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Farmed Atlantic salmon (Salmo salar) welfare and performance can be strongly influenced by stress episodes caused by handling during farming practices. To better understand the changes occurring after an acute stress response, we exposed a group of Atlantic salmon parr to an acute stressor, which involved netting and transferring fish to several new holding tanks. We describe a time-course response to stress by sampling parr in groups before (T0) and 10, 20, 30, 45, 60, 120, 240, 300, and 330 min post-stress. A subgroup of fish was also subjected to the same stressor for a second time to assess their capacity to respond to the same challenge again within a short timeframe (ReStressed). Fish plasma was assessed for adrenocorticotropic hormone (ACTH), cortisol, and ions levels. Mucus cortisol levels were analyzed and compared with the plasma cortisol levels. At 5 selected time points (T0, 60, 90, 120, 240, and ReStressed), we compared the head kidney transcriptome profile of 10 fish per time point. The considerably delayed increase of ACTH in the plasma (60 min post-stress), and the earlier rise of cortisol levels (10 min post-stress), suggests that cortisol release could be triggered by more rapidly responding factors, such as the sympathetic system. This hypothesis may be supported by a high upregulation of several genes involved in synaptic triggering, observed both during the first and the second stress episodes. Furthermore, while the transcriptome profile showed few changes at 60 min post-stress, expression of genes in several immune-related pathways increased markedly with each successive time point, demonstrating the role of the immune system in fish coping capacity. Although many of the genes discussed in this paper are still poorly characterized, this study provides new insights regarding the mechanisms occurring during the stress response of salmon parr and may form the basis for a useful guideline on timing of sampling protocols.

Kelp forests are dynamic coastal habitats that generate large amounts of carbon-rich detritus. The fate of this detritus is largely unknown and considered a missing link in global carbon budgets. Kelp detritus can serve as food for benthic invertebrates and pelagic invertebrate larvae, but we know close to nothing about the role of kelp detritus as food source for other zooplankton. Lipid-rich pelagic copepods constitute a key link from primary producers to higher trophic levels in marine boreal and arctic ecosystems, and they transport vast amounts of carbon into the deep sea. We conducted feeding experiments to test if the copepodCalanus finmarchicuscan feed on fragments of two dominant kelp species,Saccharina latissimaandLaminaria hyperborea. Such feeding would constitute an undescribed pathway from blue forests to pelagic consumers and deep-sea carbon sequestration. The experiment indicated thatC. finmarchicuscan ingest kelp particles, but the digestion is limited compared to a regular phytoplankton diet. Moreover, the results provide initial evidence thatL. hyperboreacontains substances that are toxic to copepods, an observation that warrants further research. Using specific qPCR assays to trace the consumption of kelp, we found that kelp DNA amplification signals were stronger for copepods fed withS. latissimathan L. hyperborea, but we were not able to conclusively separate consumed kelp from DNA attached to the outside of the copepods.

BACKGROUND Dietary nucleotides (NTs) have been reported to affect hepatic function and composition. However, the effects on hepatic lipid deposition are less studied. OBJECTIVES We aimed to identify the regulatory role of dietary NTs in hepatic lipid deposition of zebrafish and elucidate the underlying mechanisms. METHODS Zebrafish (60 ± 1.69 mg; 1 mo old) were fed control diet (16.2% energy as fat) or diet supplemented with 0.1% NTs or 0.02% AMP in feeding experiments 1 and 2. Experiment 3 was conducted with zebrafish larvae. In experiment 4, 1-mo-old zebrafish were fed a high-fat diet (HFD, 38.2% energy as fat) or an HFD supplemented with 0.1% NTs or 0.02% AMP. Hepatic lipid deposition was evaluated by triglyceride (TG) content and staining. Phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) was assayed by immunoblotting. Zebrafish liver (ZFL) cells were treated with exogenous adenosine. Small interfering RNA was used to knock down AMPK or nucleoside transporter SLC28a1 in ZFL cells. Vivo-morpholino was used to knock down AMPK in zebrafish larvae. RESULTS Dietary 0.1% NTs or 0.02% AMP reduced hepatic TGs by 62% and 32%, respectively, compared with control (P

BackgroundWelfare challenges in salmon farming highlights the need to improve understanding of the fish’s response to its environment and rearing operations. This can be achieved by monitoring physiological responses such as heart rate (HR) for individual fish. Existing solutions for heart rate monitoring are typically based on Electrocardiography (ECG) which is sensitive to placement and electrode orientation. These factors are difficult to control and affects the reliability of the principle, prompting the desire to find an alternative to ECG for heart rate monitoring in fish. This study was aimed at adapting an optical photoplethysmography (PPG) sensor for this purpose. An embedded sensor unit measuring both PPG and ECG was developed and tested using anesthetized Atlantic salmon in a series of in-vivo experiments. HR was derived from PPG and compared to the ECG baseline to evaluate its efficacy in estimating heart rate.ResultsThe results show that PPG HR was estimated with an accuracy of 0.7 ± 1.0% for 660 nm and 1.1 ± 1.2% for 880 nm wavelengths, respectively, relative to the ECG HR baseline. The results also indicate that PPG should be measured in the anterior part of the peritoneal cavity in the direction of the heart.ConclusionA PPG/ECG module was successfully adapted to measure both ECG and PPG in-vivo for anesthetized Atlantic salmon. Using ECG as baseline, PPG analysis results show that that HR can be accurately estimated from PPG. Thus, PPG has the potential to become an alternative to ECG HR measurements in fish.

