Intestinal immunity in crustaceans must control invading pathogens while maintaining a stable microbial community, yet the molecular mechanisms that connect immune recognition to tissue-specific signaling outputs remain poorly understood. In the Chinese mitten crab (Eriocheir sinensis), Dscam generates vast receptor diversity via alternative splicing across three extracellular exon clusters, two transmembrane (TM) exons, and three intracellular variable exons. However, how transmembrane and intracellular domain splice variants (ICD) are paired in the intestine and how these combinations influence antibacterial defense remain unknown. Here, we developed an oral intestinal infection model in the Chinese mitten crab using bacteria Vibrio parahaemolyticus and Staphylococcus aureus. Oral challenge led to broad induction of antimicrobial peptides (AMPs) and increased intestinal transmembrane Dscam expression. Systemic double-stranded RNA (dsRNA) knockdown of transmembrane Dscam resulted in higher culturable bacterial loads in the gut and significantly decreased survival after infection. Splicing analysis showed a persistent bias toward the second transmembrane exon (TM2), whereas bacterial challenge selectively remodeled ICD splicing to produce ICD-truncated isoforms, thereby altering the ICD composition of the predominantly TM2-containing receptor pool in the gut. Isoform-specific silencing demonstrated that TM2-containing Dscam isoforms dominate immune responses by promoting nuclear translocation of Dorsal, a nuclear factor κB (NF-κB) family transcription factor, and by inducing stronger AMP expression than TM1-containing isoforms. Lastly, TM-specific knockdown disrupted gut microbial structure and reduced diversity, with the greatest dysbiosis observed following TM2 depletion. Overall, these results reveal that TM-ICD combinatorial splicing acts as a dynamic regulatory layer linking intestinal Dscam isoform composition to NF-κB-dependent antibacterial immunity and microbiota homeostasis in a decapod crustacean.

Integrins are evolutionarily conserved adhesion receptors that play pivotal roles in phagocytosis across metazoans, yet the specific αβ heterodimers involved in crustacean cellular immunity remain poorly defined. In this study, we identified and characterized an integrin α subunit, designated integrin αPS3 (EsITGAPS3), from the economically important Chinese mitten crab, Eriocheir sinensis. Bioinformatic analyses revealed that EsITGAPS3 shares structural similarity with arthropod integrin αPS3/αPS4/αPS5 subfamily members but exhibits low sequence conservation across distant taxa. EsITGAPS3 was ubiquitously expressed in crab tissues, with the highest levels in immune-relevant organs, including hemocytes, hepatopancreas, and gills. Its transcription was significantly induced in hemocytes following in vitro challenge with Vibrio parahaemolyticus. Functional knockdown of EsITGAPS3 via RNA interference markedly impaired hemocyte phagocytosis of FITC-labeled bacteria, demonstrating its important role in antibacterial cellular immunity. Using AlphaFold-based structural prediction, we identified integrin βPS-like subunit integrin βPS1 (EsITGBPS1) as the top-ranked candidate β partner for EsITGAPS3, and co-immunoprecipitation confirmed their physical interaction and heterodimer formation. Importantly, RNAi-mediated silencing of EsITGBPS1 similarly compromised bacterial phagocytosis, indicating that the integrin αPS3βPS1 (EsITGAPS3-EsITGBPS1) heterodimer functions as a key receptor complex in crab hemocytes. Our findings define a specific integrin αPS3βPS1 pair required for efficient phagocytosis in E. sinensis, providing mechanistic insight into integrin-mediated immune adaptation in crustaceans and highlighting a potential target for enhancing disease resistance in aquaculture.

The Down syndrome cell adhesion molecule (Dscam) is a highly diverse immune receptor produced through extensive alternative splicing, but whether specific bacterial surface ligands can guide stimulus-specific Dscam splicing and subsequent immune signaling in crustaceans remains unclear. In this study, we examined the role of the Chinese mitten crab (Eriocheir sinensis) Dscam in hemocyte responses to two outer membrane proteins of Vibrio parahaemolyticus, OmpA0764 and OmpA1186. Bacterial challenge increased Dscam expression in hemocytes. Both recombinant proteins significantly elevated Dscam and multiple antimicrobial peptide (AMP) gene levels and also promoted Dorsal nuclear translocation. Conversely, RNA interference-mediated knockdown of Dscam markedly reduced these responses. Exon-targeted amplicon sequencing further revealed that OmpA0764 and OmpA1186 triggered distinct exon usage patterns within the extracellular exon 4 and exon 6 variable clusters of Dscam. OmpA0764 favored isoforms containing Exon4.25 and Exon6.15, while OmpA1186 favored Exon4.17 and Exon6.41. Functional studies showed that silencing these ligand-enriched exons significantly impaired Dorsal nuclear accumulation and downstream AMP expression. Collectively, these results suggest that bacterial outer membrane proteins not only induce Dscam expression but also modulate ligand-biased extracellular isoform profiles that are associated with immune signaling efficiency. This research highlights extracellular Dscam alternative splicing as a potential mechanism linking bacterial ligand-induced isoform remodeling to Dorsal activation and humoral antibacterial defense in the Chinese mitten crab.

