Investigation of putative antimicrobial peptides in Carassius gibel, revealing a practical approach to screening antimicrobials.

Antimicrobial peptides (AMPs) and their mimics are rapidly gaining attention as a new class of antimicrobials due to their clinical potential. AMPs are widely distributed throughout nature and participate in the innate host defense. In this study, 18 AMPs, including 3 ß-defensins, 3 hepcidins, 4 liver-expressed antimicrobial peptide 2 (LEAP-2) compounds, 4 g-type lysozymes, 2 c-type lysozymes, and 2 NK-lysins, were identified from the genome of Carassius auratus by a homologous search and were further classified based on their fundamental structural features and molecular phylogeny. C. auratus AMPs were found to be ubiquitously distributed in all tested tissues and showed similar expression profiles, with the exception of ß-defensins, when RT-qPCR was used to investigate the tissue distribution of AMPs in healthy Carassius gibel. In addition, the expression levels of NK-lysin genes in the tested tissues tended to be upregulated upon bacterial and viral infection when representative NK-lysins were chosen to examine their relative expression levels in various tissues. Importantly, the synthetic peptide caNKL2102-119, which targets the functional domain of saposin B in caNK-lysins, could effectively counter Aeromonas hydrophila, Staphylococcus aureus, and Escherichia coli with minimum inhibitory concentration (MIC) values of 3-6 µg/mL, as well as inhibit the proliferation of spring viraemia of carp virus (SVCV). These results provide potential targets for antibiotic-free breeding in the aquaculture industry.

ROS induced by spring viraemia of carp virus activate the inflammatory response via the MAPK/AP-1 and PI3K signaling pathways.

Spring viraemia of carp virus (SVCV) can cause a high mortality in common carp (Cyprinus carpio), and its main pathological processes include the inflammatory response. However, the detailed mechanism is still unclear. Reactive oxygen species (ROS) have been shown to play critical roles in the immune response, including inflammation, in different models. Our previous studies have demonstrated that SVCV infection results in the accumulation of ROS, including H2O2, in epithelioma papulosum cyprini (EPC) cells. In this study, we aimed to explore the relationship between H2O2 accumulation and inflammation during SVCV infection. After EPC cells were infected with SVCV, the expression levels of the inflammatory factors tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and interleukin (IL)-8 were up-regulated, while the expression of the anti-inflammatory factor interleukin (IL)-10 was down-regulated, compared with that in mock-infected EPC cells. The antioxidant N-acetyl-l-cysteine (NAC) could dampen the increased TNF-ɑ and COX-2 expression induced by SVCV and H2O2, suggesting a relationship between ROS accumulation and inflammation during SVCV infection. Dual luciferase reporter assays demonstrated that SVCV could not activate the NF-κB pathway. In addition, inhibition of NF-κB by pyrrolidine dithiocarbamate (PDTC) treatment had no effect on the expression of inflammatory factors. Furthermore, inhibition of the ERK, JNK, and p38MAPK signaling pathways by U0126, SP600125, and SB203580, respectively, reduced the expression of TNF-ɑ, COX-2, and IL-8, indicating that these three signaling pathways were all involved in the inflammatory response after SVCV infection. In addition, the PI3K signaling pathway was involved in the expression of the chemokine IL-8 in the SVCV-induced inflammatory response. We also showed that inhibition of the MAPK or PI3K signaling pathway facilitated the expression of SVCV-G as well as increased the SVCV viral titer. Altogether these results reveal the mechanism of the SVCV-mediated inflammatory response. Thus, targeting these signaling pathways may provide novel treatment strategies for SVCV-mediated diseases.

Heme Oxygenase-1 inhibits spring viremia of carp virus replication through carbon monoxide mediated cyclic GMP/Protein kinase G signaling pathway.

