Salinity is a key environmental factor associated with the gut microbiome of the Pacific white shrimp, Litopenaeus vannamei. In this study, metagenomic sequencing was used to investigate gut microbial composition, predicted functional potential, carbohydrate-active enzyme (CAZyme) profiles, and antibiotic resistance gene (ARG) profiles across the salinity gradient from 0 to 30 parts per thousand (ppt). Salinity was associated with marked shifts in microbial community composition. The freshwater group showed higher microbial diversity and enrichment of Rhodobacteraceae-related genera such as Ruegeria and Sedimentitalea, whereas Vibrio was enriched under low-salinity conditions and Enterocytozoon increased under mid- to high-salinity conditions. Functional annotation indicated that the gut microbiome retained a broadly conserved metabolic framework, while specific Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and CAZyme classes varied across salinity treatments, suggesting salinity-associated changes in predicted microbial functional potential and carbohydrate-utilization capacity. ARG profiles also varied across the salinity gradient and involved multiple resistance categories, including multidrug, β-lactam, aminoglycoside, sulfonamide, rifamycin, and tetracycline resistance. ARG–host association analysis linked representative ARGs to putative bacterial hosts. In particular, Bifidobacterium_adolescentis_rpoB was linked to Bifidobacterium adolescentis and enriched in the freshwater group, whereas TXR and CARB-18 were associated with Vibrio species and were more abundant under saline conditions. Overall, these findings suggest that salinity is an important ecological factor linked to taxonomic structure, predicted functional potential, CAZyme composition, ARG profiles, and putative ARG–host associations in the shrimp gut microbiome.

Acute hepatopancreatic necrosis disease (AHPND), caused by a specific strain of Vibrio parahaemolyticus (VpAHPND), disrupts host immune function by promoting the accumulation of lipid droplets (LDs). However, the underlying mechanisms and physiological consequences of LDs accumulation during VpAHPND infection in shrimp remain poorly characterized. In this study, we investigate the role of Penaeus vannamei glycerol-3-phosphate acyltransferase 3 (PvGPAT3), a key enzyme in triglyceride and LDs biosynthesis, during VpAHPND infection. Transcriptomic analysis and quantitative real-time PCR (qRT-PCR) revealed that PvGPAT3 expression is significantly upregulated following VpAHPND infection. Functional assays demonstrated that silencing PvGPAT3 via RNA interference (RNAi) improved shrimp survival and restricted bacterial proliferation, whereas its knockdown attenuated infection-induced LDs accumulation. Conversely, overexpression of PvGPAT3 in High Five cells significantly promoted LDs formation. Mechanistic studies further indicated that PvGPAT3-mediated LDs accumulation contributes to mitochondrial dysfunction and subsequent apoptosis. Collectively, these findings suggest that PvGPAT3 exacerbates VpAHPND pathogenesis by inducing mitochondrial dysfunction and apoptosis through the promotion of LDs accumulation.

Hemocyanins function not only as oxygen carriers in the hemolymph of arthropods and molluscs but also as an important multifunctional immune molecule. In this study, we observed a significant increase in hemocyanin expression and a decrease in PO activity in Penaeus vannamei infected with acute hepatopancreatic necrosis disease (AHPND). Using gas chromatography-mass spectrometry (GC-MS)-based metabolomics, we found that knockdown of the hemocyanin gene (PvHMC) led to significant reductions in phenylalanine and tyrosine levels. Subsequent qPCR, PO activity assays, and LC-MS analyses revealed that PvHMC knockdown significantly elevated the transcript levels of key enzymes in the phenylalanine-tyrosine metabolic pathway, accompanied by increased L-Dopa levels and enhanced PO activity. In contrast, exogenous supplementation of phenylalanine or tyrosine downregulated these four enzymes and reduced PO activity. Furthermore, after PvHMC knockdown, inhibiting phenylalanine hydroxylase with 4-Chloro-DL-phenylalanine (PCPA) or tyrosine hydroxylase with 3-Iodo-L-tyrosine (3-IT) increased phenylalanine or tyrosine levels, decreased the expression of the corresponding enzymes, and reduced both L-Dopa content and PO activity. Collectively, these results demonstrate that during Vibrio parahaemolyticus (VpAHPND) infection, PvHMC is significantly downregulated and negatively regulates PO activity via the phenylalanine-tyrosine metabolism, thereby enhancing immune defense. This study elucidates a novel metabolic mechanism through underlying PvHMC-mediated regulation of the PO cascade regulation, providing new insights into the interplay between metabolism and immunity in shrimp.

