Non-invasive sampling of water-borne hormones demonstrates individual consistency of the cortisol response to stress in laboratory zebrafish (Danio rerio).

Glucocorticoid (GC) stress hormones are well-known for their impact on phenotypic traits ranging from immune function to behaviour and cognition. For that reason, consistent aspects of an individual's physiological stress response (i.e. GC responsiveness) can predict major elements of life-history trajectory. Zebrafish (Danio rerio) emerge as a promising model to study such consistent trait correlations, including the development of individual stress coping styles, i.e. consistent associations between physiological and behavioral traits. However, consistency in GC responsiveness of this popular animal model remains to be confirmed. Such a study has so far been hampered by the small-bodied nature and insufficient blood volume of this species to provide repeated measurements of circulating GCs. Here, we adopted a technique that allows for repeated, non-invasive sampling of individual zebrafish by quantifying GCs from holding water. Our findings indicate consistency of the magnitude of post-stress GC production over several consecutive stress events in zebrafish. Moreover, water-borne GCs reflect individual variation in GC responsiveness with the strongest consistency seen in males.

Contrasting associations between breeding coloration and parasitism of male Arctic charr relate to parasite species and life cycle stage.

Conspicuous carotenoid ornamentation is considered a signal of individual "quality" and one of the most intensely studied traits found to co-vary with parasitism. Since it has been suggested that only "high quality" individuals have enough resources to express excessive sexual ornaments and resist parasites, current theory struggles to explain cases where the brightest individuals carry the most parasites. Surprisingly little emphasis has been put on the contrasting routes to fitness utilized by different parasite species inhabiting the same host. Using Arctic charr (Salvelinus alpinus) as model species, we hypothesized that skin redness and allocation of carotenoids between skin and muscle (redness ratio) will be positively and negatively associated with parasites using the fish as an intermediate and final host, respectively. Both pigment parameters were indeed positively associated with abundances of parasites awaiting trophic transmission (Diplostomum sp. and Diphyllobothrium spp.) and negatively associated with the abundance of adult Eubothrium salvelini tapeworms. These empirical data demonstrate that contrasting associations between carotenoid coloration and parasite intensities relates to the specific premises of different parasite species and life cycle stages.

Consistent melanophore spot patterns allow long-term individual recognition of Atlantic salmon Salmo salar.

The present study shows that permanent melanophore spot patterns in Atlantic salmon Salmo salar make it possible to use images of the operculum to keep track of individual fish over extended periods of their life history. Post-smolt S. salar (n = 246) were initially photographed at an average mass of 98 g and again 10 months later after rearing in a sea cage, at an average mass of 3088 g. Spots that were present initially remained and were the most overt (largest) 10 months later, while new and less overt spots had developed. Visual recognition of spot size and position showed that fish with at least four initial spots were relatively easy to identify, while identifying fish with less than four spots could be challenging. An automatic image analysis method was developed and shows potential for fast match processing of large numbers of fish. The current findings promote visual recognition of opercular spots as a welfare-friendly alternative to tagging in experiments involving salmonid fishes.

Behavioural responses in a net restraint test predict interrenal reactivity in Arctic charr Salvelinus alpinus.

In this study, a 1 min net restraint test was evaluated as a method to predict stress-coping style in Arctic charr Salvelinus alpinus, by investigating the relationship between behaviour during the test and levels of plasma cortisol sampled after 30 min confinement. In two separate groups of S. alpinus, general linearized model revealed significant correlations between cortisol levels and principal component scores extracted from principal component analysis, combining measures of activity in the tests. With the use of glmulti, the model selection ruled out any effects of size, sex and order of capture on interrenal reactivity. In general, S. alpinus that were more active in the net restraint test also had low levels of circulating cortisol, suggesting a proactive coping style. The results from two repeated runs were not correlated, but both runs, performed eight days apart, show a negative correlation between post-stress cortisol level and activity in the net. The lack of consistency could be explained by different treatments before each run and individual differences in behavioural plasticity. The net restraint test is thus predictive of stress-coping style in S. alpinus, and has the benefit of being less time-consuming than the commonly used confinement stress test.

Brain-encysting trematodes and altered monoamine activity in naturally infected killifish Fundulus parvipinnis.