The Down syndrome cell adhesion molecule (Dscam) is a highly diverse immune receptor produced through extensive alternative splicing, but whether specific bacterial surface ligands can guide stimulus-specific Dscam splicing and subsequent immune signaling in crustaceans remains unclear. In this study, we examined the role of the Chinese mitten crab (Eriocheir sinensis) Dscam in hemocyte responses to two outer membrane proteins of Vibrio parahaemolyticus, OmpA0764 and OmpA1186. Bacterial challenge increased Dscam expression in hemocytes. Both recombinant proteins significantly elevated Dscam and multiple antimicrobial peptide (AMP) gene levels and also promoted Dorsal nuclear translocation. Conversely, RNA interference-mediated knockdown of Dscam markedly reduced these responses. Exon-targeted amplicon sequencing further revealed that OmpA0764 and OmpA1186 triggered distinct exon usage patterns within the extracellular exon 4 and exon 6 variable clusters of Dscam. OmpA0764 favored isoforms containing Exon4.25 and Exon6.15, while OmpA1186 favored Exon4.17 and Exon6.41. Functional studies showed that silencing these ligand-enriched exons significantly impaired Dorsal nuclear accumulation and downstream AMP expression. Collectively, these results suggest that bacterial outer membrane proteins not only induce Dscam expression but also shape ligand-biased extracellular isoform profiles that influence immune signaling efficiency. This research highlights extracellular Dscam alternative splicing as a mechanism linking bacterial ligand recognition to Dorsal activation and humoral antibacterial defense in the Chinese mitten crab.

Phagocytosis by circulating hemocytes is crucial for antibacterial defense in crustaceans, but how intracellular trafficking GTPases coordinate this process remains poorly understood. Here, we identified and characterized a Rab6 ortholog (EsRab6) from the Chinese mitten crab, Eriocheir sinensis, and defined its role in hemocyte phagocytosis and resistance to Vibrio parahaemolyticus. EsRab6 encodes a typical Rab6 GTPase with conserved nucleotide-binding and Rab family motifs and clusters within the invertebrate Rab6 clade. Transcripts were widely expressed, with higher levels in the hepatopancreas and eyestalk, yet EsRab6 was rapidly and transiently upregulated in hemocytes following V. parahaemolyticus challenge. RNA interference-mediated knockdown of EsRab6 resulted in a significant reduction in FITC-labeled bacterial uptake by hemocytes, as shown by microscopy and flow cytometry. Immunofluorescence and LysoTracker staining showed that EsRab6 relocates from a diffuse cytosolic pattern to punctate vesicles that colocalize with lysosomes during infection. Simultaneously, EsRab6 knockdown significantly decreased lysosome-associated signals, indicating a role for EsRab6 in lysosome formation or stability. In vivo, dsRNA-mediated silencing of EsRab6 results in higher hemolymph bacterial loads and significantly lower survival rates following V. parahaemolyticus infection. Overall, these findings identify EsRab6 as a conserved trafficking regulator that links phagosome-lysosome biogenesis to adequate bacterial clearance in crab hemocytes and suggest that Rab6-dependent pathways could be targeted to enhance disease resistance in crustacean aquaculture.

