Eriocheir Sinensis Research Articles

Exploration of Synergistic Regulation Mechanisms of Cerebral Ganglion and Muscle in Eriocheir sinensis Activated in Response to Alkalinity Stress.

The cerebral ganglion and muscle are important regulatory tissues in Eriocheir sinensis. Therefore, it is of great significance to explore their synergistic roles in this organism's anti-stress response. In this study, proteomics, metabolomics, and combination analyses of the cerebral ganglion and muscle of E. sinensis under alkalinity stress were performed. The cerebral ganglion and muscle played a significant synergistic regulatory role in alkalinity adaptation. The key regulatory pathways involved were amino acid metabolism, energy metabolism, signal transduction, and the organismal system. They also played a modulatory role in the TCA cycle, nerve signal transduction, immune response, homeostasis maintenance, and ion channel function. In conclusion, the present study provides a theoretical reference for further research on the mechanisms regulating the growth and development of E. sinensis in saline-alkaline environments. In addition, it provides theoretical guidelines for promoting the vigorous development of the E. sinensis breeding industry in saline-alkaline environments in the future.

The involvement of tumor necrosis factor receptor-associated factor 6 in regulating immune response by NF-κB at pre-molt stage of Chinese mitten crab (Eriocheir sinensis)

Molting is a crucial biological process of crustaceans. Crustaceans go through three separate stages throughout their molting process, including pre-molt, post-molt and inter-molt. However, the exact mechanism of immunological modulation during molting remains unclear. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been extensively documented to participate in immune defense. In the present study, a TRAF6 gene with two TRAF-type zinc finger domains was identified from Eriocheir sinensis (designed as EsTRAF6), and its role in regulating immune response during molting process was explored. The mRNA expression level of EsTRAF6 at pre-molt stage was higher than that at post-molt stage and inter-molt stage. After Aeromonas hydrophila stimulation, the expression levels of EsTRAF6, EsRelish and anti-lipopolysaccharide factors (ALFs) genes exhibited a considerable increase at three molting stages. Subsequently, the expression patterns of EsTRAF6 and EsRelish in response to the treatment with 20-hydroxyecdysone (20E) were examined. The mRNA expression of EsTRAF6 and EsRelish were significantly increased at 12 h after 20E injection. Additionally, the protein expression level of TRAF6 was also up-regulated in 20E group compared to control group. Furthermore, the role of EsTRAF6 in regulating the anti- ALFs expression at pre-molt stage post A. hydrophila stimulation was investigated. Following the inhibition of the EsTRAF6 transcript using RNAi or the injection of inhibitor (TMBPS), there was a notable decrease of the EsALF1, EsALF2 and EsALF3 transcripts. Moreover, a significant reduction in the phosphorylation level of NF-κB at pre-molt stage was observed after A. hydrophila stimulation in TRAF6-inhibited crabs. Collectively, our results suggest that EsTRAF6 could be induced by 20E and promoted the EsALFs expression by activating NF-κB at pre-molt stage, which provides a novel insight into the research of immune regulatory mechanism during the process of molting of crustaceans.

Dietary monosodium glutamate affects the growth and feed utilization of Eriocheir sinensis by regulating the appetite related genes expression

This study assessed the impacts of monosodium glutamate (MSG) on the growth performance, feed utilization, feed intake, and appetite regulation of Chinese mitten crab, Eriocheir sinensis. A total of 240 juvenile crabs (9.16 ± 0.21 g) were assigned to six experimental diets supplemented with MSG at 0 %, 0.20 %, 0.39 %, 0.59 %, 0.78 % and 0.98 %. The results demonstrated that 0.59 % MSG supplementation significantly enhanced the weight gain rate (WGR), specific growth rate (SGR), and feed utilization of juvenile E. sinensis by increasing feed intake (FI) (p < 0.05). Moreover, dietary moderate MSG improved the mid-intestine morphology, such as height of microvillus (HMV), width of microvillus (WMV), and thickness of muscularis (TM). The qRT-PCR analysis demonstrated that the mRNA expression levels of the appetite-stimulating neural signals agouti related protein (agrp) and ghrelin were increased significantly by dietary moderate MSG (p < 0.05). Conversely, the mRNA levels of protein kinase B (akt), target of rapamycin (tor) and 4E-binding protein (4e-bp), as well as appetite-suppressing neural signals pro-opiomelanocortin (pomc), corticotropin-releasing factor (crf) and peptide yy (pyy) were significantly down-regulated (p < 0.05). The two-slope broken-line regression analysis of SGR in relation to dietary MSG levels suggested that the optimum dietary MSG level for juvenile E. sinensis was 5.6 g/kg. Collectively, MSG may potentially stimulate or suppress the appetite regulation neural signals/hormones, thereby modulating the feeding behavior of E. sinensis.