Dihydrodiol dehydrogenase (DHDH) participates in the cytochrome P450 pathway and ROS regulation in mammals, but its role in shrimp immunity remains unclear. Here, we characterized PvDHDH in Penaeus vannamei and explored its function in ROS homeostasis and anti AHPND causing Vibrio parahaemolyticus (V.pAHPND) infection. Sequence analysis revealed PvDHDH contains a conserved GFO_IDH_MocA domain, with high homology to crustacean and vertebrate DHDH, and is predominantly expressed in the hepatopancreas. Under physiological conditions, PvDHDH knockdown dysregulated cytochrome P450 pathway genes (upregulating PvCYP1A, PvCYP3A4, and PvCBR1; downregulating PvUGT) and increased total ROS. Under V.pAHPND stress, PvDHDH expression was specifically upregulated, and its knockdown further perturbed P450 pathway genes, elevated ROS/H2O2, and increased V.pAHPND abundance. These results indicate PvDHDH modulates ROS via affecting cytochrome P450 pathway genes to contributes to resist V.pAHPND, providing novel insights into shrimp innate immunity.

Vibrio parahaemolyticus-induced acute hepatopancreatic necrosis disease (AHPND) has become a prominent limiting factor in the global shrimp farming industry. Hypoxia-inducible factor 1α (HIF-1α) coordinates oxygen sensing with immune and metabolic reprogramming, yet its contribution to antibacterial defense in Penaeus vannamei remains poorly understood. Here, we identified and characterized P. vannamei HIF-1α, which encodes a 1050-aa protein containing the conserved HLH-PAS-PAC domains. HIF-1α transcripts were detected in all examined tissues, with the highest abundance in the gill. RNAi-mediated HIF-1α silencing increased bacterial load and mortality and exacerbated intestinal and hepatopancreatic lesions after V. parahaemolyticus challenge. Consistently, hemolymph analyses indicated that HIF-1α depletion accelerated hemocyte loss, augmented mitochondrial membrane depolarization, elevated reactive oxygen species (ROS) and malondialdehyde (MDA) accumulation, and reduced glutathione (GSH) content, peroxidase (POD) and lysozyme activities. Transcriptome profiling further revealed 79 core genes downregulated upon HIF-1α silencing, which were enriched in immune response, redox homeostasis, autophagy, mitochondrial function, and amino acid/lipid metabolism. Moreover, HIF-1α positively regulated mannose-binding lectin (MBL), and HIF-1α silencing reduced the expression of hemocyte antimicrobial effectors, including anti-lipopolysaccharide factors (ALFs), lysozymes (LYZs), crustins (CRUs), and penaeidins (PENs). Notably, silencing either HIF-1α or MBL independently reduced survival to 0% at 24 h post-V. parahaemolyticus challenge, significantly lower than that of controls (40%). These results indicate that HIF-1α contributes to resistance against AHPND-associated V. parahaemolyticus infection by supporting antimicrobial effector expression, redox homeostasis, immune resilience, and MBL-associated antibacterial resistance, highlighting HIF-1α as a potential target for AHPND control and disease-resistance breeding in shrimp.

Acute hepatopancreatic necrosis disease (AHPND) is one of the most severe threats to global shrimp aquaculture. Recent studies report a significant reduction in hepatopancreas hemocyanin levels in Penaeus vannamei (PvHMC) by AHPND. To investigate the functional implications, we performed liquid chromatography-tandem mass spectrometry-based metabolomics and found that PvHMC knockdown caused a marked accumulation of phosphatidylcholine (PC). qPCR and ELISA analyses further showed that PvHMC suppression upregulated the mRNA expression, protein abundance, and enzymatic activity of PvCPT1, a key enzyme in PC synthesis. Co-immunoprecipitation and confocal immunofluorescence analyses confirmed a direct interaction between PvHMC and PvCPT1, indicating that PvHMC regulates PC metabolism by binding to PvCPT1. Functional assays demonstrated that exogenous PC supplementation significantly increased reactive oxygen species (ROS) production, whereas simultaneous knockdown of PvHMC and PvCPT1 reduced plasma PC levels and ROS activity. Under pathogenic challenge, PC administration inhibited Vibrio parahaemolyticus (VpAHPND) proliferation by enhancing intracellular ROS-mediated antimicrobial responses, ultimately improving shrimp survival. In summary, PvHMC regulates PC metabolism to modulate ROS levels, strengthening shrimp's immune defense against pathogenic infections. These findings reveal a novel metabolic regulatory role of PvHMC in response to VpAHPND infection and suggest potential strategies for controlling AHPND in shrimp aquaculture.

