Acute stress response on Atlantic salmon: a time-course study of the effects on plasma metabolites, mucus cortisol levels, and head kidney transcriptome profile.

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.

Triploid Atlantic salmon Salmo salar have a higher dietary phosphorus requirement for bone mineralization during early development.

The effect of a dietary phosphorus regime in freshwater on vertebra bone mineralization was assessed in diploid and triploid Atlantic salmon, Salmo salar. Fish were fed either a low phosphorus (LP) diet containing 10.5 g kg-1 total phosphorus or a normal phosphorus (NP) diet containing 17.4 g kg-1 total phosphorus from ∼3 to ∼65 g (day 126) in body weight. Two further groups were fed the NP diet from ∼3 g in body weight, but were then switched to the LP diet after 38 (∼10 g in body weight) or 77 (∼30 g in body weight) days. Growth, vertebral ash content (% ash) and radiologically detectable vertebra pathologies were assessed. Triploids were initially smaller than diploids, and again on day 77, but there was no ploidy effect on days 38 or 126. Vertebral ash content increased with increasing body size and those fish fed the NP diet had higher vertebral ash content than those groups fed the LP diet during the intervening time period, but this diet effect became less apparent as fish grew, with all groups having relatively equal vertebral ash content at termination. In general, triploids had lower vertebral ash content than diploids on day 38 and this was most evident in the group fed the LP diet. On day 77, those triploids fed the LP diet during the intervening time period had lower vertebral ash content than diploids. At termination on day 126, the triploids had the same vertebral ash content as diploids, irrespective of diet. There was a ploidy × diet interaction on vertebral deformities, with triploids having higher prevalences of fish with ≥1 deformed vertebra in all dietary groups except continuous NP. In conclusion, between days 0 and 77 (3-30 g body size), triploids required more dietary phosphorus than diploids in order to maintain similar vertebral ash content. A possible link between phosphorus feeding history and phosphorus demand is also discussed.

Omission of expected reward agitates Atlantic salmon (Salmo salar).

The evolutionary background for cognition and awareness is currently under ardent scrutiny. Poikilothermic vertebrates such as teleost fishes are capable of classical conditioning and have long-term memories, but it remains unknown to what degree such capabilities are associated with affective states. Here, we investigate whether the concept of frustration may apply to Atlantic salmon. In mammals, this paradigm comprises the omission of an expected reward (OER), which elicits behavioural and physiological coping responses (e.g. aggression and stress reactions). Six groups with 200 fish in each were conditioned to associate a flashing light (CS) with feeding. Conditioning over 22 days led to a change from aversion to attraction to the CS. Subsequently, 3 groups served as control, and 3 groups were subjected to an OER paradigm for 9 days, in which the expected food reward was delayed for 30 min during two out of three daily meals. Compared to controls, OER groups displayed higher levels of aggression and more heterogeneous growth rates, indicating a more pronounced social hierarchy. Cortisol levels did, however, not differ between treatments and both groups responded similarly to acute stress. These results indicate that teleost fishes, like mammals, respond aggressively to OER. The capacity to respond behaviourally to frustrating conditions thus likely reflects an adaptive response to environmental unpredictability, which has been conserved throughout vertebrate evolution.

Effect of cleaner fish on sea lice in Norwegian salmon aquaculture: a national scale data analysis.

The salmon aquaculture industry has adopted the use of invertivorous 'cleaner fishes' (CF) for biological control of sea louse infestations on farmed salmon. At present, ~50 million CF are used annually in Norway alone, with variable success in experimental and industrial contexts. We used a national scale database of louse counts, delousing treatments and CF stocking events on Norwegian salmon farms to test for evidence of CF efficacy at 488 sites that completed a grow-out cycle within 2016-2018. Our analysis revealed that sites using more CF over the duration of a grow-out cycle did not have fewer lice on average, likely because CF use is reactive and in proportion to the scale of the louse problem. Over time within sites, we found that (i) sites using more CF early in the grow-out cycle were able to wait slightly longer (conservatively, a 5.2 week delay with 5000 CF stocked week-1) before conducting the first delousing treatment, and (ii) CF stocking events were followed, on average, by a small reduction in louse population growth rates. However, both effects were small and highly variable, and louse population growth rates remained positive on average, even when large numbers of CF were used (tens of thousands per site). Moreover, effects of CF on louse density tended to be short-lived, likely reflecting mortality and escape of stocked CF. Overall, the data indicate that while some sites consistently obtain good results from CF, there is also widespread suboptimal use. A better understanding of factors affecting CF efficacy in commercial sea cages is required to inform legislation and drive more efficient and ethical use of CF by the salmon aquaculture industry.

Differential effects of internal tagging depending on depth treatment in Atlantic salmon: a cautionary tale for aquatic animal tag use.

