knowledge about the toxic effects of GBH on the reproductive system of crustaceans.

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.

Transcriptome analysis reveals antioxidant defense mechanisms in the red swamp crayfish Procambarus clarkia after exposure to chromium.

Chromium (Cr) as a chromate anion has a strong redox capacity that seriously threatens the ecological environment and human health. Cr can contaminate water and impart toxicity to aquatic species. Procambarus clarkii is an important food source that once represented a large proportion of the aquaculture industry due to its rapid reproduction and high economic value. However, there have been reports on the death of P. clarkii due to heavy metal pollution. The underlying mechanism regarding heavy metal toxicity was studied in this paper. The transcriptome data of hemocytes extracted from P. clarkii injected with Cr were analyzed by high-throughput sequencing and compared to the control group. In total, 48,128,748 clean reads were obtained in the treatment group and 56,480,556 clean reads were obtained in the control group. The reads were assembled using Trinity and the identified unigenes were then annotated. Then, 421 differentially-expressed genes (DEGs) were found, 170 of which were upregulated and 251 downregulated. Many of these genes were found to be related to glutathione metabolism and transportation. The glutathione metabolic pathway of P. clarkii was thus activated by Cr exposure to detoxify and maintain body function. Validation of DEGs with quantitative real-time PCR confirms the changes in gene expression. Thus, this study provides data supporting a glutathione-focused response of P. clarkii to exposure to heavy metals.

Micro-algal astaxanthin could improve the antioxidant capability, immunity and ammonia resistance of juvenile Chinese mitten crab, Eriocheir sinensis.

Green alga Haematococcus pluvialis is an important source of natural astaxanthin (Ast), which have been shown to be beneficial for the color formulation, survival, antioxidation, immunity and stress resistance of many crustacean. This study was conducted to investigate the effects of dietary supplementation of H. pluvialis meal on growth, antioxidant status, ammonia resistance, color parameters, and carotenoids composition of juvenile Chinese mitten crab Eriocheir sinensis. Five diets were formulated to contain 0, 30, 60, 90 and 120 mg/kg dry diets of natural Ast (defined as Diet 1-5) using H. pluvialis meal as astaxanthin source. The results showed that: (1) Although all treatments with Ast supplementation had the relatively higher growth performance and survival than the control (Diet 1 treatment), no significant differences were found on growth performance, feed conversion ratio and hepatosomatic index among all treatments. (2) The highest total antioxidant capacity (T-AOC) in hepatopancreas and hemolymph were observed in Diet 4 and 3 treatments respectively, while the lowest malondialdehyde (MDA) contents in hepatopancreas and hemolymph were also found in these two treatments. Furthermore, the significantly positive relationships were detected on acid phosphatase (ACP) activities and dietary Ast contents for hepatopancreas and hemolymph. (3) Diet 3 treatment had the highest mRNA levels of EsLecA, EsTrx, and EsPrx6 in hepatopancreas, while both Diet 3 and 4 treatments reached the peaks for mRNA expression levels of EsMyd88 and EsHc, respectively. (4) The stress test with ammonia-N indicated Diet 1 treatment had the highest mortality among all treatments, and the lowest mortality was found on Diet 3 treatment during the stress test. (5) Dietary Ast significantly improved the redness (a*) of carapace and hepatopancreas, which were consistent with the Ast contents in these tissues from the different treatments. Ast concentrations in carapace reached the plateau for Diet 3 treatment while hepatopancreatic Ast concentration kept increasing with elevating dietary Ast contents. In conclusion, natural astaxanthin could enhance the antioxidative capability, non-specific immunity, tissue Ast contents and stress resistance to ammonia-N, and these results suggested the optimal diet micro-algal astaxanthin was around 60 mg/kg for juvenile E. sinensis.

Transcriptome analysis reveals the potential mechanism of dietary carotenoids improving antioxidative capability and immunity of juvenile Chinese mitten crabs Eriocheir sinensis.

