OBJECTIVE:The highly lethal Vibrio disease (HLVD) caused by highly lethal Vibrio parahaemolyticus (VpHLVD) represents a significant threat to Pacific white shrimp Litopenaeus vannamei aquaculture. The existing disease control approaches often rely on antibiotics, leading to drug resistance and environmental pollution, thereby limiting the sustainable development of shrimp farming. Probiotics are recognized for their ability to enhance host disease resistance and to inhibit pathogenic bacteria with high safety and environmental friendliness. METHODS:We evaluated the potential of a probiotic bacterial dietary supplement for HLVD prevention. Bacillus altitudinis strain JSHY-074, isolated from coastal aquaculture environments in Jiangsu, China, was identified based on morphological characterization, 16S ribosomal DNA sequencing, and whole-genome analysis. Its antagonistic activity against V. parahaemolyticus strain SHY1669 was determined by co-culture inhibition tests, and the probiotic effect was verified through a VpHLVD challenge experiment on Pacific white shrimp fed with JSHY-074. RESULTS:The strain JSHY-074 exhibited potent antagonistic activity against V. parahaemolyticus strain SHY1669, with inhibition zones expanding from 12 ± 0.31 mm (mean ± SD) at 6 h of co-culture to 30 ± 0.84 mm at 96 h of co-culture. At an inoculum density of 1.0 × 108 CFU/mL, the inhibition rate reached 88.58% at 72 h. Whole--genome analysis revealed nine gene clusters associated with secondary metabolite production, with lichenysin preliminarily identified as the primary active compound. The VpHLVD challenge experiment demonstrated that feeding Pacific white shrimp with strain JSHY-074 achieved a 70% survival rate within 5 d and a 5,942-fold reduction in hepatopancreas pathogen load (from 6.12 × 107 to 1.03 × 104 copies/ng). CONCLUSIONS:This is the first report of B. altitudinis exhibiting antagonistic activity against VpHLVD. The high technological readiness level of B. altitudinis JSHY-074 makes it a versatile probiotic feed additive, and its safety and effectiveness make it well suited for fast deployment and use in shrimp farming, offering a nutritional approach to reducing antibiotic dependency and promoting sustainable aquaculture practices.

Ecytonucleospora hepatopenaei (EHP) is an obligate intracellular microsporidian that causes severe growth retardation in Penaeus vannamei (P. vannamei), yet its pathogenic mechanisms remain incompletely clear. As the spore wall is essential for resisting environmental stress and mediating host invasion, characterising its protein components is critical for elucidating EHP biology. In this study, we identified and cloned a novel spore wall protein, EhSWP26. Sequence analysis revealed that EhSWP26 contains a 687 bp open reading frame encoding a 228-amino-acid polypeptide, lacking a signal peptide and transmembrane domain but harbouring a conserved MICSWaP (Microsporidia Spore Wall and Associated Proteins) domain characteristic of the microsporidian MICSWaPs family. Immunoelectron microscopy and indirect immunofluorescence assays demonstrated that EhSWP26 localises to the mature spore wall, where it is distributed in both the exospore and endospore layers. RNA interference further confirmed that silencing EhSWP26 significantly reduced its transcript level and led to a marked decrease in EHP spore load in shrimp hepatopancreas. Collectively, these findings indicate that EhSWP26 is a key structural and functional component associated with spore wall formation and host infection, likely playing an essential role in the EHP life cycle. This study enhances our understanding of EHP spore wall composition and function, provides a foundation for investigating its proliferation and infection mechanisms, and highlights EhSWP26 as a promising target for diagnostic development and therapeutic intervention in EHP disease of P. vannamei.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VpAHPND) is a major concern in shrimp farming, leading to substantial economic losses. In our study Bacillus subtilis JSHY-K3 have shown promise in controlling VpAHPND. Therefore, the objective of this study is to explore the mechanism of JSHY-K3 in inhibiting VpAHPND. A mutant strain JSHY-K331 with 96.38% similarity to JSHY-K3 (accession number: CP123994.1) that lost antagonistic activity was obtained using ARTP mutagenesis and resulted in mutations in two key JSHY-K331 genes, rpoB and polyribitol phosphate β-glucosyltransferase. Morphological analysis revealed that JSHY-K3 exhibited enhanced biofilm formation and significant wrinkling compared to JSHY-K331, indicating higher production of Surfactin. JSHY-K3 also displayed stronger predatory behavior, possibly mediated by Skf and Sdp toxins, which could be its way of antagonizing VpAHPND. Morphological analysis revealed that JSHY-K3 exhibited enhanced biofilm formation and significant wrinkling compared to JSHY-K331, indicating higher production of Surfactin. High production of Skf and Sdp toxins, to kill its own cells to overcome nutrient limitation, which is also a manifestation of high Surfactin production. Metabolomic analysis indicated disrupted metabolism in JSHY-K331, with a focus on cell growth rather than secondary metabolite production. And we suggest that Pro-Val-Leu, and Pro-Pro-Tyr amino acids are important precursors for Surfactin synthesis. Additionally, phospholipids were found to regulate Surfactin penetration and degradation, potentially mediated by yerP. These findings shed light on the antagonistic mechanism of JSHY-K3 against VpAHPND and offer insights into enhancing its biocontrol ability through gene modification.

