Rising water temperatures pose a major challenge to the physiological performance and survival of aquatic ectotherms. To investigate long-term responses to thermal stress, largemouth bass (Micropterus salmoides) were exposed to 34 °C for 180 days, followed by integrated analyses of histopathology, ultrastructure, multi-tissue transcriptomics, and intestinal microbiota. Chronic heat exposure induced marked pathological damage in the liver, spleen, and intestine, accompanied by mitochondrial remodeling, mitophagy, elevated TUNEL signals, and lysosomal accumulation, indicating persistent cellular stress. Transcriptomic analysis identified 105 conserved differentially expressed genes across tissues. Functional enrichment revealed tissue-specific responses, including metabolic reprogramming in the liver, immune activation in the spleen, and enhanced ribosome biogenesis in the intestine. Cross-tissue Gene Set Enrichment Analysis (GSEA) further revealed recurrent activation of the TNF and JAK-STAT signaling pathways across all three tissues, suggesting conserved molecular responses to chronic heat stress. Weighted Gene Co-expression Network Analysis (WGCNA), integrated with cross-tissue GSEA and protein-protein interaction analyses, identified three candidate key genes (rpl8, uba52, and ccr3l) associated with these conserved pathways as potential markers of long-term thermal adaptation. Concurrently, 16S rRNA sequencing revealed significant gut microbial dysbiosis, and correlation analysis suggested potential interactions between microbial composition and host gene expression. Collectively, these findings demonstrate that chronic thermal stress induces coordinated physiological and molecular adaptations along the gut-liver-spleen axis through tissue-specific responses superimposed on conserved stress-response pathways. This study provides a comprehensive framework for understanding long-term thermal adaptation in largemouth bass and informs strategies to evaluate thermal resilience in aquaculture species under climate warming.
Optimizing metabolic health in farmed fish is increasingly important as aquaculture production intensifies. Gracilaria lemaneiformis-derived polysaccharide (GLP), a natural sulfated polysaccharide, has emerged as a promising candidate for mitigating lipid metabolic disorders. To elucidate the mechanisms through which GLP modulates hepatic lipid deposition, we established complementary in vivo feeding trial in rabbitfish (Siganus canaliculatus) under standard culture conditions and in vitro palmitic acid-induced steatosis model. A total of 450 fish (initial body weight: 3.11 ± 0.01 g) were randomly distributed into three experimental groups with three replicate cages per group (50 fish per cage). Fish were fed a basal diet (control) or basal diet supplemented with 1.0 g GLP/kg diet (GLP1) or 1.5 g GLP/kg diet (GLP2) for 60 days. Results showed that the GLP supplementation promoted growth performance, with the GLP1 achieving the greatest final body mass, weight gain, and thermal growth coefficient (P < 0.05), whereas GLP2 yielded superior economic conversion efficiency and improved population uniformity (P < 0.05). In parallel, GLP reduced circulating total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels (P < 0.05). Histological assessment further indicated that GLP effectively attenuated hepatic injury and lipid droplet accumulation. Transcriptomic profiling demonstrated that differentially expressed genes were primarily enriched in pathways related to lipid metabolism, amino acid metabolism, and signal transduction. Mostly, GLP enhanced lipid catabolism and β-oxidation by upregulating genes such as acbd4 and pnpla2, while concurrently suppressing lipogenic genes including sqle and lss. Consistent with the in vivo findings, in vitro assays showed that GLP mitigated hepatocyte steatosis in parallel with downregulation of sqle and its downstream lipogenic regulators (acc, fas, and ppar-γ). While pharmacological inhibition of SQLE with terbinafine recapitulated the lipid lowering effects of GLP, identifying SQLE as a transcriptional correlate of GLP action rather than establishing direct mechanistic targeting. Collectively, these results establish GLP as an effective lipid-lowering feed additive that simultaneously promotes lipid breakdown and inhibits lipid synthesis, providing a practical nutritional strategy to enhance metabolic health and production efficiency in aquaculture.