Yorkie negatively regulates the Crustin expression during molting in Chinese mitten crab, Eriocheir sinensis

Molting is a key biological process of crustaceans, which is mainly regulated by 20-hydroxyecdyone (20E). The molting cycle could be divided into three main stages including pre-molt, post-molt and inter-molt stages. The mechanism of immune regulation during molting process still requires further exploration. Yorkie (Yki) is a pivotal transcription factor in the Hippo signaling pathway, and it plays an essential role in regulating cell growth and immune response. In the present study, a Yki gene was identified from Eriocheir sinensis (designed as EsYki), and the regulatory role of EsYki in controlling the expression of antimicrobial peptide genes throughout the molting process was investigated. The mRNA expression level of EsYki was higher at the pre-molt stage compared to the post-molt stage and inter-molt stage. Following the injection of 20E, there was a notable and consistent rise in the EsYki mRNA expression in haemocytes. The increase was observed from 3 h to 48 h with the maximum level at 12 h. And the phosphorylation of Yki in the haemocytes was also significantly up-regulated at 3 h post 20E injection. Moreover, the levels of EsYki mRNA expression at three molting stages were significantly increased post Aeromonas hydrophila stimulation. The maximum level was detected at post-molt stage following A. hydrophila stimulation, while the lowest level was observed at inter-molt stage. The expression pattern of EsCrus was in contrast to EsCrus. After EsYki mRNA transcripts were inhibited by Yki inhibitor (CA3), the mRNA expression levels of EsCrus1 and EsCrus2 following A. hydrophila stimulation were significantly elevated. Furthermore, the phosphorylation level of NF-κB was also increased following the inhibition of Yki. Collectively, our findings indicated that EsYki could be induced by 20E and has a suppressive effect on the expression of EsCrus via inhibiting NF-κB during molting process. This research contributes to the understanding of the immunological regulation mechanism during molting process in crustaceans.

Exploration of the Synergistic Regulation Mechanism in Cerebral Ganglion and Heart of Eriocheir sinensis on Energy Metabolism and Antioxidant Homeostasis Maintenance under Alkalinity Stress.

(1) The development and utilization of the vast saline-alkali land worldwide is an important way to solve the worsening food crisis. Eriocheir sinensis, due to its strong osmotic regulation capability and its characteristics of being suitable for culturing in alkaline water, has become a potential aquaculture species in saline-alkali water. The brain and heart are the key tissues for signal transduction and energy supply under environmental stress. (2) This study is the first to explore the synergistic regulatory molecular mechanism by integrated analysis on cerebral ganglion proteomics and heart metabolomics of Eriocheir sinensis under alkalinity stress. (3) The results indicate that the cerebral ganglion and heart of E. sinensis were closely related in response to acute alkalinity stress. The differential regulatory pathways mainly involved regulation of energy metabolism, amino acid metabolism, and homeostasis maintenance. Importantly, alkalinity stress induced the regulation of antioxidants and further adjusted longevity and rhythm in the cerebral ganglion and heart, reflecting that the cerebral ganglion and heart may be the key tissues for the survival of Eriocheir sinensis under an alkalinity environment. (4) This study provides a theoretical reference for research on the regulation mechanism of E. sinensis under alkalinity condition and contributes to the development of aquaculture in saline-alkali water.