Spring viremia of carp virus (SVCV) is the etiological agent of spring viremia of carp (SVC) and causes mass mortality in common carp (Cyprinus carpio). Currently, no effective treatments or commercial vaccines against SVCV are available. Heme oxygenase-1 (HO-1), an enzyme that catalyzes the degradation of heme to produce carbon monoxide (CO), biliverdin and ferrous iron (Fe2+), exerts anti-oxidant, antiinflammatory and anti-apoptotic properties. Previous studies demonstrated that nuclear factor-erythroid 2 related factor 2 (Nrf2) functions as an important upstream regulator of HO-1 and exhibits robust activity against SVCV infection. In this study, we further examined the antiviral activity of HO-1 against SVCV infection. The elevated expression of HO-1 was induced upon cobalt protoporphyrin (CoPP) treatment in EPC cells without affecting cell viability and thus inhibited SVCV replication in a dose dependent manner. Knocking down of HO-1 rescued SVCV replication. Thereby, the antiviral activity of ROS/Nrf2/HO-1 axis was confirmed in EPC cells. Furthermore, HO-1 enzymatic products CO, but not biliverdin, markedly inhibited SVCV replication via the activation of cyclic GMP/protein kinase G signaling pathway. Collectively, these findings suggest potential drug or therapy that induced the Nrf2/HO-1/CO/cGMP/PKG signaling pathway as a promising strategy for treating SVC.

SVCV impairs mitochondria complex â ¢ resulting in accumulation of hydrogen peroxide.

Spring viraemia of carp virus (SVCV) is a deadly pathogen of common carp. SVCV infection is found to be associated with excess reactive oxygen species (ROS) generation and induces oxidative stress in EPC and FHM cells, which contributes to its pathogenesis. In this study, ROS production and mitochondria function as well as antioxidant enzymes in mitochondria were investigated during SVCV infection in EPC cells. Dysfunction of mitochondria and inactivation of mitochondria electron transport chain complex Ⅲ to augment O2-∙ and H2O2 accumulation were observed in SVCV infected EPC cells. Treatment of Antimycin A reduced the activity of mitochondria complex Ⅲ in EPC cells, which also inhibited the transcription of SVCV glycoprotein gene (SVCV-G) and production of SVCV. Our studies explain the production of ROS following SVCV infection and also suggest that integrate mitochondrial function is important for SVCV infection.

Pathogenic Elizabethkingia miricola Infection in Cultured Black-Spotted Frogs, China, 2016.

Multiregional outbreaks of meningitis-like disease caused by Elizabethkingia miricola were confirmed in black-spotted frog farms in China in 2016. Whole-genome sequencing revealed that this amphibian E. miricola strain is closely related to human clinical isolates. Our findings indicate that E. miricola can be epizootic and may pose a threat to humans.

Spring viraemia of carp virus: recent advances.

Spring viraemia of carp is an environmentally and economically important disease affecting cyprinids, primarily common carp (Cyprinus carpio). The causative agent of this disease is Spring viraemia of carp virus (SVCV) - a member of the genus Vesiculovirus of the family Rhabdoviridae. The disease is presently endemic in Europe, America and several Asian countries, where it causes significant morbidity and mortality in affected fish. SVCV infection is generally associated with exophthalmia; abdominal distension; petechial haemorrhage of the skin, gills, eyes and internal organs; degeneration of the gill lamellae; a swollen and coarse-textured spleen; hepatic necrosis; enteritis; and pericarditis. The SVCV genome is composed of linear, negative-sense, ssRNA containing five genes in the order 3'-N-P-M-G-L-5', encoding a nucleoprotein, phosphoprotein, matrix protein, glycoprotein and RNA-dependent RNA polymerase, respectively. Fully sequenced SVCV strains exhibit distinct amino acid substitutions at unique positions, which may contribute to as-yet unknown strain-specific characteristics. To advance the study of SVCV and the control of spring viraemia of carp disease in the future, this review summarizes our current understanding of SVCV in terms of its genomic characteristics, genetic diversity and pathogenesis, and provides insights into antiviral immunity against SVCV, diagnosis of SVCV and vaccination strategies to combat SVCV.

Spring viraemia of carp virus induces autophagy for necessary viral replication.

