Anthropogenic noise generated by vessel traffic is a pervasive source of disturbance in marine environments, overlapping with the hearing-sensitive range of many fish species, including the European sea bass (Dicentrarchus labrax). Although behavioural responses to the specific nature of the noise have been documented, the influence of stimulus familiarity and the temporal dynamics of noise onset on these responses remains poorly explored. This study investigated how familiarity with the stimulus and onset abruptness shape acute behavioural responses to noise in European sea bass. Groups of five fishes were exposed to one of different acoustic conditions combining familiarity (familiar vs. unfamiliar boat noise) and onset profile (progressive vs. sudden), or to background noise only. Behavioural data were collected on videos across five time-intervals. Results showed that progressive familiar noise produced minimal behavioural disruption, consistent with habituation to stimuli encountered at the rearing facility. Sudden onset consistently elicited stronger acute reactions than progressive onset, and familiarity moderated both the magnitude of those reactions and the speed of recovery. Familiarity also influenced the quality of the response: sudden familiar noise triggered organized, coordinated group behaviour with rapid return to baseline, while sudden unfamiliar noise led to persistent disruption of social synchronicity, pointing to a shift toward more individualistic, less predictable responses. These findings demonstrate that familiarity and onset abruptness interact to determine both the magnitude and the quality of behavioural responses, shifting patterns from organized, group-level reactions to individualized, startle-like, disorganized responses as novelty and suddenness increase. Condition-specific fluctuations were also observed in side-tilting, suggesting a potential role of this behaviour as a displacement activity and as a component of welfare assessment in farmed fish. Taken together, these results indicate that temporal dynamics and prior acoustic experience are critical determinants of the impact of noise. The findings of this study can help define practical strategies to minimize noise-related distress and promote welfare in aquaculture settings.

CONTEXT:Traditional fixatives such as buffered formaldehyde (BF) and paraformaldehyde (PFA) are widely used but raise health concerns because of their toxicity. Their use is increasingly subject to regulatory restrictions, calling for the development of non-toxic alternatives. Glyoxal acid-free (GAF®) has emerged as a potential substitute. AIMS:This study evaluated GAF® for preserving gonadal tissues, oocyte, sperm, and larvae of the European seabass (Dicentrarchus labrax). METHODS:GAF formulations - standard and an optimized, marine-adapted formulation - were compared to conventional fixatives or fresh samples. KEY RESULTS:The standard GAF® led to tissue shrinkage and poor gamete preservation, whereas the optimized version effectively preserved oocyte, sperm, gonadal tissue, and delicate larval structures. Although 10% formalin still outperformed GAF® for certain larval tissues, extended fixation in optimized GAF® improved results. CONCLUSIONS:Overall, optimized GAF® represents a promising non-toxic alternative for histological applications in marine fish, although further refinement is needed for certain tissues and for the use in the oocyte cytoplasmic clarification applied in aquaculture broodstock selection. IMPLICATIONS:Non-toxic fixatives are fundamental to ensure operator safety, and identifying solutions that effectively preserve samples is essential; therefore, studies such as this one have important implications for both field and laboratory settings.

Rainbow trout (Oncorhynchus mykiss) is increasingly employed as an experimental model, yet knowledge of its behaviour in laboratory settings is limited. This study examined short-term (24hours), mid-term (1 week), and circadian changes in behavioural and spatial patterns of individually-housed trout under two experimental conditions differing in the extent of underwater current: control vs water flow. Moreover, the effects of feeding and tank cleaning were evaluated to identify potential behavioural indicators of welfare of the species in this context. Short-term effects revealed that flow rate impacted on swimming behaviour, with trout showing less species-specific behavior in the absence of water flow. Over time, trout exhibited a marked reduction of behavioral expression, interpretable as behavioural stabilization but likely reflecting suboptimal welfare. Trout behaviors were affected by the day time being less active in mornings. Event-related findings indicated an increased in active behaviors and a decrease in maintenance behaviours after both husbandry procedures, with that of cleaning being more pronounced and lasting remarkably longer, suggesting that the two reactions were fundamentally different. This study highlights the need to critically evaluate behavioural expression, and the need to provide species-specific husbandry conditions. We also provide foundational insights into rainbow trout behaviour valuable for refining research practices and improving welfare assessment of this experimental fish.