A better understanding of carotenoid dynamics (transport, absorption, metabolism, and deposition) is essential to develop a better strategy to improve astaxanthin (Ax) retention in muscle of Atlantic salmon. To achieve that, a comparison of post-smolt salmon with (+ Ax) or without (− Ax) dietary Ax supplementation was established based on a transcriptomic approach targeting pyloric, hepatic, and muscular tissues. Results in post-smolts showed that the pyloric caeca transcriptome is more sensitive to dietary Ax supplementation compared to the other tissues. Key genes sensitive to Ax supplementation could be identified, such as cd36 in pylorus, agr2 in liver, or fbp1 in muscle. The most modulated genes in pylorus were related to absorption but also metabolism of Ax. Additionally, genes linked to upstream regulation of the ferroptosis pathway were significantly modulated in liver, evoking the involvement of Ax as an antioxidant in this process. Finally, the muscle seemed to be less impacted by dietary Ax supplementation, except for genes related to actin remodelling and glucose homeostasis. In conclusion, the transcriptome data generated from this study showed that Ax dynamics in Atlantic salmon is characterized by a high metabolism during absorption at pyloric caeca level. In liver, a link with a potential of ferroptosis process appears likely via cellular lipid peroxidation. Our data provide insights into a better understanding of molecular mechanisms involved in dietary Ax supplementation, as well as its beneficial effects in preventing oxidative stress and related inflammation in muscle.

Nephrocalcinosis is a common disorder in farmed Atlantic salmon, but the consequences for the fish physiology are not well understood. We performed a transcriptome study in kidneys of Atlantic salmon (Salmo salar) smolts without and with severe chronic nephrocalcinosis (NC). The study revealed that numerous genes are differentially expressed in fish with NC compared with healthy salmon. The most evident changes in gene expression patterns in the NC group were a massive downregulation of metabolism and energy production, upregulation of signalling pathways important for tissue repair and function maintenance and upregulation of inflammatory responses. Overall, the extensive tissue damage and the gene regulation responses that affect salmon with severe nephrocalcinosis are highly likely to have dramatic consequences on fish survival.

Aquaculture, the farming of fish and seafood, is recognized as a highly efficient system for producing protein for human consumption. In contrast, many terrestrial animal protein production systems are inefficient, impacting land use and exacerbating climate change. Humankind needs to adopt a more plant-centric diet, the only exception being fish consumed as both a source of protein and essential dietary nutrients such as omega-3 fatty acids. Here we consider the implications of such a transition, and the challenges that aquaculture must overcome to increase productivity within planetary boundaries. We consider how agriculture, specifically crops, can provide solutions for aquaculture, especially the sectors that are dependent on marine ingredients. For example, agriculture can provide experience with managing monocultures and new technologies such as genetically modified crops tailored specifically for use in aquaculture. We propose that a closer connection between agriculture and aquaculture will create a resilient food system capable of meeting increasing dietary and nutritional demands without exhausting planetary resources. Aquaculture must develop within planetary boundaries. Experience from agriculture, such as in managing monocultures and using genetically modified crops, can inform sustainable solutions for aquaculture.

The effects of two stunning methods (carbon monoxide asphyxia, CO, and electroshock, E) on blood plasma parameters, rigor index, fillet pH and shape changes, ATP breakdown and Adenylate Energy Charge (AEC) in muscle immediately post mortem were investigated in rainbow trout (Oncorhynchus mykiss) kept in tanks containing water set at 8 °C or 12 °C. Both the methods here adopted induced a stress-response which, however, was not able to affect the rigor mortis development and fillet pH. The fillets from the E group showed the strongest length contraction and width increase at 48 h post mortem. The CO stunning method exhibited the highest levels of both ATP and AEC in the muscle immediately after death, equal to 2.28 µmol/g and 0.83, respectively, while 1.12 µmol/g and 0.64 values were found in the E group. In addition, we found that the water temperature might interact with the stunning method and minimise the stress response. In the present trial, the most suitable use of the CO stunning method would be coupled with 12 °C of rearing water temperature to better preserve ATP and AEC in muscles.Highlights CO stunning preserves ATP and AEC in trout muscle better than electrical stunning Water temperature might interact with the stunning and minimise the stress response CO stunning is suggested to be coupled with 12 °C of water temperature Electricity should be avoided at 8 °C of water temperature