Outbreaks of spring viraemia of carp virus (SVCV) in several carp species and other cultivated fish can cause significant mortality and jeopardize the billion-dollar worldwide fish industry. Spring viraemia of carp virus, also known as Rhabdovirus carpio, is a bullet-shaped RNA virus that enters and amplifies in gill epithelium and later spreads to internal organs. Young fish under stressed conditions (spring cold water, etc.) are more vulnerable to SVCV-induced lethality because of their lack of a mature immune system. Currently, the host response of SVCV remains largely unknown. Here, we observed that autophagy is activated in SVCV-infected epithelioma papulosum cyprini (EPC) cells. We demonstrated that the SVCV glycoprotein, rather than viral replication, activates the autophagy pathway. In addition, SVCV utilized the autophagy pathway to facilitate its own genomic RNA replication and to enhance its titres in the supernatants. Autophagy promoted the survival of SVCV-infected cells by eliminating damaged mitochondrial DNA generated during viral infection. We further showed that SVCV induces autophagy in EPC cells through the ERK/mTOR signalling pathway. Our results reveal a connection between autophagy and SVCV replication and propose autophagy suppression as a novel means to restrict SVCV viral replication.

A novel L-type lectin was required for the multiplication of WSSV in red swamp crayfish (Procambarus clakii).

L-type lectins are involved in glycoproteins secretory pathways and are associated with many immune responses. There is growing evidence that L-type lectins are also involved in viral replication. In this study, a novel L-type lectin (named as PcL-lectin) was identified from red swamp crayfish (Procambarus clakii). Gene sequencing and phylogenetic tree analysis results showed that the PcL-lectin was a kind of endoplasmic reticulum Golgi intermediate compartment-53 (ERGIC-53). The expression level of PcL-lectin was significantly down regulated in crayfish after challenged with white spot syndrome virus (WSSV). Recombinant PcL-lectin protein facilitated the replication of WSSV in crayfish. In addition, WSSV replication was decreased when endogenous PcL-lectin was knocked down by RNA interference in crayfish. Furthermore, PcL-lectin may interact with VP24, an envelope protein of WSSV. Our results suggest that PcL-lectin may be required for the multiplication of WSSV, and will pave a new way for the developing of strategies against WSSV infection.

Up-regulation of nuclear factor E2-related factor 2 (Nrf2) represses the replication of SVCV.

Generation of reactive oxygen species (ROS) and failure to maintain an appropriate redox balance contribute to viral pathogenesis. Nuclear factor E2-related factor 2 (Nrf2) is an important transcription factor that plays a pivotal role in maintaining intracellular homoeostasis and coping with invasive pathogens by coordinately activating a series of cytoprotective genes. Previous studies indicated that the transcription and expression levels of Nrf2 were up-regulated in SVCV-infected EPC cells with the unknown mechanism(s). In this study, the interactions between the Nrf2-ARE signalling pathway and SVCV replication were investigated, which demonstrated that SVCV infection induced accumulation of ROS as well as protein carbonyl groups and 8-OHdG, accompanied by the up-regulation of Nrf2 and its downstream genes. At the same time, the activation of Nrf2 with D, l-sulforaphane (SFN) and CDDO-Me could repress the replication of SVCV, and knockdown of Nrf2 by siRNA could promote the replication of SVCV. Taken together, these observations indicate that the Nrf2-ARE signal pathway activates a passive defensive response upon SVCV infection. The conclusions presented here suggest that targeting the Nrf2 pathway has potential for combating SVCV infection.

Development of monoclonal antibodies against polar filaments and spore valves of Myxobolus honghuensis (Myxosporea: Bivalvulida).

Myxobolus honghuensis infects the pharynx of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) and can cause high mortality. Only morphology-based diagnostic methods are currently available for clinical samples, but these methods are laborious and have low efficiency of detection. To overcome this problem, we designed a more sensitive diagnostic method. Two monoclonal antibodies (MAbs 1C7 and 3B7) were prepared by immunizing mice with soluble protein from sonicated M. honghuensis spores. Immunofluorescence analysis revealed that MAb 1C7 specifically reacts with polar filaments from spores, whereas MAb 3B7 identified protein localized on the spore valves. The isotypes of MAb 1C7 and MAb 3B7 were IgM and IgG1, respectively. Results of Western blot analysis revealed that MAb 1C7 recognized 2 prominent protein bands with molecular weights of 130 and 180 kDa, while MAb 3B7 recognized a protein band of 28 kDa. Thus, in this study we have developed 2 MAbs that have the potential for efficient detection of M. honghuensis. Moreover, identification of MAb 1C7 and MAb 3B7 allows for further studies of the functions and biochemical composition of polar filament and spore surface antigens.

