Effect of Noxious Stimulation Upon Antipredator Responses and Dominance Status in Rainbow Trout

A potentially painful experience may modify normal behavioural responses. To gauge the importance of pain relative to predation or social status, we presented competing stimuli, a predator cue or an unfamiliar social group, to two groups of noxiously treated rainbow trout, Oncorhynchus mykiss. In the predator cue experiment, fish were classified as bold or shy. Noxiously stimulated fish did not show antipredator responses, suggesting that pain is the imperative. In the social status experiment, noxiously stimulated fish held individually and undisturbed showed an increase in respiration rate and plasma cortisol. As a comparison, we used the dominant or subordinate fish in a group as the noxiously stimulated fish. After the noxious treatment, we returned this test fish to a familiar or unfamiliar social group. Neither dominants nor subordinates showed a negative change in physiology compared to their controls. However, in a familiar group the dominant was much less aggressive, suggesting a behavioural impairment in response to noxious stimulation. In an unfamiliar group, no reduction of aggression was seen, suggesting that maintaining dominance status took priority over showing signs of pain. These findings may reflect an ability to prioritize motivational drivers in fish, and as such provides evidence for central processing of pain rather than merely showing a nociceptive reflex. In animal models of pain, exposure to a new circumstance or a potentially fear-inducing or stressful situation reduces pain reactivity (e.g. Lester & Fanselow 1985; Harris & Westbrook 1994; Kavaliers & Colwell 1994; Gentle & Corr 1995; Gentle & Tilston 1999; Del Seppia et al. 2003; Nakama-Kitamura & Doe 2003; Smith et al. 2003). For example, chickens, Gallus gallus domesticus, with gouty arthritis exposed to a novel environment showed a reduction in their pain-related responses including the severity of inflammation in the affected joint (Gentle & Corr 1995; Gentle & Tilston 1999). Rodents exposed to predator odour also show reduced behavioural responses to pain, and anxiety tests have the same effect (Lester & Fanselow 1985; Kavaliers & Colwell 1991; Nakama-Kitamura & Doe 2003; Geerse et al. 2006). In humans, pain takes priority when it is chronic or particularly intense, and concurrent tasks are poorly performed (Kuhajda et al. 2002). Therefore, if pain is a high priority, it will affect behavioural responses to other stimuli (Eccleston 1995; Moseley & Arntz 2007). These studies suggest that measuring the responses to competing stimuli during a painful event could be exploited as a tool to determine the relative importance of pain or nociception to an animal. Direct assessment of pain in animals is impossible owing to its subjective nature. However, many studies have identified behavioural and physiological responses to a potentially painful event in a variety of animals including fish (Sneddon et al. 2003a), amphibians (Willenbring & Stevens 1995), birds (Machin 2005) and mammals (Flecknell & Roughan 2004). Pain in animals is a contentious issue, especially for the coldblooded vertebrates because of differences in their neurobiology from that of higher vertebrates (Chandroo et al. 2004). However, nociceptors have been identified in fish and these are similar to those found in mammals (Sneddon 2002, 2003a; Sneddon et al. 2003a; Ashley et al. 2006, 2007). Moreover, studies have shown that the brain of the fish is active during noxious stimulation and that this activity differs from the response to neutral stimuli (Dunlop & Laming 2005; Reilly et al. 2008a). Negative changes in behaviour and physiology have also been recorded (Sneddon et al. 2003a, b), suggesting an aversive affective state, and these are reduced by administering an analgesic (Sneddon 2003b). However, the real significance of this experience to the fish has not been explored. We tested this by providing noxiously stimulated rainbow trout, Oncorhynchus mykiss, with one of two different contexts to examine whether pain is the imperative. One context was the presentation of an antipredator cue to gauge responses to this threatening stimulus and the other was social novelty where fish were placed into an unfamiliar social situation. Previous work demonstrated that noxiously stimulated fish do not show the classic neophobia when a fear-inducing stimulus, a novel object, is introduced (Sneddon et al. 2003b), indicating that pain dominates their attention in this context. However, it is possible that the novel objects used in that study were not sufficiently threatening to divert the fish’s attention. In this study, we used more ecologically relevant and important distractors such as predation and social groupings to test the relative importance of noxious stimulation in these scenarios. Avoiding being eaten by predators is a strong motivational drive for all prey animals and is, therefore, likely to be a strong stimulus to use as a competing stimulus. Fish are very sensitive to the presence of predators and predator-naïve fish show antipredator behaviours if they are given the odour of a predator in combination with the odour obtained from a damaged prey fish (Mirza & Chivers 2003; Zhao et al. 2006). This latter odour is called alarm substance or pheromone and is produced by damaged fish skin to elicit antipredator responses in conspecifics (Brown 2003; Scott et al. 2003). Antipredator behaviour can consist of freezing where the fish remains motionless, erratic escape swimming, increased refuge or cover use, sinking and spending more time in the bottom of the tank and a reduction in feeding attempts (Scheurer et al. 2007). Because these behaviours assist in avoiding detection, we used an antipredator cue to determine whether noxiously stimulated fish perform appropriate antipredator behaviour. Rainbow trout are territorial and naturally form dominance hierarchies, usually including a bold individual that restricts the behaviour of subordinates (Sneddon et al. 2005). Individuals of lower status have higher stress levels as a result of losing contests with the dominant and have reduced access to food (Gilmour et al. 2005). Therefore, dominance status influences the probability of survival and overall fitness. As such, maintaining status is likely to be an important motivational driver in the behaviour of this species. We tested the behavioural and physiological responses of the top-ranked fish (dominant) and the lowestranked fish (subordinate) in a group to determine whether they show signs of pain or nociception when held in familiar groups where their dominance is already established. This was compared to returning the noxiously stimulated fish to unfamiliar groups where their dominance had not been established. Behavioural analysis of large groups can be confounded by individual variation linked to boldness and this can affect responses to noxious stimulation (L. U. Sneddon, K. L. Edwards, S. Ringrose, L. J. Ashley & C. R. McCrohan, unpublished data). Rainbow trout can be either bold and aggressive or shy and timid, as reflected in their behaviour (Sneddon 2003c; Frost et al. 2007). Bold fish tend to take more risks, are more active, spend more time in open water, learn conditioning tasks faster and dominate shy fish (Sneddon 2003c; Frost et al. 2007). Therefore, we first determined the degree of boldness of our experimental subjects to investigate whether the response to noxious stimulation was affected by the ‘personality’ of the fish. We hypothesized that bold fish may be more likely to recover from a noxious event and respond to concurrent stimuli, such as exploring a new environment. We also hypothesized that, if a potentially painful stimulus is important to the fish, noxiously stimulated fish will not respond in an appropriate manner to a predator cue. This may also be affected by the degree of boldness of the fish whereby bold fish may show more risk-prone behaviours during the presentation of the predator cue than shy fish. Finally, we tested the hypothesis that noxiously stimulated fish held in social groups may appear less affected by pain than individually held fish as they need to maintain their social status.