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.

Genetic Diversity Analysis of the Red Swamp Crayfish Procambarus clarkii in Three Cultured Populations Based on Microsatellite Markers.

With the increasing scale of crayfish breeding, the self-propagation and "catch large and keep small" breeding patterns have led to serious degradation of the fry, so the selection and breeding of high-quality fry is very important. Selecting a population with a high genetic diversity as the base population for breeding can greatly improve the breeding efficiency. Fifteen microsatellite loci were used to understand the genetic structure and diversity of three Procambarus clarkii populations in Chongming, Shanghai; Gaoyou, Jiangsu; and Xuancheng, Anhui. The results indicated that the three populations were diverse and the number of alleles, observed heterozygosity, expected heterozygosity, Shannon information index, and polymorphic information content ranged from 4.8 to 6.2, 0.5567 to 0.6257, 0.6166 to 0.7086, 1.1292 to 1.3987, and 0.5446 to 0.6452, respectively. The Xuancheng population had the highest genetic diversity. The genetic differentiation coefficient and gene flow of the three populations were between 0.0553 and 0.1068 and 2.0908 and 4.2708, respectively, and there was extensive genetic exchange between the Chongming and Xuancheng populations. Analyses of molecular variance indicated that the genetic variation was mainly within the population (91.51%) and inter-population genetic variation accounted for 8.49%. The unweighted pair group method with an arithmetic mean clustering map was utilised based on the genetic distance between groups, and the results showed that the Gaoyou group was grouped alone, while the Chongming and Xuancheng groups were clustered together. The structural results indicated that the Chongming and Xuancheng groups had the same origin, although the Xuancheng group possessed a more complex genetic structure. This study indicated that all three populations had a high genetic diversity, with the Xuancheng population exhibiting the highest diversity. The results of the study provide a reference for the selection of base populations in breeding programs and confirm that the Xuancheng population in Anhui has a better genetic background. The selection of the Xuancheng population as one of the base populations for genetic breeding will be more efficient to accumulate superior traits.

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.

Effects of copper exposure on lipid metabolism and SREBP pathway in the Chinese mitten crab Eriocheir sinensis.

Copper (Cu) is not only a common metal pollutant in the aquatic environment but also an essential trace element for aquatic organisms such as the Chinese mitten crab (Eriocheir sinensis). Cu is known to regulate lipid metabolism yet exert toxic effects if ingested in excess. However, the molecular regulatory roles of Cu in the lipid metabolism of crabs remains unclear. Thus, this study investigated the potential regulatory mechanism of Cu onto lipid metabolism of E. sinensis following acute Cu exposure. Crabs were exposed to environmental concentration of Cu (50 µg/L) for 96 h, and the expression of sterol regulatory element binding protein (SREBP) was knocked down by RNA interference (RNAi) to test its effect on Cu exposure. The results showed that RNAi significantly attenuated the Cu exposure-induced increase in lipid synthesis and triglycerides (TG) hydrolysis, while significantly inhibited the Cu exposure-induced decrease in fatty acid ß-oxidation, suggesting that SREBP is involved in Cu-induced lipid metabolism. Subsequent analyses of the transcriptome results further revealed potential responsive genes of SREBP that were linked to lipid metabolism and immune regulation. Moreover, Cu may affect lipid metabolism through the TOR-SREBP pathway in E. sinensis. This work provides a reference for exploring the effects of Cu on lipid metabolism disorders in crustaceans.

Label-free quantification proteomics analysis reveals acute hyper-osmotic responsive proteins in the gills of Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab (Eriocheir sinensis) is a typical euryhaline crustacean to study osmotic regulation of crustaceans. Osmotic-regulation of Chinese mitten crab is a complex process. In order to study the osmotic-regulation related proteins of Chinese mitten crab, we domesticated Chinese mitten crab for 144 h with 25 salinity sea water (SW) and 0 salinity fresh water (FW) respectively, and then analyzed the proteome of its posterior gills. A total of 1453 proteins were identified by label free proteomics. Under the threshold of 2 fold change (FC), 242 differentially expressed proteins (DEPs) were screened, including 122 up-regulated DEPs and 120 down-regulated DEPs. GO database and KEGG database were used to annotate and enrich DEPs. It was found that DEPs were significantly enriched in energy metabolism, signal transduction, ion transport, actin cytoskeleton, immunity, lipid metabolism, amino acid metabolism and other biological functions. After 144 h of high salinity stress, the energy metabolism of Chinese mitten crab decreased and the expression of actin and cytoskeleton protein increased. In order to cope with oxidative damage caused by high salinity, Chinese mitten crab improved its immunity and antioxidant capacity.

