This study investigated the effects of acute cold stress on metabolic, antioxidant, and immune responses in the skin tissue of juvenile yellowfin tuna (Thunnus albacares). Fish were exposed to three temperature treatments (30, 24, and 18 °C), with sampling conducted at 0, 12, 24, and 36 h. During early cold exposure, the relative enzymatic capacities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) increased under standardized assay conditions, accompanied by upregulation of hexokinase (hk) and ATPase Na[+]/K[+]-transporting subunit beta 1a (atp1b1a) gene expression. Cold stress was associated with oxidative stress-related responses, as indicated by elevated malondialdehyde (MDA) content, increased catalase (CAT) activity and transient upregulation of superoxide dismutase 2 (sod2), glutathione peroxidase 1a (gpx1a), and nuclear respiratory factor 1 (nrf1) gene expression. Immune-related genes, including interleukin-1 beta (il-1β), nuclear factor kappa B subunits 1 and 2 (nfkb1 and nfkb2), and tumor necrosis factors alpha and beta (tnf-α and tnf-β), were also upregulated during early exposure. As low-temperature exposure was prolonged, various key indicators, including the relative enzymatic capacities of LDH, ALT, CAT, and glutathione peroxidase (GSH-Px), as well as gpx1a, il-1β, and tnf-α/β expression, declined or fluctuated, suggesting that sustained low temperature alters skin physiological status. This study provides physiological, biochemical, and molecular data that contribute to understanding low-temperature responses in juvenile yellowfin tuna.

The adipokinetic hormone/corazonin-like peptide (ACP) functions as a hormone in many arthropod species. The ACP-encoding transcript is expressed in the neural tissues and sperm duct of the ornate spiny lobster, Panulirus ornatus, a species of importance to the aquaculture industry. In this study, the presence of the ACP precursor peptide was verified in the medial sperm duct and spermatophore of P. ornatus through peptide fragments identified by proteomic analysis. We examined the proteome of three tissues: the medial sperm duct, its spermatophoric contents, and a deposited 'tarspot', identifying other proteins involved with multiple functions including immune response, amelioration of oxidative stress, antimicrobial action, energy usage, and muscle function. To understand a potential role for ACP precursor peptides in these tissues, a screen targeting nine bacterial strains was carried out however no antimicrobial activity was found for ACP or related peptides. A 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to identify potential mild antioxidant activity in a peptide from the ACP precursor providing a putative function in the sperm duct. The histology of the medial sperm duct of P. ornatus, together with the protein constituents of the medial sperm duct, its contents, and tarspot are presented to provide a greater understanding of the male reproductive system of the ornate spiny lobster.

Fish skin mucus covers the body surface, serving as a physical and chemical first line of defense at the interface with the aquatic environment. Epidermal mucus provides a protective cushioning layer and contains numerous bioactive molecules involved in immune defense, antimicrobial activity, and tissue repair. During body-surface injury, direct contact between the plasma components and the mucus layer may influence plasma clotting. However, the role of epidermal mucus in this process remains poorly understood. This study investigated, under in vitro conditions, the effects of epidermal mucus from Japanese flounder (Paralichthys olivaceus) on plasma coagulation and examined coagulation-modulating components through biochemical fractionation. The addition of crude mucus to the flounder plasma shortened the fibrin clotting time and accelerated thrombin substrate cleavage under both prothrombin time-like and activated partial thromboplastin time-like conditions. Affinity fractionation of crude mucus using benzamidine-Sepharose showed that both the bound and unbound fractions retained procoagulant activity, suggesting that the observed activity was not solely attributable to serine protease-like components. Furthermore, size-based fractionation of the crude mucus using an ultrafiltration membranes and size-exclusion chromatography showed that procoagulant activity was distributed across multiple fractions in the approximate 29 to >2000 kDa range, whereas lower-molecular-weight fractions showed anticoagulant activity. These findings suggest that modulation of coagulation by mucus may contribute to the balance between local clot formation and the prevention of excessive coagulation at the epithelial surface.