Carotenoids are known to be involved in the regulation of the antioxidative capability, immune response and stress resistance in crustacean species; however, very limited information is available on their underlying molecular mechanisms. This study performed transcriptome sequencing of hemolymph and hepatopancreas of juvenile Chinese mitten crabs (Eriocheir sinensis) that fed with three diets, i.e. diet A containing 90 mg kg-1 dry weight of astaxanthin, diet B containing 200 mg kg-1 dry weight of ß-carotene and control diet without supplementation of dietary carotenoids. The results showed that there were 2955 and 497 differentially expressed genes (DEGs) in the hemolymph between the astaxanthin treatment and control groups, and between the ß-carotene treatment and control groups, respectively. Moreover, compared with the control group, 833 and 1886 DEGs were obtained in the hepatopancreas of the astaxanthin treatment and the ß-carotene treatment groups, respectively. The DEGs in the three groups were enriched in 255 specific KEGG metabolic pathways according to KEGG enrichment analysis. Through this study, a series of key genes involved in Nrf2 signalling, ROS production, intracellular antioxidant enzymes and chaperones were significantly affected by dietary carotenoids. Dietary carotenoids also significantly altered the expression levels of immune-related molecules associated with signal transduction, prophenoloxidase cascade, apoptosis, pattern recognition proteins/receptors and antimicrobial peptides. In conclusion, this transcriptomic study provides valuable information for understanding the molecular mechanism and potential pathway of dietary carotenoids improved the antioxidative capability and immunity of juvenile E. sinensis.

TMT-based quantitative proteomic analysis of Eriocheir sinensis hemocytes and thoracic ganglion during Spiroplasma eriocheiris infection.

Spiroplasma eriocheiris, a novel pathogen of Chinese mitten crab Eriocheir sinensis tremor disease, has led into catastrophic economic losses in aquaculture. S. eriocheiris invaded the hemocytes in the early stage, then invaded nerve tissue and caused typically paroxysmal tremors of pereiopod in the late stage of infection. The purpose of this study was to detect the infection mechanism of hemocytes in the early stage and thoracic ganglion in the late stage of S. eriocheiris infection at the protein level. Hemocytes and thoracic ganglion were collected at 24 h and 10 d after injection (the crabs with typical paroxysmal tremors of the pereiopod), respectively. TMT was performed with isobaric markers, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). In hemocytes, 127 proteins were up-regulated and 85 proteins were down-regulated in 2747 quantified proteins. Many proteins and process including proPO system proteins, hemolymph coagulation system proteins and lectins were differently expressed in hemocytes and involved in the early immune process of E. sinensis against S. eriocheiris infection. Meanwhile, 545 significantly different expression proteins (292 down-regulated and 253 up-regulated protein including a number of immune-associated, nervous system development and signal transmission related proteins) were identified in thoracic ganglion in the late stage of S. eriocheiris infection. The qRT-PCR analysis results shown that the selected significantly changed proteins in hemocytes and thoracic ganglion were consistent with the TMT proteomics. This paper reported for the first time to study the responses of crab hemocyte and thoracic ganglion against the S. eriocheiris infection at different stages. These findings help us understand the infection mechanism of S. eriocheiris at different stage with the different tissue.

Functional expression patterns of four ecdysteroid receptor isoforms indicate their different functions during vitellogenesis of Chinese mitten crab, Eriocheir sinensis.

In arthropods, alternative splicing of ecdysteroid receptor gene (EcR) leads to multiple functions of different EcR isoforms during metamorphosis, growth and ovarian development via ecdysteroid signaling pathway. This study was conducted to investigate the expression patterns of four EcRs of Eriocheir sinensis (EsEcRs) and the changes of haemolymph ecdysteroid titer during the ovarian development. The results showed that four EsEcR isoforms had the tissue-specific expression among 12 examined tissues, and the highest transcript levels of the four EsEcR isoforms were detected in Y-organ or sinus gland. During the ovarian development, EsEcR1 showed the highest transcript abundance of the four EsEcR isoforms. The expression profiles of all the EsEcR isoforms were similar in the hepatopancreas during the ovarian maturation cycle of E. sinensis with a trend of "high-low-high-low". In ovary, the highest expression levels of EsEcR1 and EsEcR4 were both found at stage V ovary, while the peaks of EsEcR2 and EsEcR3 were found on stage III ovary and stage IV ovary, respectively. Meanwhile, the ecdysteroid titer in haemolymph decreased gradually during ovarian maturation cycle. Further regression analysis revealed significant negative correlations were found between the ovarian EsEcR3/ EsEcR4 expression levels and haemolymph ecdysteroid titer during part or whole ovarian development cycle. These results together indicated that four EsEcR isoforms may have different functions during ovary maturation of E. sinensis. All EcR isoforms and ecdysteroid seemed to have important roles in the hepatopancreas during early ovarian development stages, while EsEcR3 and EsEcR4 were closely related to the mid-late vitellogenesis stages.