Interspecific hybridisation may improve the farm performance of sterile triploid salmonids via heterosis (i.e. hybrid vigour). We assessed growth over the final 293 days in seawater, and harvest quality, in diploid and triploid Atlantic salmon (Salmo salar) × brown trout (Salmo trutta) hybrids compared to diploid and triploid Atlantic salmon. We measured vertebral deformities, cataracts, flesh colour, gut mass, and body shape at harvest. In triploids, hybridisation had no effect on harvest size, vertebral deformities, cataracts, or body shape, but did improve fillet colouration (Mean digital SalmoFan™ score [95% CI]: 24.6 [24.4–24.9] and 26.0 [25.7–26.2] for triploid salmon and triploid hybrids, respectively) and lower relative gut size (34% lower). Compared to diploid salmon, triploid salmon were significantly heavier at harvest, triploid hybrids tended to be heavier (Post-hoc, least square means, p = 0.08), whereas diploid hybrids were 83% lighter (Mean mass [g] at harvest [95% CI]: 2676 [2470–2898], 3395 [3134–3679], 462 [401–534], and 3086 [2832–3363] for diploid salmon, triploid salmon, diploid hybrids, and triploid hybrids, respectively). However, both triploid groups had a significantly higher incidence of fish with one or more deformed vertebra (Mean % [95% CI]: 23 [14–35], 60 [47–71], 38 [20–60], and 44 [31–57] % in diploid salmon, triploid salmon, diploid hybrids, and triploid hybrids, respectively), more severe cataracts (Mean cataract score [95% CI]: 3.0 [2.7–3.3], 3.5 [3.2–3.8], 2.2 [1.7–2.6], 3.6 [3.3–4.0] for diploid salmon, triploid salmon, diploid hybrids, and triploid hybrids, respectively), and a smaller relative gut size (21% smaller) compared to diploid counterparts. In conclusion, triploid hybrids have no growth advantage over triploid salmon and suffer from similar welfare issues while only benefiting from increased fillet colour.

Nephrocalcinosis is a common disorder in nurseries in Norway (Klykken, Reed, et al., 2022) and was reported as one of the main welfare challenges in farmed salmon by The Norwegian Fish Health Report of 2019 (Sommerset et al., 2020). Nephrocalcinosis is described as deposits of minerals within the kidneys (Bruno, 1996), that can disturb kidney function, which in turn can have dramatic consequences on fish performance and survival. The aetiology is most likely related to environmental factors, and suboptimal water quality has been indicated in several studies as the main risk factor (Fivelstad et al., 1999; Fivelstad et al., 2003; Khan et al., 2018; Fivelstad et al., 2015; Good et al., 2010; Lewisch et al., 2013; Chen et al., 2001). Newly conducted research suggested that osmoregulatory stress may be the trigger for nephrocalcinosis (Boissonnot et al., 2022). Regardless of the severity of the condition, fish rarely present external signs, and it is thus challenging to monitor its prevalence and development. Present diagnostic methods require euthanasia as they consist of visually scoring the accumulation of deposits and the severity of lesions. Macroscopic assessments of necropsied fish are often imprecise, since small deposits are rarely visible to the naked eye, and histopathology is therefore considered as the best existing diagnostic method (Klykken, Boissonnot, et al., 2022). Research on, and monitoring of, nephrocalcinosis has been greatly hampered by the lack of non-invasive methods of assessing the presence and severity of this condition, as it is not possible to follow the development of the disease in single individuals, and as the number of sampled fish is limited due to ethical reasons. Radiology has previously been used for assessing vertebrae deformities in Atlantic salmon (Drábiková et al., 2021; Holm et al., 2020), based on the classification scheme developed by Witten et al. (2009), and there has been a rapid development of the technology (Ou et al., 2021) including portable systems, which allow efficient in situ diagnosis. Nephrocalcinosis in Atlantic salmon is mainly identified as amorphous carbonate apatite, a calciumdominated mineral (Klykken, Reed, et al., 2022), and it has previously been demonstrated that this mineral composition is suitable for xray detection (Smith & Lehr, 1966). Radiology is non-invasive and can be performed on anaesthetized fish, enabling assessment of nephrocalcinosis without euthanazing the fish. A non-invasive method for assessing nephrocalcinosis would allow for monitoring of the condition over time on an individual level and would be ethically and economically preferable. We have therefore explored radiology as a possible tool to detect and evaluate the severity of nephrocalcinosis, comparing it with histological scoring.