Zebrafish (Danio rerio) is a widely used model teleost in biomedical and reproductive research, yet knowledge of male reproductive management remains fragmented across basic and applied disciplines. This review provides an integrated synthesis of male reproductive biology, from spermatogenesis to practical sperm handling and evaluates how zebrafish-derived mechanisms and management principles may inform reproductive research in cultured teleosts. Acute high temperature exposure (≥34 °C) can impair male reproduction by increase Leydig-cell apoptosis and reduced steroidogenic activity, while circadian clocks regulate spermatogonial differentiation through retinoic acid signaling. Nutritional factors, including phospholipids and polyunsaturated fatty acids, influence sperm membrane function and motility. Sperm collection by stripping is particularly vulnerable to urine contamination, which triggers premature Ca2+ dependent motility activation; immediate collection into immobilizing extenders helps maintain sperm quiescence. Motility activation involves hypo-osmotic and ionic signaling, inducing K+ efflux, membrane hyperpolarization, and Ca2+ dynamics. For short-term storage, alkaline extenders (pH 7.2–7.9, 300–400 mOsmol/kg) at 0–4 °C are recommended, with a 28 °C re-acclimatization step that increase post-storage motility. Key knowledge gaps include the molecular mechanisms of sperm maturation, strain-specific sperm quality, optimization of storage and cryopreservation protocols, and paternal molecular inheritance. Overall, zebrafish provides a mechanistic and methodological framework for teleost reproductive research, sperm preservation, and aquaculture-related applications; however, direct transfer of specific temperatures, extenders, activation media, and storage protocols to cultured fish requires species-specific validation.
The sea cucumber (Apostichopus japonicus) is a key mariculture species of high economic value in East Asia. However, Ocean acidification, resulting from the increased uptake of anthropogenic CO₂ by seawater, severely impairs its growth, survival, and reproductive performance, thereby threatening the sustainable development of this aquaculture industry. The gut microbiota play a critical role in host adaptation to environmental stress; however, their involvement in mediating A. japonicus responses to seawater acidification remains unclear. Therefore, this study first compared the gut microbial communities of juvenile and adult A. japonicus under acidified and ambient seawater conditions. The results showed that Pseudoalteromonas abundance was significantly enriched under seawater acidification, from which the dominant strain, Pseudoalteromonas carrageenovora P1, was screened and isolated. This strain, when supplied as a dietary supplement under seawater acidification, improved A. japonicus growth performance and intestinal morphology, activated the Keap1-Nrf2-ARE and NF-κB pathways, enhanced antioxidant capacity and immune function, and increased resistance against Vibrio splendidus 21915 infection. Moreover, P. carrageenovora P1 modulated gut microbial structure by promoting beneficial bacteria such as Lutibacter while suppressing potential pathogens, including Vibrio, thereby maintaining intestinal homeostasis under acidification stress. This study reveals the important value of the gut microbiota of A. japonicus in regulating environmental tolerance of benthic invertebrates, and also provides a scientific reference for stress resistance regulation and health maintenance in A. japonicus aquaculture under seawater acidification.
N6-methyladenosine (m6A) plays important roles in post-transcriptional gene regulation, but its function in antiviral responses of teleosts remains largely unknown. In this study, we identified 19 conserved m6A regulatory genes in Nile tilapia (Oreochromis niloticus). These genes were highly expressed in immune-related and nervous tissues, and showed higher abundance in innate immune subsets within head kidney lymphocytes (HKLs). Using poly(I:C) to mimic viral stimulation, we performed transcriptome-wide m6A methylome analysis by MeRIP-Seq in tilapia HKLs. A total of 23,362 and 21,131 m6A peaks were identified in the control and stimulated groups, respectively, which were mainly enriched in coding regions and around stop codons, and contained the conserved RRACH motif. After poly(I:C) stimulation, 1076 differentially methylated peaks were detected, with hypermethylated peaks accounting for the vast majority (875 peaks). Integrative analysis revealed that significant peaks with coordinated changes in m6A methylation and gene expression slightly outnumbered those with discordant changes. Notably, genes in the hyper-up quadrant (hypermethylated and upregulated) were significantly enriched in antiviral pathways such as the RIG-I-like receptor pathway and cytokine-cytokine receptor interaction. This quadrant contained 25 ubiquitination-related candidate genes; these genes were transcriptionally activated upon poly(I:C) stimulation, accompanied by increased global protein ubiquitination signals in HKLs. Together, this study identified m6A regulators in Nile tilapia and revealed positive m6A methylation associated with antiviral responses in head kidney lymphocytes, suggesting a potential involvement of ubiquitination-associated processes.
