Mucosal organs exhibit distinct response signatures to hydrogen sulphide in Atlantic salmon (Salmo salar).

Hydrogen sulphide (H2S) is considered an immunotoxicant, and its presence in the water can influence the mucosal barrier functions of fish. However, there is a significant knowledge gap on how fish mucosa responds to low environmental H2S levels. The present study investigated the consequences of prolonged exposure to sub-lethal levels of H2S on the mucosal defences of Atlantic salmon (Salmo salar). Fish were continuously exposed to two levels of H2S (low: 0.05 µM; and high: 0.12 µM) for 12 days. Unexposed fish served as control. Molecular and histological profiling focused on the changes in the skin, gills and olfactory rosette. In addition, metabolomics and proteomics were performed on the skin and gill mucus. The gene expression profile indicated that the gills and olfactory rosette were more sensitive to H2S than the skin. The olfactory rosette showed a dose-dependent response, but not the gills. Genes related to stress responses were triggered at mucosal sites by H2S. Moreover, H2S elicited strong inflammatory responses, particularly in the gills. All mucosal organs demonstrated the key molecular repertoire for sulphide detoxification, but their temporal and spatial expression was not substantially affected by sub-lethal H2S levels. Mucosal barrier integrity was not considerably affected by H2S. Mucus metabolomes of the skin and gills were unaffected, but a matrix-dependent response was identified. Comparing the high-concentration group's skin and gills mucus metabolomes identified altered amino acid biosynthesis and metabolism pathways. The skin and gill mucus exhibited distinct proteomic profiles. Enrichment analysis revealed that proteins related to immunity and metabolism were affected in both mucus matrices. The present study expands our knowledge of the defence mechanisms against H2S at mucosal sites in Atlantic salmon. The findings offer insights into the health and welfare consequences of sub-lethal H2S, which can be incorporated into the risk assessment protocols in salmon land-based farms.

Mucosal and systemic physiological changes underscore the welfare risks of environmental hydrogen sulphide in post-smolt Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) might encounter toxic hydrogen sulphide (H2S) gas during aquaculture production. Exposure to this gas can be acute or chronic, with heightened levels often linked to significant mortality rates. Despite its recognised toxicity, our understanding of the physiological implications of H2S on salmon remains limited. This report details the mucosal and systemic physiological consequences in post-smolt salmon reared in brackish water at 12 ppt after prolonged exposure to elevated H2S levels over 4 weeks. The fish were subjected to two concentrations of H2S: 1 µg/L (low group) and 5 µg/L (high group). An unexposed group at 0 µg/L served as the control. Both groups exposed to H2S exhibited incremental mortality, with cumulative mortality rates of 4.7 % and 16 % for the low and high groups, respectively. Production performance, including weight and condition factors, were reduced in the H2S-exposed groups, particularly in the high group. Mucosal response of the olfactory organ revealed higher tissue damage scores in the H2S-exposed groups, albeit only at week 4. The high group displayed pronounced features such as increased mucus cell density and oedema-like vacuoles. Transcriptome analysis of the olfactory organ unveiled that the effects of H2S were more prominent at week 4, with the high group experiencing a greater magnitude of change than the low group. Genes associated with the extracellular matrix were predominantly downregulated, while the upregulated genes primarily pertained to immune response. H2S-induced alterations in the metabolome were more substantial in plasma than skin mucus. Furthermore, the number of differentially affected circulating metabolites was higher in the low group compared to the high group. Five core pathways were significantly impacted by H2S regardless of concentration, including the phenylalanine, tyrosine, and tryptophan biosynthesis. The plasma levels of phenylalanine and tyrosine were reduced following exposure to H2S. While there was a discernible distinction in the skin mucus metabolomes among the three treatment groups, only one metabolite - 4-hydroxyproline - was significantly impacted by H2S. Furthermore, this metabolite was significantly reduced in the plasma and skin mucus of H2S-exposed fish. This study underscores that prolonged exposure to H2S, even at concentrations previously deemed sub-lethal, has discernible physiological implications that manifest across various organisational levels. Given these findings, prolonged exposure to H2S poses a welfare risk, and thus, its presence must be maintained at low levels (<1 µg/L) in salmon land-based rearing systems.

Comparative basal transcriptome profiles of the olfactory rosette and gills of Atlantic salmon (Salmo salar) unveil shared and distinct immunological features.