Cyclins are core regulators of cell-cycle progression and also participate in transcriptional regulation and cellular stress responses. However, the cyclin family has not been systematically characterized in the Chinese mitten crab Eriocheir sinensis, and its potential role in hemocyte proliferation during antibacterial responses remains unclear. In this study, 18 cyclin family members were identified from the E. sinensis genome and designated as EsCyclins (abbreviated as EsCyc plus the subtype letter). Comparative analyses showed that these family members are distributed across 13 chromosomes and can be assigned to the major cyclin subfamilies on the basis of phylogenetic relationships, conserved motifs, gene structures, and domain organization. Representative cyclins, including EsCycA, EsCycB, EsCycB3, EsCycD, and EsCycE, displayed conserved sequence features and similar predicted core three-dimensional structures relative to invertebrate and vertebrate homologs. Tissue expression analysis further showed that these genes are broadly expressed but tissue biased, with relatively high transcript levels in hepatopancreas, testis, and, depending on the gene, stomach or muscle. After stimulation with heat-killed Vibrio parahaemolyticus, representative EsCyclin genes showed dynamic, gene-specific temporal expression patterns in synchronized primary hemocytes. Functional assays demonstrated that siRNA-mediated knockdown of EsCycA, EsCycD, or EsCycE significantly reduced hemocyte proliferation under bacterial stimulation, as assessed by flow cytometry and EdU incorporation. Together, this study provides the first systematic characterization of the cyclin family in E. sinensis and identifies EsCycA, EsCycD, and EsCycE as positive regulators of bacteria-induced hemocyte proliferation in vitro. These findings extend our understanding of cyclin-family evolution in crustaceans and provide a foundation for investigating how cell-cycle regulators participate in crustacean antibacterial cellular responses.

Antimicrobial peptides (AMPs) are major effectors of crustacean intestinal immunity, but the discovery of novel crustacean AMP families is hampered by rapid sequence divergence and low homology to well-characterized insect AMPs. Here, we report the identification and functional characterization of an Attacin family member from the Chinese mitten crab Eriocheir sinensis, designated EsAttacin (EsAtt), which, to our knowledge, represents the first Attacin described in any crustacean species. Using our previously established Vibrio parahaemolyticus (Vp)-induced intestinal inflammation model in E. sinensis, we found that transcriptomic profiling of the infected intestine at an early time point revealed a strongly upregulated yet functionally unannotated transcript. Although this transcript showed minimal primary-sequence identity with known insect Attacins, structural alignment of its AlphaFold 3-predicted model against the Protein Data Bank using Foldseek revealed clear three-dimensional conservation with Drosophila Attacins, supporting its classification as an Attacin family member. EsAtt was predominantly expressed in the intestine and was strongly induced after oral Vp infection. Recombinant EsAtt (rEsAtt) bound lipopolysaccharide (LPS) and peptidoglycan (PGN) in a dose-dependent manner. rEsAtt also exhibited direct antibacterial activity against both Gram-negative (Vp, Escherichia coli) and Gram-positive (Staphylococcus aureus, Bacillus subtilis) bacteria, with a minimum inhibitory concentration (MIC) lowest against Vp (1.25 μM). RNAi-mediated silencing of EsAtt significantly increased the intestinal bacterial load and accelerated host mortality, whereas oral administration of rEsAtt reduced bacterial burden and improved survival. Histopathological analysis showed that EsAtt silencing exacerbated Vp-induced intestinal damage, characterized by thinning of the intestinal wall, disrupted tissue architecture, and loss of epithelial integrity. Exogenous rEsAtt supplementation substantially rescued these pathological changes. RNAi-mediated knockdown of either EsImd or EsRelish significantly suppressed EsAtt expression under both basal and Vp-challenged conditions, indicating a conserved IMD-Relish-Attacin regulatory axis from insects to crustaceans. Collectively, these findings identify EsAtt as the first functionally characterized crustacean member of the Attacin family, extend the known phylogenetic distribution of Attacins from insects to crustaceans, and illustrate the value of structure-guided strategies for identifying divergent immune effectors in non-model aquatic species.

CDK genes play crucial roles in key biological processes such as cell cycle progression and transcriptional regulation. However, the specific number of CDK gene family members in the Chinese mitten crab (Eriocheir sinensis) remained unclear. In this study, we identified 19 CDK genes in E. sinensis through genome-wide analysis. Phylogenetic analysis classified them into 13 major subfamilies, all containing a relatively conserved S_TKc domain. The majority of these 19 genes showed high expression levels in epidermal and muscle tissues. Furthermore, six of them were significantly upregulated upon bacterial challenge. Since bacterial stimulation promotes cell proliferation, we knocked down the highly expressed CDK genes and found that depletion of CDK2-L1 or CDK4 abolished the pro-proliferative phenotype, indicating their essential role in regulating cell proliferation. Additionally, we demonstrated that CDK2-L1 interacts with CyclinE and Cyclin A, and CDK4 binds to Cyclin D-both cyclins being key regulators of cell proliferation. Thus, this study reveals that CDK2-L1 and CDK4 regulate cell proliferation by forming functional complexes with their respective cyclin partners.