Nitrogen to protein conversion factors (NPCF) are used to estimate the crude protein content based on the nitrogen content of protein sources. Throughout agriculture and including aquaculture, the most used NPCF is 6.25 which relies on two assumptions, (1) all nitrogen comes from proteins and (2) all proteins contain 16% nitrogen. These assumptions do not account for non-protein nitrogen including nucleotides, ammonia, urea, and chitin, or that some proteins have higher or lower nitrogen content than 16% nitrogen. Recognition of flaws in the assumptions has led to the production of species-specific NPCFs; however, NPCFs specific to crustaceans are lacking. There are various NPCFs calculatable for a substance however the present study focused on the NPCFs KA’ and KP. KA’ accounts for assumption two whilst KP accounts for both assumptions therefore KA’ is most comparable to the NPCF 6.25 whereas KP may be more practically applicable. Two NPCFs, KA’ and KP, were calculated for tropical spiny rock lobster (Panulirus ornatus), tiger prawn (Penaeus monodon), and brine shrimp (Artemia salina). Crustacean KA’ values ranged from 4.91–5.58 whereas KP ranged from 3.39 to 5.12. The interspecific deviation from 6.25 highlights the need for species-specific NPCFs to more accurately represent protein compositions.
Mechanical stress and injuries during handling and treatment are major risks to fish welfare in today's salmon industry. Inert sensor packages can track motion and impacts during pipe passage and could be used to assess handling systems such as delousing units. However, to relate these measurements to actual mechanical impacts experienced by live and potentially moving fish, verification is needed.This field study used gastric data storage tags (DST) in live and euthanized Atlantic salmon (Salmo salar) passing through a delousing system at three flow rates to locate areas of the pipe system where fish are particularly exposed to impacts and shocks (e.g., valves, splitters). The tags registered passage time as well as frequency and intensity of mechanical impacts. Additionally, welfare scorings related measured impact to observed injuries. In parts of the system the number of impacts increased significantly with faster flow rate for live and dead individuals. For live fish, increased water flow rate also resulted in increased impact intensity. While welfare scores showed mild eye damage as a risk of system passage, effects of flow rate could not be assessed in detail due to methodical issues and limited sample size.This study confirms the potential for using inert sensor packages in dead or “sensor fish” (artificial dummy fish) as objective welfare documentation and as a tool for detecting critical areas in pipe-based handling systems. Replacing live fish in welfare documentation could reduce the number of experimental animals while providing valuable insights into the treatment system.
The performance of Arctic charr (Salvelinus alpinus) was monitored following transfer from low-salinity water (LSW, 3.6 ppt) to brackish water (BW, 20 ppt) at two juvenile stages. The early-entry group (EE) was transferred at ∼8 g, whereas the late-entry group (LE) entered BW 151 days later at 176 g. Mortality did not differ between groups. Growth was initially slower in BW than in LSW, but after the LE group was transferred, the EE group gradually caught up, and from day 328 to harvest on day 502 no differences in body length were detected, although the LE group had significantly higher final weight. Length growth was relatively stable, whereas weight growth followed cyclical patterns of reduced and accelerated growth. At harvest, the LE group showed a significantly higher incidence of skeletal deformities, suggesting that the timing of transfer to BW may influence bone health. Fish with elevated gonadosomatic index (GSI) at harvest showed contrasting growth trajectories between sexes. Males with slightly elevated GSI (Mean GSI ≈ 0.13%) were heavier than males with lower GSI (< 0.1%), whereas females with slightly elevated GSI (mean GSI ≈ 0.47%) exhibited reduced somatic growth during the latter part of the production cycle. Overall, the study suggests that Arctic charr juveniles of the Hólar strain can be transferred to BW earlier than is currently practiced, without compromising welfare.