Probiotics provide an efficient and relatively safe method for preventing disease and increasing production in aquaculture. During the screening of beneficial bacteria in the economically important mud crab (Scylla paramamosain), an indigenous gut strain, Cetobacterium somerae R9, was isolated for the first time as a butyrate producer. This study aimed to evaluate the probiotic potential of C. somerae R9 both in vitro and in vivo. In vitro assays revealed that C. somerae R9 exhibited grow at pH 7-9, NaCl concentrations of 0.5-2.5%, and bile salt concentrations of 0.4-1.0%, and displayed susceptibility to most of the antibiotics tested. In the in vivo study, dietary supplementation with C. somerae R9, either alone or in combination with prebiotics (resistant starch and galactooligosaccharides), enhanced growth, improved antioxidative status (evidenced by elevated SOD and CAT activity and reduced MDA content), reduced hepatopancreatic damage (reduced AST activity), and maintained intestinal integrity. Supplementation also selectively enriched beneficial gut microbiota (e.g., members of Fusobacteriota). Transcriptome analysis showed that C. somerae R9 appeared to activate the PI3K-Akt signaling pathway, focal adhesion, and ECM-receptor interaction, while the combination of C. somerae R9 and prebiotics activates complement and coagulation cascades, amino sugar and nucleotide sugar metabolism, and protein digestion and absorption. Furthermore, mud crabs fed diets supplemented with C. somerae R9 exhibited significantly higher survival after challenge with Vibrio parahaemolyticus, with the synbiotic providing greater benefits than the probiotic alone. These findings collectively suggest that C. somerae R9 is a promising probiotic candidate (used either alone or in combination with prebiotics) for mud crab aquaculture.

In aquatic invertebrates, hemolymph is critical for host-environment interaction. Although maintaining microbial homeostasis in hemolymph is significant for disease pathogenesis, the key regulatory mechanisms remain elusive. This study investigates the essential role of the respiratory glycoprotein hemocyanin (HMC) in modulating hemolymph microbial composition in penaeid shrimp. Hemocyanin-depleted shrimp infected with Vibrio parahaemolyticus (Vp) exhibited disruptions in the niacinamide (NAM) salvage pathway, attenuated plasma niacinamide and nicotinamide adenine dinucleotide (NAD) levels, and reduced transcripts of key enzymes. These alterations correlated with an increased total bacterial load and a higher abundance of opportunistic pathogens like Vibrio and Shewanella. Hemocyanin likely modulates niacinamide metabolism through interactions with Litopenaeus vannamei nicotinamide riboside kinase 1 (LvNRK1), sirtuin 2 (LvSIRT2), and sirtuin 6 (LvSIRT6). Remarkably, niacinamide supplementation after hemocyanin knockdown restored hemolymph microbial balance, reduced Vibrio dominance, and improved shrimp survival against V. parahaemolyticus infection, but not Gram-positive Staphylococcus aureus (Sa). These findings provide novel insights into the pivotal role of niacinamide metabolism in regulating shrimp hemolymph microbial composition via hemocyanin.

Immunometabolic reprogramming is a hallmark of host defense. Recent studies have shown that hemocyanin regulates the tricarboxylic acid (TCA) cycle in shrimp to enhance immune competence, yet its role in glycolytic regulation remains unclear. Here, we show that RNA interference-mediated silencing of hemocyanin in Penaeus vannamei markedly reduces the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme. Hemocyanin knockdown also decreases GAPDH protein abundance and enzymatic activity, accompanied by reduced levels of 1,3-bisphosphoglycerate and NADH. Although no direct physical interaction between hemocyanin and GAPDH was detected, co-expression of PvHMC was associated with increased total GAPDH enzymatic activity in the heterologous HEK-293T system, suggesting an indirect functional association. Upon pathogen challenge, both hemocyanin and GAPDH responded dynamically, and exogenous NADH supplementation partially restored the expression of representative glycolytic and TCA-cycle enzymes in hemocyanin-silenced shrimp. Importantly, NADH supplementation reduced Vibrio parahaemolyticus burden and improved host survival after infection. Together, these findings support a model linking hemocyanin with GAPDH-related glycolytic metabolism and NADH homeostasis.