This paper presents novel evidence to address mechanisms by which trematode parasites effect behavioural changes in naturally infected fish hosts. California killifish Fundulus parvipinnis infected with the brain-encysting trematode Euhaplorchis californiensis display conspicuous swimming behaviours that render them 30 times more likely to be eaten by birds, the parasite's final host. Prevalence of E. californiensis reaches nearly 100% in most F. parvipinnis populations, with parasite biomass constituting almost 2% of F. parvipinnis biomass in some locations. Despite having thousands of cysts on their brains, infected fish grow and mature at rates comparable to those of uninfected populations. The lack of general pathology combined with the specificity of the altered behaviours suggests that the behavioural changes are due to parasite manipulation. The monoamine neurotransmitters serotonin and dopamine, which control locomotion and social behaviour in fishes and other vertebrates, were examined to explore the underlying mechanisms of this behaviour modification. Whereas previous studies were similarly conducted with experimentally infected fish, in this study, brain dopaminergic and serotonergic activity were analysed in naturally infected fish to assess how E. californiensis may alter F. parvipinnis monoamines in a naturally occurring system. A parasite density-associated decrease in serotonergic activity occurred in the hippocampus of naturally infected fish, as well as a decrease in dopaminergic activity in the raphe nuclei, suggesting that E. californiensis inhibits serotonin and dopamine signaling in naturally infected F. parvipinnis. The neurochemical profile of infected fish is consistent with the hypothesis that E. californiensis affects brain monoaminergic systems in order to induce impulse-driven, active, and aggressive behaviour in its hosts.

Fish welfare and genomics.

There is a considerable public and scientific debate concerning welfare of fish in aquaculture. In this review, we will consider fish welfare as an integration of physiological, behavioral, and cognitive/emotional responses, all of which are essentially adaptative responses to stressful situations. An overview of fish welfare in this context suggests that understanding will rely on knowledge of all components of allostatic responses to stress and environmental perturbations. The development of genomic technologies provides new approaches to this task, exemplified by how genome-wide analysis of genetic structures and corresponding expression patterns can lead to the discovery of new aspects of adaptative responses. We will illustrate how the genomic approach may give rise to new biomarkers for fish welfare and also increase our understanding of the interaction between physiological, behavioral, and emotional responses. In a first part, we present data on expression of candidate genes selected a priori. This is a common avenue to develop molecular biomarkers capable of diagnosing a stress condition at its earliest onset, in order to allow quick corrective intervention in an aquaculture setting. However, most of these studies address isolated physiological functions and stress responses that may not be truly indicative of animal welfare, and there is only rudimentary understanding of genes related to possible cognitive and emotional responses in fish. We also present an overview on transcriptomic analysis related to the effect of aquaculture stressors, environmental changes (temperature, salinity, hypoxia), or concerning specific behavioral patterns. These studies illustrate the potential of genomic approaches to characterize the complexity of the molecular mechanisms which underlies not only physiological but also behavioral responses in relation to fish welfare. Thirdly, we address proteomic studies on biological responses to stressors such as salinity change and hypoxia. We will also consider proteomic studies developed in mammals in relation to anxiety and depressive status which may lead to new potential candidates in fish. Finally, in the conclusion, we will suggest new developments to facilitate an integrated view of fish welfare. This includes use of laser microdissection in the transcriptomic/proteomic studies, development of meta-analysis methods for extracting information from genomic data sets, and implementation of technological advances for high-throughput proteomic studies. Development of these new approaches should be as productive for our understanding of the biological processes underlying fish welfare as it has been for the progress of pathophysiological research.

Effect of nutritional status and sampling intensity on recovery after dorsal aorta cannulation in free-swimming Atlantic salmon (Salmo salar L.).

Recovery from implantation of a cannula in the dorsal aorta (DA) of Atlantic salmon (Salmo salar) was studied in relation to nutritional status and sampling intensity. The incentive for the study was the inconsistency between published reports and our own experience of recovery and longevity of fish exposed to this protocol. In two studies using starved fish, blood (0.3 ml) was sampled 0, 1 and 24 h after DA-cannulation, and thereafter at 48 and 72 h and thereafter once weekly for four weeks. In a third study using fed fish, four consecutive samples (0, 3, 6 and 12 h after a meal) were obtained twice a week over a four-week period. All fish displayed a sharp increase in pCO(2) and haematocrit (Hct) during surgery, followed by a marked raise in cortisol, glucose, sodium and potassium (1 h). pCO(2), pH and Hct approached baseline levels as early as the 1 h post-surgery sample, while this was not the case for cortisol and electrolytes before the 24 h post-surgery sample. Glucose did not display any significant changes post surgery. From then on, all variables displayed minor but non-significant (P > 0.05) changes indicating a steady state close to baseline values for unstressed fish. This pattern was independent of sampling procedure, i.e. repeated single or multiple samples and thus volume of blood removed. Nutritional status (fed vs. starved) did not affect post-surgical recovery pattern. Only K(+) and Hct displayed consistent and significant post-prandial patterns. We found marked differences between baseline level of cannulated fish and uncannulated control fish, in pH, K(+) and Hct indicating that cannulation may be the preferred method to obtain representative resting values in fish.

Consistent boldness behaviour in early emerging fry of domesticated Atlantic salmon (Salmo salar): Decoupling of behavioural and physiological traits of the proactive stress coping style.