The molecular repertoire of the mucosa-associated lymphoid tissue (MALT) in the olfactory rosette in most teleost fish is unknown. Here we present the basal transcriptome of the olfactory rosette of Atlantic salmon (Salmo salar). To investigate its mucosal immune features, we performed a comparative transcriptomic analysis with the gills, one of the most studied organs possessing MALT. Pathway enrichment revealed that cytokine-cytokine interaction and the neuroactive ligand-receptor interaction pathways were at the core of the shared similarity between the two organs. The immunological features of the two organs were further characterised by the overrepresentation of several immune-related pathways, particularly important for pathogen recognition. The immunological differences between the two organs were underlined with the differential regulation of markers for interleukins, extracellular matrix, antimicrobial peptides, and complement. The basal transcriptome of Atlantic salmon olfactory rosette is a valuable molecular toolbox that will advance our understanding of nasal immunity in teleost fish.

Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua).

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12 °C or 17 °C. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by elevated temperature and infection, except for the number of mucus cells in the 12 °C group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17 °C. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.

Embracing prospects for reducing the numbers of animals used in aquaculture research.

The principles of three Rs-REPLACEMENT, REDUCTION, and REFINEMENT-govern the protection and use of animals, including fish, for research purposes in the European Union and Norway. In this paper, we discuss some straightforward steps to simplify the delivery of these principles at the idea stage and adapt some of these examples for conducting fish trials related to health and welfare. Although some of the approaches are well established in other animal science arenas, we believe there can be a timely recap of their key facets. We discuss a number of simple strategies to emphasize how a reduction in fish numbers can be achieved from initial project conception to implementation, highlighting not only their advantages but also their limitations. We also highlight the role that funding agencies can play in the implementation of the 3R principles in aquaculture research. These simple points can be used in frameworks to initiate a broader and dynamic intersectoral dialogue among stakeholders of aquaculture research on how to promote ethics and embrace opportunities for this within the tenets of the 3Rs.

Skin mucus metabolomics provides insights into the interplay between diet and wound in gilthead seabream (Sparus aurata).

The molecular processes underlying skin wound healing in several fish species have been elucidated in the last years, however, metabolomic insights are scarce. Here we report the skin mucus metabolome of wounded and non-wounded gilthead seabream (Sparus aurata) fed with silk fibroin microparticles, a functional additive considered to accelerate the wound healing process. The three experimental diets (commercial diet enriched with 0 mg (control), 50 mg or 100 mg of silk fibroin microparticles Kg-1) were administered for 30 days and thereafter, a skin wound was inflicted. Skin mucus was collected on day 30 of feeding and 7 days post-wounding and subjected to metabolomic analysis by Ultra Performance Liquid Chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometry. The most enriched metabolite class was amino acids and derivatives, followed by nucleotides, nucleosides and analogues and carbohydrates and their derivatives. Metabolomic profiles revealed that the diet had a more profound effect than wounding in skin mucus. Metabolic pathway analysis of significantly affected metabolites revealed perturbations in the aminoacyl t-RNA biosynthesis in the skin. In particular, skin wound resulted in a decreased methionine level in mucus. Further, silk fibroin supplementation increased methionine level in skin mucus, which correlated with several wound morphometric parameters that characterized the epithelial healing capacity in seabream. The results provided new insight into the physiological consequences of skin wounds and how these processes could be influenced by dietary manipulation.

The skin mucosal barrier of lumpfish (Cyclopterus lumpus L.) is weakened by exposure to potential aquaculture production-related stressors.

Various cleaner fish species, such as the lumpfish (Cyclopterus lumpus L.), are used in the sea cage production of Atlantic salmon (Salmo salar L.) as a control measure against the ectoparasitic salmon louse (Lepeophtheirus salmonis). Nonetheless, during severe lice infestation, alternative treatments are required to control parasitic burden. The aim of this study was to gain insight into how lumpfish skin responds to different chemicals used to treat parasites. The authors collected skin from lumpfish from both research facilities (tank-reared fish) and commercial production (cage-reared fish) and used operational welfare indicators, in vitro models, histology and transcriptomics to study how the skin responded to two anti-parasitic oxidative chemicals, hydrogen peroxide (H2 O2 ) and peracetic acid. Lumpfish sampled from the farm were classified as clinically healthy or weak based on their morbidity status, and fish from each category were used to gain insight into how the therapeutics affect the skin barrier. Differences between healthy and weakened (moribund) fish, and between treated fish from each of the two groups, were observed. Histological examination showed an overall reduced skin quality in fish characterized as moribund, including different grades of exposed bony plates. In vitro oxidant-treated lumpfish skin had reduced the migration capacity of keratocytes, a weakened epidermal barrier, and altered gene transcription, changes that are known predisposing factors to secondary infections. Skin from non-treated, healthy fish sampled from commercial farms exhibited similar features and attributes to oxidant-exposed tank-reared fish from a research facility, suggesting that apparently healthy cage-held lumpfish exhibited stress responses in the epidermal barrier. The results of the study outline the risks and consequences lumpfish can face if accidentally subjected to potential anti-parasitic oxidant treatments aimed at Atlantic salmon. It also strengthens the evidence behind the requirement that lumpfish should be removed from the cages before being potentially exposed to this type of treatment and outlines the potential risks of differing husbandry practices upon lumpfish health, welfare and resilience.