Abstract Background Understanding the role of non-coding genomic variation in speciation remains a major challenge in evolutionary biology. Here, we investigated whether regulatory elements contribute to this process between Atlantic and Mediterranean lineages of European sea bass ( Dicentrarchus labrax ), a well-characterized case-study near speciation where barriers to introgression exist in the presence of connectivity between diverging populations. Results We generated a novel, highly contiguous genome assembly, which was annotated at the epigenomic level using ATAC-seq and ChIP-seq with six embryonic developmental stages and five tissue types in adult fish, identifying thousands of promoters, enhancers, and open chromatin regions. Integrating this annotation with whole-genome sequence data from 65 individuals across three geographically distinct populations, we identified 57,505 outlier SNPs and 332 structural variants (SVs) showing elevated differentiation between Atlantic and East Mediterranean lineages. Outlier SVs affected key regulatory elements and coding genes, while outlier SNPs were enriched in regulatory elements, particularly enhancers active in adult tissues. Local genomic divergence correlated positively with regulatory element density, especially on chromosomes 1, 9, and 18, which are enriched in genes related to osmoregulation, immune response, and oxidative stress — processes relevant to adaptation across contrasting marine environments. Conclusions These findings support a major role for regulatory variation in driving deep lineage divergence through local adaptation.

The induction of triploidy, a strategy to mitigate unwanted pre-harvest sexual maturation and a genetic containment measure for escaped farmed Atlantic salmon (Salmo salar), may give rise to challenges because of the distinct environmental and dietary requirements of sterile triploid fish. Smoltification is a critical phase in the life cycle of Atlantic salmon, so knowledge about parr–smolt transformation in triploids is important for the salmon farming industry. This study covered an investigation of hepatic expression of growth hormone receptor (GHrec) and insulin-like growth factor-I (IGF-I) genes, both of which are intimately involved in the regulation of osmoregulation and growth. Additionally, hepatic presence and location of IGF-I mRNA were examined using RNAscope®, an advanced in situ hybridization technique. Triplicate groups of juvenile diploid and triploid salmon were reared at low temperature (10 °C) and fed either a standard diet or one enriched with hydrolyzed fish proteins from the start of feeding onwards. Liver samples were collected from three fish per tank each month from October to December (2454–3044 degree-days post-start feeding), the period encompassing smoltification, and hepatic expression of IGF-I and GHrec genes was quantified by real-time PCR. The results indicated that neither ploidy nor diet significantly influenced IGF-I or GHrec gene expression, suggesting that, under our conditions, triploidy and diet did not adversely affect this molecular pathway linked to growth and osmoregulation. IGF-I gene expression exhibited significant temporal variation, correlating with the progression of smoltification, while GHrec gene expression showed a similar, albeit non-significant, trend. Triploids exhibited IGF-I and GHrec gene expression patterns comparable to diploids, and both the temporal changes and lack of difference between triploids and diploids were mirrored in the quantification of IGF-I mRNA within the liver cells. The potential applicability to a commercial aquaculture setting requires further investigation.

MicroRNAs (miRNAs) are key post-transcriptional regulators of antiviral immunity, controlling gene expression by targeting 3’ UTRs of immune-related transcripts. Despite their importance, the role of miRNAs in viral nervous necrosis (VNN) resistance in European seabass ( Dicentrarchus labrax ) is unexplored. Here, we characterized for the first time the brain miRNome of seabass from three VNN-resistance genotypes (susceptible, intermediate, resistant) across two genetically distinct seabass clusters. Differential expression analyses revealed cluster-specific patterns, with susceptible fish consistently showing overexpression of the differently expressed miRNAs (DEmiRNAs) as compared to the resistant fish. Considering the two genetic clusters in the study, miR–199–5p was differentially expressed between the VNN susceptible and resistant fish. This miRNA was found to be less expressed in the resistant individuals. Functional characterization of the miRNA predicted that it binds to two distinct miRNA recognition elements (MREs) within the ifi27l2a 3’ UTR. These MREs flank a SNP (Chr3:10,082,380) previously associated with VNN survival. A strong negative correlation (r= -0.840) between miR–199–5p expression and ifi27l2a mRNA abundance further supports a post–transcriptional repression mechanism. Together, these results propose a regulatory model in which miR–199–5p modulates ifi27l2a expression, contributing to phenotypic variation in VNN resistance and positioning it as a promising biomarker for seabass aquaculture breeding.

