Differential metabolic responses in bold and shy sea anemones during a simulated heatwave.

As climate change-induced heatwaves become more common, phenotypic plasticity at multiple levels is a key mitigation strategy by which organisms can optimise selective outcomes. In ectotherms, changes to both metabolism and behaviour can help alleviate thermal stress. Nonetheless, no study in any ectotherm has yet empirically investigated how changing temperatures affect among-individual differences in the associations between these traits. Using the beadlet anemone (Actinia equina), an intertidal species from a thermally heterogeneous environment, we investigated how individual metabolic rates, linked to morphotypic differences in A. equina, and boldness were related across changing temperatures. A crossed-over design and a temporal control were used to test the same individuals at a non-stressful temperature, 13°C, and under a simulated heatwave at 21°C. At each temperature, short-term repeated measurements of routine metabolic rate (RMR) and a single measurement of a repeatable boldness-related behaviour, immersion response time (IRT), were made. Individual differences, but not morphotypic differences, were highly predictive of metabolic plasticity, and the plasticity of RMR was associated with IRT. At 13°C, shy animals had the highest metabolic rates, while at 21°C, this relationship was reversed. Individuals that were bold at 13°C also exhibited the highest metabolic rates at 21°C. Additional metabolic challenges during heatwaves could be detrimental to fitness in bold individuals. Equally, lower metabolic rates at non-stressful temperatures could be necessary for optimal survival as heatwaves become more common. These results provide novel insight into the relationship between metabolic and behavioural plasticity, and its adaptive implications in a changing climate.

The impact of personality, morphotype and shore height on temperature-mediated behavioural responses in the beadlet anemone Actinia equina.

Between-individual variation in behavioural phenotype, termed personality, is an important determinant of how populations cope with acute environmental fluctuation related to climate change. Personality in the beadlet sea anemone Actinia equina is linked to genetically distinct morphotypes, which are associated with different heights on the shore. In the intertidal zone, high-shore environments experience more environmental fluctuation due to longer periods of exposure, and animals adapted to live in these environments are predicted to deal more effectively with environmental perturbation than their low-shore counterparts. We collected beadlet anemones of two different morphotypes from three different shore heights. We investigated variation in two behaviours at three different temperatures and in a temporal control treatment where the temperature was not changed: startle response time, the time it took an anemone to re-extend its tentacles after a threatening stimulus, and immersion response time, the time to re-extend tentacles after simulated tidal immersion. These behaviours reflect risk-taking and allow individuals to be categorized as bold, shy or intermediate based upon response times. Both behaviours showed significant changes as the temperature increased. For immersion response, the morphotype associated with the low-shore-lengthened response times at high temperatures. For startle response, all animals lengthened their response times at high temperatures but animals collected from the low-shore lengthened theirs to the greatest degree. At the individual level, although control individuals exhibited temporal changes in their response times, a clear effect of temperature was present in both behaviours. Shy and bold individuals became more intermediate at higher temperatures in immersion response (this effect was present to a lesser degree in control individuals), while intermediate individuals raised their response times at higher temperatures for startle response. Given that prolonged tentacle retraction reduces foraging opportunities and can negatively impact respiratory efficiency, our data suggest that some individuals within a single population of A. equina, particularly those associated with the lower shore, may exhibit less effective behavioural responses to temperature shifts than others. These findings demonstrate that acute temperature changes influence risk-taking, and could have profound short and long-term implications for survival in the face of climate change.

Acute and chronic stress prevents responses to pain in zebrafish: evidence for stress-induced analgesia.

The state of an animal prior to the application of a noxious stimulus can have a profound effect on their nociceptive threshold and subsequent behaviour. In mammals, the presence of acute stress preceding a painful event can have an analgesic effect whereas the presence of chronic stress can result in hyperalgesia. While considerable research has been conducted on the ability of stress to modulate mammalian responses to pain, relatively little is known about fish. This is of particular concern given that zebrafish (Danio rerio) are an extensively used model organism subject to a wide array of invasive procedures where the level of stress prior to experimentation could pose a major confounding factor. This study, therefore, investigated the impact of both acute and chronic stress on the behaviour of zebrafish subjected to a potentially painful laboratory procedure, the fin clip. In stress-free individuals, those subjected to the fin clip spent more time in the bottom of the tank, had reduced swimming speeds and less complex swimming trajectories; however, these behavioural changes were absent in fin-clipped fish that were first subject to either chronic or acute stress, suggesting the possibility of stress-induced analgesia (SIA). To test this, the opioid antagonist naloxone was administered to fish prior to the application of both the stress and fin-clip procedure. After naloxone, acutely stressed fin-clipped zebrafish exhibited the same behaviours as stress-free fin-clipped fish. This indicates the presence of SIA and the importance of opioid signalling in this mechanism. As stress reduced nociceptive responses in zebrafish, this demonstrates the potential for an endogenous analgesic system akin to the mammalian system. Future studies should delineate the neurobiological basis of stress-induced analgesia in fish.

Assessment of behaviour in groups of zebrafish (Danio rerio) using an intelligent software monitoring tool, the chromatic fish analyser.

