Long-term water quality data explain interpopulation variation in responsiveness to stress in sticklebacks at both wastewater effluent-contaminated and uncontaminated sites

The magnitude of the corticosteroid response to a standardised stressor varied in proportion to the concentration of effluent in three-spined sticklebacks (Gasterosteus aculeatus L.) captured downstream of 10 wastewater treatment works (WWTW). However, at 9 sites with no upstream WWTW input inter-population variation in the reactivity of the stress axis occurred across a similar range to that seen for fish at impacted sites, suggesting that the factor(s) responsible for modulating stress responsiveness in sticklebacks are not unique to sites receiving WWTW effluent. Physicochemical data from a long-term monitoring programme were employed to investigate whether variation in water quality contributed to between-site variation in stress axis reactivity. Between-site variation in fourteen water quality determinands explained between 30% and 60% of the variation in stress reactivity, and fish size, for sticklebacks at both WWTW-contaminated and uncontaminated sites. At uncontaminated sites the mean mass and length of sticklebacks increased with total oxidised N concentration (as an indicator of anthropogenic input) whereas at WWTW-contaminated sites fish size decreased with increasing effluent concentration, suggesting that factors adversely affecting growth were present predominantly at WWTW-contaminated sites. In contrast, at contaminated and uncontaminated sites the magnitude of the corticosteroid response to a standardised stressor increased with anthropogenic input (effluent concentration or total oxidised N respectively), indicating that factor(s) modulating the reactivity of the stress axis may be present at both WWTW-contaminated and uncontaminated sites

Disruption of the stress response in wastewater treatment works effluent-exposed three-spined sticklebacks persists after translocation to an unpolluted environment

The hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis plays a key role in responding to biotic and abiotic challenges in all vertebrates. Recent studies have shown that the apical response of the HPI axis to stressors in three-spined sticklebacks varies in proportion to the concentration of wastewater treatment works (WWTW) effluent to which the fish are exposed. This study was conducted to determine whether between-site variation in stress responsiveness among WWTW effluent-exposed sticklebacks is persistent or reversible. Sticklebacks from eight sites in north-west England affected by WWTW effluent and exhibiting between-population variation in HPI axis reactivity, were moved to a clean-water aquarium environment. After five months in the contaminant-free environment the responsiveness of these fish to a standardised stressor was determined, by measuring the rate of stress-induced cortisol release across the gills, and compared with the responses of fish newly sampled from the eight original capture sites. Inter-site differences in the reactivity of the HPI axis, proportional to the effluent concentration at each site, persisted among the translocated female sticklebacks for at least 5 months. In male fish however, the direct relationship between stress responsiveness and site-specific effluent was not evident 5 months post-translocation. These results support previous observations that the HPA/I axis, a non-reproductive endocrine system, is vulnerable to modulation by anthropogenic factors in fish and show for the first time that, in female fish at least, this modulation is not transient. The mechanisms underlying these observations, and the implications for the fitness and resilience of affected populations, requires investigation

ACTH does not mediate divergent stress responsiveness in rainbow trout

Two lines of rainbow trout selected for high (HR) and low (LR) responsiveness to a standardised confinement stressor displayed a sustained divergence in plasma cortisol levels during a 3 h period of confinement (max.: HR: 167 ± 13 ng ml-1; LR: 103 ± 8 ng ml-1; P < 0.001). However, no significant difference in plasma ACTH levels was evident (max: HR: 153 ± 9 pg ml-1; LR: 142 ± 7 pg ml-1). Dexamethasone (DEX) was administered to HR and LR fish to block endogenous adrenocorticotropin (ACTH) release. Administration of a weight-adjusted dose of ACTH to the DEX-blocked fish elevated plasma cortisol levels to a significantly greater extent in HR (233 ± 24 ng ml-1) than LR (122 ± 14 ng ml-1) fish (P < 0.001). Plasma cortisol levels in DEX-blocked HR and LR fish after sham injection were low but also significantly different (HR: 6.7 ± 1 ng ml-1; LR: 2.2 ± 0.2 ng ml-1; P < 0.001). These results indicate that modulation of cortisol responsiveness to stressors in HR and LR fish resides, at least in part, downstream of the hypothalamic-pituitary axis