Moderate levels of intestinal damage and inflammation are often seen in intensive fish aquaculture. The causes may be due to antinutrients from plant meals, stress or other causes. There is currently a lack of good models to explore these effects and so how to mitigate the consequences. Most studies have used full-fat soy or other compounds that cause intestinal damage that are likely not reversible. In this study we have explored the possibility of using soybean HP48, made from solvent extracted peeled soybeans, as a low-inflammation model in post-smolt Atlantic salmon, and then investigated whether supplementation of the probiotic Pediococcus acidilactici CNCM I-4622 – MA 18/5 M (Bactocell) could diminish this effect. The fish were fed triplicate diets. A Control diet containing 18.08% soy protein concentrate (SPC), a HP48 diet where most of the SPC was replaced by HP48 (5.00% SPC and 17.68% HP48), and a Bactocell diet that was identical to the HP48 diet but contained 0.03% Bactocell. After 10 weeks of feeding, the mid- and hind-intestinal health were assessed by histology, integrity (Ussing chamber) and gene expression (RNAseq). Transcriptomic and integrity data suggests that HP48 led to a disturbed mid-intestinal homeostasis with impaired cellular integrity and increased inflammation and cell turnover. Most of the transcriptomic effects were reversed with Bactocell including downregulation of immune genes and upregulation of transmembrane proteins such as type IV collagen, which is important in restoring epithelial homeostasis. In the hind-intestine, the HP48 diet led to deleterious morphological changes such as widening of lamina propria and stratum granulosum, disrupted mucosal folds, loss of absorptive vacuoles, and upregulation of several immune regulated genes and downregulation of genes involved in solute- and water transport. The intestinal integrity assessed by Ussing chamber was not affected. Bactocell supplementation did alleviate several of the morphological effects. However, it was not able to completely reverse the expression of immune- or transport related genes, suggesting a higher effect of probiotic supplement in the mid-intestine compared to the hind-intestine. This study demonstrates that the level of HP48 used here is sufficient to create low-level intestinal changes in Atlantic salmon, which is within range for functional feed ingredients to reverse.