Electronic tags are widespread tools for studying aquatic animal behavior; however, tags risk behavioral manipulation and negative welfare outcomes. During an experiment to test behavioral differences of Atlantic salmon Salmo salar in different aquaculture cage types, including ones expected to elicit deeper swimming behavior, we found negative tagging effects depending on whether cages were depth-modified. In the experiment, data storage tags implanted in Atlantic salmon tracked their depth behavior and survival in unmodified sea-cages and depth-modified sea-cages that forced fish below or into a narrow seawater- or freshwater-filled snorkel tube from a 4 m net roof to the surface. All tagged individuals survived in unmodified cages; however, survival was reduced to 62% in depth-modified cages. Survivors in depth-modified cages spent considerably less time above 4 m than those in unmodified cages, and dying individuals in depth-modified cages tended to position in progressively shallower water. The maximum depth that fish in our study could attain neutral buoyancy was estimated at 22 m in seawater. We calculated that the added tag weight in water reduced this to 8 m, and subtracting the tag volume from the peritoneal cavity where the swim bladder reinflates reduced this further to 4 m. We conclude that the internal tag weight and volume affected buoyancy regulation as well as the survival and behavior of tagged fish. Future tagging studies on aquatic animals should carefully consider the buoyancy-related consequences of internal tags with excess weight in water, and the inclusion of data from dying tagged animals when estimating normal depth behaviors.

Thermal injuries in Atlantic salmon in a pilot laboratory trial.

Thermal delousing is a new method for removing sea lice from farmed Atlantic salmon (Salmo salar L). We investigated thermally-related tissue injuries in Atlantic salmon in a pilot laboratory trial to describe the acute effect of high water temperatures (34-38 °C). Acute tissue injuries in gills, eyes, brain and possible also nasal cavity and thymus were seen in salmon exposed to water temperatures of 34 - 38 °C in 72 to 140 s. This implies that exposing salmon to such water temperatures is a welfare risk, not only due to the direct tissue injuries that may also be dependent on exposure time, but also due to risk of thermal pain and aversion, including flight reactions.

Lowering treatment temperature reduces salmon mortality: a new way to treat with hydrogen peroxide in aquaculture.

BACKGROUND: Hydrogen peroxide (H2 O2 ) baths are widely used to reduce numbers of salmon lice on farmed Atlantic salmon. Fish mortalities often occur after baths, with warmer temperatures increasing lethality. We tested whether mortality could be reduced and lice removal efficacy maintained by lowering bath temperatures relative to ambient temperatures. Post-smolt salmon infected with lice were held at 10, 13 or 16 °C, and treated with 1.5 g/L H2 O2 for 20 min at equal or lower bath temperatures of 7, 10 or 13 °C. RESULTS: Salmon mortality decreased as ambient and bath temperatures decreased. No mortality occurred when fish at 13 °C were treated at 7 °C. For ambient temperatures of 16 °C, the number of lice remaining was reduced by four-fold when treated at 7 °C compared with 13 °C. All treatments in which mortality was zero had similar efficacies regardless of bath temperature. CONCLUSION: We took salmon from warmer to colder temperatures to determine the optimum bathing temperature to prevent mortality. A temperature of 7 °C was optimal when treating with 1.5 g/L of H2 O2 , as mortality was zero and pre-adult lice removal was unchanged. By manipulating temperature, we developed a new method of H2 O2 bathing that reduces mortality. When ambient temperatures are >10 °C, we recommend that the industry decrease H2 O2 bath temperatures. © 2017 Society of Chemical Industry.

Sea lice infestation levels decrease with deeper 'snorkel' barriers in Atlantic salmon sea-cages.

BACKGROUND: Salmon lice (Lepeophtheirus salmonis) are the most important parasites of farmed salmon. Infective larvae position themselves in the upper part of the water column to increase encounter probabilities with potential hosts. Previous studies have shown that a 'snorkel' sea-cage technology protects salmon from infection in surface waters. We tested whether deep snorkels would more effectively reduce lice infestation than shallow snorkels and still uphold adequate conditions for the fish. Five sea-cages (12 m × 12 m) each holding approximately 3000 Atlantic salmon (Salmo salar) (53 ± 10 g) were fitted with snorkels that gave protection from infection for 0, 4, 8, 12 or 16 m. We tested whether reductions in the settlement of new salmon lice copepodids were consistent among four separate infection periods. RESULTS: Lice infestation decreased exponentially with depth in all time periods. Infection levels in shallow snorkels (0 and 4 m) were consistently 4-10 times higher than those in deep snorkels (12 and 16 m). Key welfare and production performance indices were similar across all snorkel depths. CONCLUSION: Deeper snorkels dramatically and consistently reduced infection levels of salmon lice compared with shallow snorkels, without consequences for fish welfare and production performance. Therefore, reducing salmon sea lice encounters using a depth-based barrier is a powerful management tool for salmon farming. © 2017 Society of Chemical Industry.

Soft texture of atlantic salmon fillets is associated with glycogen accumulation.