Underwater sound levels in coastal environments are increasing due to expanding maritime traffic. These habitats are dominated by low-frequency ship and boat noise (<1 kHz), which propagates efficiently over long distances and overlaps the hearing sensitivity of many fish species. During the spawning season, Atlantic salmon (Salmo salar) are exposed to this anthropogenic noise both during migration in the wild and when held in coastal aquaculture systems. We experimentally investigated behavioral and physiological responses of pre-spawn Atlantic salmon of wild genetic lineage exposed to synthesized boat noise in tanks. An acute experiment consisted of a 1-h continuous exposure across a gradient of sound levels to determine the onset of responses and examine links between behavioral and physiological changes. Under control conditions, fish displayed active and disorganized swimming. In contrast, noise-exposed salmon reduced mobility, increased group cohesion, and exhibited freezing behavior that intensified with sound level. Plasma cortisol concentrations were higher in control fish and reduced under noise exposure, whereas no other physiological changes (glucose, lactate, oxidative stress) were observed. Behavioral responses were more sensitive than physiological metrics in detecting disturbance. Together, these responses indicate a shift toward an antipredator-like state, suggesting that boat noise is perceived as a risk cue and triggers behavioral inhibition rather than a classical physiological stress response. Such responses may alter how energy is allocated during a critical life stage, with potential consequences for performance and condition in both wild and farmed salmon exposed to repeated noise.

The eye is a sensitive target of sublethal stress in aquaculture-reared fish due to its direct exposure to the aquatic environment. This study tested a photoelectrocatalytic (PEC) water treatment system, integrated into a standard recirculating aquaculture system (RAS), to improve water quality and evaluated ocular health in Oncorhynchus mykiss (rainbow trout) reared at 30 kg/m3 for 28 days, with particular emphasis on the cornea as an indicator of fish welfare. Ocular analyses focused on the cornea and retina, two anatomically and functionally distinct structures. PEC significantly reduced ammonia levels and modulated nitrate concentrations compared to the control group (CTR), represented by a standard RAS. No differences in growth performance or body condition were observed between groups. Corneal integrity was assessed using optical coherence tomography, histology, and mucous cell staining to evaluate epithelial structure and protective responses. Corneal tissue was examined to detect local oxidative effects through morphological analysis and immunohistochemistry for 8-hydroxy-2'-deoxyguanosine (8-OHdG). Alcian Blu-Periodic Acid-Schiff (AB-PAS) staining did not reveal significant differences in mucin-producing cells among groups. CTR fish exhibited epithelial disruption and increased 8-OHdG immunoreactivity, whereas fish reared in the RAS equipped with the PEC system, ensuring improved water quality, showed preserved corneal architecture despite mild oxidative stress. Molecular analysis of ocular tissues revealed no differential expression of oxidative stress-related genes, such as GPx1, GR, or sod1, in the two groups. Overall, these findings support the use of the cornea as a sensitive indicator of sublethal environmental stress in farmed fish and suggest that PEC treatment may contribute to improved water quality management and welfare monitoring in intensive aquaculture systems.

Fish welfare is increasingly gaining attention in both aquaculture and research, aligning with the "One Welfare" principles that connect animal welfare, human wellbeing, and environment. Here we evaluate whether short-term feed withdrawal, a common practice used in research and aquaculture prior to handling, transportation, and sedation determines a stress response in the adult zebrafish (Danio rerio), through the assessment of key biomarkers across different biological matrices. Adult zebrafish were assigned either to a control group, fed with standard diet twice daily, or to a group subjected to 96 h of complete food deprivation. Cortisol levels were measured in fins, while advanced oxidation protein products (AOPP) and malondialdehyde (MDA) levels were analyzed in muscle tissue. Cellular and oxidative stress markers were assessed through the immunohistochemical localization of Heat Shock Protein-70 (HSP70) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). The levels of cortisol did not have significant differences by treatment, sex, or their interaction. AOPP levels exhibited a significant interaction between treatment and sex, with a 10 % increase in fasted males compared to control females, while MDA levels were unchanged. Consistently, immunohistochemical analyses revealed no significant differences between groups in HSP70 and 8-OHdG positivity. These findings suggest that short-term feed withdrawal does not significantly affect key physiological stress markers in zebrafish. Finally, we validated the use of the caudal fin as a matrix for measuring individual cortisol levels in zebrafish, providing an alternative to terminal whole-body analyses as well as to group-level water-borne cortisol.