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.

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 Transcriptome Analysis Reveals the Process of Ovarian Development and Nutrition Metabolism in Chinese Mitten Crab, Eriocheir Sinensis.

Ovarian development is a key physiological process that holds great significance in the reproduction of the Chinese mitten crab (Eriocheir sinensis), which is an economically important crab species for aquaculture. However, there is limited knowledge for the regulatory mechanisms of ovarian development. To study the molecular mechanisms of its ovarian development, transcriptome analysis was performed in the ovary and hepatopancreas of E. sinensis during ovarian stages I (oogonium proliferation), II (endogenous vitellogenesis), and III (exogenous vitellogenesis). The results showed that 5,520 and 226 genes were differentially expressed in the ovary and hepatopancreas, respectively. For KEGG enrichment analysis, the differentially expressed genes in the ovary were significantly clustered in phototransduction-fly, phagosome, and ECM-receptor interaction. Significantly enriched pathways in the hepatopancreas included fatty acid biosynthesis, fatty acid metabolism, and riboflavin metabolism. Further analysis showed that 25 genes and several pathways were mainly involved in oogenesis, including the ubiquitin-proteasome pathway, cyclic AMP-protein kinase A signaling pathway, and mitogen-activated protein kinase signaling pathway. Twenty-five candidate genes involved in vitellogenesis and endocrine regulation were identified, such as vitellogenin, vitellogenin receptor, estrogen sulfotransferase, ecdysone receptor, prostaglandin reductase 1, hematopoietic prostaglandin D synthase and juvenile hormone acid O-methyltransferase. Fifty-six genes related to nutritional metabolism were identified, such as fatty acid synthase, long-chain-fatty-acid-CoA ligase 4, 1-acyl-sn-glycerol-3-phosphate acyltransferase 4, fatty acid-binding protein, and glycerol-3-phosphate acyltransferase 1. These results highlight the genes involved in ovarian development and nutrition deposition, which enhance our understanding of the regulatory pathways and physiological processes of crustacean ovarian development.

Polystyrene microplastics increase Pb bioaccumulation and health damage in the Chinese mitten crab Eriocheir sinensis.

Microplastics may be potential vectors for environmental contaminants such as heavy metals in the aquatic ecosystem due to their highly hydrophobic surfaces and fugacity property. To investigate the combined effects of microplastics with Pb, we exposed juvenile Chinese mitten crabs Eriocheir sinensis to different Pb concentrations (0, 5 and 50 µg/L) combined with microplastics (0 and 400 µg/L) for 21 days to determine the Pb bioaccumulation, oxidative stress, lipid anabolism, and histopathology of hepatopancreas. In general, the results showed that compared to single Pb exposure, the combination of MPs and Pb significantly increased the bioaccumulation of Pb, activities/content of antioxidant biomarkers and lipid metabolism enzymes, and liver injury parameters in crabs, indicating MPs are potential vector of heavy metals and co-exposure exerts more severe effects on crabs. This study provides the insights into the oxidative defense and preliminary lipid anabolism of economic crustaceans in response to combined stress of Pb and MPs.

Moderate acidification mitigates the toxic effects of phenanthrene on the mitten crab Eriocheir sinensis.

Freshwater acidification and phenanthrene may result in complex adverse effects on aquatic animals. Juvenile Chinese mitten crabs (Eriocheir sinensis) were exposed to different pH levels (7.8, 6.5, and 5.5) under phenanthrene (PHE) (0 (control) and 50 µg/L) conditions for 14 days. Antioxidant and transcriptomic responses were determined under stress conditions to evaluate the physiological adaptation of crabs. Under the control pH 7.8, PHE led to significantly reduced activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione S-transferase (GST), but increased glutathione peroxidase (GSH-Px), 7-ethoxyresorufin-o-deethylase (EROD) activities, and malondialdehyde (MDA) levels. However, moderate acidification (pH 6.5) changed PHE effects by increasing antioxidant enzymes. Acidification generally reduced SOD, GPx, GST and EROD activities, but increased CAT, GR, MDA. Compared with pH7.8 group, pH7.8 × PHE and pH6.5 × PHE groups had 1148 and 1498 differentially expressed genes, respectively, with "Biological process" being the main category in the two experimental groups. pH7.8 × PHE treatment caused significant enrichment of disease and immune-related pathways, while under pH6.5 × PHE, more pathways related to metabolism, detoxification, environmental information processing, and energy supply were significantly enriched. Thus, PHE had a significant inhibitory effect on antioxidant performance in crabs, while moderate acidification (pH6.5) mitigated the toxic effects of PHE. Overall, moderate acidification has a positive effect on the defense against the negative effects of PHE in Chinese mitten crabs, and this study provides insights into the defense mechanism of crustaceans in response to combined stress of acidification and PHE.