Enterocytozoon hepatopenaei (EHP), causing hepatopancreatic microsporidiosis (HPM), significantly impacts Litopenaeus vannamei, leading to economic losses. Using bioinformatics and machine learning, this study characterized EHP infection stages and host-pathogen interactions. Consensus clustering of 2,613 metabolism-related genes from 36 shrimp samples identified four subclasses: healthy (HG), heavily (HEG), moderately (MEG), and lightly infected groups (LEG). Gene Set Variation Analysis (GSVA) revealed subclass-specific metabolic and immune patterns, with HEG showing impaired carbohydrate metabolism and upregulated amino acid degradation, MEG indicating recovery, and LEG demonstrating metabolic normalization. Weighted Gene Co-expression Network Analysis (WGCNA) linked infection subclasses to pathways like Hippo, JAK-STAT, steroid biosynthesis, and calcium signaling. Machine learning identified 52 characteristic genes involved in EHP proliferation (e.g., RAPTOR), host invasion (e.g., cell surface glycoprotein 1), and host defense (e.g., mucin-5AC). A stacked classifier model predicted infection severity with high accuracy. EHP severely impacts immunity, autophagy, and oxidative stress in early infection, with host responses evolving from detoxification to metabolic recovery and adaptation. Key pathways and genes, including polar tube protein (PTP) and mucin-5AC, were identified as critical to host-pathogen interactions, offering insights into EHP infection dynamics and potential intervention strategies.

Ecytonucleospora hepatopenaei (EHP) is an obligate intracellular parasitic pathogen of shrimp, causing growth retardation and increased susceptibility to opportunistic infections. Currently, there are few reports on the prevention and treatment of EHP infection in shrimp, and research on its drug targets is limited. This article utilizes RNA interference (RNAi) technology to conduct knockdown verification studies on potential drug targets involved in EHP’s invasion mechanism and life cycle. Three target gene sequences from genome, and annotated as EhAQP1 (aquaporin 1), EhPTP2 (polar tube protein), and EhTK (thymidine kinase) were used to synthesize specific small interfering RNA (siRNA). These siRNAs were injected into EHP‐infected shrimp, and the hepatopancreas was sampled at 12, 24, 48, and 72 h postinjection to verify the interference effect and spore load. The results showed that RNAi exhibits varying inhibitory effects on different genes and is time‐dependent. For EhAQP1, interference was most effective in the early stages (12 to 24 h), while the interference effects for EhTK were more pronounced at 48 and 72 h, the expression of EhPTP2 was significantly downregulated ( p < 0.001) only at 24 h. In the EhAQP1 and EhPTP2 interference group, the spore load decreased from 12 to 48 h, but increased at 72 h. In contrast, the EhTK interference group showed a continuous decrease in spore load from 12 to 72 h. In summary, this study provides a new strategy for prevention and treatment of shrimp EHP infection and lays the foundation for subsequent research on RNAi–based prevention and treatment of EHP infection.