Background: Antimicrobials revolutionized human as well as animal medicine in the 20th century by providing effective treatment of diseases caused by pathogenic microorganisms. However, microorganisms have the ability to develop antimicrobial resistant strains. This occurs when microorganisms mutate or when resistance genes are exchanged between them. The use of antimicrobial drugs accelerates the emergence of drug-resistant strains. A priority is to safeguard the efficacy of antimicrobial drugs we depend on for treatment of infectious diseases in humans. Use of antimicrobials in food animals can create a source of antimicrobial resistant bacteria that can spread to humans both by direct contact and through the food supply. Coccidiosis is an intestinal disease in animals caused by unicellular parasites called coccidia. As most of the damage of this infection is done by the time signs of the disease are widespread, preventive measures are preferred. Coccidiostats are animal feed additives used to prevent coccidiosis by inhibiting or killing coccidia. There are two major groups of coccidiostats; ionophores and non-ionophores, the latter also referred to as “non-ionophore coccidiostats” (but also called chemicals). One main difference between these groups is that ionophores also inhibit or kill some bacterial species, whereas non-ionophore coccidiostats do not. Consequently, some bacterial infections may also be controlled by ionophore coccidiostats, e.g. the poultry disease necrotic enteritis caused by the bacterium Clostridium perfringens (C. perfringens). Eleven different coccidiostats have been authorised for use in the EU, both ionophores and non-ionophore coccidiostats. Norway has been exempted from the EEA Agreement in this field and has approved only five; all ionophores. The two ionophore coccidiostats currently used in Norway are narasin for broilers and monensin for turkeys. Resistance to coccidiostats in coccidia and bacteria: Development of resistance in coccidia to all eleven coccidiostats has been described in the scientific literature, but the prevalence of resistance is unknown. Cross-resistance between various ionophore coccidiostats has also been shown, i.e. development of resistance to one ionophore may also render the coccidia resistant to another ionophore. Various rotation and shuttle programmes with exchange between ionophores and non-ionophore coccidiostats are believed to prevent or delay development of resistance in coccidia. In Norway, such programmes will have little effect as long as only ionophores and not non-ionophore coccidiostats are approved for use. Development of resistance against ionophores has also been observed in bacteria. In the Norwegian surveillance programme NORM-VET during the years 2002 - 2013, between 50 - 80% of the tested flocks had narasin resistant faecal enterococci, which are bacteria that are part of the normal intestinal microbiota. However, the pathogenic bacterium C. perfringens has not been shown to be resistant against any ionophore. Cross-resistance in bacteria to more than one ionophore has been observed. In addition, a limited amount of data may indicate an association between narasin and resistance to the antibacterials bacitracin and vancomycin. As these are antibacterials used for treatment in humans, more research should be performed to validate these results. Non-ionophore coccidiostats, which do not have antibacterial effect, are not approved in Norway. If such coccidiostats were approved in Norway, coccidiostats with negligible probability of inducing resistance in bacteria would be available. Human exposure to resistant bacteria and coccidiostats: Humans may theoretically be exposed to coccidiostat resistant bacteria from poultry in a number of ways, e.g. by handling live animals and their manure, through slaughtering and processing, and by preparation and consumption of poultry meat. Furthermore, bacteria of the human normal microbiota, which cover all skin and mucosal surfaces, might develop resistance if they are exposed to coccidiostats. In this assessment, the probabilities of exposure are classified as: Negligible (extremely low), Low (possible, but not likely), Medium (likely), High (almost certain) and Not assessable. The Panel has estimated the following probabilities of human exposure: Handling manure from coccidiostat fed poultry without sufficient risk-reducing measures entails a high probability of exposure to both resistant bacteria and coccidiostats. Without proper protection, the probability of exposure to coccidiostats is also high when handling coccidiostat premixes and feeds containing coccidiostats without proper protection measures. Various treatments, e.g. composting, of the manure may reduce the probability. The probability of exposure to resistant bacteria is medium for workers handling carcasses and raw meat on a daily basis if risk-reducing measures are not applied, whereas the probability of exposure to coccidiostats is negligible. For consumers, the probability of exposure to coccidiostats is negligible. The probability for exposure to resistant bacteria is also negligible in heat treated food since heat treatment kills the bacteria. The probability of exposure to coccidiostat resistant bacteria is low to medium if handling raw meat without proper hygienic procedures, because raw meat may harbour resistant bacteria. Risk-reducing measures will lower the probabilities. However, little is known concerning the consequences of human exposure to coccidiostat resistant bacteria or to to coccidiostats. There is little information in scientific literature indicating whether such bacteria in fact will colonize the human body, either transitionally or permanently. Furthermore, there is no information on the probability of exchange of resistance genes from transferred bacteria to bacteria of the human natural microbiota or to pathogens. Likewise, the Panel has no information on the level of exposure, e.g. the amount of coccidiostats and their metabolites, or the time period, necessary for the various bacteria to give rise to resistant variants. As coccidiostats are not used to treat infectious diseases in humans, concern of resistance is related to possible cross- or co-resistance with antibacterials considered important in human medicine. Such resistance has so far not been confirmed. Use of therapeutic antibacterials for poultry: If the ionophore coccidiostats used in Norway are replaced by one or more non-ionophore coccidiostat with no antibacterial effect and no other changes are done, the coccidiostats used will no longer inhibit the bacterium Clostridium perfringens, which is the cause of necrotic enteritis. Over time this will likely to lead to a need for intermittent or continuous use of higher levels of therapeutic antibacterials due to increased incidence of this desease in poultry production. The magnitude of the increase is difficult to predict. Alternatives to in-feed antimicrobials: Eradication from the birds’ environment of coccidia causing coccidiosis is difficult to achieve because the coccidia form oocysts that survive outside the host and resist commonly used disinfectants. Vaccination with non-pathogenic vaccines is now used increasingly in commercial Norwegian broiler farms, instead of in-feed coccidiostats. So far coccidiosis has not been reported as a problem in this transition process to broiler rearing without in-feed coccidiostats in Norway. Non-antimicrobial feed additives with purported health-promoting benefits, i.e. acid-based products, probiotics, prebiotics, synbiotics, yeast-based products, plant-derived products, combinations of these, and other products have been developed and used in feed. These products have been tested for efficacy against coccidia with conflicting, non-consistent or non-convincing results. The majority of these products appear to target the bacterial microbiota rather than coccidia. The Panel has not assessed possible effects of other types of management changes.

Probiotics are widely used in aquafeeds and exhibited beneficial effects on fish by improving host health and resisting pathogens. However, probiotics applied to aquaculture are mainly from terrestrial sources instead of the host animal. The purpose of the work was to evaluate the effects of stabilized fermentation product of commensal Cetobacterium somerae XMX-1 on gut, liver health and antiviral immunity of zebrafish. A total of 240 zebrafish were assigned to the control (fed a basal diet) and XMX-1 group (fed a basal diet with 10 g XMX-1/kg diet). After four weeks feeding, growth performance, feed utilization, hepatic steatosis score, TAG, lipid metabolism related genes and serum ALT were evaluated. Furthermore, serum LPS, the expression of Hif-1α, intestinal inflammation score, antioxidant capability and gut microbiota were tested. The survival rate and the expression of antiviral genes were analyzed after challenge by spring viremia of carp virus (SVCV). Results showed that dietary XMX-1 did not affect growth of zebrafish. However, dietary XMX-1 significantly decreased the level of serum LPS, intestinal inflammation score and intestinal MDA, as well as increased T-AOC and the expression of Hif-1α in zebrafish intestine (p 0.05). Furthermore, XMX-1 supplementation decreased the relative abundance of Proteobacteria and increased Firmicutes and Actinobacteria. Additionally, XMX-1 supplementation significantly decreased hepatic steatosis score, hepatic TAG, serum ALT and increased the expression of lipolysis genes versus control (p 0.05). Zebrafish fed XMX-1 diet exhibited higher survival rate after SVCV challenge. Consistently, dietary XMX-1 fermentation product increased the expression of IFNφ2 and IFNφ3 after 2 days of SVCV challenge and the expression of IFNφ1, IFNφ2 and MxC after 4 days of SVCV challenge in the spleen in zebrafish versus control (p 0.05). In conclusion, our results indicate that dietary XMX-1 can improve liver and gut health, while enhancing antiviral immunity of zebrafish.