The circulating plasma metabolome of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar).

Metabolomics can provide insights into the dynamic small-molecule fluctuations occurring in response to infection and has become a valuable tool in studying the pathophysiology of diseases in recent years. However, its application in fish disease research is limited. Here, we report the circulating plasma metabolome of Atlantic salmon (Salmo salar) experimentally infected with Neoparamoeba perurans-the causative agent of amoebic gill disease (AGD). Plasma samples were collected from fish with varying degrees of infection inferred from an external gross morphological score of gill pathology (i.e., gill score [GS] 1 -- GS3), where a higher GS indicates advanced infection stage. Uninfected fish (GS0) served as the control. Typical pathologies associated with AGD infection, such as hyperplastic lesions and lamellar fusion, were evident in infected gill samples. Plasma metabolites were identified by ultra-performance liquid chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometer. Identification of compounds were performed at four levels of certainty, where level 1 provided the most accurate compound identity. A total of 900 compounds were detected in the samples of which 143 were annotated at level 3, 68 on level 2b, 74 on level 2a, and 66 on level 1. Versus GS0, GS1 showed the highest number of significantly affected metabolites (104), which decreased with a higher GS. Adrenaline and adenosine were the two Level 1 compounds significantly affected by AGD regardless of GS, with the former increasing and the latter decreasing in infected fish. Hippuric acid significantly increased in GS1 and GS2, while the tryptophan metabolite indole-3-lactic acid decreased in response to the initial stage of infection but returned to basal levels at a higher GS. There were ten significantly affected metabolic pathways: Eight of which were significantly downregulated while two were downregulated in GS1 relative to GS0. The super-pathway of purine nucleotide salvage was enriched both within the upregulated metabolites in GS1vsGS0 and the down-regulated metabolites in GS3vsGS1. This is the first report on the circulating plasma metabolome of AGD infected salmon, and the results show that low infection levels resulted in a more dramatic metabolomic dysregulation than advanced infection stages. The metabolites identified are potential biological markers for the systemic physiological impact of AGD.

Exogenous sulphide donors modify the gene expression patterns of Atlantic salmon nasal leukocytes.

Hydrogen sulphide (H2S) is a known mediator of immunity, but the regulatory function of its exogenous form is not well understood in fish particularly in the mucosa. Here we report transcriptomic changes in the nasal leukocytes of Atlantic salmon (Salmo salar) following exposure to two forms of H2S donors - the salt sodium hydrosulfide (NaHS) and the organic analogue morpholin-4-ium 4-methoxyphenyl (morpholino) phosphinodithioate (GYY4137). Nasal leukocytes were exposed to three concentrations (1, 10 and 100 µM) of either of the two H2S forms for 24 h before the cells were checked for viability and collected for microarray analysis. Though cellular viability was minimally affected by the exposure to two H2S donors, GYY4137-exposed cells exhibited reduced viability compared with the NaHS group at the highest dose. The H2S-induced transcriptomic changes in the nasal leukocytes were concentration-dependent regardless of the sulphide forms. However, a larger number of differentially expressed genes (DEGs) were identified in the NaHS-exposed versus GYY4137-exposed groups across concentrations. In all comparisons, at least 53% of the DEGs identified were significantly upregulated. Gene ontology (GO) terms enriched in the lists of upregulated DEGs at higher concentrations included ferric iron binding. A comparison of the two H2S forms showed a clear grouping of different GO terms relative to concentrations. Pathway enrichment analysis revealed a significant influence in VEGF ligand-receptor interactions, oxidative stress, innate and adaptive immunity, and interleukin signalling especially at higher concentrations. Congruence analysis demonstrated that there were 16 GO terms overlapping; of these, 12 were upregulated by both sulphide donors including several involving iron binding and transport. The study offers the first molecular insights into how fish nasal leukocytes respond to exogenous H2S, and the results will be vital in resolving the regulatory function of H2S on mucosal immunity in fish.