Identification and Characterization of MicroRNAs in Snakehead Fish Cell Line upon Snakehead Fish Vesiculovirus Infection.

MicroRNAs (miRNAs) play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection. Snakehead fish Vesiculovirus (SHVV) has caused mass mortality in snakehead fish during the past few years. To identify specific miRNAs involved in SHVV infection, we performed microRNA deep sequencing on a snakehead fish cell line (SSN-1) with or without SHVV infection. A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs, and nine novel miRNAs were identified using MiRDeep2 software. Eighteen and 143 of the 205 known miRNAs were differentially expressed at three and 24 h post-infection (poi), respectively. From the differentially-expressed miRNAs, five were randomly selected to validate their expression profiles using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and their expression profiles were consistent with the microRNA sequencing results. In addition, the target gene prediction of the SHVV genome was performed for the differentially-expressed host miRNAs, and a total of 10 and 58 differentially-expressed miRNAs were predicted to bind to the SHVV genome at three and 24 h poi, respectively. The effects of three selected miRNAs (miR-130-5p, miR-214 and miR-216b) on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting. The results showed that all three miRNAs were able to inhibit the multiplication of SHVV; whereas the mechanisms underlying the SHVV multiplication inhibited by the specific miRNAs need to be further characterized in the future.

Isolation and Characterization of a Novel Dicistrovirus Associated with Moralities of the Great Freshwater Prawn, Macrobrachium rosenbergii.

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean and is farmed in many countries. Since 2009, a larval mortality syndrome of M. rosenbergii has broken out and spread widely in the main breeding area, including Zhejiang, Jiangsu, Guangxi, and Guangdong Provinces in mainland China. A novel virus, named Macrobrachium rosenbergii Taihu virus (MrTV), was isolated from the moribund larvae and was determined to be the causative agent of the M. rosenbergii larval mortality syndrome by experimental infection. Further genomic sequencing suggested that the MrTV genome is monopartite, 10,303 nt in length, and dicistronic with two non-overlapping open reading frames (ORFs) separated by an intergenic region (IGR) and flanked by untranslated regions (UTRs). Phylogenetic analysis using the full-length genomic sequence and the putative amino acid sequences of the capsid protein revealed that MrTV was more closely related to the taura syndrome virus (TSV) than to any other viruses. According to these molecular features, we proposed that MrTV is a new species in the genus Aparavirus, family Dicistroviridae. These results may shed light on controlling larval mortality syndrome in M. rosenbergii.

Molecular characterisation and prevalence of a new genotype of Cyprinid herpesvirus 2 in mainland China.

Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) has been confirmed as a causative agent of the acute haematopoietic necrosis disease outbreak in farmed goldfish (Carassius auratus L.) and gibel carp (Carassius auratus gibelio Bloch). In this study, we present the genomic characteristics of a variant CyHV-2 strain (SY-C1) isolated from farmed gibel carp in mainland China and its comparative genomics analysis with the CyHV-2 reference strain ST-J1. Overall, the full-length genome of SY-C1 shares 98.8% homology with that of ST-J1. Sequence comparisons between SY-C1 and ST-J1 indicate that the variations include single-nucleotide mutations, insertions, deletions, and rearrangements, which suggested that SY-C1 is different from ST-J1 and represents a new genotype. Therefore, we propose that the identified CyHV-2 can be divided into 2 different genotypes and be named China genotype (C genotype) and Japan genotype (J genotype) according to their isolation loci. Furthermore, epidemiological surveys indicate that the dominant genotype of CyHV-2 circulating in mainland China is closer to the China genotype than the Japan genotype.

Transcriptome analysis of epithelioma papulosum cyprini cells after SVCV infection.