Long-chain polyunsaturated fatty acids (LC-PUFA) are essential nutrients for membrane function, development, and immunity in animals. Polychaetes are promising sources of LC-PUFA due to their endogenous biosynthetic capacity, but optimal feeding strategies to enhance their nutritional value remain poorly understood. We investigated the nutritional regulation of LC-PUFA biosynthesis in the marine polychaete Platynereis dumerilii fed three diets differing in fatty acid (FA) profiles: spinach, yeast, and commercial fish feed. Juveniles were cultured for 37 days at 35‰ salinity, and survival, growth, total lipids, FA profiles, and expression of elongases, desaturases, and putative transcription factors were analysed. Survival rates were high across all diets (93.3-95.0%), while growth was significantly reduced in spinach-fed worms, which showed a lower specific growth rate (1.5 ± 0.1% day[-1]) than worms fed yeast (2.2 ± 0.1% day[-1]) or fish feed (2.0 ± 0.1% day[-1]). Diet strongly affected FA composition: fish feed increased n-3 LC-PUFA, spinach elevated α-linolenic acid, and yeast enhanced n-6 LC-PUFA. Despite low dietary LC-PUFA, eicosapentaenoic acid levels remained stable, indicating active endogenous synthesis. Gene expression responses were diet-dependent, with elongase Elovl2/5 expression higher in fish-feed-fed worms (0.8 ± 0.3) than in yeast-fed worms (0.4 ± 0.1), while the front-end desaturase Fed1 (Δ5 activity) and methyl-end desaturase ω des1 (Δ12 activity) showed their highest expression in spinach-fed worms (3.9 ± 1.8 and 2.4 ± 1.0, respectively). Overall, P. dumerilii adjusted its lipid metabolism in response to dietary inputs, demonstrating transcriptional plasticity and reinforcing its potential as a sustainable source of LC-PUFA.

The permanently sub-zero temperatures and high oxygen solubility of the Southern Ocean promote the formation of reactive oxygen species (ROS) in the tissues of resident organisms, imposing persistent oxidative stress. Antarctic notothenioid fish have evolved efficient antioxidant systems to counter this pressure. This study reports the first molecular characterisation of the catalase (cat) transcript and deduced protein (CAT) sequence, together with tissue-specific CAT activity, in two Antarctic species with contrasting physiological traits: the red-blooded notothenioid Trematomus bernacchii and the haemoglobin-less icefish Chionodraco hamatus. Integrating cDNA sequencing with transcriptomic data, we reconstructed full-length cat coding sequences, performed Bayesian and maximum-likelihood phylogenetic analyses, and compared electrostatic surface properties using structural modelling. The cat transcript levels and CAT activity were quantified in liver, gills, heart, and skeletal muscle. Catalytic, NADPH-binding, and substrate-channel residues were fully conserved across Antarctic and non-Antarctic teleosts, consistent with strong purifying selection. T. bernacchii displayed a markedly more negative electrostatic surface than the icefish and non-Antarctic species, a pattern consistent with the increased surface acidity typical of cold-adapted proteins and with the higher oxidative burden of the red-blooded condition. The icefish showed a near-neutral electrostatic profile, consistent with its haemoglobin-less physiology. The liver was the principal site of both cat transcript accumulation and CAT activity in both species. Non-hepatic tissues showed a translation index below unity, consistent with post-transcriptional and/or post-translational regulation, with maximum translational efficiency shifting from the liver in T. bernacchii to the heart in C. hamatus. These findings suggest that enzyme surface properties and tissue-specific translational regulation may jointly contribute to antioxidant homeostasis under extreme marine conditions.