Exploring the effects of dietary cholesterol on growth, immunity, and lipid metabolism of juvenile Chinese mitten crabs (Eriocheir sinensis)

This study aims to investigate the nutritional needs and physiological functions of dietary cholesterol in juvenile Chinese mitten crabs (Eriocheir sinensis). In the 8-week feeding experiment, we evaluated the effects of various cholesterol levels (0 control, 0.6, 1.3, 2.6, 5.9, 12.0 g/kg diet) on growth, immune capacity, lipid metabolism, molting, and cholesterol and bile acid metabolism. Crabs (1.06 ± 0.04 g) were divided into six groups with four replicates of 40 crabs per tank. The highest weight gain was found in crabs fed 5.9 g/kg cholesterol (P < 0.05), while the highest molting frequency was observed in the 0.6 g/kg group (P < 0.05). Cholesterol significantly changed fatty acid composition in the hepatopancreas, and 5.9 g/kg cholesterol increased immune enzyme activities (acid phosphatase, alkaline phosphatase, and polyphenol oxidase) (P < 0.05). A 5.9 g/kg cholesterol significantly upregulated lipogenesis- and catabolism-related genes, while higher levels of cholesterol (12.0 g/kg) downregulated lipogenesis-related genes significantly (P < 0.05). Low cholesterol levels upregulated chitinase expression, while ecdysone receptor and molt-inhibiting hormone expressions were upregulated with cholesterol levels (P < 0.05). Cholesterol also upregulated the gene in bile acid metabolism. The total bile acids, the proportion of primary bile acids, and the content of primary bile acids increased significantly compared to the control. Based on quadratic regression model analysis, the optimal dietary cholesterol level for juvenile E. sinensis is 5.74–5.77 g/kg. The study suggests that cholesterol promotes growth and immunity in juvenile E. sinensis by regulating molting frequency, improving lipid and HUFA utilization, and increasing bile acids.

Effects of dietary defatted black soldier fly (Hermetia illucens) larvae meal substituting fish meal on growth, antioxidative capacity, immunity, intestinal histology and microbiota of juvenile Chinese mitten crab (Eriocheir sinensis)

This study evaluated the effects of replacing dietary fish meal (FM) with defatted black soldier fly (Hermetia illucens) larvae meal (DBSFLM) on growth, non-specific immunity, antioxidative capacity, intestinal histology and microbiota of juvenile Chinese mitten crab (Eriocheir sinensis). A control diet was designed to contain 200 g/kg FM (Con), then 25 %, 50 %, 75 %, and 100 % of dietary FM was replaced by DBSFLM, referring to DBSFLM25, DBSFLM50, DBSFLM75 and DBSFLM100, respectively. The five isoproteic and isolipidic diets were fed to crab (7.15 ± 0.10 g) for 8 weeks. The results indicated that the final weight, weight gain, feed conversion ratio and survival of DBSFLM25 and DBSFLM50 groups all reached the same level as Con (P > 0.05). The proper DBSFLM replacement on WG and FCR from broken line model were 49.70 % and 52.90 %, respectively. For immune enzyme activity, ACP, ALP, and LZM activities showed a trend of first increasing and then decreasing, the DBSFLM50 group had the highest activity, and the DBSFLM100 group was significantly lower than the DBSFLM50 group (P < 0.05). In terms of antioxidant capacity, the T-AOC, T-SOD, GSH-Px activities and MDA content of DBSFLM25 DBSFLM50 and DBSFLM75 groups all shows no significant difference with Con (P > 0.05), the DBSFLM100 group has the lowest antioxidant enzyme activities and highest MDA content. The intestinal number of folds and folds height in DBSFLM25 and DBSFLM50 groups reached the same level as Con (P > 0.05). The abundance of intestinal harmful bacteria Vibrio was enhanced with the increase of dietary DBSFLM level (P < 0.05). In conclusion, the recommended level of replacing FM with DBSFLM was at 50 % (100 g/kg) without negative effect on growth, non-specific immunity, antioxidative capacity, intestinal histology and microbiota of Chinese mitten crab.

