Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Linking Fearfulness and Coping Styles in Fish

  • Catarina I. M. Martins ,

    cimartins@ualg.pt

    Affiliations Centro de Ciências do Mar (CCMAR), Universidade do Algarve, Faro, Portugal, Aquaculture and Fisheries Group, Wageningen University, Wageningen, The Netherlands

  • Patricia I. M. Silva,

    Affiliations Centro de Ciências do Mar (CCMAR), Universidade do Algarve, Faro, Portugal, Institute of Aquatic Resources, Section for Aquaculture, North Sea Center, Danish Technical University, Hirtshals, Denmark, Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, Ås, Norway

  • Luis E. C. Conceição,

    Affiliation Centro de Ciências do Mar (CCMAR), Universidade do Algarve, Faro, Portugal

  • Benjamin Costas,

    Affiliations Centro de Ciências do Mar (CCMAR), Universidade do Algarve, Faro, Portugal, Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Universidade do Porto, Porto, Portugal

  • Erik Höglund,

    Affiliation Institute of Aquatic Resources, Section for Aquaculture, North Sea Center, Danish Technical University, Hirtshals, Denmark

  • Øyvind Øverli,

    Affiliation Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, Ås, Norway

  • Johan W. Schrama

    Affiliation Aquaculture and Fisheries Group, Wageningen University, Wageningen, The Netherlands

Linking Fearfulness and Coping Styles in Fish

  • Catarina I. M. Martins, 
  • Patricia I. M. Silva, 
  • Luis E. C. Conceição, 
  • Benjamin Costas, 
  • Erik Höglund, 
  • Øyvind Øverli, 
  • Johan W. Schrama
PLOS
x

Abstract

Consistent individual differences in cognitive appraisal and emotional reactivity, including fearfulness, are important personality traits in humans, non-human mammals, and birds. Comparative studies on teleost fishes support the existence of coping styles and behavioral syndromes also in poikilothermic animals. The functionalist approach to emotions hold that emotions have evolved to ensure appropriate behavioral responses to dangerous or rewarding stimuli. Little information is however available on how evolutionary widespread these putative links between personality and the expression of emotional or affective states such as fear are. Here we disclose that individual variation in coping style predicts fear responses in Nile tilapia Oreochromis niloticus, using the principle of avoidance learning. Fish previously screened for coping style were given the possibility to escape a signalled aversive stimulus. Fearful individuals showed a range of typically reactive traits such as slow recovery of feed intake in a novel environment, neophobia, and high post-stress cortisol levels. Hence, emotional reactivity and appraisal would appear to be an essential component of animal personality in species distributed throughout the vertebrate subphylum.

Introduction

Individual variation in the physiological and behavioural responses to aversive stimuli is increasingly viewed as adaptive responses that are crucial for survival in a continuously changing environment [1]. In contrast to the presumed advantages of flexible responses, when faced with changing environmental conditions, individuals of the same species or population show consistent responses in stressful and dangerous situations [2], [3], [4]. This phenomenon is referred to as animal personality [5], behavioural syndrome [6], temperament [7], or coping style [2]. In general, some individuals show a proactive behavioural pattern, consistently being more aggressive, more explorative, more neophilic, and more actively avoiding danger than their reactive counterparts. In addition to consistent differences in behavioural traits that correlate among each other, proactive and reactive individuals also differ in neuro-endocrine traits. Proactive individuals have a low hypothalamus-pituitary adrenal/ interrenal (HPA, HPI in fish) axis responsiveness, but high sympathetic reactivity, while the opposite is true for reactive individuals [2], [3], [8]. There is evidence that the physiological traits correlated to animal personality are heritable (e.g. [9], [10]), and contrasting personalities are associated with different fitness consequences [5], which suggests that personality is subjected to evolutionary processes. Likewise, emotions are thought to confer survival advantages by giving animals the ability to avoid harm/punishments and seek valuable resources/reward (e.g. [11], [12]). Under an evolutionary point of view, therefore, emotions - by being functional and adaptive - are unlikely to have evolved spontaneously in the recent human lineage. In addition, the capacity for emotions is likely to differ substantially between species as a consequence of both evolutionary lineage and selective pressures associated with life history [13]. Fear, for example, as a negative emotion increases precautionary behaviour, allowing individuals to avoid potential threat or danger and, therefore has an adaptive value [14].