While early-life probiotics supplementation is widely practiced in shrimp larviculture, its long-term efficacy and underlying mechanisms remain poorly understood. Specifically, it is unclear whether probiotics achieve stable intestinal colonization or merely exert transient effects, and whether such early interventions confer lasting benefits through the grow-out phase. This study investigated the effects of a probiotics mixture administered during the larval stage of Penaeus vannamei on survival, microbial communities, and subsequent long-term health. Our findings revealed that larval probiotics supplementation significantly enhanced postlarval survival rate of P. vannamei. 16S rRNA sequencing showed that the administered probiotics were detected during the supplementation period (from Zoeal I to Mysis II stage) but were no longer detected after cessation of supplementation. Although the supplemented probiotics did not persist in the intestinal tract, they markedly altered the larval intestinal and rearing water microbiomes, as evidenced by increased species diversity, reduced relative abundance of potential pathogens (e.g., Vibrio), and enrichment of beneficial taxa (e.g., NS3a_marine_group and Maritalea) in the water. The intestinal microbiota structure in the probiotics group showed a positive shift, as evidenced by increased relative abundances of health- and immunomodulation- ralated genera such as Rhodobacteraceae_unclassified and Leisingera. Meanwhile, the abundance of the potential pathogen Aquibacter in the larval intestine was effectively suppressed early on. Predicted functional profiling indicated significant upregulation of quorum sensing and ABC transporter pathways. Remarkably, this early-life probiotics intervention induced strong carry-over effects during the grow-out phase. Under challenge with Vibrio parahaemolyticus, shrimp from the probiotics group exhibited significantly enhanced immune response characterized by a lower intestinal Vibrio load, elevated serum alkaline phosphatase activity, and upregulation of the mucosal immune gene mucin 1 (Muc-1) and the intestinal barrier gene integrin. Histological analysis confirmed a healthier hepatopancreas with increased populations of R-cells and B-cells. Microbiome analysis further revealed enrichment of the beneficial genus Halocynthiibacter and reduction of the potentially pathogenic genus Ruegeria in the probiotics group, accompanied by higher predicted abundance of energy metabolism pathways, including oxidative phosphorylation and pyruvate metabolism. In conclusion, our study demonstrated that early probiotics supplementation, even without permanent colonization, could critically reshape the initial microbial succession. This “early-life microbial remodeling” improved larval performance and conferred lasting enhancements in immune competence and immune response in shrimp, providing a novel theoretical foundation for optimizing probiotics strategies in sustainable aquaculture.
Warming-associated heat stress and aquatic hypoxia frequently co-occur in aquaculture and natural aquatic environments, posing serious threats to the physiological stability and aquaculture sustainability of cold-water fish species. However, compared with single stressors, the effects of combined heat and hypoxia on fish remain insufficiently understood. In this study, rainbow trout (Oncorhynchus mykiss) were exposed to four treatments, including control (16 °C, 8 mg/L dissolved oxygen [DO]), heat stress (24 °C, 8 mg/L DO), hypoxia (16 °C, 3 mg/L DO), and combined heat and hypoxia (24 °C, 3 mg/L DO), to characterize shared and stressor-specific responses in hepatic ultrastructure and transcriptomic profiles. Transmission electron microscopy revealed distinct ultrastructural alterations among treatments, with the endoplasmic reticulum and mitochondria being among the most prominently affected organelles. The combined treatment was associated with more pronounced ultrastructural alterations, suggesting greater disturbance of organelle homeostasis under multiple stressors. RNA-seq further identified a shared transcriptional signature across heat stress, hypoxia, and their combination, characterized by significant enrichment of differentially expressed genes in pathways related to endoplasmic reticulum (ER) stress, unfolded protein response (UPR), protein quality control, and endoplasmic reticulum-associated degradation (ERAD), together with upregulation of chaperone- and folding-related genes, including hsp47, hsp90aa1, hsp90b1, and hspa5. These findings suggest that increased protein folding demand and protein quality control are central components of the hepatic response to these stressors. Beyond this common core, heat stress was more closely associated with folding-related catalytic functions and molecular chaperone networks, which may help buffer protein instability caused by elevated temperature, whereas hypoxia showed stronger associations with redox-related processes, heme binding, and metabolic adjustment under oxygen and energy limitation. The combined treatment induced the most extensive transcriptomic changes, while factorial analysis revealed pathway-dependent temperature × dissolved oxygen interaction effects involving ER proteostasis, oxidative phosphorylation, protein degradation, and stress-signaling pathways.