Effect of dietary L-tryptophan on the survival, immune response and gut microbiota of the Chinese mitten crab, Eriocheir sinensis.

This study investigated the influence of L-tryptophan (L-trp) on the survival, immune response and gut microbiota of the Chinese mitten crab, Eriocheir sinensis (with an average weight of 16.58 ±â€¯2.20 g). After 30 days of feeding with diets supplemented with L-trp at 0.36%, 0.47%, 0.73% and 1.05% (groups 1, 2, 3 and 4, respectively), the survival rate and bacterial challenge (Aeromonas hydrophila) were evaluated, the activities of antioxidant and phosphatase enzymes in the serum were assessed, and the gut microbiota were measured via high-throughput Illumina sequencing. The results showed that the supplementation of L-trp significantly improved the survival rate of crabs (P < 0.05). After feeding for 7 days, it was observed that a high L-trp diet significantly increase the survival rate relative to a basal diet after a 96-h post-challenge with A. hydrophila (P < 0.05). The activity of CAT and AKP in the serum were increased by the addition of L-trp. The activity of CAT and AKP in the serum in group 4 were higher than those in group 1 (P < 0.05). Furthermore, we observed that adjunction of the L-trp can significantly increase the richness and diversity of the gut microbiota. The dominant phylum in the intestine of the Chinese mitten crab were Tenericutes, Proteobacteria, Firmicutes, Chloroflexi and Actinobacteria. The L-trp in the diets increased the richness of Proteobacteria, Firmicutes and Actinobacteria in the intestine significantly. These bacteria were all dominant bacteria and had a specific role in promoting the immunity of E. sinensis. Therefore, it could be inferred that L-trp supplementation is beneficial in the diet of E. sinensis. Based in these results, the dietary 0.47% or 0.73%L-trp supplemented is found to be optimum to improve E. sinensis survival.

Effect of estradiol on hepatopancreatic lipid metabolism in the swimming crab, Portunus trituberculatus.

Estradiol is an important sex steroid hormone that involved in regulation of animal lipid metabolism. However, the effect of estradiol on lipid metabolism in swimming crab (Portunus trituberculatus) is unclear. The present study investigated the effect of four concentrations of exogenous estradiol (0, 0.01, 0.1 and 1 µg g-1 crab weight) on the expression levels of lipid metabolism-related genes, lipid composition and histology of hepatopancreas in the P. trituberculatus. The results showed that the mRNA levels of carnitine palmitoyltransferase I and II (CPT-I and CPT-II) increased significantly at the low concentrations (0.01 µg g-1 and 0.1 µg g-1), while decreased significantly in the highest concentration (1 µg g-1). The mRNA levels of acyl-CoA oxidase (ACOX), fatty acid transport protein (FATP), fatty acid-binding protein (FABP), diacylglycerol acyltransferase 1 (DGAT1) and acetyl-CoA carboxylase (ACC) were significantly down-regulated. The transcripts of fatty acid synthase (FAS) and fatty acyl desaturase (FAD) decreased significantly only in 1 µg g-1 treatment. All estradiol treatments (0.01, 0.1 and 1 µg g-1) had significantly higher percentages of 20:4n6, 20:5n3 and 22:6n3, but lower percentages of total monounsaturated fatty acids and polar lipids than the control treatment (0 µg g-1). Histological observations indicated the size of B cell became larger under estradiol treatment. The results indicated that estradiol promoted lipid catabolism in the hepatopancreas of P. trituberculatus.

Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation.

Tissue remodeling is a crucial process in animal development and disease progression. Coordinately controlled by the two main insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), tissues are remodeled context-specifically during insect metamorphosis. We previously discovered that two matrix metalloproteinases (Mmps) cooperatively induce fat body cell dissociation in Drosophila However, the molecular events involved in this Mmp-mediated dissociation are unclear. Here we report that JH and 20E coordinately and precisely control the developmental timing of Mmp-induced fat body cell dissociation. We found that during the larval-prepupal transition, the anti-metamorphic factor Kr-h1 transduces JH signaling, which directly inhibited Mmp expression and activated expression of tissue inhibitor of metalloproteinases (timp) and thereby suppressed Mmp-induced fat body cell dissociation. We also noted that upon a decline in the JH titer, a prepupal peak of 20E suppresses Mmp-induced fat body cell dissociation through the 20E primary-response genes, E75 and Blimp-1, which inhibited expression of the nuclear receptor and competence factor ßftz-F1 Moreover, upon a decline in the 20E titer, ßftz-F1 expression was induced by the 20E early-late response gene DHR3, and then ßftz-F1 directly activated Mmp expression and inhibited timp expression, causing Mmp-induced fat body cell dissociation during 6-12 h after puparium formation. In conclusion, coordinated signaling via JH and 20E finely tunes the developmental timing of Mmp-induced fat body cell dissociation. Our findings shed critical light on hormonal regulation of insect metamorphosis.