It is well established that farmed Atlantic salmon (Salmo salar) need n3-highly unsaturated fatty acids (HUFA) in their diet to thrive and grow. However, the biological functions to the individual HUFAs may differ, implying that future supplementation could require fixed ratios for maximum benefit. The intestinal barrier is essential to f ish health, and any disruption of the barrier can have detrimental effects. The current experiment was designed to examine the response of the intestinal hindgut when fed a low HUFA diet with 8.5 g/kg EPA + DHA (4.5% total fatty acid) or two high HUFA diets, with either high DHA (28.7 g/kg and 5.9% total fatty acid) or high EPA (25 g/kg and 14.2% total fatty acid). The diets were fed to Atlantic salmon post-smolt over 10 weeks and thereafter exposed to 3 weeks of chronic stress. After 10 weeks of feeding there were no differences in intestinal permeability and integrity, but intestinal morphology indicated increased intestinal health in the high EPA group. Gene expression also suggest that fish fed the high EPA diet had more regulation of pathways related to protein turnover compared to the high DHA fed fish. There was also indication of lower energy utilization in the low HUFA fed fish than high HUFA. Subjecting fish to 3 weeks of chronic stress led to a reduction in transepithelial resistance, increased ion flux and active L-lysine transport across the intestinal barrier in addition to a decrease in mucosal fold, enterocyte height and supranuclear vacuole density and an increase in thickness of the intestinal muscularis. After stress, the low HUFA group showed signs of inflammation with increased infiltration of MHCII positive cells. Gene expression also showed that low HUFA fed fish had a lower response to chronic stress compared to the high HUFA groups. Comparing fish fed either high DHA or EPA exposed to chronic stress showed few physical effects, but a lower density of supranuclear vacuoles and upregulation in immune-related gene expression indicate inflammation in the high DHA group.

Summary Propionate and propionyl-CoA accumulation have been associated with the development of mitochondrial dysfunction. In this study, we show that propionate induces intestinal damage in zebrafish when fed a high-fat diet (HFD). The intestinal damage was associated with oxidative stress owing to compromised superoxide dismutase 2 (Sod2) activity. Global lysine propionylation analysis of the intestinal samples showed that Sod2 was propionylated at lysine 132 (K132), and further biochemical assays demonstrated that K132 propionylation suppressed Sod2 activity. In addition, sirtuin 3 (Sirt3) played an important role in regulating Sod2 activity via modulating de-propionylation. Finally, we revealed that intestinal oxidative stress resulting from Sod2 propionylation contributed to compositional change of gut microbiota. Collectively, our results in this study show that there is a link between Sod2 propionylation and oxidative stress in zebrafish intestines and highlight the potential mechanism of intestinal problems associated with high propionate levels., Graphical abstract, Highlights • Propionate supplementation in high-fat diet induces intestinal damage • Propionate induces oxidative stress via Sod2 propionylation at 132 lysine site • Increased Sod2 propionylation is associated with reduced expression of Sirt3 • Intestinal oxidative stress alters gut microbiota composition, Gastroenterology; Molecular physiology; Cell biology

Increased cortisol production and oxygen consumption are commonly used as indicators for stress in most fish species. For comparative studies measuring acute stress responsiveness, it is often assumed that the cortisol is sampled at peak response. However, the effect of acclimated temperature on cortisol response is largely unknown, and so is also the dynamics of oxygen uptake following stress. In the present study we acclimated Atlantic salmon post-smolt to four different temperature within the species relevant temperature spectrum (4, 8, 12 and 17 °C), and applied an acute stressor of 60 s of confinement. Cortisol, as measured in water, was sampled before and up to 360 min following the stressor, while oxygen consumption rates was estimated per minute. Basal cortisol production increased significantly with temperature, which must be considered when calculating the cortisol response per se. The cortisol response followed a Gaussian pattern, where the amplitude (max value) increased with temperature while the curve width (time before peak and recovery) decreased. Also, the total amount of cortisol released in response to the stressor increased with temperature. The cortisol peak at 4, 8, 12 and 17 °C was respectively measured after 123.75 ± 7.2, 67.5 ± 9.7, 56.25 ± 3.75 and 33.74 ± 3.75 min. The stress induced peak in oxygen uptake increased with temperature in an exponential manner, and both peak value and the total oxygen uptake above baseline levels was three times higher at 17 than at 12 °C, while the total oxygen uptake was similar between 4, 8, 12 °C. This strong difference in oxygen uptake between 12 and 17 °C was not reflected by the cortisol levels, and suggests that salmon are extra vulnerable for creating hypoxic conditions when stressed at high temperature. The current work suggests when to sample cortisol for attaining peak values at a given acclimated temperatures in salmon post-smolt. This should allow for better comparison of cortisol values between studies.