Regulation of the molecular repertoires of oxidative stress response in the gills and olfactory organ of Atlantic salmon following infection and treatment of the parasite Neoparameoba perurans.

The present study investigated the involvement of key molecular regulators of oxidative stress in amoebic gill disease (AGD), a parasitic infestation in Atlantic salmon. In addition, the study evaluated how these molecular biomarkers responded when AGD-affected fish were exposed to a candidate chemotherapeutic peracetic acid (PAA). Atlantic salmon were experimentally infected with the parasite Neoparameoba perurans, the causative agent of AGD, by bath exposure and after 2 weeks, the fish were treated with three commercial PAA products (i.e., Perfectoxid, AquaDes and ADDIAqua) at a dose of 5 ppm. Two exposure durations were evaluated - 30 min and 60 min. Sampling was performed 24 h and 2 weeks after PAA treatment (equivalent to 2- and 4-weeks post infection). At each sampling point, the following parameters were evaluated: gross gill pathology, gill parasitic load, plasma reactive oxygen species (ROS) and total antioxidant capacity (TAC), histopathology and gene expression profiling of genes with key involvement in oxidative stress in the gills and olfactory organ. AGD did not result in systemic oxidative stress as ROS and TAC levels remained unchanged. There were no clear patterns of AGD-mediated regulation of the oxidative stress biomarkers in both the gills and olfactory organ; significant changes in the expression were mostly related to time rather than infection status. However, the expression profiles of the oxidative stress biomarkers in AGD-affected salmon, following treatment with PAA, revealed that gills and olfactory organ responded differently - upregulation was prominent in the gills while downregulation was more frequent in the olfactory organ. The expression of catalase, glutathione S-transferase and thioredoxin reductase 2 was significantly affected by the treatments, both in the gills and olfactory organ, and these alterations were influenced by the duration of exposure and PAA product type. Parasitic load in the gills did significantly increase after treatment regardless of the product and exposure duration; the parasite was undetectable in some fish treated with AquaDes for 30 mins. However, PAA treated groups for 30 min showed lower macroscopic gill scores than the infected-untreated fish. Histology disclosed the classic pathological findings such as multifocal hyperplasia and increased number of mucous cells in AGD-affected fish. Microscopic scoring of gill injuries showed that AGD-infected-PAA-treated fish had lower scores, however, an overall trend could not be established. The morphology and structural integrity of the olfactory organ were not significantly altered by parasitism or PAA treatment. Collectively, the results indicate that AGD did not affect the systemic and mucosal oxidative status of Atlantic salmon. However, such a striking profile was changed when AGD-affected fish were exposed to oxidative chemotherapeutics. Moreover, the gills and olfactory organ demonstrated distinct patterns of gene expression of oxidative stress biomarkers in AGD-infected-PAA-treated fish. Lastly, PAA treatment did not fully resolve the infection, but appeared not to worsen the mucosal health either.

Vitamin C Mitigates Oxidative Stress and Behavioral Impairments Induced by Deltamethrin and Lead Toxicity in Zebrafish.

Environmental contamination from toxic metals and pesticides is an issue of great concern due to their harmful effects to human health and the ecosystems. In this framework, we assessed the adverse effects when aquatic organisms are exposed to toxicants such as deltamethrin (DM) and lead (Pb), alone or in combination, using zebrafish as a model. Moreover, we likewise evaluated the possible protective effect of vitamin C (VC) supplementation against the combined acute toxic effects of the two toxicants. Juvenile zebrafish were exposed to DM (2 µg L-1) and Pb (60 µg L-1) alone and in combination with VC (100 µg L-1) and responses were assessed by quantifying acetylcholinesterase (AChE) activity, lipid peroxidation (MDA), some antioxidant enzyme activities (SOD and GPx), three-dimension locomotion responses and changes of elements concentrations in the zebrafish body. Our results show that VC has mitigative effects against behavioral and biochemical alterations induced by a mixture of contaminants, demonstrating that it can be used as an effective antioxidant. Moreover, the observations in the study demonstrate zebrafish as a promising in vivo model for assessing the neuroprotective actions of bioactive compounds.