Correction to: Immunolocalization and changes of 17beta-estradiol during ovarian development of Chinese mitten crab Eriocheir sinensis.

The published online version contains mistake in Title and in Acknowledgement section. Please find below for the needed corrections.

Immunolocalization and changes of 17beta-estradiol during ovarian development of Chinese mitten crab Eriocheir sinensis' [corrected].

17beta-estradiol (E2) is important for crustacean ovarian development. This study aims to investigate the distribution and change pattern of E2 in the ovary, hepatopancreas, thoracic ganglion and brain ganglion as well as Vg-mRNA expression level during ovarian development of Chinese mitten crab Eriocheir sinensis. Results showed that strongly positive signals of E2 were mainly distributed in follicle cells of ovaries for all developmental stages as well as oocyte cytoplasm of stages III to V ovaries. In hepatopancreas, the E2-positive signal was mainly detected in the cytoplasm and nucleus of fibrillar cells and the nucleus of resorptive cells, while the maximum fluorescence intensity was observed in stage III hepatopancreas. On the contrary, the E2 immunoreactivities in nervous tissues were relatively stable during ovarian development. Moreover, the changing pattern of E2 concentration was similar within hemolymph, ovary and hepatopancreas during the ovarian development. From stages I to III, the E2 content in three tissues increased significantly, then decreased gradually until stage V. As for the Vg-mRNA expression level in hepatopancreas and ovaries, an increasing trend was found in ovaries but no significant difference was detected during the period of ovarian stages III to V. Hepatopancreatic Vg-mRNA expression level increased significantly during stages I to IV and dramatically decreased at stage V. In conclusion, our study suggests that ovary, hepatopancreas, hemolymph and nervous tissues are the target organs of E2 in E. sinensis and E2 concentrations in different tissues are closely related to vitellogenesis in ovary and hepatopancreas during ovarian development.

De novo transcriptome sequencing and analysis of male and female swimming crab (Portunus trituberculatus) reproductive systems during mating embrace (stage II).

BACKGROUND: The swimming crab Portunus trituberculatus is one of the most commonly farmed crustaceans in China. As one of the most widely known and high-value edible crabs, it crab supports large crab fishery and aquaculture in China. Only large and sexually mature crabs can provide the greatest economic benefits, suggesting the considerable effect of reproductive system development on fishery. Studies are rarely conducted on the molecular regulatory mechanism underlying the development of the reproductive system during the mating embrace stage in this species. In this study, we used high-throughput sequencing to sequence all transcriptomes of the P. trituberculatus reproductive system. RESULTS: Transcriptome sequencing of the reproductive system produced 81,688,878 raw reads (38,801,152 and 42,887,726 reads from female and male crabs, respectively). Low-quality (quality <20) reads were trimmed and removed, leaving only high-quality reads (37,020,664 and 41,021,030 from female and male crabs, respectively). A total of 126,188 (female) and 164,616 (male) transcripts were then generated by de novo transcriptome assembly using Trinity. Functional annotation of the obtained unigenes revealed that a large number of key genes and some important pathways may participate in cell proliferation and signal transduction. On the basis of our transcriptome analyses and as confirmed by quantitative real-time PCR, a number of genes potentially involved in the regulation of gonadal development and reproduction of P. trituberculatus were identified: ADRA1B, BAP1, ARL3, and TRPA1. CONCLUSION: This study is the first to report on the whole reproductive system transcriptome information in stage II of P. trituberculatus gonadal development and provides rich resources for further studies to elucidate the molecular basis of the development of reproductive systems and reproduction in crabs. The current study can be used to further investigate functional genomics in this species.

Effects of the complete replacement of fish oil with linseed oil on growth, fatty acid composition, and protein expression in the Chinese mitten crab (Eriocheir sinensis).