Phosphorylation plays a critical role in regulating immune responses in invertebrates. In Penaeus vannamei, hemocyanin, a multifunctional immune protein, is cleaved to produce antimicrobial peptides (AMPs) essential for pathogen defense. This study identifies Ser548 phosphorylation of the hemocyanin small subunit (PvHMCs) as a key regulator of trypsin-mediated hemocyanin degradation and antimicrobial peptides production. Dephosphorylation of Ser548, controlled by PvCK2α kinase and PvPP2AC phosphatase, enhances cleavage, generating peptides with strong antibacterial activity against Vibrio parahaemolyticus and Streptococcus iniae. Phosphorylation inhibits this process, reducing peptide production and immune efficacy. In vivo, Ser548 dephosphorylation improves bacterial clearance and enhances shrimp survival. These findings reveal a critical molecular mechanism underlying shrimp immunity and suggest targeting hemocyanin phosphorylation to boost disease resistance in aquaculture.

The sustainable development of shrimp aquaculture is significantly compromised by Vibrio parahaemolyticus infections. Identifying host resistance genes and characterizing their immunological roles are essential for developing effective disease control strategies. In this study, we conducted a comparative transcriptomic analysis of intestinal tissues from Penaeus vannamei exhibiting varying degrees of pathological damage post-V. parahaemolyticus challenge to identify key resistance genes. KEGG enrichment analysis revealed that the ABC transporter pathway was markedly enriched among upregulated genes in both the 9 h vs 0 h and 48 h vs 0 h comparison groups. Based on the expression profiles and domain characteristics of genes within this pathway, the full transporter PvABCA3, half transporter PvABCC1, and soluble protein PvABCF2 were selected for RNAi assays. The result indicated that silencing PvABCF2, but not PvABCA3 and PvABCC1, significantly increased mortality, tissue damage, and Vibrio load in V. parahaemolyticus-challenged shrimp. Further investigation revealed that PvABCF2 silencing substantially suppressed the expression of antimicrobial peptides (AMPs), components of the proPO-activating system, and key genes involved in the JAK-STAT and NF-κB signaling pathways. These findings suggested that the increased susceptibility of shrimp to V. parahaemolyticus following PvABCF2 silencing may be associated with downregulation of these specific immune-related genes. Moreover, one SNP within PvABCF2 was found to be markedly associated with resistance to V. parahaemolyticus via SNP association analysis. Collectively, these results suggested that PvABCF2 was involved in defense response against V. parahaemolyticus and identified a potential molecular marker for disease-resistant breeding.

Butyrate-producing bacteria (BPB) are widely used as additives in aquaculture because of the beneficial effects of the butyrate they produce. However, most available BPB currently are of terrestrial origin, which are quite different from aquatic animals, resulting in profoundly unstable probiotic performance. This study isolated a novel, indigenous BPB, Clostridium moniliforme G18, from the gut of mud crabs, Scylla paramamosain. The strain demonstrated robust butyric acid production, tolerance to gastrointestinal conditions (pH 4–9, 0–3