Ecytonucleospora hepatopenaei (EHP), an emerging microsporidian, has caused significant economic losses to the Asian aquaculture industry. This study investigates the infection dynamics of EHP through deep transcriptomic sequencing of infected shrimp samples at different time points. The analysis revealed distinct periodic changes in metabolism, immune response, and DNA repair during EHP infection. Shrimp rapidly mobilize energy metabolism and activate immune pathways in the early stage (day 2), followed by immune suppression and metabolic regulation in the mid-stage (days 4-6), and enhanced immune responses by day 8. Trend analysis supports an 8-day infection cycle, with periodic changes in gene expression profiles, especially in energy metabolism, protein synthesis and degradation, and immune responses. Host gene expression displays a phased response aligning with EHP proliferation and its life cycle. EHP's gene expression also shows periodicity, with upregulation of proliferation-associated genes on days 4 and 10. Weighted Gene Co-expression Network Analysis (WGCNA) identified key gene modules related to metabolic adaptation and immune suppression, indicating EHP's adaptation to the host environment. The study reveals a cyclical pattern of interaction between EHP and its host, providing insights into EHP's pathogenic mechanisms.

Acute hepatopancreatic necrosis disease (AHPND), caused by the toxin-producing Vibrio sp., has become a serious threat to shrimp aquaculture. Egg yolk immunoglobulin(IgY) has the advantage of low cost and good protection in the treatment of diseases caused by specific pathogens in crustaceans. This study evaluated the protective effect of IgY against AHPND infection in Penaeus vannamei. IgY was isolated from eggs laid by hens immunized with recombinant PirAB toxin, which prepared with the PirAB gene as the target gene. Freeze-dried egg powders with anti-PirAB-IgY were mixed with basal diets at 20 % and 10 % concentrations and IgY from non-immunized hen (control-IgY) was mixed with basal diets at 20 % concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirAB-IgY(20 %), anti-PirAB-IgY(10 %), and control-IgY diets were 70 %, 50 % and 13 %, respectively. The parameters including β-1,3-d-glucan-binding protein and Heat shock proteins 70 (Hsp70) had increased in the experimental groups fed with anti-PirAB-IgY compared with the positive control group, and the expression levels of crustin were significantly(P < 0.05) higher in the positive control group than in the other groups. This study provides an effective prophylactic method against AHPND infection in shrimp.

Ecytonucleospora hepatopenaei (EHP) is an obligate intracellular microsporidian that causes severe disease in Litopenaeus vannamei but whose infection mechanism remains elusive. Because the spore wall is a critical interface between the parasite and its environment, characterizing the functionally unstudied protein SWP12 is essential for elucidating host - microsporidian interactions. In this study, we cloned the full-length open reading frame (ORF) of EHP SWP12, designated EhSWP12, which is 735 bp in length and encodes a 244-amino-acid polypeptide. Bioinformatic analyses revealed that EhSWP12 contains no signal peptide or transmembrane domain but possesses an AH/BAR domain, with a predicted isoelectric point (pI) of 9.05 and a molecular mass (Mw) of 28.7 kDa. The EhSWP12 gene was subsequently expressed in Escherichia coli, and indirect immunofluorescence assays (IFA) demonstrated that the protein localizes to the mature spore wall. Further subcellular localization studies identified its presence in both the exospore and endospore layers. Our findings indicate that EhSWP12 is a membrane-associated protein, suggesting that it plays a pivotal role in the EHP life cycle. These results enhance our understanding of EHP biology and provide a theoretical and technical foundation for the future investigation of its proliferation and infection mechanisms.

Ecytonucleospora hepatopenaei (EHP) is a microsporidian parasite that infects Penaeus vannamei. To study the effects of EHP infection on shrimp microRNA (miRNA) expression, we deep-sequenced the miRNA expression profiles of P. vannamei before and after EHP infection. A total of 86 miRNAs were differentially expressed in P. vannamei infected by EHP, potentially targeting over 15,600 genes involved in protein binding, cellular components, intracellular parts, and biological regulation. Moreover, the target genes were significantly enriched in several KEGG pathways, including Endocytosis and Apoptosis. Additionally, the study found that differentially expressed miRNAs, such as miR-315-x, miR-92-y, and miR-317-z, are involved in multiple regulatory processes during EHP infection, influencing growth, immune regulation, and metabolic balance. These findings reflect the mechanisms by which shrimp coordinate their defense response and metabolic regulation through miRNA expression in response to EHP infection. The study provides new insights into the molecular mechanisms of shrimp response to microsporidian infection.