Quantitative genetic analysis indicate non-heritability of major fatty acid in the muscle of Chinese mitten crab (Eriocheir sinensis)

The Chinese mitten crab (Eriocheir sinensis) is a commercially important aquaculture species, and crab meat generally has the high percentage of long-chain polyunsaturated fatty acids (LC-PUFAs) in the total fatty acids, which contributes the unique flavor and high nutrition value for adult crab. Muscle is the largest edible tissue in crustaceans, containing a variety of fatty acids, and understanding the genetic parameters of fatty acids in the meat of E. sinensis would be useful for the aquaculture of this species. This study investigated the heritability, genetic and phenotypic correlations of major fatty acids in the muscle of E. sinensis. Progeny from 25 full-sib families were reared under a common environment until they reached market size, and 429 mature crabs were selected for analysis. The results showed that the heritabilities of most fatty acids were low ranged from 0.01 (n-3PUFA) to 0.21 (MUFA), but the high heritabilities were recorded for C20:3n3 and C20:3n6 with values of 0.99 and 0.32, respectively. Furthermore, the strongest positive genetic correlations were found between C20:5n3 and C22:6n3(0.99), n-3PUFA(0.99), and PUFA and LC- PUFAs (0.99), n-3PUFA(0.99), whereas the strongest phenotype correlation was found between C20:5n3 and n-3 PUFA (0.96); C20:3n6 showed a significant negative genetic correlation with C20:3n3(−0.86) and C18:0 (−0.98), whereas C20:3n6 showed significant negative phenotypic correlation with C20:3n3(−0.14), C20:5n3(−0.15) and C22:6n3(−0.18). In conclusion, the percentages of major fatty acids, particularly C20:5n3, C22: 5n3 and C20:4n6, in E. sinensis muscle have very low heritabilities, suggesting that these fatty acids contents/composition in the muscle are ineffective targets for the selective breeding programs of E. sinensis.

Effects of Dietary Glutamate on the Growth Performance and Antioxidant Capacity of Juvenile Chinese Mitten Crab (Eriocheir sinensis)

In order to explore the effects of glutamate on the growth performance, antioxidant capacity and protein metabolism of juvenile Chinese mitten crab, 0%, 1% and 2% glutamate were supplemented to low protein (30%) and normal protein (35%) diets, respectively. There were 5 parallel tanks in each treatment, and the feeding duration was 8 weeks. The results showed that dietary glutamate did not significantly affect the weight gain of Chinese mitten crab. Diets supplemented with 2% glutamate significantly decreased the crude protein of crabs. The T-AOC of crabs fed the 30% protein diets was significantly lower than crabs fed the 35% protein diets. At 30% protein level, the superoxide dismutase (SOD) activity significantly increased with the increase in glutamate content. Dietary glutamate significantly down-regulated the relative expressions of mTOR, PI3K, S6K1 and 4EBP at 35% protein level. In conclusion, dietary glutamate cannot significantly increase the growth of Chinese mitten crab, but it can improve the antioxidant capacity in Chinese mitten crab under low protein conditions.

Bacterial recognition and inhibition activities of an LRR-only protein in the Chinese mitten crab, Eriocheir sinensis

LRR-only protein (LRRop) is an important class of immune molecules that function as pattern recognition receptor in invertebrates, however, the bacterial inhibitory activity of this proteins remain largely unknown. Herein, a novel LRRop was cloned from Eriocheir sinensis and named as EsLRRop2. The EsLRRop2 consists of six LRR motifs and formed a horseshoe shape three-dimension structure. EsLRRop2 was mainly expressed in intestine and hepatopancreas. The transcripts of EsLRRop2 in the intestine and hepatopancreas were induced by Vibrio parahaemolyticus and Staphylococcus aureus, and displayed similar transcriptional profiles. The expression levels of EsLRRop2 responded more rapidly and highly to V. parahaemolyticus than S. aureus in the intestine and hepatopancreas. Although the basal expression level of EsLRRop2 in hemocytes was relatively low, its transcripts in hemocytes were significantly induced by V. parahaemolyticus and S. aureus. The recombinant proteins of EsLRRop2 (rEsLRRop2) displayed a wide range of binding spectrum against vibrios, including V. parahaemolyticus, V. alginolyticus, and V. harveryi. The rEsLRRop2 showed dose- and time-dependent inhibitory activity against V. parahaemolyticus and S. aureus, and it could agglutinate the two bacteria. Furthermore, the inhibitory activities of rEsLRRop2 against V. parahaemolyticus, V. alginolyticus, V. harveryi and S. aureus was slightly affected by pH and salinity, and the rEsLRRop2 displayed the strongest inhibitory activity against all the three vibrios when the salinity was 20 ‰ and pH was 8.0. Collectively, these results elucidate the bacterial binding and inhibitory activities of EsLRRop2, and provide theoretical foundations for the application of rEsLRRop2 in prevention and control of vibrio diseases in aquaculture.