Light pollution poses significant ecological challenges for nocturnal animals reliant on natural light for migration, orientation, and circadian rhythms. The physiological effects of abrupt exposure to artificial light at night (ALAN) on migratory fish, such as the light experienced passing near illuminated infrastructures, remain poorly understood. This study investigates the physiological responses of brown trout (Salmo trutta) smolts to low-intensity (0.02 lx) and short-term (30 s) ALAN, simulating nocturnal migration light conditions near illuminated bridges. To evaluate the influence of social dynamics, trout were tested individually (solitary) or in groups of six. Using continuous cardiac monitoring with data storage tags, alongside analyses of oxidative stress markers and adenylate kinase (AK) activity in the heart, we identified distinct patterns of physiological responses. Solitary fish exhibited significant heart rate variability (HRV) increases following repeated ALAN exposure, suggesting impaired physiological regulation under repeated ALAN exposure. In contrast, trout in groups displayed consistently lower HRV over the entire 90-min experiment, implying that social dynamics likely influenced a sustained oxidative stress response, corroborated by increased AK activity. Oxidative stress markers further reflected social effects, with significant upregulation of key antioxidant enzymes (sod1, sod2, gpx1, gpx4) and elevated lipid peroxidation, identifying lipids as primary oxidative targets. The observed divergence between superoxide dismutase (SOD) activity and sod gene expression suggests adaptive post-transcriptional regulation to maintain redox balance under combined environmental and social stress. These findings reveal that social dynamics under ALAN can amplify physiological stress, potentially affecting migratory outcomes.

Viral nervous necrosis (VNN) is an important viral disease threatening global aquaculture sustainability and affecting over 50 farmed and ecologically important fish species. A major QTL for resistance to VNN has been previously detected in European sea bass, but the underlying causal gene(s) and mutation(s) remain unknown. To identify the mechanisms and genetic factors underpinning resistance to VNN in European sea bass, we employed integrative analyses of multiple functional genomics assays in European sea bass. The estimated heritability of VNN resistance was high (h2 0.40), and a major QTL explaining up to 38

Nervous Necrosis Virus (NNV) is a major threat to aquaculture, causing high mortality in farmed fish, including European sea bass. The genetic basis of host response to NNV has been well characterised, suggesting the potential role of interferon-induced genes in resistance to the virus, although the molecular mechanisms underlying NNV infection in sea bass are still debated. The time- and tissue-specific dynamics of gene expression is crucial for understanding host response to NNV. Here, we report on a time-course transcriptome analysis of brain and head kidney in NNV-infected bass that integrated the statistical evidence of differential expression with the information on temporal profiles (i.e. 6, 12, 24, 48, and 72 h post-infection (hpi)) compared to mock-infected controls. Results revealed substantial changes in gene expression over time, particularly for brain, with downregulation of genes involved in nervous system functions and upregulation of immune and inflammatory response genes from 24 to 48 hpi onwards, mostly associated with the interferon (IFN) response. The study highlights tissue-specific differences in the timing and magnitude of the innate immune response compared to other fish species and provides a comprehensive view of the dynamic host response, emphasizing the need for time-course studies in understanding viral pathogenesis.