BACKGROUND: Spring viraemia of carp virus (SVCV) has been identified as the causative agent of spring viraemia of carp (SVC) and it has caused significant losses in the cultured common carp (Cyprinus carpio) industry. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood. In this study, deep RNA sequencing was used to analyse the transcriptome and gene expression profile of EPC cells at progressive times after SVCV infection. This study addressed the complexity of virus-cell interactions and added knowledge that may help to understand SVCV. RESULTS: A total of 33,849,764 clean data from 36,000,000 sequence reads, with a mean read length 100 bp, were obtained. These raw data were assembled into 88,772 contigs. Of these contigs, 19,642 and 25,966 had significant hits to the NR and Uniprot databases where they matched 17,642 and 13,351 unique protein accessions, respectively. At 24 h post SVCV infection (1.0 MOI), a total of 623 genes were differentially expressed in EPC cells compared to non-infected cells, including 288 up-regulated genes and 335 down-regulated genes. These regulated genes were primarily involved in pathways of apoptosis, oxidative stress and the interferon system, all of which may be involved in viral pathogenesis. In addition, 8 differentially expressed genes (DEGs) were validated by quantitative PCR. CONCLUSIONS: Our findings demonstrate previously unrecognised changes in gene transcription that are associated with SVCV infection in vitro, and many potential cascades identified in the study clearly warrant further experimental investigation. Our data provide new clues to the mechanism of viral susceptibility in EPC cells.

Up-regulation of nuclear factor E2-related factor 2 upon SVCV infection.

Nuclear factor E2 - related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant response element (ARE)-dependent genes, including heme oxygenase-1 (HO-1) and superoxide dismutase 1 (SOD1). The Nrf2/ARE pathway has been regarded as a critical switch in the initiation of cellular defence systems for surviving oxidative insults and viral infection. In this study, the Nrf2 gene of EPC cells, which is originally derived from Pimephales promelas, was cloned, and an investigation on the interactions between Nrf2 and spring viraemia of carp virus (SVCV) was performed. These results demonstrated that the virus facilitated the nuclear accumulation of Nrf2 and up-regulated its transcriptional and protein profiles in EPC cells. In addition, exogenous activation of Nrf2 conferred EPC cells with a higher cellular total antioxidant capacity via an increase in the expression of HO-1 and SOD1, but did not suppress the replication of SVCV.

Bi-directional regulation of type I interferon signaling by heme oxygenase-1.

Moderate activation of IFN-I contributes to the body's immune response, but its abnormal expression, stimulated by oxidative stress or other factors causes pathological damage. Heme oxygenase-1 (HO-1), induced by stress stimuli in the body, exerts a central role in cellular protection. Here we showed that HO-1 could promote IFN-1 under Spring Viremia of Carp virus (SVCV) infection and concomitantly attenuate the replication of SVCV. Further characterization of truncated mutants of HO-1 confirmed that intact HO-1 was essential for its antiviral function via IFN-I. Importantly, HO-1 inhibited the IFN-I signal by degrading the IRF3/7 through the autophagy pathway when it was triggered by H2O2 treatment. The iron ion-binding site (His28) was critical for HO-1 to degrade IRF3/7. HO-1 degradation of IRF3/7 is conserved in fish and mammals. Collectively, HO-1 regulates IFN-I positively under viral infection and negatively under oxidative stress, elucidating a mechanism by which HO-1 regulates IFN-I signaling in bi-directions.

Proteomic analysis of epithelioma papulosum cyprini cells infected with spring viremia of carp virus.

Spring viremia of carp (SVC), caused by spring viremia of carp virus (SVCV) is an important disease due to its drastic effects on carp fisheries in many countries. To better understand molecular responses to SVCV infection, two dimensional electrophoresis (2-DE) and MALDI-TOF/TOF were performed to investigate altered proteins in epithelioma papulosum cyprini cells (EPCs). Differentially expressed proteins in mock-infected EPCs and SVCV-infected EPCs were compared. A total of 54 differentially expressed spots were successfully identified (33 up-regulated spots and 21 down-regulated spots) which include cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism, and ubiquitin proteasome pathway-associated proteins. Moreover, 7 corresponding genes of the differentially expressed proteins were quantified using real time RT-PCR to examine their transcriptional profiles. The presence of four selected cellular proteins (beta-actin, gamma1-actin, heat shock cognate 71 kDa protein and annexin A2) associated with the spring viremia of carp virus (SVCV) particles was validated by Western blot assay. This study provides dynamic and useful protein-related information to further understand the underlying pathogenesis of SVCV infection.

Dysbiosis of intestinal homeostasis contribute to Whitmania pigra edema disease.