Chemically and Green Synthesized ZnO Nanoparticles Alter Key Immunological Molecules in Common Carp (Cyprinus carpio) Skin Mucus.

This study was conducted to compare the effects of commercially available (C) and green synthesized (GS) Zinc oxide nanoparticles (ZnO-NPs) on immunological responses of common carp (Cyprinus carpio) skin mucus. GS ZnO-NPs were generated using Thymus pubescent and characterized by UV-vis diffuse reflectance spectroscopy (DRS), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscope (SEM), and energy-dispersive X-ray spectroscopy (EDX). Fish (n = 150) were randomly allocated into five groups in triplicate and received a waterborne concentration of 0% (control), 25%, and 50% of LC50 96 h of commercially available (C1 and C2) and green synthesized ZnO-NPs (GS1 and GS2) for 21 days. Results from XRD displayed ZnO-NPs with 58 nm in size and UV-vis DRS, EDX, and FT-IR analysis showed that some functional groups from plant extract bonded to the surface of NPs. The SEM images showed that ZnO-NPs have conical morphology. Acute toxicity study showed a higher dose of LC5096h for green synthesized ZnO-NPs (78.9 mg.L-1) compared to the commercial source (59.95 mg.L-1). The highest activity of lysozyme and alternative complement activity (ACH50) were found in control and GS1 groups. A significant decrease in alkaline phosphatase activity (ALP) was found in C1 and C2 groups compared to other treatments. Protease activity (P) was significantly decreased in the C2 group compared to the control and GS groups. Total immunoglobulin (total Ig) content was the highest in the control. In addition, total Ig in the GS1 group was higher than GS2. The exposure to ZnO-NPs lowered total protein content in all experimental groups when compared to control. Present findings revealed lower induced immunosuppressive effects by green synthesized ZnO-NPs on key parameters of fish skin mucus.

Dietary inclusion of Antarctic krill meal during the finishing feed period improves health and fillet quality of Atlantic salmon (Salmo salar L.).

There is an urgent need to find alternative feed resources that can further substitute fishmeal in Atlantic salmon diets without compromising health and food quality, in particular during the finishing feeding period when the feed demand is highest and flesh quality effects are most significant. This study investigates efficacy of substituting a isoprotein (35 %) and isolipid (35 %) low fishmeal diet (FM, 15 %) with Antarctic krill meal (KM, 12 %) during 3 months with growing finishing 2·3 kg salmon (quadruplicate sea cages/diet). Final body weight (3·9 (se 0·04) kg) was similar in the dietary groups, but the KM group had more voluminous body shape, leaner hearts and improved fillet integrity, firmness and colour. Ectopic epithelial cells and focal Ca deposits in intestine were only detected in the FM group. Transcriptome profiling by microarray of livers showed dietary effects on several immune genes, and a panel of structural genes were up-regulated in the KM group, including cadherin and connexin. Up-regulation of genes encoding myosin heavy chain proteins was the main finding in skeletal muscle. Morphology examination by scanning electron microscopy and secondary structure by Fourier transform IR spectroscopy revealed more ordered and stable collagen architecture of the KM group. NEFA composition of skeletal muscle indicated altered metabolism of n-3, n-6 and SFA of the KM group. The results demonstrated that improved health and meat quality in Atlantic salmon fed krill meal were associated with up-regulation of immune genes, proteins defining muscle properties and genes involved in cell contacts and adhesion, altered fatty acid metabolism and fat deposition, and improved gut health and collagen structure.

The Effects of Ozone on Atlantic Salmon Post-Smolt in Brackish Water-Establishing Welfare Indicators and Thresholds.