Individual variation in the way animals cope with stressors has been documented in a number of animal groups. In general, two distinct sets of behavioural and physiological responses to stress have been described: the proactive and the reactive coping styles. Some characteristics of stress coping style seem to be coupled to the time to emerge of fry from spawning redds in natural populations of salmonid fishes. In the present study, behavioural and physiological traits of stress coping styles were compared two and five months after emergence in farmed Atlantic salmon (Salmo salar), using individuals with an early or late time to emerge. Initially, compared to late emerging individuals, early emerging individuals showed a shorter time to resume feeding after transfer to rearing in isolation. Resumption of feeding after isolation was suggested to be related to boldness behaviour, rather than hunger, in the present study. This observation was repeated five months after emergence, demonstrating behavioural consistency over time in this trait. However, in other traits of proactive and reactive stress coping styles, such as social status, resting metabolism or post stress cortisol concentrations, early and late emerging individuals did not differ. Therefore, this study demonstrates that boldness in a novel environment is uncoupled from other traits of the proactive and reactive stress coping styles in farmed salmonids. It is possible that this decoupling is caused by the low competitive environment in which fish were reared. In natural populations of salmonids, however, the higher selection pressure at emergence could select for early emerging individuals with a proactive coping style.

Melanin-based skin spots reflect stress responsiveness in salmonid fish.

Within animal populations, genetic, epigenetic and environmental factors interact to shape individual neuroendocrine and behavioural profiles, conferring variable vulnerability to stress and disease. It remains debated how alternative behavioural syndromes and stress coping styles evolve and are maintained by natural selection. Here we show that individual variation in stress responsiveness is reflected in the visual appearance of two species of teleost fish; rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). Salmon and trout skin vary from nearly immaculate to densely spotted, with black spots formed by eumelanin-producing chromatophores. In rainbow trout, selection for divergent hypothalamus-pituitary-interrenal responsiveness has led to a change in dermal pigmentation patterns, with low cortisol-responsive fish being consistently more spotted. In an aquaculture population of Atlantic salmon individuals with more spots showed a reduced physiological and behavioural response to stress. Taken together, these data demonstrate a heritable behavioural-physiological and morphological trait correlation that may be specific to alternative coping styles. This observation may illuminate the evolution of contrasting coping styles and behavioural syndromes, as occurrence of phenotypes in different environments and their response to selective pressures can be precisely and easily recorded.

Parasite manipulation of brain monoamines in California killifish (Fundulus parvipinnis) by the trematode Euhaplorchis californiensis.

California killifish (Fundulus parvipinnis) infected with the brain-encysting trematode Euhaplorchis californiensis display conspicuous swimming behaviours rendering them more susceptible to predation by avian final hosts. Heavily infected killifish grow and reproduce normally, despite having thousands of cysts inside their braincases. This suggests that E. californiensis affects only specific locomotory behaviours. We hypothesised that changes in the serotonin and dopamine metabolism, essential for controlling locomotion and arousal may underlie this behaviour modification. We employed micropunch dissection and HPLC to analyse monoamine and monoamine metabolite concentrations in the brain regions of uninfected and experimentally infected fish. The parasites exerted density-dependent changes in monoaminergic activity distinct from those exhibited by fish subjected to stress. Specifically, E. californiensis inhibited a normally occurring, stress-induced elevation of serotonergic metabolism in the raphae nuclei. This effect was particularly evident in the experimentally infected fish, whose low-density infections were concentrated on the brainstem. Furthermore, high E. californiensis density was associated with increased dopaminergic activity in the hypothalamus and decreased serotonergic activity in the hippocampus. In conclusion, the altered monoaminergic metabolism may explain behavioural differences leading to increased predation of the infected killifish by their final host predators.

Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness.

This paper investigates whether two lines of rainbow trout displaying genetically determined variation in stress responsiveness and behavior also show differences in brain monoaminergic activity. In several brain regions, strains of rainbow trout selected for consistently high or low post-stress cortisol levels displayed differences in tissue concentrations of monoamines and/or monoamine metabolites, or in metabolite/monoamine ratios. High-responsive trout reacted to stress by an increase in the concentrations of both serotonin (brain stem), dopamine (brain stem), and norepinephrine (optic tectum, telencephalon), whereas low-responsive fish did not. Brain stem and optic tectum concentrations of monoamine metabolites were also elevated after stress in high responders, but not in low-responsive fish. The simultaneous increase in the concentration of monoamines and their metabolites suggests that both synthesis and metabolism of these transmitters were elevated after stress in high-responsive trout. A divergent pattern was seen in the hypothalamus, where low-responsive fish displayed elevated levels of 5-hydroxyindoleacetic acid (a serotonin metabolite) and 3-methoxy-4-hydroxyphenylglycol (a norepinephrine metabolite). In the telencephalon, both populations had elevated concentrations of these metabolites after stress. These results clearly suggest that selection for stress responsiveness in rainbow trout is also associated with changes in the function of brain monoaminergic systems. The possible functional significance of these observations is discussed with respect to the physiological and behavioral profile of these strains of fish. Literature is reviewed showing that several factors affecting brain monoaminergic activity might be altered by selection for stress responsiveness, or alternatively be under direct influence of circulating glucocorticoids.