Vibrio parahaemolyticus causes mass mortality in global penaeid shrimp aquaculture worldwide, with lethality exceeding 90 %, threatening global food security and economic sustainability. As alternatives to antibiotics, bacteriophages and their lytic enzymes offer target specificity, minimal resistance development, and high bactericidal efficiency. In this study, ten phages were isolated from Litopenaeus vannamei aquaculture ponds and adjacent estuarine areas using Vp499 as the host, which were isolated by our lab. Among these, phage SHY-Vp8 exhibited the highest titer against the host Vp499.Its optimal multiplicity of infection (MOI) was determined to be 1. Notably, SHY-Vp8 demonstrated a superior lytic capacity, with a high burst size (96 PFU/cell) and a short latent period (30 min), representing an 18–41 % improvement in lytic efficiency over previously reported Vibrio phages. Furthermore, it exhibited exceptional environmental resilience, tolerating temperatures up to 60 °C and a broad pH range (3–12), surpassing the stability thresholds of most known vibriophages. Whole-genome sequencing indicated a double-stranded DNA genome of 58,525 bp with 46.38 % GC content. Bioinformatic annotation identified 85 predicted genes, of which 31 encoded functionally characterized proteins. No tRNA or virulence genes were detected, demonstrating potential for therapeutic applications in aquaculture. Transmission electron microscopy confirmed an icosahedral capsid and a long non-contractile tail, classifying SHY-Vp8 within the Siphoviridae family. Notably, the gp59 gene was predicted to encode an endolysin. The gene was amplified cloned and expressed. The lytic activity of Lys59 exhibited a concentration-dependent increase, with peak activity observed at 50 μg/mL. Lys59 alone was capable of lysing Vp499 without the aid of EDTA; however, pretreatment with EDTA significantly enhanced its lytic efficiency. Results indicate that both SHY-Vp8 and Lys59 exhibit promising potential for controlling V. parahaemolyticus infections in L. vannamei, offering a novel and sustainable strategy for disease management in aquaculture—particularly in pond water treatment and seafood safety enhancement. These excellent in vitro activities and characteristics provide a solid foundation for further development of in vivo infection models and eco-friendly biocontrol agents.