Effects of Dietary L-TRP on Immunity, Antioxidant Capacity and Intestinal Microbiota of the Chinese Mitten Crab (Eriocheir Sinensis) in Pond Culture.

L-tryptophan (L-TRP) is an essential amino acid for the normal growth of crustaceans. As a nutritional supplement and antioxidant, L-TRP has the function of immune and antioxidant capacity regulation. From July to November, the effects of L-TRP on the immunity, antioxidant capacity and intestinal microflora of the Chinese mitten crab (Eriocheir sinensis) in pond culture were investigated. After feeding an L-TRP diet for 30 (named as August), 60 (named as September) and 106 (named as November) days, respectively, the activities of the immune and antioxidant enzymes in the hepatopancreas and hemolymph were evaluated, and the intestinal microbiota were profiled via high-throughput Illumina sequencing. The results showed that supplementation of L-TRP significantly increased the activities of AKP in the hepatopancreas in September, and significantly increased the activities of ACP in the hepatopancreas in August and September, and the hemolymph's ACP activities also significantly increased in August and November (p < 0.05). Similarly, the activities of SOD, AOC and POD in the hepatopancreas significantly increased in September and November (p < 0.05) after feeding the L-TRP diet; meanwhile, the activities of SOD and AOC in the hemolymph also significantly increased in August (p < 0.05). However, in August, the L-TRP diet resulted in a significant increase in MDA activity in the hepatopancreas and hemolymph (p < 0.05). In addition, the results of the intestinal microbiota analysis showed that Firmicutes, Bacteroidetes and Proteobacteria were the dominant phyla in August, September and November, and Patescibacteria was the dominant phylum in September and November. After feeding the L-TRP diet, the richness of Cyanobacteria and Desulfobacterota significantly increased in August (p < 0.05), and the richness of Actinobacteriota significantly decreased in September (p < 0.05). Moreover, the L-TRP supplementation significantly reduced the abundance of ZOR0006 in the Firmicutes in September (p < 0.05). In conclusion, dietary L-TRP could improve the immunity and antioxidant ability and impact the intestinal health of E. sinensis at the early stage of pond culturing. However, long-term feeding of an L-TRP diet might have no positive impact on the activities of the immune, antioxidant enzymes and intestinal microbiota.

Reproductive performance and semen characteristics of pond-reared and wild-caught large-sized male broodstock of the Chinese mitten crab Eriocheir sinensis.

There has been a recent emphasis on production of large-sized Eriocheir sinensis broodstock. In China, aquaculturists generally prefer wild-caught (WC) crabs from the Yangtze River as broodstock because offspring performance is superior to that of pond-reared (PR) broodstock. Currently, however, there is a ban on fishing in the Yangtze River, and effects on E. sinensis breeding have not been ascertained. There was comparison in the present study of reproductive performance and semen characteristics of male broodstock of PR and WC groups. After copulation, sperm quantity in the vas deferens of crabs in specimens of both groups was large, although there was a consistent decrease in vaso-somatic index. Although sperm density of PR crabs was less, that of WC specimens remained relatively constant. Specimens of neither group, however, had changes in the hepatopancreas index or condition factor, and sperm survival was close to 100%. Although the acrosome reaction was detected in response to cold-temperature induction, there were differences in extent of reaction to cold temperatures. Importantly, in as many as 98% of sperm from female spermathecae, the reaction was completed, which was considerably greater than 15% for sperm of males post-mating. It is concluded there was no difference between PR and WC crabs with respect to reproductive performance or semen characteristics, and, notably, sperm from PR crabs were of sufficient quality for use in E. sinensis aquaculture enterprises. Accordingly, it is predicted the Yangtze River fishing ban would only have a limited effect on supply of male E. sinensis broodstock.

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.