Ozone is a strong oxidant, and its use in aquaculture has been shown to improve water quality and fish health. At present, it is predominantly used in freshwater systems due to the high risk of toxic residual oxidant exposure in brackish water and seawater. Here, we report the effects of ozone on Atlantic salmon (Salmo salar) post-smolts (~100 g), in a brackish water (12 ppt) flow-through system. Salmon were exposed to oxidation reduction potential concentrations of 250 mV (control), 280 mV (low), 350 mV (medium), 425 mV (high) and 500 mV (very high). The physiological impacts of ozone were characterized by blood biochemical profiling, histopathologic examination and gene expression analysis in skin and gills. Fish exposed to 425 mV and higher showed ≥33% cumulative mortality in less than 10 days. No significant mortalities were recorded in the remaining groups. The skin surface quality and the thickness of the dermal and epidermal layers were not significantly affected by the treatments. On the other hand, gill histopathology showed the adverse effects of increasing ozone doses and the changes were more pronounced in the group exposed to 350 mV and higher. Cases of gill damages such as necrosis, lamellar fusion and hypertrophy were prevalent in the high and very high groups. Expression profiling of key biomarkers for mucosal health supported the histology results, showing that gills were significantly more affected by higher ozone doses compared to the skin. Increasing ozone doses triggered anti-oxidative stress and inflammatory responses in the gills, where transcript levels of glutathione reductase, copper/zinc superoxide dismutase, interleukin 1ß and interleukin were significantly elevated. Heat shock protein 70 was significantly upregulated in the skin of fish exposed to 350 mV and higher. Bcl-2 associated x protein was the only gene marker that was significantly upregulated by increasing ozone doses in both mucosal tissues. In conclusion, the study revealed that short-term exposure to ozone at concentrations higher than 350 mV in salmon in brackish water resulted in significant health and welfare consequences, including mortality and gill damages. The results of the study will be valuable in developing water treatment protocols for salmon farming.

The gut mucosal barrier of zebrafish (Danio rerio) responds to the time-restricted delivery of Lobosphaera incisa-enriched diets.

Recent studies in mammalian models revealed compelling evidence that along with the intrinsic characteristics of diets, the time of their delivery could have a profound impact on their benefits. In this study, we explored a time-dependent modulation of the gut mucosal barrier by delivering diets enriched with the green microalga (Lobosphaera incisa) either in a time-restricted regime or randomly to zebrafish (Danio rerio). The basal diet was enriched with microalgal biomass through two inclusion levels (i.e., 10% and 15% w/w), and the feeding trial lasted for six weeks. The control group was fed with the basal diet. After collection of tissue samples at week 6, the remaining fish were challenged by intraperitoneal injection of Streptococcus inaie. A histological analysis of the gut structure revealed that the fish that received the microalgae randomly exhibited shorter villi length. Genes coding for immunity were modulated in the gut by dietary treatments. Notably, the transcript levels of lysozyme, ß-defensin and hepcidin were significantly higher in the group subjected to the time-restricted feeding regime. Dietary microalgae affected the fatty acid content in the gut, particularly the level of arachidonic acid (ARA), and the time-restricted feeding influenced its accumulation. Groups that received diets enriched with 15% microalgae, regardless of the feeding strategy, displayed a significantly higher resistance to S. inaie 16 days post-infection, though differences between the delivery strategies were pronounced during the early stage of infection. In conclusion, the dietary inclusion of L. incisa modulated some of the features of the gut mucosal barrier of zebrafish, and the time of delivery appeared to have a considerable influence on immunomodulatory functions.

Atlantic salmon (Salmo salar) mounts systemic and mucosal stress responses to peracetic acid.

Peracetic acid (PAA), a strong organic peroxide, is considered a relatively sustainable disinfectant in aquaculture because of its broad effectivity against many pathogens at low concentrations and because it degrades spontaneously to harmless residues. The impacts of PAA on fish health must be determined before its use as either a routine disinfectant or chemotherapeutant. Here we investigated the systemic and mucosal stress responses of Atlantic salmon (Salmo salar) to PAA. In experiment 1, salmon were exposed to different nominal concentrations (0, 0.6, and 2.4 ppm) of PAA for 5 min, followed by a re-exposure to the same concentrations for 30 min 2 weeks later. Sampling was performed before exposure to PAA and at 2 h, 48 h, and 2 w after exposures. In experiment 2, fish were subjected to crowding stress prior to PAA exposure at 4.8 ppm for 30 min. The fish were sampled before exposure and 1 h, 4 h, and 2 w after. The two trials were performed in a recirculation system. Both systemic (i.e., plasma cortisol, glucose, lactate, total antioxidant capacity) and mucosal (i.e., expression of antioxidant coding genes in the skin and gills) stress indicators were affected by the treatments at varying levels, and it was apparent that the fish were able to mount a robust response to the physiological demands of PAA exposure. The cortisol levels increased in the early hours after exposure and returned to basal level afterwards. Prior exposure history to PAA did not markedly affect the levels of plasma lactate and glucose when fish were re-exposed to PAA. Crowding stress before PAA treatment, however, did alter some of the stress indicators (i.e., lactate, glucose and expression of antioxidant genes in the gills), suggesting that stress history serves as both a confounding and compounding factor on how stress responses to PAA are mobilised. Nonetheless, the changes were not substantial. Gene expression profile analyses revealed that the antioxidant system was more responsive to PAA in the gills than in the skin. The increased antioxidant capacity in the plasma, particularly at 2.4 ppm and higher, indicates that antioxidants were produced to neutralise the internal redox imbalance resulting from PAA exposure. In conclusion, the results show that salmon were able to mount a robust adaptive response to different PAA doses and exposure times, and a combined exposure to stress and PAA. These results underscore the potential of PAA as a chemotherapeutant for salmon at PAA concentrations commonly applied to control parasitic infestations.