Atlantic salmon (Salmo salar L.) with soft fillets are not suited for manufacturing high quality products. Therefore fillets with insufficient firmness are downgraded, leading to severe economic losses to the farming and processing industries. In the current study, morphological characteristics of salmon fillets ranging from soft to hard were analysed. Different microscopic techniques were applied, including novel methods in this field of research: morphometric image analysis, periodic acid Schiff staining, immunofluorescence microscopy, transmission electron microscopy and fourier transform infrared microscopy. The results showed that the myocytes of soft muscle had detached cells with mitochondrial dysfunctions, large glycogen aggregates and enlarged inter cellular areas, void of extracellular matrix proteins, including lower amounts of sulfated glycoproteins. Myofibre-myofibre detachment and disappearance of the endomysium in soft muscles coincided with deterioration of important connective tissue constituents such as Collagen type I (Col I), Perlecan and Aggrecan. In summary our investigations show for the first time an association between soft flesh of Atlantic salmon and massive intracellular glycogen accumulation coinciding with degenerated mitochondria, myocyte detachment and altered extracellular matrix protein distribution. The results are important for further understanding the etiology of soft salmon.

Coping with unpredictability: dopaminergic and neurotrophic responses to omission of expected reward in Atlantic salmon (Salmo salar L.).

Comparative studies are imperative for understanding the evolution of adaptive neurobiological processes such as neural plasticity, cognition, and emotion. Previously we have reported that prolonged omission of expected rewards (OER, or 'frustrative nonreward') causes increased aggression in Atlantic salmon (Salmo salar). Here we report changes in brain monoaminergic activity and relative abundance of brain derived neurotrophic factor (BDNF) and dopamine receptor mRNA transcripts in the same paradigm. Groups of fish were initially conditioned to associate a flashing light with feeding. Subsequently, the expected food reward was delayed for 30 minutes during two out of three meals per day in the OER treatment, while the previously established routine was maintained in control groups. After 8 days there was no effect of OER on baseline brain stem serotonin (5-HT) or dopamine (DA) activity. Subsequent exposure to acute confinement stress led to increased plasma cortisol and elevated turnover of brain stem DA and 5-HT in all animals. The DA response was potentiated and DA receptor 1 (D1) mRNA abundance was reduced in the OER-exposed fish, indicating a sensitization of the DA system. In addition OER suppressed abundance of BDNF in the telencephalon of non-stressed fish. Regardless of OER treatment, a strong positive correlation between BDNF and D1 mRNA abundance was seen in non-stressed fish. This correlation was disrupted by acute stress, and replaced by a negative correlation between BDNF abundance and plasma cortisol concentration. These observations indicate a conserved link between DA, neurotrophin regulation, and corticosteroid-signaling pathways. The results also emphasize how fish models can be important tools in the study of neural plasticity and responsiveness to environmental unpredictability.

Duration of effects of acute environmental changes on food anticipatory behaviour, feed intake, oxygen consumption, and cortisol release in Atlantic salmon parr.

We compared behavioural and physiological responses and recovery time after different acute environmental challenges in groups of salmon parr. The fish were prior to the study conditioned to a flashing light signalling arrival of food 30 s later to study if the strength of Pavlovian conditioned food anticipatory behaviour can be used to assess how salmon parr cope with various challenges. The effect on anticipatory behaviour was compared to the effect on feed intake and physiological responses of oxygen hyper-consumption and cortisol excretion. The challenges were temperature fluctuation (6.5C° over 4 h), hyperoxia (up to 380% O(2) saturation over 4 h), and intense chasing for 10 min. Cortisol excretion was only elevated after hyperoxia and chasing, and returned to baseline levels after around 3 h or less. Oxygen hyper-consumption persisted for even shorter periods. Feed intake was reduced the first feeding after all challenges and recovered within 3 h after temperature and hyperoxia, but was reduced for days after chasing. Food anticipatory behaviour was reduced for a longer period than feed intake after hyperoxia and was low at least 6 h after chasing. Our findings suggest that a recovery of challenged Atlantic salmon parr to baseline levels of cortisol excretion and oxygen consumption does not mean full recovery of all psychological and physiological effects of environmental challenges, and emphasise the need for measuring several factors including behavioural parameters when assessing fish welfare.

Food anticipatory behaviour as an indicator of stress response and recovery in Atlantic salmon post-smolt after exposure to acute temperature fluctuation.

In this study we evaluated Pavlovian conditioned food anticipatory behaviour as a potential indicator for stress in groups of Atlantic salmon, and compared this with the physiological stress responses of cortisol excretion into water and hyper-consumption of oxygen. We hypothesised that environmental stress would result in reduced feeding motivation. To assess this, we measured the strength of anticipatory behaviour during a period of flashing light that signalled arrival of food. Further, we expected that fish given a reduced food ration would be less sensitive to environmental stress than fish fed full ration. The fish responded to an acute temperature fluctuation with hyper-consumption of oxygen that decreased in line with the temperature, and elevated cortisol excretion up to 1h after the stressor. These physiological responses did not differ significantly between the food ration groups. The anticipatory behaviour was significantly reduced after the stressor and returned to control levels after 1 to 2 h in the reduced ration group, but not until after 3 to 4 h in the full ration group. Our results show that acute stress can be measured in terms of changes to feeding motivation, and that it is a more sensitive indicator of stress that influences the fish over a longer time period than measures of change in cortisol excretion.