The present study aimed to evaluate the effects of self-loading training on the overall stress response during pre-slaughter transportation in slaughter horses. Thirty-two slaughter horses were divided into two groups: the control group (CG) and the Trained Group (TG). For six weeks, the TG horses were trained to self-load using a method based on target training and shaping. Animals from both groups were transported to the same slaughterhouse in small groups on different days using the same truck along the same route. The baseline and post-transportation values of the eye temperature and fecal cortisol metabolites were determined for all the animals. All the horses were video-recorded while being transported and unloaded. The horses’ behavior was analyzed using a focal animal sampling method. During transportation, the presence of head shaking, licking, and chewing was significantly higher in the CG than the TG. During unloading, walking forward tended to be more frequent in the TG. The fecal cortisol metabolites and eye temperature were higher after transportation than at the baseline, but no differences between the groups were found. Overall, the results confirm that transportation for less than one hour caused detectable stress in slaughter horses. Based on these results, self-loading training appears to be somewhat useful to mitigate the overall transport stress.

Glucocorticoids like cortisol are widely used to assess stress in fish, but their interpretation can be limited. Incorporating dehydroepiandrosterone (DHEA), a hormone with anti-glucocorticoid and neuroprotective effects, may provide a broader understanding of hypothalamic-pituitary-inter-renal (HPI) axis activity. As DHEA data in fish remain scarce, this study examined its role in rainbow trout (Oncorhynchus mykiss) subjected to acute stress (30 min confinement), considering sex and sexual maturity. Cortisol and DHEA were quantified by optimized radioimmunoassay (RIA) in serum, muscle, fin, and scales, and gonadal histology was performed to confirm reproductive status. Both hormones were successfully measured in all matrices, with serum DHEA levels notably higher than those reported to date in fish. As expected, serum cortisol increased markedly after stress and correlated with levels in muscle and fin but not in scales, which appears to reflect chronic rather than acute exposure. In contrast, serum DHEA showed no stress-induced changes and only minor sex- and maturity-related differences, although alternative matrices displayed variable patterns, particularly in muscle and fin. The cortisol/DHEA ratio in serum mirrored cortisol dynamics, suggesting limited utility for acute stress assessment, while tissue-specific DHEA variation may integrate longer-term influences. Further research is needed to clarify the role of DHEA under chronic stress and its potential origins in inter-renal tissue, gonads, or the brain.

This study examines the potential in vitro application of different concentrations of titanium dioxide (TiO2) nanoparticles (NPs) irradiated with UV light for the sanitation of recirculating aquaculture systems (RASs) and their antiviral activity. The diverse effects of Nodavirus on immune gene expression (i.e., pro-inflammatory and anti-inflammatory genes, cellular response genes, humoral response genes, and stress genes) were studied using RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction). In addition, the viability and cytopathic effect in E-11 fish cells were also investigated. The results obtained did not show a clear cytopathic effect under the reversed-phase microscope observation at different TiO2 concentrations. A significant decrease in viral coat protein gene expression was observed when using 2.5 and 1.25 g/L TiO2 suspensions under UV irradiation. TiO2 at 1.25 g/L induced an inflammatory response to Nodavirus by increasing the expression of all target genes. Thus, this work suggests that TiO2 NPs can strengthen the immune system of fish to fight virus infection and make aquaculture a greener and more sustainable activity.

Reducing the environmental impact of poultry farming aligns with the European Green Deal’s goal of climate neutrality and sustainable food production. Local chicken breeds and low-input diets are promising strategies to achieve this goal. This study evaluated the effects of diet (standard vs. low-input, formulated with reduced soybean meal in favour of local ingredients) on the morphological characteristics of the jejunum in fast-growing chickens (Ross 308), local breeds (Bionda piemontese, BP; Robusta maculata, RM), and their crosses with Sasso (SA) hens (BP × SA, RM × SA). Histological samples from the jejunum were collected at slaughter (47 days for Ross 308, 105 days for others). Jejunal morphology was assessed focusing on villi height, crypt depth, goblet cell density, and immune markers (CD3+ and CD45+ cells). Local breeds, particularly RM, exhibited superior villus height-to-crypt depth ratios, related to better nutrient absorption compared to fast-growing genotypes. Ross chickens had higher goblet cell densities, reflecting greater sensitivity to environmental stress. Although the low-input diet reduced villi height and villus-to-crypt ratio, it tended to increase CD3+ cell density. These effects may be ascribed to the replacement of soybeans with fava beans and their antinutritional factors. These findings highlight the resilience of local breeds to dietary changes, supporting their suitability for alternative poultry production systems.