Interplay between daily rhythmic serum-mediated bacterial killing activity and immune defence factors in rainbow trout (Oncorhynchus mykiss).

Circadian rhythm is emerging as an important regulator of immune functions. However, there is a paucity of information on the influence of this biological phenomenon in the antimicrobial factors in teleost fish. This study investigated the dynamics and interplay of serum-mediated bacterial killing activity and immune defence factors throughout the light:dark (LD) cycle in rainbow trout (Oncorhynchus mykiss). The juvenile fish came from two different emergence time fractions (i.e., late and early) that were believed to exhibit behavioural and physiological differences. Serum collected during the day from fish (mean ± SD: 39.8 ± 6.3 g) reared under 14L:10D photoperiod demonstrated bactericidal activity against Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida of varying magnitude, but no significant differences between the emergence fractions were observed. A day-night comparison in the same batch of fish revealed time-of-day dependence in the bactericidal activity against F. psychrophilum and Y. ruckeri amongst emergence fractions. A group of fish (63.3 ± 4.7 g) from each fraction was entrained to 12L:12D photoperiod for 21 days to investigate whether serum bactericidal activity exhibited daily rhythm. Serum-mediated bacterial killing activity against F. psychrophilum and Y. ruckeri displayed significant daily rhythm in both emergence fractions, where the peak of activity was identified during the light phase. Moreover, several serum defence factors manifested variations during the LD cycle, where anti-protease (ANTI) and myeloperoxidase (MPO) activities exhibited significant daily oscillation. However, there were no remarkable differences in the daily changes of serum factors amongst emergence fractions. Acrophase analysis revealed that the peaks of activity of alkaline phosphatase (only in late fraction), ANTI, lysozyme (only in early fraction) and MPO were identified during the light phase and corresponded with the period when serum-mediated bacterial killing activity was also at its highest. The daily dynamics of bactericidal activity and immune defence factors displayed positive correlation, particularly between MPO and, the two pathogens (i.e., F. pyschrophilum and Y. ruckeri). Taken together, the study revealed that serum-mediated bacterial killing activity and immune defence factors remarkably varied during the LD cycle in rainbow trout. In addition, the two emergence fractions displayed nearly comparable immunological profiles.

Rhythmicity and plasticity of digestive physiology in a euryhaline teleost fish, permit (Trachinotus falcatus).

Digestive physiology is considered to be under circadian control, but there is little evidence in teleost fish. The present study explored the rhythmicity and plasticity to feeding schedules of enzymatic digestion in a candidate aquaculture fish, the permit (Trachinotus falcatus). The first experiment identified the rhythms of digestive factors throughout the light-dark (LD) cycle. Gastric luminal pH and pepsin activity showed significant daily variation albeit not rhythmic. These dynamic changes were likewise observed in several digestive enzymes, in which the activities of intestinal protease, chymotrypsin and lipase exhibited significant daily rhythms. In the second experiment, the existence of feed anticipatory activity in the digestive factors was investigated by subjecting the fish to either periodic or random feeding. Anticipatory gastric acidification prior to feeding was identified in periodically fed fish. However, pepsin activity did not exhibit such anticipation but a substantial postprandial increase was observed. Intestinal protease, leucine aminopeptidase and lipase anticipated periodic mealtime with elevated enzymatic activities. Plasma melatonin and cortisol demonstrated robust daily rhythms but feeding time manipulations revealed no significant impact. Plasma ghrelin level remained constant during the LD cycle and appeared to be unaffected by differing feeding regimes as well. Taken together, the digestive factors of permit were highly dynamic during the LD cycle. Periodic feeding entrained digestive physiology and mediated anticipatory gastric acidification and intestinal enzymatic activities. This knowledge will be essential in developing feeding protocols and husbandry-related welfare strategies that will further advance this candidate finfish as an aquaculture species.