Salinity is a key environmental factor affecting gonadal development in euryhaline crustaceans, yet the underlying molecular mechanisms remain unclear. Although Litopenaeus vannamei can survive across a wide salinity range, its ovarian development requires optimal salinity. To investigate the mechanisms by which salinity affects ovarian maturation, shrimp were cultured at different salinities (3%, 2.5%, 2%, and 1.5%). Ovarian maturation was observed, and TMT-labeled quantitative proteomic analyses were performed on ovarian, eyestalk, hepatopancreas, and gill tissues. Results showed that low salinity significantly reduced the ovarian maturation rate, with progressively lower rates at decreasing salinities. Proteomic analysis identified 3240 differentially expressed proteins (DEPs). GO and KEGG enrichment analyses revealed that under low salinity stress, up-regulated DEPs were primarily involved in energy metabolism, oxidative stress response, and nutrient transport, while down-regulated DEPs were associated with pathways related to immunity, stress response, and apoptosis. Notably, two proteins critical for ovarian development—vitellogenin and chorion peroxidase—exhibited significantly reduced expression abundance in the eyestalk, hepatopancreas, and gills of low salinity groups (particularly at 1.5% and 2%). qRT-PCR validation experiments yielded largely consistent results at the transcriptional level, confirming that low salinity stress inhibited the expression of these key reproductive genes in peripheral tissues. In summary, this study shows from a proteomic perspective that a low salinity environment may force shrimp to allocate more energy to basic physiological activities like osmoregulation, and suppressing the expression of crucial reproductive proteins like vitellogenin and chorion peroxidase, ultimately impairing ovarian development in L. vannamei. These findings offer an important basis for understanding of the molecular mechanisms by which salinity regulates reproduction in crustaceans.

Vibrio parahaemolyticus is a major threat to Penaeus vannamei aquaculture industry. Uncovering V. parahaemolyticus resistance gene and utilizing them to breed disease-resistant shrimp varieties represent an effective strategy to resolve this issue. In this study, we performed a genome wide association study (GWAS) on an F₂ shrimp population (n = 200) utilizing specific locus amplified fragment sequencing (SLAF-seq) to identify V. parahaemolyticus resistance genes. A total of 207,767 high-quality SNPs were detected, 12 of which were significantly associated with resistance to V. parahaemolyticus. Within 100 kb regions flanking of these significant SNPs, 20 candidate genes were identified. By integrating the GWAS results with prior transcriptomic data, PvPdcd6, PvDnajc22, and PvGlyctk were selected for further functional research. The results indicated that silencing PvPdcd6 and PvDnajc22, but not PvGlyctk significantly increased mortality of shrimp, Vibrio load and pathological damage in hemolymph, hepatopancreas and intestines following V. parahaemolyticus challenge. Further analysis demonstrated that inhibition of PvPdcd6 downregulated the expression of genes involved in the NF-κB pathway, antimicrobial peptides (AMPs), and autophagy, suggesting that PvPdcd6 contributed to host defense by activating autophagy and upregulating NF-κB signaling pathway, thereby promoting AMPs expression. Moreover, SNP association analysis identified four SNPs in PvPdcd6 and two in PvDnajc22 notably linked to V. parahaemolyticus resistance. Collectively, this study enhances our comprehension of the mechanisms underlying V. parahaemolyticus tolerance and lays a theoretical foundation for marker-assisted breeding of disease-resistant varieties.

Nitrite is a key environmental challenge in intensive shrimp aquaculture, adversely affecting physiological regulation and survival. Although tolerant Penaeus vannamei families have been established by selective breeding, the basis of family-level variation in tolerance has yet to be clarified. In this study, nitrite-tolerant and nitrite-sensitive families were compared using survival analysis, transcriptomics, targeted qPCR validation, physiological assays, and RNA interference of representative transport-related genes. Under nitrite exposure, the tolerant family exhibited significantly higher survival and a distinct gill transcriptional response, characterized by stronger induction of acid-base and ion-transport genes, including carbonic anhydrase 2 (CA2), the Na+/K+-ATPase subunits ATP1A and ATP1B, as well as several V-type H+-ATPase-related genes. These transcriptional changes were accompanied by elevated ATP content and Na+/K+-ATPase activity, improved hemolymph pH stability, and reduced nitrite accumulation in both gill and hemolymph. RNAi-mediated knockdown of CA2 or ATP1B attenuated the nitrite-induced transport response, decreased ATP content and NKA activity, exacerbated hemolymph acidification, promoted internal nitrite accumulation, and ultimately reduced shrimp survival under nitrite stress. Family-based validation further showed that the tolerant family displayed higher survival than the sensitive family in the dsEGFP group, whereas this advantage was markedly reduced after CA2 or ATP1B knockdown under nitrite stress. These findings highlight that strengthened branchial ion transport and acid-base regulation represent key physiological mechanisms underlying nitrite tolerance in resistant shrimp families.