Whitmania pigra is widely used in traditional Chinese medicine. However, W. pigra is being threatened by an edema disease with unknown causes (WPE). In this study, a comprehensive exploration of virome, microbiome, and metabolome aberrations in the intestine of W. pigra was performed to address the aetiology of WPE. Virome analysis indicated that eukaryotic viruses did not contribute to WPE, whereas an expansion of Caudovirales was observed in WPE. Compared to the control, the microbial richness and diversity in diseased W. pigra decreased remarkably. Nine genera, including Aeromonas, Anaerotruncus, Vibrio, Proteocatella, Acinetobacter, and Brachyspira were overrepresented in WPE, whereas eleven genera, including Bifidobacterium, Phascolarctobacterium, Lactobacillus, Bacillus and AF12, were enriched in healthy individuals. Furthermore, certain metabolites, especially amino acids, short-chain fatty acids, and bile acids, were found to be linked to intestinal microbiota alterations in WPE. An integration of the microbiome and metabolome in WPE found that dysbiosis of the gut microbiota or metabolites caused WPE. Notably, W. pigra accepted intestinal microbiota transplantation from WPE donors developed WPE clinical signs eventually, and the dysbiotic intestinal microbiota can be recharacterized in this recipient W. pigra. Strikingly, pathological features of metanephridium and uraemic toxin enrichment in the gut indicated a putative interconnection between the gut and metanephridium in WPE, which represents the prototype of the gut-kidney axis in mammals. These finding exemplify the conservation of "microecological Koch's postulates" from annelids to insects and other vertebrates, which provides a direction of prevention and treatment for WPE and opens a new insight into the pathogenesis of aquatic animal diseases from an ecological perspective.

Novel supramolecular deep eutectic solvent-enabled in-situ lignin protection for full valorization of all components of wheat straw.

Lignin is usually deemed as an inhibitor to enzymatic hydrolysis of cellulose due to its physical barrier, non-productive adsorption, and steric hindrance. Herein, a novel supramolecular deep eutectic solvent (SUPRADES), comprising ethylene glycol and citric acid in 5:1 M ratio, and ß-cyclodextrin (ß-CD) in a concentration of 3.5% (w/w), was developed to be efficient for pretreating wheat straw. The delignification rate, cellulose enzymatic digestibility, and hemicellulose removal reached 90.45%, 97.36% and 87.24%, respectively, which may be attributed to the introduction of ß-CD with superior ability of both adsorption and in-situ lignin protection to efficiently remove lignin with intact structure from cellulose surface. The mechanisms of high-efficiency lignin extraction/protection were systematically illustrated by adsorption kinetics. Moreover, Trichosporon cutaneum grown on the hemicellulose and cellulose fractions after pretreatment afforded 8.8 g total lipids from 100 g wheat straw. The green SUPARDES pretreatment strategy offers a new avenue for upgrading lignocellulose to biofuels.

Sub-acute toxicity of the herbicide glufosinate-ammonium exposure in adult red swamp crayfish (Procambarus clarkii).

Glufosinateammonium (GLA) is one of the most widely used agricultural herbicides. It is frequently detected in surface waters near farmland and may pose a risk to non-target aquatic species. This study aimed to explore the toxicity of subacute GLA exposure in crayfish. Adult red swamp crayfish were exposed to GLA (0, 1, 10, and 100 mg/L) for 21 days. Bioaccumulation, oxidative stress, nonspecific immunity, and the expression of genes encoding xenobiotic detoxification-related enzymes were examined. The results showed GLA accumulation and hepatopancreatic histopathological changes (dilation of hepatic tubules and vacuolation of hepatocytes) in the exposed crayfish. GLA exposure induced ROS production, inhibited glutathione expression, and catalase activity in the crayfish hepatopancreas, as well as inhibited immunoenzyme expression (acid phosphatase, alkaline phosphatase, and lysozyme) in the hemolymph. In addition, the total hemocyte number decreased, and the proportion of hemocyte subsets changed significantly. Superoxide dismutase first increased and then decreased with increasing GLA dosage. GLA promoted the expression of biotransformation enzymes (cypb5, gst) in the hepatopancreas. Our results suggest that subacute GLA exposure caused structural damage to the hepatopancreatic tissue and decreased antioxidant capacity and non-specific immunity in crayfish. These findings provide insight into the toxicity of herbicides on non-target organisms.