Serine racemase (SR) catalyzes the interconversion of l-serine and d-serine and can also catalyze β-elimination of serine to pyruvate and ammonia. In mammals, however, the presence and enzymatic function of SR have been reported mainly in mouse, rat, and human. It is therefore unclear whether functional SR is broadly conserved across mammals or whether its enzymatic properties differ among lineages. To address this question, we produced and purified eleven SRs from representative eutherians, marsupials, and a monotreme and compared their catalytic properties. All enzymes examined showed both racemization and β-elimination activities. Together with the phylogenetic relationships, these activities suggest that functional SR was present before the monotreme-therian divergence and retained in the lineages examined. Specific activities and kinetic parameters nevertheless differed among species. Residues involved in cofactor and substrate binding were broadly conserved, whereas variation at the substrate-recognition region around positions 150-152 may contribute to the observed kinetic differences. Analysis of public transcriptome data further suggested species differences in tissue expression patterns. Together, these findings show conservation of SR function across the mammalian lineages examined, with species-dependent differences in catalytic properties and expression.

Fluctuations in oxygenation and reoxygenation rates are common in aquaculture and natural environments and can cause oxidative stress and contribute to pathological conditions. This study evaluated how reoxygenation rate and temperature influence antioxidant defence and oxidative stress in pacu (Piaractus mesopotamicus) exposed to hypoxia. Juvenile fish (61.7 g) were exposed to 12 h of hypoxia (2 mg O2 L[-1]) at three temperatures (18, 23, and 28 °C), followed by rapid or gradual reoxygenation (0.5 or 5 h). A normoxic group (7 mg O2 L[-1]) was maintained at each temperature. At 1 and 12 h after reoxygenation, total antioxidant capacity against peroxyl radicals (ACAP), lipid peroxidation (LPO), and protein thiols (PSH) were determined in the gills and liver. ACAP was lowest at 18 °C after 1 h and at 28 °C after 12 h of recovery, compared with the other two temperatures at the same times (p < 0.05). At both recovery times, gill LPO was lower at 18 °C than at 23 and 28 °C with rapid reoxygenation, while liver LPO was higher at 23 °C than at 28 °C (p < 0.05). Gill PSH was lower at 18 °C than at 23 °C, whereas liver PSH was higher at 23 °C than at 28 °C after recovery (p < 0.05). These organ-specific patterns indicate that intermediate and high temperatures intensify oxidative stress during reoxygenation, whereas 18 °C mitigates it by reducing metabolic demand and reactive oxygen species (ROS) formation. In conclusion, pacu juveniles are hypoxia-tolerant and respond more effectively to rapid reoxygenation at 18 °C.

Environmental changes affect microplastic (MP) biofilms, but how the resulting bacterial adsorption influences infection rates and physiological responses in fish remains unclear. This study investigated the combined effects of temperature (18 °C, 22 °C, and 26 °C) and MP size (102 and 220 μm) on biofilm formation and the subsequent effects on Vibrio harveyi infection and physiological responses in Korean rockfish Sebastes schlegelii. Biofilms were formed on MPs over 21 days under different temperature conditions, followed by a 5-day co-exposure experiment with V. harveyi. Biofilm formation markedly increased with rising temperature and was more pronounced on smaller MPs owing to their larger specific surface area, with the highest levels observed in the 26 °C small MP group. Correspondingly, the abundance of V. harveyi on MPs and its accumulation in fish tissues were markedly elevated, indicating enhanced bacterial transport and internalization. Co-exposure to MPs and V. harveyi induced oxidative stress, as evidenced by increased reactive oxygen species production in the liver, along with upregulation of antioxidant enzymes. Immune responses were also activated, with marked increases in the mRNA expression levels of interleukin-1 beta (il-1β), tumor necrosis factor alpha (tnf-α), and c-c chemokine ligand 25 (ccl25), particularly under high-temperature and small MP conditions. In situ hybridization further confirmed increased il-1β expression in liver tissues. Overall, elevated temperature and smaller MPs promoted biofilm formation, facilitating bacterial infection and inducing oxidative stress and immune responses. These findings suggest that climate-driven warming and MP pollution may synergistically increase disease risks in marine fish, with important implications for aquaculture and marine ecosystem health.