The Veneto Region (Italy) experienced one of the heaviest contaminations by per- and polyfluoroalkyl substances (PFAS), pollutants of emerging concern due to their environmental persistence and bioaccumulation potential in animal tissues. Hence, there is a need to study their impact on freshwater fish inhabiting contaminated rivers, particularly at the level of the antioxidant system, since PFAS are known to cause an imbalance in reactive oxygen species (ROS) production, thereby increasing the risk of oxidative stress. This study examines the physiological responses triggered by chronic exposure to three distinct environmental concentrations of PFAS in the European chub (Squalius cephalus). The sites were classified as "control" (with a PFAS concentration < 5 ng/L), "low polluted" (5.64 ng/L) and "highly polluted" (582.6 ng/L). Biochemical and molecular analyses were performed on the kidney, one of the main organs for xenobiotic bioaccumulation. The catalase (CAT) and selenium-dependent glutathione peroxidases (Se-GPXs) expression was quantified at both active protein and mRNA transcript levels. Results confirm the activation of antioxidant defences against the risk of PFAS-induced oxidative stress. There is a differential induction in the biosynthesis of enzymes inside specific intracellular compartments: CAT in peroxisomes in the "low polluted" site and Se-GPXs in the cytoplasm in the "highly polluted" one. The gene gpx1 was the only isoform whose mRNA level corresponded to that of the active protein, suggesting the highest contribution to the biosynthesis of Se-GPXs at high PFAS concentrations. Conversely, gpx4 increased its transcription level in the "low polluted" site, which didn't match with an increase in protein content, leading us to hypothesise an involvement of specific cytoplasmic mRNA-protein complexes, called stress granules, acting in the temporary silencing of gpx4.

Downstream guidance of migrating fish is a major conservation challenge in fragmented riverine systems, where orientation depends on fine-scale environmental cues and physiological readiness. Flow manipulation is increasingly explored to improve behavioral guidance at hydropower facilities, particularly for salmonids during the smolt stage. We investigated the effects of laterally injected jet flow on stress physiology and behavior of Atlantic salmon (Salmo salar) smolts in a large-scale flume. Fish were exposed to three treatments: jet flow from the left (Left Jet), from the right (Right Jet), and no flow (Control). Physiological analysis revealed that the Right Jet treatment significantly elevated malondialdehyde (MDA) levels and increased the activity of the antioxidant enzymes catalase (CAT) and glutathione peroxidase (GPx) in erythrocytes, indicating the presence of oxidative stress. No significant effects were detected for cortisol (plasma), hemoglobin and lactate (whole blood), or advanced oxidation protein products (AOPP, erythrocytes). Behaviorally, smolts showed a consistent left-side preference and swam predominantly near the bottom, regardless of treatment. Nevertheless, jet flow did not significantly alter lateral or vertical swimming distribution. Together, these results demonstrate that sublethal physiological strain can occur without overt behavioral change, underscoring the value of integrative, non-lethal approaches for evaluating fish responses to hydrodynamic conditions in guidance systems.

Given the pervasive detection of perfluoroalkyl substances (PFAS) in several environmental matrices and their known toxicological effects, it is imperative to investigate their impact on the physiological responses of freshwater organisms. This research is crucial for developing effective strategies to protect aquatic ecosystems by directly addressing how PFAS influences aquatic species' health and survival. In this study, we conducted a biomonitoring analysis to evaluate the effects of naturally occurring PFAS, specifically perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), on the physiology of common chub (Squalius cephalus), a freshwater fish native to the Veneto region. We measured oxidative damage in the kidney and skeletal muscle, with results showing that low PFAS contamination is sufficient to increase protein oxidation in both tissues. Conversely, even high PFAS levels did not induce lipid peroxidation in either tissue. We also examined the expression of peroxiredoxin isoform 4 (prdx4) in the kidney, finding its down-regulation with increasing PFAS pollution, which demonstrates the minor function of Prdx4 against oxidative stress. Instead, its down-regulation plays an important role in increasing lipid accumulation in the cell, creating a hydrophobic environment that limits PFAS bioaccumulation and their capacity to bind proteins, thus preserving them from further damage.