Light color has increasingly been recognized as significant factor in aquaculture, influencing fish behavior, physiology, and consequently production efficiency. The Chinese longsnout catfish (Leiocassis longirostris), an economically important freshwater species in China, is known for its pronounced sensitivity to environmental stimuli, particularly light. This study systematically investigated the effects of green (516.9 nm, 115 lx), and red (630.9 nm, 115 lx),as well as white (full spectrum, 115 lx) LED light conditions on the feeding behavior and growth performance of L. longirostris over a 21-day period. Eighteen fish (mean weight: 32.3 +/- 5.4 g; mean total length: 15.6 +/- 0.8 cm) were randomly assigned to tanks (n = 6 per treatment), and both feeding behavior (latency, frequency, and motivation) and growth parameters (total food intake, specific growth rate, feed conversion ratio, and condition factor) were quantitatively analyzed. Results showed that red light significantly increased feeding motivation, reducing latency to feed (similar to 12.6 s to similar to 7.8 s) and boosting feeding frequency by 20 % and an 11.5 % reduction feed conversion efficiency. Green light enhanced feeding behavior compared to white light, although to a lesser extent than red light. This suggests that while spectral manipulation enhances feeding behavior and feed intake, other factors are needed for substantial growth. Overall, these findings provide critical insights into species-specific visual and behavioral adaptations, offering practical guidance for aquaculture optimization through tailored LED lighting strategies that enhance feeding efficiency without compromising production outcomes.

Hypoxia-caused spermatogenesis impairment may contribute to male infertility. FOXA2 has been found to be abundant in spermatogonial stem cells and critical for spermatogenesis. Here we aimed to explore the roles of FOXA2 in regulating spermatogonial cells against hypoxia stimulation. Our results showed that FOXA2 expression was downregulated in hypoxia-stimulated spermatogonial cells. Overexpression of FOXA2 prevented hypoxia-induced endoplasmic reticulum (ER) stress with decreased expression levels of associated markers including GRP78, CHOP, and ATF-4. FOXA2 overexpression caused a decrease in MDA content and an increase in activities of SOD, CAT, and GSH-Px in spermatogonial cells under hypoxic conditions, implying its inhibitory effect on oxidative stress. Besides, cell apoptosis under hypoxic conditions was also prevented by FOXA2 overexpression, as shown by reduced apoptotic rate and caspase-3 activity. Moreover, we found that hypoxia stimulation inactivated the Nrf2 pathway, which could be prevented by FOXA2 overexpression. Nrf2 knockdown attenuated the effects of FOXA2 overexpression on hypoxia-induced ER stress, oxidative stress, and apoptosis in spermatogonial cells. In conclusion, FOXA2 exerted protective effects on spermatogonial cells against hypoxia-induced ER stress, oxidative stress, and apoptosis via regulating Nrf2/HO-1 signaling. These findings suggested that FOXA2 might be a therapeutic target for treating hypoxia-induced spermatogenesis impairment.

Small GTPase RhoA is a pivotal regulator of cytoskeletal dynamics and phagocytosis in mammalian phagocytes, yet its functional role in crustacean immunity remains poorly characterized. In this study, we identified and characterized RhoA from Eriocheir sinensis (designated EsRhoA), demonstrating its essential role in hemocyte phagocytosis and antibacterial defense. The EsRhoA gene encodes a 257-amino-acid protein containing a conserved RHO domain and displays over 90 % sequence similarity to orthologs in both vertebrates and invertebrates. Transcriptional analysis revealed widespread expression of EsRhoA across various tissues, with a significant upregulation observed in hemocytes following infection with Vibrio parahaemolyticus. RNA interference (RNAi)-mediated knockdown of EsRhoA in hemocytes led to decreased expression of phagocytosis-related effectors ROCK2 and Arp2/3, resulting in a 62 % reduction in bacterial phagocytosis as measured by flow cytometry. Immunocytochemistry analysis confirmed that EsRhoA protein translocates to the hemocyte membrane during pathogen-induced phagocytosis, suggesting a mechanistic similarity to integrin-mediated internalization in mammalian cells. Importantly, in vivo knockdown of EsRhoA significantly increased bacterial loads in hemolymph and reduced crab survival after infection. Collectively, these findings establish EsRhoA as an evolutionarily conserved regulator of hemocyte phagocytosis that provides critical protection against bacterial pathogens in crustaceans, offering novel insights into the evolution of immune cell phagocytosis mechanisms and potential applications in crustacean aquaculture disease management.