The increasing replacement of fish oil (FO) by vegetable oils (VO) in aquafeeds for marine carnivorous species may lead to modifications of the dietary arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) levels and ratios that may compromise the fish immune status. Therefore, this study aimed to assess how different dietary ratios of ARA, EPA, and DHA modulate the immune response of gilthead sea bream juveniles. For that purpose, four isoproteic and isolipidic plant-based diets were formulated to include different dietary Long-Chain-Polyunsaturated Fatty Acids (LC-PUFA) ratios (%DM): Diet A (2.0% ARA: 0.2% EPA: 0.1% DHA); Diet B (1.0% ARA: 0.4% EPA: 0.4% DHA); Diet C (0% ARA: 0.6% EPA: 0.6% DHA); Diet D (0% ARA: 0.3% EPA: 1.5% DHA). Fish were fed the experimental diets for 56 days and then the haematological profile, humoral innate immune parameters, and expression of immune-related genes in the gut were determined. Our results showed that fish fed diet B exhibited an increase in the monocyte numbers, alternative complement activity, and bactericidal activity against Photobacterium damselae compared to the values found in fish fed other diets. Contrarily, peroxidase, antiproteases, and proteases activity and nitric oxide levels were not affected by the dietary treatments. Immune-related gene expression in the distal intestine did not show differences between dietary treatments. Overall, a diet with a balanced n-6 (ARA) and n-3 (EPA+DHA) LC-PUFA ratio seemed to better modulate the fish innate immune response, thus better preparing the fish innate immune system against potential immunological insults.

Propionate and propionyl-CoA accumulation have been associated with mitochondrial dysfunction. In this study, we observed that propionate induced intestinal damage in the context of high fat diet (HFD) in zebrafish. The intestinal damage was associated with oxidative stress and mitochondrial dysfunction owing to compromised SOD2 activity and the resultant impairment of antioxidant capacity. Global lysine propionylation analysis of the intestinal samples showed that SOD2 was propionylated at lysine 132, and further biochemical assays demonstrated that lysine 132 propionylation suppressed SOD2 activity. In addition, SIRT3 played an important role in regulating SOD2 activity via modulating depropionylation, and the enhanced SOD2 propionylation in zebrafish fed high fat plus propionate diet was attributable to reduced SIRT3 expression. Finally, we reveal that intestinal oxidative stress resulting from SOD2 propionylation contributed to the compositional change of gut microbiota, which further deteriorated intestinal oxidative stress independent of SIRT3. Collectively, the results in this study reveal a link between SOD2 propionylation and oxidative stress, and highlight the potential mechanism of intestinal problems associated with high propionate level.

Late developmental stages of the marine copepods in the genus Calanus can spend extended periods in a dormant stage (diapause) that is preceded by the accumulation of large lipid stores. We assessed how lipid metabolism during development from the C4 stage to adult is altered in response to predation risk and varying food availability, to ultimately understand more of the metabolic processes during development in Calanus copepods. We used RNA sequencing to assess if perceived predation risk in combination with varied food availability affects expression of genes associated with lipid metabolism and diapause preparation in C. finmarchicus. The lipid metabolism response to predation risk differed depending on food availability, time and life stage. Predation risk caused upregulation of lipid catabolism with high food, and downregulation with low food. Under low food conditions, predation risk disrupted lipid accumulation. The copepods showed no clear signs of diapause preparation, supporting earlier observations of the importance of multiple environmental cues in inducing diapause in C. finmarchicus. This study demonstrates that lipid metabolism is a sensitive endpoint for the interacting environmental effects of predation pressure and food availability. As diapause may be controlled by lipid accumulation, our findings may contribute towards understanding processes that can ultimately influence diapause timing. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and Permissions

Propionate is a commonly used preservative in various food and feedstuffs and has been regarded as a food additive without safety concerns. However, we observed that dietary propionate supplementation induced intestinal damage in the context of high fat diet (HFD) in zebrafish. The intestinal damage was attributable to oxidative stress owing to impaired antioxidant capacity, which was caused by compromised SOD2 activity in the intestine. Global lysine propionylation analysis of the intestinal samples showed that SOD2 was propionylated at K132, and further biochemical assays demonstrated that K132 propionylation suppressed SOD2 activity. In addition, SIRT3 could directly interact with SOD2 and played an important role in regulating SOD2 activity via modulating depropionylation, and the enhanced SOD2 propionylation in zebrafish fed high fat plus propionate diet was attributable to reduced SIRT3 expression. Finally, we reveal that intestinal oxidative stress resulting from SOD2 propionylation contributed to the compositional change of gut microbiota, which further deteriorated intestinal oxidative stress independent of SIRT3. Collectively, the results in this study reveal a link between protein propionylation and intestine health, and suggest potential risk of a widely used food preservative in HFD context.