Comparative proteomics elucidates the dynamics of ovarian development in the Chinese mitten crab Eriocheir sinensis.

Ovarian development is a complex physiological process for crustacean reproduction that is divided into the oogonium proliferation stage, endogenous vitellogenic stage, exogenous vitellogenic stage, and oocyte maturation stage. Proteomics analysis offers a feasible approach to reveal the proteins involved in the complex physiological processes of any organism. Therefore, this study performed a comparative proteomics analysis of the ovary and hepatopancreas at three key ovarian stages, including stages I (oogonium proliferation), II (endogenous vitellogenesis) and IV (exogenous vitellogenesis), of the Chinese mitten crab Eriocheir sinensis using a label-free quantitative approach. The results showed that a total of 2,224 proteins were identified, and some key proteins related to ovarian development and nutrition metabolism were differentially expressed. The 26 key proteins were mainly involved in the ubiquitin/proteasome pathway (UPP), cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway during oogenesis. Fifteen differentially abundant proteins (DAPs) were found to participate in vitellogenesis and oocyte development, such as vitelline membrane outer layer protein 1 homolog, vitellogenin, vitellogenin receptor, heat shock 70 kDa protein cognate 3 and farnesyl pyrophosphate synthase. Forty-seven DAPs related to nutrition metabolism were identified, including the protein digestion, fatty acid metabolism, prostaglandin metabolism, lipid digestion and transportation, i.e. short-chain specific acyl-CoA dehydrogenase, acyl-CoA desaturase, fatty acid-binding protein, long-chain fatty acid CoA ligase 4, and hematopoietic prostaglandin D synthase. These results not only indicate proteins involved in ovarian development and nutrient deposition but also enhance the understanding of the regulatory pathways and physiological processes of crustacean ovarian development.

The effects of ammonia-N stress on immune parameters, antioxidant capacity, digestive function, and intestinal microflora of Chinese mitten crab, Eriocheir sinensis, and the protective effect of dietary supplement of melatonin.

Ammonia nitrogen pollution seriously affects the economic benefits of Chinese mitten crab (Eriocheir sinensis) farming. In this study, we first evaluated the protective effects of melatonin (MT) on immune parameters, antioxidant capacity, and digestive enzymes of E. sinensis under acute ammonia nitrogen stress. The results showed that ammonia-N stress significantly decreased the antibacterial ability of crabs, nevertheless MT could significantly improve it under ammonia-N stress (P < 0.05). Ammonia-N group hemolymph antioxidant capacity indicators (T-AOC, T-SOD, GSH-Px) were significantly decreased than control (p < 0.05), while the MT ammonia-N group hemolymph T-SOD activity significantly increased than ammonia-N group (p < 0.05). For hepatopancreas, ammonia-N group GSH-PX activity significantly decreased than control group, but MT ammonia-N group was significant increased than ammonia-N (p < 0.05). Ammonia-N stress has significantly increased the content of MDA in hemolymph and hepatopancreas (p < 0.05), but MT ammonia-N treatment significantly decreased than ammonia-N group (p < 0.05). Compared with the control group, ammonia-N significantly reduced the activities of Trypsin in the intestine and hepatopancreas (p < 0.05), while MT ammonia-N group can significantly improve the intestinal trypsin activity than ammonia-N (p < 0.05). The intestinal microbiota of E. sinensis results showed that ammonia-N stress significantly decreased the relative abundance of Bacteroidetes (p < 0.05). Ammonia-N stress significantly decreased the Dysgonomonas and Rubellimicrobium, and the Citrobacter significantly increased. In summary, melatonin has a protective effect on E. sinensis under ammonia-N stress. Acute ammonia-N stress may lead to the decrease of probiotics and the increase of pathogenic bacteria, which may be closely related to the impairment of digestive function and immune function.

Effects of cadmium alone and in combination with pH on bioaccumulation, tissue structure, and enzyme activity of the Chinese mitten crab, Eriocheir sinensis.