BACKGROUND: The finite marine resources make it difficult for us to obtain enough fish oil (FO) used in aquatic feeds. Another sustainable ingredients should be found to substitute FO. The effects of replacing FO with vegetable oil have been studied in a variety of crustaceans, but most studies have focused on the phenotypic effects. Little is known about the mechanisms of the effects. METHODS: To understand the molecular responses during the replacement of FO in Eriocheir sinensis, we investigated the effects of feeding FO or linseed oil (LO) on growth performance, digestive enzyme activity, fatty acid composition and protein expression in E. sinensis. Twenty-four juvenile crabs were fed diets containing FO or LO for 112 days. Weight, carapace length and width were recorded. Fatty acid composition of the diets and the hepatopancreas and protein expression in the hepatopancreas were analyzed. RESULTS: Growth performance and molting interval were unchanged by diet. Crabs fed FO and LO had same activity of lipase and amylase, but comparing with crabs fed LO, crabs fed FO had higher trypsin activity and lower pepsin activity. Hepatopancreas fatty acid composition changed to reflect the fatty acid composition of the diets. In total, 194 proteins were differentially expressed in the hepatopancreas between the diets. Expression of heat shock proteins was higher in crabs fed LO. Expression of fatty acid synthase, long-chain fatty acid transport protein 4, acyl-CoA delta-9 desaturase, and fatty acid-binding protein 1, was higher in crabs fed FO. CONCLUSIONS: The substitution of FO with LO didn't have any effects on the growth and molting of mitten crab, but could significantly decrease the ability of mitten crab to cope with stress. The high content of HUFAs in the hepatopancreas of mitten crab fed FO is due to the high abundance of the proteins relative to the transport of the HUFAs. These findings provide a reason of the high content of EPA and DHA in crabs fed with FO, and provide new information for the replacement of FO in diets of mitten crab.

Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation.

In the pond culture of Eriocheir sinensis, high limb-autotomy seriously affects the quality and culture's economic efficiency. Based on our previous studies, limb autotomy can induce the changes of hematological immune response in E. sinensis hemolymph. Eyestalk ablation can accelerate the regeneration of limbs after autotomy. To detect the important functional genes related to the hematological molecular immunity of E. sinensis, we compared and analyzed the hemolymph transcriptome data of the intact crab, left cheliped autotomized crabs and bilateral eyestalk ablation crabs with high-throughput sequencing techniques. The results showed that the three groups obtained 62 172 414, 68 143 682, and 67 811 618 clean reads, respectively. A total of 9567 differentially expressed genes were obtained by multiple comparison of the three groups' libraries. Gene ontology (GO) functional classification analysis shows that the differential genes belong to 42 categories of biological process, cellular components and molecular function. The differentially expressed genes in the three libraries were enriched to 344 specific KEGG metabolic pathways by KEGG enrichment analysis, such as the up-regulated gene (dual oxidase (Duox), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAQ)) in MAPK signaling pathway, the up-regulated gene (aldehyde dehydrogenase 1 (ALDH 1)) and down-regulated gene (UDP-glucuronosyltransferase 2 (UGT 2)) in metabolism of the xenobiotics by cytochrome P450 pathway, the down-regulated gene (actin gene (AG), heat shock protein 90 (HSP 90)) in fluid shear stress and atherosclerosis pathway. To verify the expression levels of DEGs identified by RNA-Seq, the above six hematological immune-related genes were selected for qRT-PCR validation, the qRT-PCR results were consistent with the DEGs results. Our research obtained abundant E. sinensis hemolymph transcriptome information by RNA-Seq, which provides multi-level information for the cloning of novel genes and the study of hemolymph molecular immunology mechanisms of 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.

Effects of glyphosate on immune responses and haemocyte DNA damage of Chinese mitten crab, Eriocheir sinensis.