The Infectious myonecrosis virus (IMNV) poses a significant threat to Litopenaeus vannamei, and there is limited research on the transcriptomic and metabolomic changes in shrimp muscle tissue post-infection. This study aims to integrate transcriptomic and metabolomic data to elucidate the gene expression and metabolic alterations in shrimp muscle tissue induced by IMNV. We present a molecular model of IMNV invasion and the mechanisms leading to muscle cell necrosis. Sequencing analyses at 30, 60, and 90 days post-infection identified 695, 2411, and 401 differentially expressed genes (DEGs) and 118, 131, and 68 significantly different metabolites (SDMs), respectively. In the early infection stage (IE, 30 days), we observed alterations in gene expression related to glucose and lipid metabolism, along with upregulation of antioxidant-related genes. The intermediate stage (IM, 60 days) exhibited significant reprogramming of metabolic pathways and signal transduction. In the persistent infection stage (IP, 90 days), we noted significant changes in energy metabolism and cell repair-related gene expression, with upregulation of genes involved in glycolysis and fatty acid biosynthesis to maintain energy supply. Our proposed model for IMNV invasion encompasses viral receptor recognition, endosomal escape, replication, and maintenance of the host cell environment. IMNV binds to host cells via the laminin receptor (Lamr), with the SMPD1 gene facilitating endosomal escape through ceramide production. The PI3K-Akt-mTOR pathway provides energy for viral replication, while the JAK-STAT pathway may be hijacked by IMNV. The virus reprograms metabolism by regulating pathways and metabolites such as D-glucose-6-phosphate, D-aspartic acid, and L-glutamic acid, while enhancing autophagy and regulating sphingolipid metabolism and antioxidant mechanisms. During viral particle assembly and release, SMPD1 promotes necroptosis and ceramide production. Additionally, IMNV modulates cytoskeletal remodeling and adhesion by regulating the Act57B, Act5C, and Act88F genes. IMNV leads to host muscle cell necrosis by suppressing the immune response, inducing cytoskeletal remodeling, oxidative stress, and activating cell death pathways such as apoptosis and ferroptosis. These processes disrupt the structure and function of muscle cells, leading to extensive necrosis. This study provides insights into the pathogenic mechanisms of IMNV and establishes a foundation for the development of anti-IMNV strategies.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus (VpAHPND) poses a significant challenge to the shrimp farming industry. Although lipopeptides produced by Bacillus subtilis have been shown to exert strong inhibitory effects against Vibrio parahaemolyticus, the underlying mechanisms remain largely unexplored. This study reveals that the lipopeptide surfactin, produced by Bacillus subtilis, significantly inhibits the VpAHPND strain JSHY-1669 through multiple mechanisms. Using antagonistic assays and transcriptomic analysis, this paper investigates the molecular mechanisms of surfactin's inhibitory action on VpAHPND strain JSHY-1669. The minimum inhibitory concentration (MIC) of surfactin against JSHY-1669 was determined to be 0.125 mg/mL, with a cumulative inhibitory effect. Exposure to surfactin caused significant structural damage to the bacterial cells, markedly inhibiting their growth and virulence gene expression. Transcriptomic analysis identified 64 genes with significant differential expression, including upregulation of genes involved in key metabolic pathways such as carbohydrate transport, and downregulation of non-essential pathways like amino acid and sulfur metabolism. Surfactin affects JSHY-1669 by disrupting key physiological processes. Specifically, it increases cell membrane depolarization, reactive oxygen species (ROS) production, and malondialdehyde (MDA) levels. These changes collectively lead to the loss of membrane integrity, which ultimately inhibits bacterial growth. Additionally, the study found downregulation of slyA, a key regulatory factor related to DNA-binding transcription, virulence regulation, and carbohydrate metabolism. Surfactin may expand its inhibitory range by affecting the slyA regulatory network, providing a basis for surfactin's broader antibacterial targets. These findings elucidate the inhibitory mechanisms of surfactin on VpAHPND strain JSHY-1669, laying a foundation for its potential applications.

Acute hepatopancreatic necrosis disease (AHPND), which is caused by a particular strain of Vibrio that carries the pirA and pirB virulence protein gene, has caused major economic losses in shrimp farming. Bacillus subtilis JSHY-K3, a bacterial strain apparently antagonistic to VpAHPND, was screened by our laboratory to better control AHPND. Our previous whole-genome sequencing analysis and untargeted metabolomics-LCMS-EMDB analysis of strain JSHY-K3 revealed that its antimicrobial material could be surfactin, aurantinin B/C/D, fengycin, sublancin 168, bacillibactin, subtilosin A, bacilysin, caffeic acid, fosfomycin, minocycline and so on. However, the specific bacteriostatic substances remain unclear and need to be further investigated. In this study, based on the whole genome sequence information obtained previously, we used CRISPR/Cas9 technology to edit the genome of B. subtilis JSHY-K3 and constructed 6 strains of gene knockout mutants, among which the bacterial solution and cell-free culture medium of the mutants ΔsrfAB-AC, ΔdhbC and ΔbacA showed a significant decrease in antagonism to VpAHPND. Therefore, we suspected that the main antibacterial substances of B. subtilis JSHY-K3 may be surfactin, bacillibactin and bacilysin. We then carried out high-resolution mass spectrometry and three-quadrupole quantitative analysis on the fermentation supernatants of strains JSHY-K3, ΔsrfAB-AC, ΔdhbC and ΔbacA, and confirmed that these three gene clusters are involved in the synthesis of surfactin, bacillibactin and bacilysin, respectively. It was also confirmed that the deletion of the srfAB-AC, dhbC, bacA genes led to a reduction in the synthesis of surfactin, bacillibactin, bacilysin, which in turn led to a decrease in the inhibitory ability of the three mutant strains against VpAHPND. In conclusion, surfactin, bacillibactin and bacilysin are the main inhibitory substances of B. subtilis JSHY-K3 to inhibit the growth of VpAHPND.