BACKGROUND: Zebrafish (Danio rerio) are an increasingly popular model species within a variety of biomedical and neurobiological contexts. Researchers are required to prevent any negative states, such as pain, when using experimental animals to optimise fish welfare but analysis tools for zebrafish are lacking. NEW METHOD: The chromatic fish analyser (CFA) is a computer-based monitoring system that has the potential to identify changes in fish behaviour via spatial chromatic analysis of video images. The CFA was used to monitor the behaviour of groups of six fish, where none, one, three or six fish were given a fin clip. Additionally a drug with pain-relieving properties, lidocaine, was administered to determine if this ameliorated any alterations in behaviour. The CFA measured hue horizontally and vertically reflecting the position of the fish in their tank. Saturation (indicates clustering distribution) and lightness were measured to reflect overall zebrafish activity. RESULTS: Changes in vertical hue demonstrated that all fin clipped animals were closer to the bottom of the tank relative to pre-treatment; this was not observed in control groups, and was alleviated in those treated with lidocaine. Saturation (clustering) and lightness alterations indicated fin clipped groups reduced activity after receiving the fin clip. Lidocaine was effective in preventing the behavioural changes when 1 or 3 fish were clipped. CONCLUSIONS: The CFA proved powerful enough to identify significant changes in behaviour taken directly from video images. With further development this monitoring tool represents a step forward in detecting behavioural changes in groups of zebrafish indicating welfare.

Automated monitoring of behaviour in zebrafish after invasive procedures.

Fish are used in a variety of experimental contexts often in high numbers. To maintain their welfare and ensure valid results during invasive procedures it is vital that we can detect subtle changes in behaviour that may allow us to intervene to provide pain-relief. Therefore, an automated method, the Fish Behaviour Index (FBI), was devised and used for testing the impact of laboratory procedures and efficacy of analgesic drugs in the model species, the zebrafish. Cameras with tracking software were used to visually track and quantify female zebrafish behaviour in real time after a number of laboratory procedures including fin clipping, PIT tagging, and nociceptor excitation via injection of acetic acid subcutaneously. The FBI was derived from activity and distance swum measured before and after these procedures compared with control and sham groups. Further, the efficacy of a range of drugs with analgesic properties to identify efficacy of these agents was explored. Lidocaine (5 mg/L), flunixin (8 mg/L) and morphine (48 mg/L) prevented the associated reduction in activity and distance swum after fin clipping. From an ethical perspective, the FBI represents a significant refinement in the use of zebrafish and could be adopted across a wide range of biological disciplines.

Plasticity of boldness in rainbow trout, Oncorhynchus mykiss: do hunger and predation influence risk-taking behaviour?

Boldness, a measure of an individual's propensity for taking risks, is an important determinant of fitness but is not necessarily a fixed trait. Dependent upon an individual's state, and given certain contexts or challenges, individuals may be able to alter their inclination to be bold or shy in response. Furthermore, the degree to which individuals can modulate their behaviour has been linked with physiological responses to stress. Here we attempted to determine whether bold and shy rainbow trout, Oncorhynchus mykiss, can exhibit behavioural plasticity in response to changes in state (nutritional availability) and context (predation threat). Individual trout were initially assessed for boldness using a standard novel object paradigm; subsequently, each day for one week fish experienced either predictable, unpredictable, or no simulated predator threat in combination with a high (2% body weight) or low (0.15%) food ration, before being reassessed for boldness. Bold trout were generally more plastic, altering levels of neophobia and activity relevant to the challenge, whereas shy trout were more fixed and remained shy. Increased predation risk generally resulted in an increase in the expression of three candidate genes linked to boldness, appetite regulation and physiological stress responses - ependymin, corticotrophin releasing factor and GABA(A) - but did not produce a significant increase in plasma cortisol. The results suggest a divergence in the ability of bold and shy trout to alter their behavioural profiles in response to internal and exogenous factors, and have important implications for our understanding of the maintenance of different behavioural phenotypes in natural populations.

Physiological and genetic correlates of boldness: characterising the mechanisms of behavioural variation in rainbow trout, Oncorhynchus mykiss.

Bold, risk-taking animals have previously been putatively linked with a proactive stress coping style whereas it is suggested shyer, risk-averse animals exhibit a reactive coping style. The aim of this study was to investigate whether differences in the expression of bold-type behaviour were evident within and between two lines of rainbow trout, Oncorhynchus mykiss, selectively bred for a low (LR) or high (HR) endocrine response to stress, and to link boldness and stress responsiveness with the expression of related candidate genes. Boldness was determined in individual fish over two trials by measuring the latency to approach a novel object. Differences in plasma cortisol concentrations and the expression of eight novel candidate genes previously identified as being linked with divergent behaviours or stress were determined. Bold and shy individuals, approaching the object within 180 s or not approaching within 300 s respectively, were evident within each line, and this was linked with activity levels in the HR line. Post-stress plasma cortisol concentrations were significantly greater in the HR line compared with the LR line, and six of the eight tested genes were upregulated in the brains of LR fish compared with HR fish. However, no direct relationship between boldness and either stress responsiveness or gene expression was found, although clear differences in stress physiology and, for the first time, gene expression could be identified between the lines. This lack of correlation between physiological and molecular responses and behavioural variation within both lines highlights the complexity of the behavioural-physiological complex.