As a broad-spectrum organophosphorus herbicide, glyphosate is widely utilized around the world. The toxic effects of glyphosate on Chinese mitten crab, Eriocheir sinensis, were assessed using immunotoxicity and genotoxicity biomarkers in this study. The results showed that 24 h and 96 h LC50 values of glyphosate for E. sinensis were estimated as 461.54 and 97.89 mg/L, respectively, and the safe concentration was 4.4 mg/L. According to the results above, glyphosate was applied at concentrations of 0, 4.4, 9.8, 44 and 98 mg/L, for 96 h in the exposure experiment. Total haemocyte count (THC) and percentage of granulocytes decreased significantly following 6 h exposure to each concentration of glyphosate and tended to gradually stabilize after 12 h except in 4.4 mg/L, which rapidly recovered to a normal level in 12 h. Phagocytic activity in all treatments decreased dramatically at 6 h and maintained stability until the 96-h mark. Comet tail has been observed early at 24 h in each treatment, and the comet ratio and percentage of DNA (% DNA) in the tail increased as the exposure experiment progressed. Immune-related enzyme activity varied during the experiment. Acid phosphatase (ACP) and alkaline phosphatase (AKP) activities in 44 and 98 mg/L treatments decreased significantly after 48 h exposure, while AKP activities in all concentrations increased markedly at the beginning of exposure. The superoxide dismutase (SOD) and peroxidase (POD) activities increased significantly after 6 h exposure to 44 and 98 mg/L of glyphosates but decreased at 24 h. In addition, the ß-glucuronidase (ß-GD) activities in the 9.8, 44 and 98 mg/L groups, increased after 6-h exposure and were significantly higher than those in the control at 96 h. These results indicated that glyphosate has evident toxic effect on E. sinensis by immune inhibition that is possibly due to the haemocyte DNA damage and a sharp decline in haemocyte numbers, which subsequently induced changes in activities of immune-related enzymes and haemocyte phagocytosis.

Effects of lactic acid bacteria on rearing water bacterial community in Eriocheir sinensis culture.

The Chinese mitten crab (CMC, Eriocheir sinensi) culture in ponds is a unique aquaculture system. Probiotics are commonly used in the maintenance of the health of pond-cultured CMCs. However, the effects of probiotics on the bacterial community of CMC-culturing water remain unclear. This study utilized 16S rRNA gene amplicon sequencing to assess changes in the bacterial community composition, diversity, assembly, and co-occurrence patterns in CMC-culturing water following probiotic application. The results indicate that the α-diversity of the bacterial community in CMC-culturing water varied with time following probiotic application. The addition of probiotics to the water resulted in an increase in the occurrence of new operational taxonomic units (OTUs). The bacterial community assembly in the CMC-culturing water was shaped by a balance between deterministic and stochastic processes, while commercial probiotics enhanced the proportion of heterogeneous selection. In addition, including OTU2953 (Burkholderiaceae) and OTU3005 (Lactobacillaceae), from the commercial probiotics served as keystone species in the bacterial network of CMC-culturing water. Overall, probiotic application had a significant impact on the bacterial ecology of CMC-culturing water.

Effect of different rice transplanting patterns on microbial community in water, sediment, and Procambarus clarkii intestine in rice-crayfish system.

Although the microbial ecology of integrated rice-crayfish farming systems is receiving increasing attention with the expanding application area in China, the effects of rice transplanting patterns on the microbial community of water, sediment and Procambarus clarkii intestine in rice-crayfish system has yet to be determined. This study explored the microbial community present in water, sediment and intestine samples from three transplant patterns (rice crayfish with wide-narrow row transplanting, rice-crayfish with normal transplanting and pond-crayfish, abbreviated as RC-W, RC, and PC, respectively) using high-throughput sequencing. The results showed that the dominant microbial taxa from sediment, surrounding water, and intestine at phylum level were Proteobacteria, Chloroflexi, Cyanobacteria, Actinobacteria, Bacteroidetes. The patterns of rice transplanting had significant effects on microbial biodiversity and species composition in surrounding water. The OTUs community richness of water under RC group was significantly higher than that of PC group and RC-W group. The OTU relative abundance of top 10 operational taxonomic units had significantly different (p < 0.05) in the water samples from the three groups. The intestinal OTU community richness of Procambarus clarkii in the three groups was positively correlated with the community richness of water. The proximity between intestinal and water samples in PCA diagram indicated that their species composition was more similar. The results also showed that rice transplanting patterns can affect intestinal microbial biodiversity of Procambarus clarkii and the intestinal microbial biodiversity correlated with water bodies. Although the intestinal microbial diversity of crayfish in RC-W group was lower than that in RC group, the relative abundance of potential pathogenic bacteria, such as Vibrio, Aeromonas, in intestine of the crayfish in the RC-W group was significantly decreased under rice wide-narrow row transplanting model. Redundancy analysis revealed that environmental parameters, such as pH, DO, nitrate, which regulate the composition of microbial community structures. This study provides an understanding for microbial response to different rice transplanting pattern in rice-crayfish farming system.