During the early stages of foot-and-mouth disease virus (FMDV) infection, a series of acute inflammatory responses occur in the host. As the disease progresses, these inflammatory responses gradually weaken until the host is nearly recovered. However, the mechanism by which FMDV participates in the negative regulation of host inflammatory responses remains unclear. In this study, we found that FMDV 3C plays a crucial role in inhibiting the inflammatory response by degrading various molecules in the TLR4 signaling pathway. Mechanistically, we discovered that this degradation is mediated by caspase activity, which is activated by 3C protease. Specifically, FMDV 3C targets TLR4, TRIF, p65, IRF3, and TBK1 for degradation through caspase-3, and degrades IRF3 and TBK1 via caspase-8. Notably, FMDV 3C targets TBK1 for degradation through caspase-3, caspase-8, and caspase-9 independently. In conclusion, this is the first report identifying FMDV 3C as an anti-inflammatory factor that mediates the degradation of various molecules to inhibit TLR4 signaling through caspase activity. This study provides a novel insight into explore the relationship between FMDV and inflammation and offers ideas for exploring the biological function of 3C and the pathogenesis of FMDV.

Nontyphoidal Salmonella (NTS) is the most commonly reported foodborne gastrointestinal infection and ranks among the top three causes of foodborne bacterial outbreaks in China. This study analyzed NTS data from the Foodborne Disease Surveillance System in China from 2013 to 2022 to summarize epidemiological features and assess reporting rate changes. We employed joinpoint regression model to calculate the annual percentage change in NTS reporting rate, sex reporting rate, as well as rural-urban reporting rate. We applied the Pearson correlation coefficient to evaluate the correlation of NTS reporting rate and the national per capita consumption of major foods and cases of COVID-19. During this period, 55,266 NTS cases were reported, with reporting rates increasing significantly (AAPC [CRR] = 21.89

The RNA N6-methyladenosine (m6A) methylation, catalyzed by methyltransferase-like 3 (METTL3), has emerged as a pivotal epitranscriptomic regulator in immune processes. However, its functional role in crustacean immunity remains poorly understood. In this study, we identified and characterized a METTL3 homolog-designated EsMETTL3-from the Chinese mitten crab Eriocheir sinensis. EsMETTL3 contains a highly conserved MT-A70 domain and shares significant sequence identity in this catalytic region across diverse species. Expression analysis revealed that EsMETTL3 is ubiquitously expressed across all tested tissues, with relatively low basal levels observed in hemocytes. Notably, its transcription in hemocytes was significantly upregulated following infection with Vibrio parahaemolyticus. Functional experiments demonstrated that bacterial infection elevates global m6A levels in hemocytes in an EsMETTL3-dependent manner. Knockdown of EsMETTL3 or pharmacological inhibition of m6A methylation led to a marked reduction in the expression of key antimicrobial peptides (AMPs), including Crustin1, Crustin2, Lysozyme, and DWD. Further mechanistic analyses revealed that EsMETTL3-mediated m6A methylation modulates the Toll signaling pathway by enhancing pelle expression and suppressing cactus expression, thereby promoting nuclear translocation of the transcription factor Dorsal. Our findings establish a critical role for EsMETTL3-mediated m6A methylation in regulating crab innate immunity through fine-tuning the Toll/Dorsal pathway and AMP production, providing novel insights into the epitranscriptomic regulation of immune responses in crustaceans.