There is an increasing interest in the capture and welfare of wild caught fish. Despite this, the methods used to catch and kill fish in commercial fisheries remain associated with significant welfare concerns. It is therefore necessary to determine neurophysiological, and endocrine responses of fish to different fishing gear in order to understand and highlight the welfare challenges affecting wild fish populations. We characterized neuronal activity, neuroendocrine, neurochemical and molecular responses of wild cod caught with Danish seine, fish traps, longline or gillnet. Fish caught by Danish seine had a significant region-specific increase in neuronal activity in the tubercular nuclei (TN) of the hypothalamus, accompanied by a reduced stress response profile in monoamine neurochemistry and plasma cortisol, in comparison to fish caught with the other fishing methods. In contrast, the fish caught with gillnet displayed the highest stress response reactivity. Total transcriptome analysis of the TN revealed that fish caught by gillnet, longline and fish traps had a neural gene expression pattern associated with energy metabolism, neuroinflammation and neurodegeneration. This suggests that the stress imposed by these fishing methods activates pathways that, over time, may lead to neural dysfunction. These results emphasize that Danish seine-caught fish had relatively better welfare compared to fish caught using the other fishing methods, whereas the gillnet-caught fish had relatively poorer welfare outcomes. Furthermore, we present several candidate genes that may be suitable as markers for stress-related neurodegenerative conditions that should be further studied to elucidate their specific functions within the fish brain.

In decapod crustaceans, red pigment-concentrating hormone (RPCH) is considered an ortholog of insect adipokinetic hormone (AKH); both belong to the conserved arthropod AKH/RPCH neuropeptide family. Research on crustacean RPCH has focused primarily on pigmentary functions, but the tissue-level roles of RPCH beyond its role in pigmentation are poorly understood. In this study, we used RNA interference to knock down the rpch gene in Litopenaeus vannamei (Lv-rpch) and integrated transcriptomic analysis, reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation, biochemical assays, and histological examination to characterize responses in the gill and hepatopancreas. The two tissues exhibited distinct differentially expressed gene (DEG) profiles, comprising 190 DEGs in the hepatopancreas and 768 in the gill. Gill DEGs were enriched mainly in defense-related processes, lysosome-associated categories, structural components, and carbohydrate metabolism pathways. In the hepatopancreas, enrichment primarily involved transport, protein processing, oxidoreductase activity, xenobiotic metabolism, and carbohydrate and lipid metabolism. Biochemical assays revealed lower superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in both tissues, higher gill lysozyme (LYS) activity, and lower hepatopancreatic lipase activity. Trypsin activity did not change. Histological examination indicated morphological alterations in both tissues after Lv-rpch knockdown. Taken as a whole, these results link Lv-rpch knockdown to tissue-specific transcriptomic, biochemical, and histological responses in L. vannamei, although the underlying causal mechanisms remain to be established.

Telomerase reverse transcriptase (tert), which encodes the major catalytic subunit of telomerase, is thought to play a central role in the regulation of telomerase activity. However, its transcriptional regulatory mechanisms remain unclear. In this study, we investigated the transcriptional regulatory mechanisms of tert in EPC (Epithelioma papulosum cyprini) cells, a teleost fish cell line derived from fathead minnow (Pimephales promelas) epithelium and the mechanisms underlying the maintenance of telomerase activity. A series of deletion mutants spanning the 2 kb upstream region of the fathead minnow tert transcription start site was inserted into the pGL3-Basic vector. Following transfection into EPC cells, the mutants were analyzed using a luciferase assay. A cellular myelocytomatosis (c-Myc) transcription factor binding site was identified in the region extending from -227 to -620 upstream of the tert transcription start site. Luciferase activity decreased significantly following introduction of site-specific mutations into the c-Myc binding site or treatment with the c-Myc inhibitor 10058-F4. In contrast, qRT-PCR analysis revealed no significant decrease in endogenous tert expression following the addition of 10058-F4 to EPC cells, suggesting that either DNA methylation in the region -1160 to -1420 upstream of tert or c-Myc binding, or both, may contribute to tert expression, and that this redundancy maintains tert expression. Furthermore, cycloheximide chase assays and real-time quantitative telomeric repeat amplification protocol suggest that high telomerase activity in EPC cells may also be maintained by the high stability of the telomerase protein.