Ammonia pollution is a major environmental stressor in shrimp aquaculture, causing tissue damage and limiting production. While the cellular mechanisms that confer resilience to ammonia toxicity remain poorly understood, hemocyanin expression is consistently upregulated under ammonia stress. This study reveals the role of hemocyanin in mitigating ammonia-induced hepatopancreas damage through DNA damage repair pathways. Hemocyanin knockdown aggravated tissue damage and elevated DNA damage levels, whereas hemocyanin supplementation alleviated both effects. Transcriptomic analysis revealed that hemocyanin silencing downregulated key DNA repair-related genes, including metallothionein 1-like, RAD9A-like, DNA damage-inducible transcript 4-like, and nicotinamide phosphoribosyltransferase-like. Proteomic analysis and interaction assays identified four DNA repair-related proteins, i.e., ADP-ribosylation factor 1, H2A-variant, putative DNA-dependent protein kinase subunit-like, and ribosomal protein RPL6, as hemocyanin-binding partners. Bioinformatics analysis and GST pull-down assays confirmed direct interactions between hemocyanin and both RPL6 and H2A-variant. RPL6 knockdown disrupted hemocyanin's nuclear localization and further increased DNA damage under ammonia stress. These findings suggest that hemocyanin is recruited to DNA damage sites via RPL6, where it facilitates repair processes to preserve hepatopancreas integrity. This study reveals a previously uncharacterized function of hemocyanin in nuclear DNA repair and provides valuable insights for developing stress-tolerant shrimp strains to enhance aquaculture sustainability.

The development of functional feed additives represents a promising strategy to enhance shrimp performance and resilience in sustainable aquaculture. This study investigated the individual and combined effects of octacosanol and Saccharomyces cerevisiae on growth performance, physiological status, antioxidant capacity, immune response, and disease resistance in Litopenaeus vannamei. A total of 360 shrimp were randomly assigned to four dietary treatments in triplicate and fed for 60 days: a control diet, octacosanol (12 mg/kg), S. cerevisiae (4 g/kg), and a combined treatment (12 mg/kg octacosanol + 4 g/kg yeast). Disease resistance was further evaluated using a waterborne immersion challenge with the pathogenic Aeromonas hydrophila, with survival monitored over 10 days. All supplemented groups exhibited significantly improved growth performance, with OCTA×SC recording the highest final body weight, weight gain, and specific growth rate, alongside the lowest feed conversion ratio, relative to control (p < 0.05). Digestive enzyme activities, including lipase and protease, were significantly enhanced, particularly in the OCTA×SC group. Fatty acid profiling of both muscle and hepatopancreas revealed a significant reduction in saturated fatty acids and a concurrent increase in n-6 and n-3 polyunsaturated fatty acids, with DHA accumulation being most pronounced in OCTA×SC. Serum biochemical analysis demonstrated significant improvements in total protein, albumin, globulin, and HDL-cholesterol, alongside significant reductions in triglycerides and hepatic transaminase activities (AST and ALT) in supplemented groups. Antioxidant enzyme activities (CAT, SOD, GPx) and total antioxidant capacity were significantly upregulated, while lipid peroxidation (MDA) was significantly reduced, most markedly under combined supplementation. Transcriptional analysis confirmed significant (p < 0.05) upregulation of immune-related genes (HMC-Lv, Pen3-Lv, LZM-Lv, ProPO-Lv) and antioxidant genes (cMnSOD-Lv, CAT-Lv, GSH-Px-Lv) in all supplemented groups, with OCTA×SC consistently yielding the greatest induction. Following bacterial challenge, OCTA×SC exhibited the highest survival rate and relative percentage of survival (RPS = 43.48%), significantly exceeding single-supplement groups (RPS = 30.43% each). In conclusion, the synergistic application of octacosanol and S. cerevisiae (12 mg/kg octacosanol + 4 g/kg yeast) effectively enhances growth, physiological stability, and disease resistance in L. vannamei. These findings support the integrated use of octacosanol and S. cerevisiae as a synergistic nutritional strategy to enhance shrimp performance and disease resilience in intensive aquaculture systems. Future work should address mechanisms, dosage optimization, and long-term impacts on health and sustainability.