Nitrite and sulfide stress affects physiological and metabolic functions of gills in crab Eriocheir sinensis

Nitrite and sulfide are environmental factors that affect crabs survival and immunity. Gills are important respiratory and immune organs of Chinese mitten crabs Eriocheir sinensis, and both nitrite and sulfide can affect the physiological function stability of gill tissues. The present study investigated the acute toxicity of 10 mg/L nitrite and 5 mg/L sulfide, single and in combination, on the morphological, immune, osmoregulatory, and metabolic functions of the gill tissues of Chinese mitten crab. Both single and combined stressors were observed to disrupt the structural integrity of gill tissues. Oxidative stress-related indicators, including MDA, H2O2, and ·O2− content, exhibited an increase in all stress groups. The activities of antioxidant enzymes such as CAT and SOD and the expression levels of antioxidant genes fluctuated to varying degrees in all stress groups. The expression levels of osmotic regulating genes such as aqp, vatp, and nhe increased, as did the expression levels of lipid metabolism-related genes such as pparγ, rxr, fabp1, gyk, and dgat1. Furthermore, nitrite and sulfide stress affected the functions of metabolic pathways related to arachidonic acid, linoleic acid, D-amino acids, phenylalanine, and other substances in Chinese mitten crabs gill tissues. Additionally, the study identified 18 potential biomarkers related to stress responses. The nitrite and sulfide stressors disrupt the structure of crab gill tissues, cause osmoregulation disturbances, immune dysfunction, induce amino acid and lipid metabolic changes in gill tissue.

A positive loop between relish and cuticle proteins and their roles in regulating AMPs expression during bacterial infection in Eriocheir sinensis

Cuticle proteins (CPs) are the vital components of the cuticle and chitin lining covering the digestive tract of crustaceans. In this study, four new CP genes (designated as EsCP3, EsCP4, EsCP5, and EsCP8) were initially cloned and identified from the Chinese mitten crab Eriocheir sinensis. EsCP3/4/5/8 included 375, 411, 381, and 570 bp open reading frame encoding 124, 136, 126, and 189 amino acid proteins, respectively. Except for EsCP8, EsCP3/4/5 all contained a Chitin_bind_4 domain. EsCP3/4/5/8 were clustered into different groups in the phylogenetic tree. Quantitative real-time PCR results indicated that four EsCP genes have different patterns of tissue distribution. Changes in the expression levels of these four EsCP genes were observed in the intestine of crabs under Vibrio parahaemolyticus challenge. RNA interference assay showed that the knockdown of EsCPs in the intestine could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. In addition, the knockdown of EsRelish in the intestine decreased the expression levels of these four EsCP genes. These results indicated that EsCPs were involved in regulating the expression of AMPs, and EsCPs were regulated by EsRelish.

The regulatory effects of arginine on ovary development in the Chinese mitten crab Eriocheir sinensis