Behavioral and neuroendocrine correlates of selection for stress responsiveness in rainbow trout - a review

In rainbow trout the magnitude of the cortisol response to stress shows both consistency over time and a moderate to high degree of heritability, and high responding (HR) and low responding (LR) lines of rainbow trout have been generated by individual selection for consistently high or low post-stress cortisol values. Using 2nd and 3rd generation fish, we tested the hypothesis that differential stress responsiveness is associated with behavioral alterations in the HR-LR trout model. LR fish showed a tendency to become socially dominant, a rapid recovery of food intake after transfer to a novel environment, and a reduced locomotor response in a territorial intrusion test. Furthermore, stress induced elevation of brain stem and optic tectum concentrations of the monoamine neurotransmitters serotonin, dopamine, and norepinephrine and their metabolites suggests that both synthesis and metabolism of these transmitters were elevated after stress to a larger degree in HR than in LR trout. A divergent pattern was seen in the hypothalamus, where LR fish displayed elevated levels of 5-hydroxyindoleacetic acid (a serotonin metabolite) and 3-methoxy-4-hydroxyphenylglycol (a norepinephrine metabolite). Thus, selection for a single trait, cortisol responsiveness, in rainbow trout is associated with concurrent changes in both behavior and central signaling systems. The apparent parallel to genetically determined stress coping styles in mammals, and the existence of similar trait associations in unselected populations of rainbow trout, suggests an evolutionarily conserved correlation between multiple traits. Continuing studies on the HR and LR trout lines are aimed at providing the physiological and genetic basis for new marker-assisted selection strategies in the rapidly developing finfish aquaculture industry, as well as increased knowledge of the function and evolution of central neuroendocrine signaling systems

The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices

Indicators of a generalised stress response (changes in cortisol, glucose, RNA:DNA ratio and total protein) when measured in whole-body preparations of individual sticklebacks display significant alterations in response to acute (hours) and chronic (days) disturbances and food withdrawal. In addition, changes in alkali-labile phosphorous, a specific biomarker of exposure to estrogenic contaminants, can be detected in whole-body preparations of estrogen-exposed sticklebacks confirming that the measurement of biomarkers normally assessed in a specific tissue can be equally possible in whole fish

Selective breeding to improve welfare in farmed fish: Modification of the stress response in rainbow trout (Oncorhynchus mykiss)

Intensive rearing of fish presents the challenge of minimising production cost without compromising the health and well-being of the stock. Most farmed fish species, (with the exception of carp) are just a few generations removed from the wild-type and consequently many relatively benign aspects of the aquaculture environment are perceived as stressors by fish. Although the neuroendocrine stress response is a key element of an animals adaptive repertoire, inappropriate activation of the response leads to poor growth, reproductive dysfunction and immunosuppression. One strategy that has been investigated to address this is to selectively breed for reduced stress responsiveness - by reducing the magnitude of response to conditions that elicit a stress response, the longer-term cumulative effects of stress on the performance and well-being of the population will be reduced. The trait associated with stress responsiveness that is most accessible to directed selection is the magnitude of the plasma cortisol response. Cortisol is known to be a causal agent in most of the adverse effects of stress in fish. This presentation will outline the results of a selective breeding programme to ameliorate stress in fish, including the finding that neuroendocrine differences arising from selection for either high or low activity of the hypothalamic-pituitary axis during stress are accompanied by a bimodality in behavioural characteristics and cognitive attributes. These suggest that the two selected lines exemplify two distinct combinations of physiological and behavioural strategies for dealing with stressful circumstances akin to the proactive and reactive coping strategies observed in mammals

Modulation of the stress response in wild fish is associated with variation in dissolved nitrate and nitrite