Suppression of aggression in rainbow trout (Oncorhynchus mykiss) by dietary L-tryptophan.

Juvenile rainbow trout Oncorhynchus mykiss were isolated in individual compartments in observation aquaria and allowed to acclimate for 1 week, during which they were fed commercial trout feed. Thereafter, the fish were tested for aggressive behaviour using a resident/intruder test. Following this first resident/intruder test, the feed was exchanged for an experimental wet feed supplemented with 0.15 % or 1.5 % L-tryptophan (by wet mass). Controls received the same feed but without L-tryptophan supplementation. The fish were fed to satiety daily, and their individual feed intake was recorded. Aggressive behaviour was quantified again after 3 and 7 days of L-tryptophan feeding using the resident/intruder test. Feeding the fish L-tryptophan-supplemented feed for 3 days had no effect on aggressive behaviour, whereas feeding the fish L-tryptophan-supplemented feed for 7 days significantly suppressed aggressive behaviour in the fish, an effect seen at both levels of L-tryptophan supplementation. Fish fed L-tryptophan-supplemented feed showed elevated plasma and brain levels of L-tryptophan. The amino acid L-tryptophan is the precursor of serotonin, and supplementary dietary L-tryptophan was found to elevate levels of 5-hydroxyindoleacetic acid (5-HIAA) and the 5-HIAA/serotonin concentration ratio in the brain. Neither feed intake nor plasma cortisol level was significantly affected by dietary L-tryptophan. Central serotonin is believed to have an inhibitory effect on aggressive behaviour, and it is suggested that the suppressive effect of dietary L-tryptophan on aggressive behaviour is mediated by an elevation of brain serotonergic activity.

Differential stress coping in wild and domesticated sea trout.

Offspring of wild and sea-ranched (domesticated) sea trout (Salmo trutta) originating from the same river, were reared under identical hatchery conditions from the time of fertilization. At one year of age individual fish were exposed to two standardized stressors; transfer to a novel environment, with or without a simultaneous predator exposure. Blood plasma concentrations of glucose and cortisol were analyzed along with brain levels of dopamine (DA), 3,4-hydroxyphenylacetic acid (DOPAC, a major DA metabolite), serotonin (5-hydroxytryptamine, 5-HT), and 5-hydroxyindoleacetic acid (5 HIAA, a major 5-HT metabolite). Transfer to a novel environment, alone as well as in combination with predator exposure, resulted in elevated plasma concentrations of glucose and cortisol. Moreover, exposure to these stressors resulted in elevated brain levels of 5-HT and 5-HIAA, as well as elevated brain 5-HIAA/5-HT and DOPAC/DA ratios. Wild trout displayed significantly higher post stress plasma glucose levels than domesticated fish. Similarly, following stress, brain 5-HIAA/5-HT and DOPAC/DA ratios were significantly higher in wild than in domesticated fish. These differences were not caused by differences in brain levels of 5-HIAA and DOPAC, but instead by differences in brain 5-HT and DA concentrations. These results suggest that domestication results in attenuated stress responses in trout, and that alterations in brain monoamine neurotransmission are part of this effect.

Short-term effects of fights for social dominance and the establishment of dominant-subordinate relationships on brain monoamines and cortisol in rainbow trout.

We report changes in brain serotonergic, noradrenergic and dopaminergic activity, along with plasma cortisol concentrations, occurring during the initial 24-h period following the establishment of dominant-subordinate relationships in pairs of rainbow trout. Immediately (within 5 min) after the termination of staged fights for social dominance, a large increase in blood plasma cortisol was observed in both fight losers (future subordinate fish) and winners (future dominant fish). In dominant fish, cortisol decreased rapidly (within 3 h) to the level of unstressed controls, while continuing to increase in subordinate fish. At 3 h following fights, the brain serotonergic system was activated in both dominant fish and subordinate fish, at least in some brain regions (telencephalon). This effect was reversed in dominant individuals within 24 h of social interaction, whereas in subordinate fish a substantial activation of the serotonergic system was manifest in all brain regions by 24 h. Similarly, a strong increase in brain catecholaminergic activation was indicated after 24 h of social interaction in subordinate fish, but not in dominant fish. Relationships between plasma cortisol and brain serotonergic and noradrenergic activity in the various experimental groups suggest that these systems influence cortisol secretion under normal conditions and during moderate or short-term stress.