Infectious myonecrosis virus (IMNV), first identified in 2003, predominantly infects the shrimp species Litopenaeus vannamei and has caused significant economic losses in major shrimp farming regions in Brazil and Southeast Asia. However, the infection mechanisms of IMNV has not been elucidated. In this study we have investigated the molecular mechanisms of IMNV-shrimp interaction and identified potential antiviral key genes to provide a theoretical reference for developing new control technologies. Transcriptome sequencing of control and infected shrimp groups at 30 (early), 60 (mid), and 90 days (late) post-infection identified 695, 2411, and 401 differentially expressed genes (DEGs), respectively. Early infection analysis revealed IMNV promotes colonization and spread by inhibiting hemagglutination and apoptosis-related genes while activating antiviral pathways like JAK-STAT, indicating it evades host defenses by interfering with the innate immune response. During mid-stage infection, glucose and amino acid metabolism pathways were enriched, suggesting IMNV induces host metabolic reprogramming to support viral replication. Late-stage analysis showed downregulated apoptosis and autophagy pathways, and upregulated cytoskeleton and extracellular matrix genes, indicating the host mitigates viral tissue damage by regulating cell fate and repair mechanisms. This transcriptomics study uncovers dynamic shrimp gene expression changes induced by IMNV infection, identifying multiple immune and metabolic pathways involved in the antiviral response. It elucidates IMNV's pathogenic mechanism and identifies key regulatory genes in pathways like PI3K-Akt-mTOR and JAK-STAT. These findings enhance our understanding of IMNV-shrimp host interaction and could inform novel antiviral strategy development.

Acute hepatopancreatic necrosis disease (AHPND), caused by certain strains of Vibrio, has resulted in substantial economic losses in global shrimp industries. Hence, developing effective and sustainable alternatives to antibiotics to control this disease is of profound importance. In this study, we isolated a strain of antagonistic bacterium, JSHY-K3, from shrimp pond sediment, evaluated its efficacy in preventing AHPND in shrimp and tentatively investigated its mechanism of defence and control against AHPND. The isolated strain exhibited strong antagonistic activity against Vibrio parahaemolyticus, the etiological agent of AHPND (VpAHPND) harboring toxin genes pirA and pirB. JSHY-K3 was identified as B. subtilis based on its morphological, physiological, biochemical characteristics and 16 S rDNA sequence analysis. In vivo challenge assays demonstrated that JSHY-K3 could significantly mitigate cumulative mortality of shrimps infected with VpAHPND, suppress the expression of VpAHPND virulence genes and modulate shrimp immune responses. To elucidate the mechanistic basis of JSHY-K3 in preventing and controlling AHPND, whole-genome sequencing and untargeted metabolomics-LCMS-EMDB analyses were conducted. Whole-genome analysis revealed 12 gene clusters involved in synthesizing of secondary metabolites including the bacteriostatic agents surfactin, aurantinin B/C/D, fengycin, sublancin 168, bacillibactin, subtilosin A and bacilysin. Untargeted metabolomics uncovered 3691 known metabolites, with caffeic acid, fosfomycin, minocycline and surfactin A documented as V. parahaemolyticus growth inhibitors. We hypothesize that the production of these bacteriostatic metabolites by JSHY-K3 underlies its antagonistic effects against VpAHPND. Collectively, these findings suggest B. subtilis JSHY-K3 could be applied as a probiotic to prevent AHPND in shrimp aquaculture.