This study aimed to investigate the effects of arginine (Arg) on the vitellogenesis and ovarian maturation of Chinese mitten crab. Six isonitrogenous and isolipidic diets (1.83, 2.52, 3.26, 3.96, 4.81 and 5.52 % Arg) were fed to adult female Chinese mitten crab (60.05 ± 4.33 g). The feeding period lasted for eleven weeks. The results showed that gonadosomatic index of crabs increased with increasing dietary Arg concentrations from 1.83 % to 2.52 % and significantly decreased from 2.52 % to 5.52 %. The quadratic regression analysis based on gonadosomatic index estimated that the optimal Arg requirement for adult female crabs was 2.60 %. Dietary supplementation with 2.52 % Arg significantly increased the crude protein concentration in the ovary. A dose of 2.52 % Arg significantly decreased the vitellogenesis-inhibiting hormone (VIH) content in the hemolymph. Arg (2.52 %) significantly upregulated the Vg and protein kinase A (PKA) gene expressions and increased the cyclic adenosine monophosphate (cAMP) concentration in the hepatopancreas. Moreover, dietary supplementation with 2.52 % Arg significantly decreased the cyclic guanosine monophosphate (cGMP) concentration and downregulated protein kinase G (PKG) and p38 mitogen-activated protein kinase (p38 MAPK) gene expression. Arg at 2.52 % significantly increased hemolymph Vg content and the gene expression of the vitellogenin receptor (VgR). In conclusion, Arg at 2.52 % improved vitellogenesis and ovarian maturation by reducing the VIH content and regulating Vg expression via the cGMP/cAMP pathway.

Single-cell RNA-sequencing reveals Eriocheir sinensis hemocyte subpopulations and their molecular responses to Spiroplasma eriocheiris infection

The hemocytes is the central part of the Eriocheir sinensis innate immune system, however, their subclasses have not been completely defined and the single-cell gene transcriptional profiles and functions are still unknown. In the present study, the single-cell mRNA sequencing (scRNA-seq) was carried out to build a single-cell gene expression map of E. sinensis hemocytes and further analysis of the interaction molecular mechanism between the crab and novel aquatic pathogens Spiroplasma eriocheiris. By scRNA-seq, we successfully identified nine cell clusters (Hem1–9) and predicted their functions according to the specific marker genes. Pseudotime inference analysis revealed that all the identified cells undergo three stages, state 1 (the initial point), state 2 and state 3 (the other two branches). After being infected by S. eriocheiris, lots of different expression genes (DEGs) were identified in each cell cluster compared with the control group (with fold change ≥2 and p-value <0.05). The bioinformatics analysis found that the Hem1 and Hem5 were the main cell clusters synthesis and release of antimicrobial peptides (AMPs) for help the host against the pathogen infection. The Hem5 also could activate the proPO system to participate in the process of host eliminate the invading S. eriocheiris. Hem7, Hem8, and Hem9 were mainly responsible for killing the invading pathogen through phagocytosis. In particular, there were also many immune-related genes identified, including β-glucan binding protein (BGBP), Clip-domain serine proteases (CLIPs), phenoloxidase 1 (PO1), anti-lipopolysaccharide factors (ALFs), syndecan, and lysozyme C, which not only played critical roles in the crab immune system but also could serve as the marker genes for identifying cell subgroup. In conclusion, the results obtained in the present study could provide pivotal insights into the crab hemocytes cell-type-specific molecular mechanisms response S. eriocheiris infection at a single-cell level.

One-Year-Old Precocious Chinese Mitten Crab Identification Algorithm Based on Task Alignment.

The cultivation of the Chinese mitten crab (Eriocheir sinensis) is an important component of China's aquaculture industry and also a field of concern worldwide. It focuses on the selection of high-quality, disease-free juvenile crabs. However, the early maturity rate of more than 18.2% and the mortality rate of more than 60% make it difficult to select suitable juveniles for adult culture. The juveniles exhibit subtle distinguishing features, and the methods for differentiating between sexes vary significantly; without training from professional breeders, it is challenging for laypersons to identify and select the appropriate juveniles. Therefore, we propose a task-aligned detection algorithm for identifying one-year-old precocious Chinese mitten crabs, named R-TNET. Initially, the required images were obtained by capturing key frames, and then they were annotated and preprocessed by professionals to build a training dataset. Subsequently, the ResNeXt network was selected as the backbone feature extraction network, with Convolutional Block Attention Modules (CBAMs) and a Deformable Convolution Network (DCN) embedded in its residual blocks to enhance its capability to extract complex features. Adaptive spatial feature fusion (ASFF) was then integrated into the feature fusion network to preserve the detailed features of small targets such as one-year-old precocious Chinese mitten crab juveniles. Finally, based on the detection head proposed by task-aligned one-stage object detection, the parameters of its anchor alignment metric were adjusted to detect, locate, and classify the crab juveniles. The experimental results showed that this method achieves a mean average precision (mAP) of 88.78% and an F1-score of 97.89%. This exceeded the best-performing mainstream object detection algorithm, YOLOv7, by 4.17% in mAP and 1.77% in the F1-score. Ultimately, in practical application scenarios, the algorithm effectively identified one-year-old precocious Chinese mitten crabs, providing technical support for the automated selection of high-quality crab juveniles in the cultivation process, thereby promoting the rapid development of aquaculture and agricultural intelligence in China.