Autophagy plays a critical role in colitis-associated colorectal cancer (CAC). However, non-autonomous regulation of macroautophagic/autophagic flux during inflammation remains largely unexplored. Here, we show that F2rl1/Par2 deficiency (F2rl1[ΔIEC]) aggravated azoxymethane-dextran sulfate sodium-induced CAC based on tumor number and burden, promoted autophagy dysfunction characterized by SQSTM1/p62 accumulation and autophagosome-lysosome fusion inhibition in IECs, and reduced lysosomal acidification by suppressing FOXA2-induced V-ATPase ATP6V0E1 transcription. FOXA2 or ATP6V0E1 overexpression rescued autophagy impairment, reactive oxygen species accumulation, and DNA damage induced by F2RL1 deficiency in vitro and in vivo. Neutrophil-derived serine proteases suppressed FOXA2 expression, causing autophagy dysfunction. F2RL1 knockout completely blocked the effects of neutrophil proteases on FOXA2 and ATP6V0E1. The correlation between neutrophil and FOXA2-ATP6V0E1 activities was validated in ulcerative colitis and colorectal carcinoma. Therefore, F2RL1 deficiency in intestinal epithelial cells suppressed FOXA2 expression, leading to V-ATPase-mediated autophagic dysfunction and exacerbating CAC. Neutrophils may contribute to impaired autophagy and promote CAC by inactivating canonical F2RL1/PAR2 signaling via its derived proteases. F2RL1/PAR2 signaling may participate in maintaining intestinal homeostasis via autophagy. These findings provide useful insights into F2RL1/PAR2 and its cleaving serine proteases in CAC and would help in developing new therapeutic strategies for this malignancy.Abbreviations: AOM: azoxymethane; ATP6V0C: ATPase H+ transporting V0 subunit c; ATP6V0E1: ATPase H+ transporting V0 subunit e1; ATP6V1C2: ATPase H+ transporting V1 subunit C2; ATP6V1F: ATPase H+ transporting V1 subunit F; CAC: colitis-associated colorectal cancer; CRC: colorectal cancer; CTSB: cathepsin B; CTSG: cathepsin G; DEGs: differentially expressed genes; DSS: dextran sulfate sodium; FOXA2: forkhead box protein A2; F2RL1: F2R like trypsin receptor 1; IBD: inflammatory bowel disease; IECs: intestinal epithelial cells; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFs: transcription factors; UC: ulcerative colitis.

Ovarian cancer is the second leading cause of gynecologic cancer death worldwide, with only 20

INTRODUCTION:Global warming is increasing interest in how aquatic animals can adjust their physiological performance and cope with temperature changes. Therefore, understanding the behavioral changes and molecular underpinnings in fish under warming is crucial for both the individual and groups survival. This could provide experimental evidence and resource for evaluating the impact of global warming. OBJECTIVE:Three genetic families of common carp (Cyprinus carpio) were generated. These juveniles were constructed short-term (4 days) and long-term (30 days) warming groups to investigate the effects of warming on behavioral responses and to elucidate the potential underlying mechanisms of warming-driven behavior. METHODS:Behavioral tests were used to explore the effects of short- and long-term exposure to warming on the swimming behavior of C. carpio. Brain transcriptome combined with measurement of nervous system activity was used to further investigated the comprehensive neuromolecular mechanisms under warming. RESULTS:Long-term warming groups had a more significant impact on the decline of swimming behavior in juvenile C. carpio. Furthermore, brain comparative transcriptomic analysis combined with measurement of nervous system activity revealed that genes involved in cytoskeletal organization, mitochondrial regulation, and energy metabolism are major regulators of behavior in the juvenile under warming. Importantly, especially in the long-term warming groups, enrichment analysis of associated gene expression suggested functional alterations of synaptic transmission and signal transduction leading to swimming function impairment in the central nervous system, as revealed by behavioral tests. CONCLUSIONS:Our study provides evidence of the neurogenomic mechanism underlying the decreased swimming activity in juvenile C. carpio under warming. These findings have important implications for understanding the impacts of climate change on aquatic ecosystems and the organisms that inhabit them.

Protein kinases of the MAPK cascade family (MAPKKK-MAPKK-MAPK) play an important role in the growth and development of organisms and their response to environmental stress. The MAPKK gene families in the Chinese mitten crab Eriocheir sinensis have never been systematically analyzed. We identified four MAPKK family genes, EsMEK, EsMAPKK4, EsMAPKK6, and EsMAPKK7, in E. sinensis and analyzed their molecular features and expression patterns. All four MAPKK genes are composed of multiple exons and introns, all have a conserved domain, and all have 10 conserved motifs (except EsMEK and EsMAPKK7 which are missing motif 10). The four MAPKK genes are on four different chromosomes and have no gene duplications, and the results of phylogenetic tree analysis indicate that the ESMAPKK gene family is highly conserved evolutionarily. The EsMAPKK genes were widely expressed in all the examined tissues with higher expression in hemocytes, hepatopancreas, and gills. Notably, EsMAPKK6 was also highly expressed in the ovary. Vibrio parahaemolyticus infection significantly increased the mRNA levels of the EsMAPKK genes in hemocytes. Further disruption of the EsMAPKK gene family expression affects the expression levels of multiple antimicrobial peptides in hemocytes. Our experimental results provide a starting point for a more in-depth study of the innate immunity functional roles of members of the MAPKK gene families in E. sinensis.