Litopenaeus vannamei represents the most cultivated crustacean species, accounting for approximately 80% of global shrimp aquaculture output. The intestinal microbiota structure and functionality are known to critically influence L. vannamei growth. While previous investigations of shrimp gut microbes emphasized the whole intestine, limited research has examined the composition and functional differences of microbiota across specific intestinal sections in L. vannamei. In this study, healthy shrimp samples were collected and divided into four sections after removing the contents: S(stomach), H (from posterior stomach to the distal end of the hepatopancreas.), G (the section from hepatopancreas’s end to the sixth abdominal segment), and R(rectum). The microbial composition and function were analyzed across locations via 16 S rRNA high-throughput sequencing. The results revealed that significant α- and β-diversity distinctions existed between sections (p < 0.05). Microbial abundance and diversity were enriched within group G and H compared to group S and R. The result of PCoA analysis revealed similarities between group S, H, and G, while group R markedly differed (p < 0.05). Three dominant phyla were identified across sections - Firmicutes (enriched in group S and H), Bacteroidota (enriched in group G) and Proteobacteria (enriched in group R). At genus level, Bacillus, Flavobacterium, Vibrio, and Mycoplasmataceae predominated (>10%) across sections. The rectum pathways including carbon, nitrogen, sulfur metabolisms were significantly elevated. Quorum sensing and two-component systems were also overrepresented in rectum. One-way network analysis showed that the dominant microbiota of group S, H and G were Bacillus, Mycoplasmataceae, and Flavobacterium, which are promoters of beneficial bacteria in the gut, while the dominant flora of rectumthe most competitive genus of bacteria in the intestinal tract was Vibrio,and Pseudofulvibacter, Alcaligenaceae, and Pseudomonas represent potential probiotic genera that could be screened as antagonists against Vibrio in the shrimp gut. In summary, we hypothesize the stomach and H sections degraded organic matter, G section absorbed and digested macronutrients, and rectum section recycled materials and participated in circular metabolism. These segment-specific analyses provide novel perspectives into section-specific microbial interactions and functions along L. vannamei intestine.

Enterocytozoon hepatopenaei (EHP) is a prevalent microsporidian pathogen responsible for hepatopancreatic microsporidiosis (HPM) in Litopenaeus vannamei. This infection not only leads to slowed growth in shrimp abut aslo inflicts substantial economic losses in the global aquaculture industry. However, the molecular mechanisms by which EHP influences the host during various infection stages remain unclear. This study employed comparative transcriptomics to examine the effects of EHP infection on Litopenaeus vannamei between early and late stage of infection groups. Utilizing transcriptomic approaches, we identified differentially expressed genes (DEGs) with notable biological significance through the COG, GO, KEGG, GSEA, and Mufzz time-series methodologies. The results reveal that EHP infection considerably influences host gene expression, with marked differences between early and late infection across distinct timeframes. Key processes such as detoxification, cell apoptosis, and lipid metabolism are pivotal during host-parasite interactions. Hexokinase and phosphatidic acid phosphatase emerge as key factors enabling invasion and sustained effects. Cytochrome P450 and glucose-6-phosphate dehydrogenase could facilitate infection progression. EHP significantly impacts growth, especially through ecdysteroids and 17β-estradiol dehydrogenase. By delineating stage-specific effects, we gain insights into interaction between EHP and Litopenaeus vannamei, showing how intracellular pathogens reprogram host defenses into mechanisms enabling long-term persistence. This study provides a deeper understanding of host-pathogen dynamics, emphasizing the interplay between detoxification, metabolism, immunity, apoptosis and growth regulation over the course of long-term symbiosis.

Salinity is an important factor in the aquatic environment, and its fluctuations always result in osmotic stress, which affects the survival, distribution, and physiological activities of crustaceans. Crustaceans counter them through osmoregulation, which consists of many mechanisms. Palaemon gravieri is an important economic species in Palaemonidae, widely distributed in the southern East China Sea and the China Yellow Sea, and has a good adaptability to salinity stress. Currently, there are only a few studies on the effects of salinity on P. graviera. Therefore, it is particularly important to study the molecular responses of P. gravieri to salinity fluctuations. In this study, P. gravieri was treated with salinities of 10, 25, and 40, and the hepatopancreas and gills of shrimp in the different salinity groups were sampled after 24 h. The samples were used for RNA extraction and transcriptome analysis. In total, 80,994 unigenes were obtained, of which 19,114 were annotated. The differences in gene expression between different tissues at the same salinity were more significant. Many metabolism-related genes were downregulated in the gills, such as beta-hexosaminidase subunit alpha (HEXA), 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), and Alcohol dehydrogenase class-3 (ADH5). Scanning transmission electron microscope analysis showed that the expression levels of some stress-(but not salinity stress) related genes changed after stress (mostly upregulated), suggesting the existence of secondary stress. Gene set enrichment analysis (GSEA) focused on the expression of transporters in osmoregulation, and the results showed that they mainly played a role in the gills, but ATP-binding cassette (ABC) transporters were more active in the hepatopancreas. This study showed that the response of P. gravieri to salinity change was different not only between the hepatopancreas and gills, but also between low salinity and higher salinity, and the ion transport-related genes were mainly expressed in the gills. Overall, these results improve our understanding of salt tolerance mechanism in P. gravieri.