Infectious Haematopoietic Necrosis Virus (IHNV) is a WOAH-notifiable pathogen causing severe economic losses in global salmonid aquaculture. While the WOAH-recommended TaqMan assay is the current standard, its diagnostic efficacy is frequently compromised by high execution costs and reduced sensitivity toward specific genotypes, particularly the JS lineage, due to single nucleotide polymorphisms (SNPs) in the probe-binding region. To address these limitations, this study developed a novel SYBR Green-based qPCR assay targeting the highly conserved nucleoprotein (N) gene. Meticulous thermodynamics of a new primer set was optimized by balancing GC content, melting temperature (Tm), and Gibbs free energy (ΔG)—to ensure broad genotype inclusivity (U, M, E, JN, and JS) while eliminating non-specific amplifications and primer-dimers. The assay demonstrated superior analytical performance, particularly for the JS genotype, yielding a significantly lower Cq value (16.69) compared to the WOAH assay (20.30), which suffered from probe-mismatch issues. The estimated LoD95% was 16.92 copies/reaction, with 100% diagnostic sensitivity and specificity in experimentally infected samples. Validation using 123 field samples from Republic of Korea and Poland revealed that the new assay identified eight IHNV-positive domestic samples initially missed by the WOAH-recommended method, all of which were subsequently confirmed via sequencing. With a coefficient of variation (CV) consistently below 5% and proven inter-laboratory reproducibility across seven facilities, this assay provides a robust, highly sensitive, and cost-effective alternative to probe-based methodologies. It is suited for large-scale surveillance and routine diagnostics, especially in resource-limited settings where detecting diverse viral mutations is critical.
Natterin-3 is a crucial immune effector that involves in antimicrobial defense in teleosts. However, the mechanisms of Natterin-3 in the innate immunity still remain unclear. In this study, we identified Natterin-3 from Cynoglossus semilaevis (CsNatterin-3) and investigated its function against Vibrio harveyi. CsNatterin-3 contains a DM9 and an aerolysin-like pore-forming (ALP) domain. CsNatterin-3 was highly expressed in the liver and mucosal tissues, and significantly upregulated expression upon bacterial infection. Recombinant CsNatterin-3 (rCsNatterin-3) exhibited a broad-spectrum binding to bacteria by recognizing lipopolysaccharide (LPS) and peptidoglycan (PGN), and induced bacterial aggregation. Following bacterial infection, rCsNatterin-3 interacted with peripheral blood leukocytes (PBLs), and enhanced cellular phagocytosis, reactive oxygen species (ROS), acid phosphatase (ACP) activity, and the expression of proinflammatory cytokines and toll-like receptors. Notably, rCsNatterin-3 protected PBLs from cytotoxicity and promoted bacterial clearance. In vivo administration showed that rCsNatterin-3 alleviated tissue damage, reduced bacterial loads, and improved survival. Conversely, knockdown of CsNatterin-3 increased bacterial colonization and mortality of fish. The above findings first suggested that CsNatterin-3 bridges pathogen recognition and cellular activation to mediate immune defense against bacterial infection.