In this study, Chinese mitten crabs (Eriocheir sinensis) were exposed to various combinations of reduced pH (7.8, 7.3, and 6.5) and cadmium (Cd; 0 and 1 mg·L-1) for 7, 14, and 21 days. The reduced pH and 1 mg·L-1 Cd treatment significantly decreased the Cd concentration in crab tissues in the order of pH 7.8 > pH 7.3 > pH 6.5. The exposure to Cd resulted in edema, tubular vacuolization in epithelial cells, and hepatic duct degeneration in the hepatopancreas and indistinct cellular structure and disconnected epithelial layer in the gills. However, low pH alleviated the toxic effects of Cd on the tissues. In gill and hepatopancreas tissues, low pH and Cd exposure caused a significant increase in superoxide dismutase and catalase activities and oxidized glutathione content, but metallothionein activity was not affected. In contrast, the activity of glutathione-S-transferase decreased. Thus, indirect effects of pH on metal accumulation and antagonistic toxicities were observed in E. sinensis, and reduced pH and Cd exposure modulated the oxidative balance via different mechanisms.

Effects of miR-143 and its target receptor 5-HT2B on agonistic behavior in the Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab (Eriocheir sinensis) as a commercially important species is widely cultured in China. However, E. sinensis is prone to agonistic behavior, which causes physical damage and wastes energy resources, negatively impacting their growth and survival. Therefore, understanding the regulatory mechanisms that underlie the switching of such behavior is essential for ensuring the efficient and cost-effective aquaculture of E. sinensis. The 5-HT2B receptor is a key downstream target of serotonin (5-HT), which is involved in regulating animal behavior. In this study, the full-length sequence of 5-HT2B gene was cloned. The total length of the 5-HT2B gene was found to be 3127 bp with a 236 bp 5'-UTR (untranslated region), a 779 bp 3'-UTR, and a 2112 bp open reading frame encoding 703 amino acids. Phylogenetic tree analysis revealed that the 5-HT2B amino acid sequence of E. sinensis is highly conserved with that of Cancer borealis. Using in vitro co-culture and luciferase assays, the miR-143 targets the 5-HT2B 3'-UTR and inhibits 5-HT2B expression was confirmed. Furthermore, RT-qPCR and Western blotting analyses revealed that the miR-143 mimic significantly inhibits 5-HT2B mRNA and protein expression. However, injection of miR-143 did not decrease agonistic behavior, indicating that 5-HT2B is not involved in the regulation of such behavior in E. sinensis.

Gonadal development and biochemical composition of Chinese mitten crabs (Eriocheir sinensis) from four sources.

The gonadal development; edible tissue ratio; and proximate, fatty acid, and free amino acid composition were examined and compared among Eriocheir sinensis samples from four sources. The gonadosomatic index (GSI) of Heilongjiang (HLJ) crabs was significantly higher than that of the other crabs from September to October (P < 0.05). Of all the edible tissues of E. sinensis, HLJ crab had the highest linolenic acid (18:3n3, LNA) and arachidonic acid (20:4n6, ARA) contents (P < 0.05), while Qinghai (QH) crab had the highest eicosapentaenoic acid (20:5n3, EPA) content (P < 0.05). The highest docosahexaenoic acid (22:6n3, DHA) and DHA/EPA contents were observed in Shandong (SD) crabs (P < 0.05), and the highest linoleic acid (18:2n6, LA) content was found in Shanghai (SH) crabs (P < 0.05). In conclusion, E. sinensis inhabiting relatively low-temperature regions (northern China) can initiate gonadal development earlier, and the quality of E. sinensis living in paddies and lakes is better than that of crabs living in ponds.

The protective effects of melatonin on survival, immune response, digestive enzymes activities and intestinal microbiota diversity in Chinese mitten crab (Eriocheir sinensis) exposed to glyphosate.

Glyphosate is one of the most widely used pesticides, which can cause toxicity to aquatic animals. In this study, the survival rate, immune response, digestive enzyme activities, and the intestinal microbiota diversity of Chinese mitten crab (Eriocheir sinensis) were evaluated after 14 days of exposure to glyphosate (48.945 mg/L from 50% 96 h LC50 value) and melatonin feeding (80 mg/kg). The results showed that MT significantly improved the survival rate, antibacterial capacity of E. sinensis (P < 0.05). After exposure to glyphosate, the expression of Hsp60, Hsp70 and Hsp90 in cranial ganglia and thoracic ganglia was decreased significantly, but MT significantly raised the expression of these proteins (P < 0.05). Glyphosate significantly decreased lipase activity compared with the control group (P < 0.05), while melatonin significantly increased the lipase, amylase and trypsin activities (P < 0.05). Melatonin significantly increased the Chao1 and Shannon index and the relative abundance of Proteobacteria and Bacteroidetes (P < 0.05). This study shows that melatonin has a protective effect on the glyphosate exposed E. sinensis.

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.