Innate immune defenses exhibit circadian rhythmicity and differential temporal sensitivity to a bacterial endotoxin in Nile tilapia (Oreochromis niloticus).

The present study investigated the daily dynamics of humoral immune defenses and the temporal influence in the sensitivity of these responses to a bacterial endotoxin in Nile tilapia (Oreochromis niloticus). The first experiment subjected the fish to two photoperiod conditions, 12L:12D (LD) and 0L:24D (DD), for 20 days to characterize the rhythms of humoral immunity. Serum alkaline phosphatase (ALP), lysozyme (LYZ), peroxidase (PER) and protease (PRO) exhibited significant rhythmicity under LD but not in DD. No significant rhythms were observed in esterase (ESA) and anti-protease (ANTI) in both photoperiod conditions. Fish reared under LD were subsequently subjected to DD while the group previously under DD was exposed to LD, and this carried on for 3 days before another set of samples was collected. Results revealed that the rhythms of LYZ, PER and PRO but not ALP persisted when photoperiod was changed from LD to DD. Nonetheless, immune parameters remained arrhythmic in the group subjected from DD to LD. Cluster analysis of the humoral immune responses under various light conditions revealed that each photic environment had distinct daily immunological profile. In the second experiment, fish were injected with bacterial endotoxin lipopolysaccharide (LPS) either at ZT3 (day) or at ZT15 (night) to evaluate the temporal sensitivity of humoral immunity to a pathogen-associated molecular pattern. The results demonstrated that responses to LPS were gated by the time of day. LPS significantly modulated serum ALP and ANTI activities but only when the endotoxin was administered at ZT3. Serum LYZ and PER were stimulated at both injection times but with differing response profiles. Modulated LYZ activity was persistent when injected at ZT3 but transient when LPS was applied at ZT15. The magnitude of LPS-induced PER activity was higher when the endotoxin was delivered at ZT3 versus ZT15. It was further shown that plasma cortisol was significantly elevated but only when LPS was administered at ZT3. On the other hand, plasma melatonin was significantly affected by LPS injection but only when exposed at ZT15. Taken together, this study shows that several key components of humoral immunity in tilapia exhibit circadian rhythms and adapt to photoperiodic changes. Further, results of the bacterial endotoxin challenge suggest that responsiveness of serum humoral factors to a biological insult is likely mediated by the time of day, highlighting the importance of circadian rhythm in the immunological functions of fish.

Dietary phytoimmunostimulant Persian hogweed (Heracleum persicum) has more remarkable impacts on skin mucus than on serum in common carp (Cyprinus carpio).

Immunostimulation through the use of sustainable and eco-friendly dietary additives is one of the current prophylactic strategies in fish husbandry. Plant-based immunostimulants are highly considered for this intent, both for their scientific and practical advantages. Persian hogweed (Heracleum persicum) is a flower-bearing herb that possesses interesting pharmacological importance due to its bioactive compounds. It is commonly used as spice, food additive, dietary supplement and traditional remedy. The present study evaluated the potential of H. persicum as a dietary phytoimmunostimulant in common carp (Cyprinus carpio). The powder form of H. persicum was incorporated in the basal diet at three different inclusion levels: 2.5, 5 and 10 g kg-1. The basal diet (0 g kg-1 of H. persicum) served as control. Experimental diets were administered to the fish for a period of 8 weeks. At the termination of the feeding experiment, impacts on fish immunity and performance were evaluated. Inclusion of H. persicum in the diet significantly elevated several immunological factors such as immunoglobulins, lysozyme, protease and alternative complement activities in carp. Interestingly, the changes were more pronounced in the skin mucus than in the serum. Performance was significantly improved in the fish groups that received the candidate phytoimmunostimulant. Specifically, final weight, weight gain, specific growth rate and feed conversion ratio were significantly improved in the fish that received dietary H. persicum at inclusion levels 5 g kg-1 and higher. This study demonstrated the potential of Persian hogweed as a candidate dietary phytoimmunostimulant in carp, impacting mainly the skin mucosal defenses. The study supports the current trend in the exploration of sustainable plant-based dietary supplements that are capable of boosting the immunological defenses of farmed fish.