Litopenaeus vannamei B lymphocyte-induced maturation protein 1 (LvBlimp1) is a transcription factor expressed in macrophage-like phagocytes that modulates immune responses via direct bactericidal activity, opsonization, and phagocytosis. However, the molecular targets and regulatory architecture by which LvBlimp1 controls shrimp disease resistance remain poorly defined. In this study, ChIP-seq was performed in shrimp hemocytes to identify genome-wide binding targets of LvBlimp1. Five core downstream genes (MAP1B, SLC46, TACC1, COL3A1, and LGMN) are directly regulated by LvBlimp1 were identified through integrative analysis of ChIP-seq data, transcriptomes following LvBlimp1 knockdown, and marker gene sets of macrophage-like phagocytes. Four targets (MAP1B, SLC46, TACC1, COL3A1) were verified as direct transcriptional targets after FIMO analysis and ChIP-qPCR. Distinct expression patterns and regulatory correlations were observed in public transcriptomic datasets under white spot syndrome virus (WSSV) infection, Vibrio alginolyticus (V. alginolyticus) infection, and low-salinity stress, indicating that stress-specific immune responses are orchestrated by LvBlimp1 selective target engagement. Significantly altered expression of LvBlimp1 and its targets was detected in Vibrio parahaemolyticus-resistant and WSSV-resistant shrimp families, with divergent expression profiles observed under distinct pathogenic challenges. This study constructs a context-dependent transcriptional regulatory network of LvBlimp1 in shrimp innate immunity. Identify MAP1B and SLC46 as candidates for dissecting antimicrobial immune mechanisms against WSSV and V. alginolyticus, and highlight LvBlimp1, COL3A1, and TACC1 as high-value targets for potential immune regulation of shrimp with improved low-salinity tolerance and pathogen resistance. These results provide mechanistic insights into stimulus-specific immune responses in crustaceans and promising LvBlimp1 direct target genes associated with immune response of disease-resistant shrimp.

Litopenaeus vannamei is an economically important shrimp species that can survive in a variety of environments; however, low salinity compromises its reproduction. To investigate whether salinity affects ovarian development in L. vannamei, we reared female shrimp under optimal (30‰) and low (25‰, 20‰, 15‰) salinity conditions. On day 26 post-induction, the ovarian maturation rates were 67.7%, 58.3%, 56.7%, and 31.7% for the 30‰, 25‰, 20‰, and 15‰ salinity groups, indicating that low-salinity compromises ovarian maturation. To provide mechanistic insight, we performed comparative transcriptomic analysis of hepatopancreas, eyestalk, gill, and ovary tissues five days post-maturation induction. GO and KEGG enrichment analyses revealed that upregulated differentially expressed genes (DEGs) under low-salinity conditions (n = 2359 total) were primarily associated with immune response, apoptosis regulation, polysaccharide metabolism, and antioxidation pathways, reflecting stress adaptation to maintain cellular homeostasis. Downregulated DEGs (n = 2008 total) were significantly enriched in pathways related to energy metabolism, lipid metabolism, and estrogen signaling, indicating the inhibition of metabolic and reproductive pathways. Trend analysis identified the ovarian development-related gene, vitelline membrane outer layer protein 1 (VMO1), whose expression was verified to decrease significantly with declining salinity. These results provide practical guidance for L. vannamei breeding and a new perspective for understanding salinity-mediated reproductive regulation.

Single-nucleus sequencing is a powerful tool for studying ovarian development. However, a standardized protocol for single-nucleus isolation in crustaceans is currently lacking. In this study, the key parameters for preparing nuclei suspension of Litopenaeus vannamei were optimized systematically for the first time, employing evaluation methods such as HE staining, trypan blue staining, scanning electron microscopy, 10 × Genomics quality control data, and t-SNE plots. The results showed that the optimal lysis efficiency of fresh ovarian tissue was achieved by manually grinding with 0.1% NP40 for 5 min, and the nuclei quality was ensured. Meanwhile, an iodixanol-assisted approach was used to reduce the mechanical damage to nuclei caused by centrifugation, significantly enhancing nuclear recovery. To capture as many nuclear types as possible, we employed a combination of high- and low-speed centrifugation, improving nuclear recovery and reducing data bias. Shrimp ovarian samples can be processed within 40 min or less. The single nuclei isolated from shrimp ovaries by the optimized method were uniformly round with clear boundaries, a significant reduction in clumping and debris, and minimal nuclear residue in the supernatant. The nuclear recovery rate increased from 4.78% to 40.56%. This study bridges the gap in single-cell research on the reproductive system of L. vannamei and provides new directions and insights for the preparation of nuclei suspension from complex tissue samples in the crustacean research.