Starvation is a common stressor in aquaculture that can markedly affect intestinal health and function in fish. This study focused on yellow cheek carp (Elopichthys bambusa, initial body weight: 221.36 ± 6.75 g; initial body length: 28.47 ± 0.56 cm) to explore how short-term (8 days) and long-term (28 days) starvation influence intestinal morphology, expression of key functional genes, and gut microbiota composition. Additionally, Spearman's rank correlation analyses were conducted to explore potential host-microbe interactions. The results showed that short-term starvation did not significantly affect intestinal muscle layer thickness or villus height, but markedly upregulated genes associated with autophagy and apoptosis such as bcl-2-associated X protein 2 (bax2), bcl-2-like protein 1 (bcl2l1), and cysteine-aspartic acid protease 8(casp8). It also increased microbial diversity and altered the composition of dominant gut microbiota. In contrast, long-term starvation significantly suppressed the expression of copper/zinc superoxide dismutase (Cu-Zn sod), casp3a, and casp9, increased the number of goblet cells, inhibited muscle layer development, and weakened the correlation between gut microbes and host gene expression. In summary, short-term starvation appears to maintain intestinal homeostasis through activation of autophagy- and apoptosis-related pathways in conjunction with microbial restructuring. However, prolonged starvation inhibited muscularis development, increased goblet cell density, downregulated antioxidant and immune-related gene expression, and weakened the associations between the host and its microbiota. These findings provide new insights into starvation-induced physiological responses and contribute to gut health management strategies in aquaculture.

Anemonia viridis is a species of sea anemone (Cnidaria:Anthozoa) currently experiencing negative environmental impacts on their natural populations in the Mediterranean Sea. This species has excellent regeneration capacity, such that asexual reproduction can be induced in captivity via longitudinal bisection, but there is no information about the impact of this procedure on the animal's physiology beyond short-term responses. The goal of this work was to assess the stress response of A. viridis during and after regeneration. We measured oxidative and immunological state markers in control and bisected anemones, at 4 weeks post-injury and 20 weeks post-injury. We then performed Principal Component Analysis (PCA) to identify trends at the multivariate level. Additionally, we carried out a histological evaluation of the wound healing process during the first 7 days post-injury. Higher antioxidant enzymatic activity was found in bisected anemones, mainly in catalase and glutathione-dependent enzymes. This response was often reverted by 20 weeks post-injury in tentacles. No differences in lipid peroxidation were observed. Phosphatase activities were unaltered within the studied time-frame. Histological analysis indicated recruitment of amoebocytes, the immune effector cells in anthozoans, and appearance of fibrillar mesoglea, detectable between 6 h and 24 h post-bisection. Results suggest the antioxidant response was effective containing oxidative damage. Longitudinal fission appears to be a viable technique for inducing asexual reproduction in A. viridis without triggering a stress response, and could reduce dependence of the market on the natural environment.

Understanding the thermal adaptation of protein function is key to predicting species distributions under climate warming. This study investigated the secondary structure of cytosolic malate dehydrogenase (cMDH) from three intertidal snails with distinct heat tolerance. Despite minimal sequence differences (2-3 amino acids) in cMDHs, their heat tolerance differed significantly. Circular dichroism spectra of wild-type and experimentally-generated mutant cMDHs confirmed that all are α-helix-rich proteins. The cMDH from the extremely heat-tolerant Echinolittorina malaccana had a significantly higher α-helix content than that from heat-tolerant E. radiata. A substitution from glycine to serine at residue 48 significantly reduced the α-helix content of cMDH (p < 0.05), however, substitutions at residues 4 and 114 did not significantly alter α-helix content (p > 0.05). Furthermore, the extremely heat-tolerant cMDH exhibited fewer salt bridges and hydrogen bonds, suggesting greater structural flexibility that may facilitate functional dynamics under heat stress. The findings demonstrate that the secondary structure content, particularly α-helix percentage, may be associated with the temperature adaptation of cMDHs.