Penaeid prawns are considered as most demanding fishery resources. The current study aims to purify and characterize lectin from the haemolymph of Penaeus semisulcatus. The semisulcatus-lectin was purified by affinity chromatography using mannose coupled Sepharose CL-4B column and purified lectin exhibited a single band of 66 kDa in SDS-PAGE. The purity and crystalline structure of purified lectin was confirmed by HPLC and X-ray diffraction analysis. Semisulcatus-lectin exhibited yeast agglutination activity against Saccharomyces cerevisiae and agglutinated human erythrocytes. Semisulcatus-lectin was evaluated for phenol oxidase activation and phagocytic activities. It was observed that semisulcatus-lectin had antibacterial activity against Gram-negative Vibrio parahaemolyticus and Aeromonas hydrophila, suggesting a potential therapeutic strategy in aquaculture industry for disease management.

BackgroundWith declining wild fish populations, farmed salmon has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA). However, the introduction of plant oil in farmed salmon feeds has reduced the content of these beneficial LC-HUFA. The synthetic capability for LC-HUFAs depends upon the dietary precursor fatty acids and the genetic potential, thus there is a need for in-depth understanding of LC-HUFA synthetic genes and their interactions with other genes involved in lipid metabolism. Several key genes of LC-HUFA synthesis in salmon belong to the fatty acid desaturases 2 (fads2) family. The present study applied whole transcriptome analysis on two CRISPR-mutated salmon strains (crispants), 1)Δ6abc/5Mtwith mutations inΔ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-cgenes,and 2)Δ6bcMtwith mutations inΔ6fads2-b and Δ6fads2-cgenes. Our purpose is to evaluate the genetic effectfads2mutations have on other lipid metabolism pathways in fish, as well as to investigate mosaicism in a commercial species with a very long embryonal period.ResultsBothΔ6abc/5MtandΔ6bcMtcrispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. TheΔ6abc/5Mtfish displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation ofsrebp1genes which are known key transcription regulators of lipid metabolism as well as a number of down-stream genes involved in fatty acidde-novosynthesis, fatty acidβ-oxidation and lipogenesis. Bothelovl5andelovl2genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation ofΔ6bcMtsurprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability.ConclusionsCRISPR-Cas9 can efficiently mutate multiplefads2genes simultaneously in salmon. The results of the present study have provided new information on the transcriptional regulations of lipid metabolism genes after reduction of LC-HUFA synthesis pathways in salmon.

With declining wild fish populations, farmed Atlantic salmon (Salmo salar) has gained popularity as a source for healthy long-chain highly unsaturated fatty acids (LC-HUFA) including 20:5n-3 and 22:6n-3. However, the introduction of plant-based oil in fish diets has reduced the content of these beneficial LC-HUFA. The capability of biosynthesis of LC-HUFAs depends on fatty acids supplied in diets and the genetic potential residing in the fish. Key proteins involved in LC-HUFA synthesis in salmon include fatty acid desaturases 2 (Fads2). In a recent study we used CRISPR/Cas9 to generate two F0 mutant strains of salmon, 1)Δ6abc/5Mtwith mutations inΔ5fads2, Δ6fads2-a, Δ6fads2-b and Δ6fads2-cgenes, and 2)Δ6bcMtwith mutations inΔ6fads2-b and Δ6fads2-cgenes. The CRISPR mutated salmon (crispants) had reduced levels of LC-HUFA and expression of targetedfads2genes. In present study we apply whole transcriptome analysis on thesefads2crispants. Our purpose is to evaluate the genetic mosaicism infads2crispants and the effect these mutations had on other lipid metabolism pathways in fish. BothΔ6abc/5MtandΔ6bcMtcrispants demonstrated high percentage of indels within all intended target genes, though different indel types and percentage were observed between individuals. Skipping of a CRISPR-targeted exon was observed inΔ6fads2-agene ofΔ6abc/5Mtsalmon. TheΔ6abc/5Mtfish also displayed several disruptive indels which resulted in over 100 differentially expressed genes (DEGs) enriched in lipid metabolism pathways in liver. This includes up-regulation ofsrebp1genes as well as genes involved in fatty acidde-novosynthesis, fatty acidβ-oxidation and lipogenesis. Bothelovl5andelovl2genes were not changed, suggesting that the genes were not targeted by Srebp1. The mutation ofΔ6bcMtsurprisingly resulted in over 3000 DEGs which were enriched in factors encoding genes involved in mRNA regulation and stability.