The sustainability of shrimp aquaculture can be achieved through the development of greenhouse and aquaponic rearing modes, which are classified as heterotrophic and autotrophic bacterial aquaculture systems. However, there have been few investigations into the discrepancies between the intestinal and water microbiota of these two rearing methods. In this study, we collected shrimp samples from greenhouse-rearing (WG) and aquaponic-rearing (YG) ponds, and water samples (WE, YE), and investigated the intestinal and water microbiota between the two rearing modes. The results, through alpha and beta diversity analyses, reveal that there was basically no significant difference between shrimp intestine WG and YG (p > 0.05) or between rearing water WE and YE (p > 0.05). At the phylum and genus levels, the common bacteria between WE and WG differed significantly from those of YE and YG. The analysis of the top six phyla shows that Proteobacteria and Patescibacteria were significantly more abundant in the WG group than those in the YG group (p < 0.05). Conversely, Actinobacteriota, Firmicutes, and Verrucomicrobiota were significantly more abundant in the YG group than those in the WG group (p < 0.05). Venn analysis between WE and WG shows that Amaricoccus, Micrococcales, Flavobacteriaceae, and Paracoccus were the dominant bacteria genera, while Acinetobacter, Demequina, and Rheinheimera were the dominant bacteria genera between YE and YG. Pathways such as the biosynthesis of secondary metabolites, microbial metabolism in different environments, and carbon metabolism were significantly more upregulated in WG than those in YG (p < 0.05). In addition, pathways such as sulfate, chloroplast, phototrophy, and the nitrogen metabolism were significantly different between the WE and YE samples. These findings suggest that the greenhouse mode, a typical heterotrophic bacterial model, contains bacterial flora consisting of Amaricoccus, Micrococcales, Flavobacteriaceae, and other bacteria, which is indicative of the biological sludge process. Conversely, the aquaponic mode, an autotrophic bacterial model, was characterized by Acinetobacter, Demequina, Rheinheimera, and other bacteria, signifying the autotrophic biological process. This research provides an extensive understanding of heterotrophic and autotrophic bacterial aquaculture systems.

为确定 2018 年冬季以来江苏省沿海地区养殖脊尾白虾患"僵尸病"的病原及流行病学特点,实验采用LB培养基和PDA培养基从病虾血淋巴中分离得到直径为 1～3 mm、边缘整齐、米黄色隆起菌落;人工回感实验结果显示,回感后的脊尾白虾表现出与自然患病脊尾白虾相同的症状,并在回感脊尾白虾体内也分离出了相同的菌株,符合科赫氏法则.对该菌株进行形态观察结合 18S rRNA序列对比及系统发育分析,发现分离菌株MQ2101具有酵母的典型形态,且与二尖梅奇酵母相似度达 99.82%,结果表明菌株MQ2101 为二尖梅奇酵母.致病性结果初步分析显示,MQ2101 对脊尾白虾的半致死浓度(LD50)为1.39×107 CFU/尾.病理学观察发现,患病脊尾白虾鳃、肌肉和肝胰腺均发生不同程度的病变,其中肝胰腺病变最为严重,肝小管呈现空泡化,管腔体积变大;在鳃和肝胰腺组织中均存在大量定殖的菌体.流行病学调查结果显示,每年 2-5 月发病迅速,发病率为5%～30%,死亡率为 3%～10%.本研究确定了二尖梅奇酵母为江苏沿海地区脊尾白虾"僵尸病"的病原,其对脊尾白虾具有较强致病性,主要侵染组织为肝胰腺和鳃.以上研究结果为脊尾白虾"僵尸病"的防控提供了相关科学依据.