Disruption of non-reproductive endocrine systems in wildlife by chemicals has received little attention but represents a potentially significant problem. Nitrate is a major anthropogenic contaminant in the freshwater aquatic environment and has been identified as a potential disrupter of endocrine function in aquatic animals. This study was conducted to investigate the relationship between the function of the neuroendocrine stress axis in fish and inorganic N loading along reaches of rivers receiving cumulative point source and diffuse chemical inputs. To accomplish this, the responsiveness of the stress axis, quantified as the rate of release of cortisol to water across the gills during exposure to a standardised stressor, was measured in three-spined sticklebacks (Gasterosteus aculeatus L.) resident at three sites on each of four rivers in north-west England. The magnitude of the stress response in fish captured at the sites furthest downstream on all rivers was more than twice that of fish captured at upstream sites. Site-specific variation in stress axis reactivity was better explained by between-site variation in concentrations of dissolved nitrate, nitrite, and ammonia than by the concentration of wastewater treatment works effluent. An increase in the magnitude of the stress response was seen among sticklebacks at sites where long-term averaged concentrations of NH3-N, NO3-N and NO2-N exceeded 0.6, 4.0 and 0.1 mg/L respectively. These data suggest that either (i) inorganic N is a better surrogate than wastewater effluent concentration for an unknown factor or factors affecting stress axis function in fish, or (ii) dissolved inorganic N directly exerts a disruptive influence on the function of the neuroendocrine stress axis in fish, supporting concerns that nitrate is an endocrine-modulating chemical

Selection of rainbow trout (Oncorhynchus mykiss) lines for divergent stress responsiveness

A rationale is provided for undertaking the targeted manipulation of the stress axis in aquacultured fish by selective breeding. The results of a long-term investigation into the consequences of selection for stress responsiveness in rainbow trout are outlined with respect to endocrine characteristics, growth, reproductive performance, behaviour, and cognition. Future and ongoing studies are summarised

Extinction of a conditioned response in rainbow trout selected for high or low responsiveness to stress

Two lines of rainbow trout (Oncorhynchus mykiss) that exhibit divergent endocrine responsiveness to stressors also display disparate behavioral traits. To investigate whether the high-responding (HR) and low-responding (LR) fish also differ in cognitive function the rate of extinction of a conditioned response was compared between the two lines. Groups of HR and LR fish were exposed to a paired conditioned stimulus (CS; water-off) and unconditioned stimulus (US; confinement stressor). After exposure to 18 CS-US pairings at least 70% of individuals of both lines acquired a conditioned response (CR), manifested as an elevation of blood cortisol levels on presentation of the CS only. Post-conditioning, the fish were tested by presentation of the CS at weekly intervals, for 4 weeks, with no further reinforcement and the extinction of the CR in the two lines was compared. The decline in mean plasma cortisol levels after exposure to the CS over successive tests suggested that the CR was retained for a shorter period among the HR (<14 days) than LR fish (<21 days). The frequency of individuals within each line whose plasma cortisol levels indicated a stress response when exposed to the CS was significantly greater among the LR than HR fish at 14 and 21 days with no HR fish falling into this category at 21 days. At 28 days post-conditioning, there were no HR fish and only three LR fish that were categorized as “stressed”. These results suggest that there are differences in cognitive function between the two lines. Possible mechanisms underlying these differences are discussed

Contrasting baseline expression of stress axis genes in rainbow trout selected for divergent stress responsiveness

The expression of eight candidate genes, with roles implicated in the stress response and associated behaviour, was quantified in the brains of lines of rainbow trout, Oncorhynchus mykiss Walbaum, that were selectively bred for divergent cortisol responses to stress. These lines represent a low responding (LR) and high responding (HR) line that are known to differ in their stress physiology and behavioural phenotype with LR always dominating HR. To understand whether there is differential gene expression in the brains of these lines fish were held individually and then sampled for brain gene expression of eight candidate genes (AANAT, calcineurin, CRF, urotensin I, glucocorticoid receptor II, mineralocorticoid receptor, melatonin receptor 1a, and melanocortin 2 receptor) to obtain a better understanding of the gene expression profile between HR and LR. Seven genes were expressed at a significantly higher level in the low-responding line compared to the high-responding line, and four genes, AANAT, CRF, UI and CaN, were positively correlated with baseline plasma cortisol concentrations. Only AANAT was not significantly differentially expressed between the two stress lines (due to high variation among individuals). Hence there is clear evidence of a tangible relationship between gene expression and stress physiology in these animals prior to stress