Reconstruction of Eriocheir sinensis Protein-Protein Interaction Network Based on DGO-SVM Method.

Eriocheir sinensis is an economically important aquatic animal. Its regulatory mechanisms underlying many biological processes are still vague due to the lack of systematic analysis tools. The protein-protein interaction network (PIN) is an important tool for the systematic analysis of regulatory mechanisms. In this work, a novel machine learning method, DGO-SVM, was applied to predict the protein-protein interaction (PPI) in E. sinensis, and its PIN was reconstructed. With the domain, biological process, molecular functions and subcellular locations of proteins as the features, DGO-SVM showed excellent performance in Bombyx mori, humans and five aquatic crustaceans, with 92-96% accuracy. With DGO-SVM, the PIN of E. sinensis was reconstructed, containing 14,703 proteins and 7,243,597 interactions, in which 35,604 interactions were associated with 566 novel proteins mainly involved in the response to exogenous stimuli, cellular macromolecular metabolism and regulation. The DGO-SVM demonstrated that the biological process, molecular functions and subcellular locations of proteins are significant factors for the precise prediction of PPIs. We reconstructed the largest PIN for E. sinensis, which provides a systematic tool for the regulatory mechanism analysis. Furthermore, the novel-protein-related PPIs in the PIN may provide important clues for the mechanism analysis of the underlying specific physiological processes in E. sinensis.

Genetic variation mining of the Chinese mitten crab (Eriocheir sinensis) based on transcriptome data from public databases.

At present, public databases house an extensive repository of transcriptome data, with the volume continuing to grow at an accelerated pace. Utilizing these data effectively is a shared interest within the scientific community. In this study, we introduced a novel strategy that harnesses SNPs and InDels identified from transcriptome data, combined with sample metadata from databases, to effectively screen for molecular markers correlated with traits. We utilized 228 transcriptome datasets of Eriocheir sinensis from the NCBI database and employed the Genome Analysis Toolkit software to identify 96 388 SNPs and 20 645 InDels. Employing the genome-wide association study analysis, in conjunction with the gender information from databases, we identified 3456 sex-biased SNPs and 639 sex-biased InDels. The KOG and KEGG annotations of the sex-biased SNPs and InDels revealed that these genes were primarily involved in the metabolic processes of E. sinensis. Combined with SnpEff annotation and PCR experimental validation, a highly sex-biased SNP located in the Kelch domain containing 4 (Klhdc4) gene, CHR67-6415071, was found to alter the splicing sites of Klhdc4, generating two splice variants, Klhdc4_a and Klhdc4_b. Additionally, Klhdc4 exhibited robust expression across the ovaries, testes, and accessory glands. The sex-biased SNPs and InDels identified in this study are conducive to the development of unisexual cultivation methods for E. sinensis, and the alternative splicing event caused by the sex-biased SNP in Klhdc4 may serve as a potential mechanism for sex regulation in E. sinensis. The analysis strategy employed in this study represents a new direction for the rational exploitation and utilization of transcriptome data in public databases.

High temperature aggravated hypoxia-induced intestine toxicity on juvenile Chinese mitten crab (Eriocheir sinensis)

High temperature and hypoxia in water due to global warming threaten the growth and development of aquatic animals. In natural or cultured environments, stress usually does not occur independently, whereas the synergistic effect of high temperature and hypoxia on Chinese mitten crab (Eriocheir sinensis) are rarely reported. In this study, 450 juvenile crabs were equally divided into control group (24 °C ± 0.5 °C, DO 6.8 ± 0.1 mg/L), hypoxia stress group (24 °C ± 0.5 °C, DO 1 ± 0.1 mg/L) and combined stress group (30 °C ± 0.5 °C, DO 1 ± 0.1 mg/L), and the intestinal health status, microbial diversity and metabolite profiles were evaluated for 24 h treatment. The results showed that hypoxia stress induced the expression level of pro-inflammatory related genes were significantly up-regulated in intestine of juvenile E. sinensis, and intestinal peritrophic membrane factor related genes were significantly down-regulated. High temperature further amplified the effects of hypoxia on pro-inflammatory and peritrophic membrane factor-related genes. Interesting, hypoxia stress induced a significant up-regulated of intestinal antioxidant-related genes, whereas high temperature reversed this trend. In addition, single stress or/and combined stress led to changes in intestinal microbiota diversity and abundance, and intestinal metabolite profiles. Compared with hypoxia stress, the synergistic effect of high temperature and hypoxia led to an increase in the abundance of pathogenic bacteria and a decrease in the abundance of probiotic bacteria. Moreover, intestinal metabolic pathways were significantly changed, especially amino acid metabolism and glycerophospholipid metabolism. Therefore, the results indicated that hypoxia stress could induce intestinal inflammatory response and oxidative stress, and lead to abnormal changes in intestinal microbiota and metabolic profiles, whereas high temperature further aggravate the toxic effects of hypoxia on the intestine. This study preliminarily revealed the synergistic toxic effects of high temperature and hypoxia on the intestine of juvenile E. sinensis.

Abamectin-induced behavioral alterations link to energy metabolism disorder and ferroptosis via oxidative stress in Chinese mitten crab, Eriocheir sinensis

The increasing application of abamectin (ABM) in agriculture has raised concerns regarding its environmental safety and potential adverse effects on aquatic environment safety. In the present study, the toxic effects of ABM exposure on the adult Chinese mitten crab, Eriocheir sinensis were investigated, with a focus on locomotion impairment, behavioral changes, oxidative stress, energy metabolism disruption, and ferroptosis. Crabs were exposed to sublethal concentrations of ABM at 2, 20 and 200 μg/L. After 21 d chronic exposure to 200 μg/L, residual ABM in hepatopancreas and muscles were detected as 12.24 ± 6.67 and 8.75 ± 5.42 μg/Kg, respectively. By using acute exposure experiments (96 h), we observed significant locomotion and behavioral alterations, alongside biochemical evidences of oxidative stress and energy metabolism impairment. The presence of ferroptosis, a form of cell death driven by iron-dependent lipid peroxidation, was notably identified in the hepatopancreas. Functional tests with N-acetylcysteine (NAC) supplementation showed restored behavioral responses and decrease of ferroptosis levels. It suggests that mitigating oxidative stress could counteract ABM-induced toxicity. Our findings highlight the critical roles of oxidative stress and ferroptosis in mediating the toxic effects of ABM on E. sinensis, underscoring the need for strategies to mitigate environmental exposure to pesticides.

Four amino acids play an important role in the allergenicity of hemocyanin allergen

To identify the key amino acids (AAs) affecting the allergenicity of hemocyanin (HC) allergens from Chinese mitten crabs, in this study, two epitopes, P1-SHFTGSKSNPEQR and P2-LSPGANTITR were employed and four potential key AAs (P1: F3 and N9 and P2: N6 and R10) were predicted. Mast cell and mouse models revealed that four mutants induced lower levels of immunoglobulin E (IgE) and Th2 type cytokines (15.47–49.89 %), proving that F3, N9, N6, and R10 were the key AAs of two epitopes. Mutants reduce allergic responses via the Th2 pathway. However, the roles of every key AA affecting allergenicity were different (P1-F3 > N9 and P2-N6 > R10). In addition, lower transport and higher efflux were observed in the mutants during transport absorption by Caco-2 cells. The allergenicity of HC was stronger when the transport absorption efficiency of epitopes and mutants was higher and their efflux was lower. Our study provides a novel method for revealing the allergenic molecular mechanisms of food allergens.