There are indications that certain stimuli are appraised as fearful in a wide variety of animal groups. This has been demonstrated by behavioural responses to direct exposure to novelty and/or predators (e.g. [15][19]). Such responses in fish have been used to describe differences in boldness, and have been interpreted in different ways, such as neophobia [19], reduced exploration or hesitancy [17] or emotional reactivity [18] including fearfulness [15], [16]. However, to which extent responses to direct exposure to aversive stimuli involves common phylogenic roots of cognitive processes involved in fear, such as appraisal, is largely unknown.

The link between personality or coping styles and emotions, including fear, has been addressed in humans, non-human mammals and birds. The individual variation in the threshold for when a stimulus becomes inhibiting rather than stimulatory, i.e. coping style (sensu [2]) is likely correlated to the individual's subjective experience of that stimulus in a given situation. Different personality types have been shown to differ in emotional reactivity [20], the reactivity to negative appraisals [21] and susceptibility to psychological illness [22]. Fear reactivity, for example, has been shown to be a dimension of temperament in humans [23], [24] influencing the susceptibility to depression and anxiety [25]. However, how evolutionary widespread these putative links between personality and the expression of fear are remains to be studied.

Utilizing a teleost fish as a comparative vertebrate model allows investigation of the link between emotions and endocrinal and behavioural dimensions of coping styles in this animal group. Further, this will add to our understanding of the evolutionary relevance and adaptive value of personality, and unravel whether emotions are an essential component of coping styles in species distributed throughout the vertebrate subphylum.

We investigated whether coping styles can predict fear responses in fish using the principle of avoidance learning (combination of classical and operant conditioning). Fish previously screened along the proactive-reactive styles continum (using 3 subsequent tests: feed recovery after transfer itno a novel environemnt, novel object and net restraining) were given the possibility to escape an aversive stimulation that was associated with a cue signalling the onset of the aversive stimuli. In this study, individuals of Nile tilapia were subjected to a signaled aversive stimulus for 7 days (conditioned stimulus, CS: stopping water inflow for 30 sec; unconditioned stimulus, US: confinement stress by lowering a frame into the tank until touching the dorsal fin). Afterwards fish were exposed to the CS only and were allowed to escape from the previous confinement area by using an escape door. The individual variation in escape behavior in this fish was registered and related with the behavior and neuro-endocrine profiling of the same fish screened for coping styles.

Nile tilapia, Oreochromis niloticus was used as a model species due to its well characterized behaviour, endocrine and physiological profiles in different behavioural paradigms, including conditioning [26], [27].

Results

Coping styles in Nile tilapia

Feed intake recovery after transfer into a novel environment was shown to predict neophobia (rs = 0.45, p = 0.027, Fig. 1). This suggests that fish recovering their feed intake faster after transfer to a novel environment show lower neophobic response when exposed to a novel object, i.e. traits typically ascribed to bold individuals.

thumbnail
Figure 1. Relationship between feed intake recovery after transfer to a novel environment and neophobia (n = 24).

https://doi.org/10.1371/journal.pone.0028084.g001

No correlation was however found between cortisol after the net restraining stress, feed intake recovery and the behaviour during the novel object test (p>0.05).

Avoidance learning

Latency to escape from the conditioned stimulus (CS, stopping the water inflow, from now on water off) decreased significantly over the 7 days of training (one-way repeated measures ANOVA, F3.10,71.3 = 14.6, p<0.001). On training day 1 fish took, on average, 513 sec to escape, and by day 7 fish were escaping in less than 30 sec (p = 0.001, Bonferroni comparison, Fig. 2). During avoidance learning, 22 fish (out of 24) learned to associate the CS (water off) with the unconditioned stimulus (US, exposure to a confinement stress); i.e. escaped even in the absence of the confinement frame on day 8. The 2 fish that did not learn were excluded from the analysis concerning the link between coping styles and avoidance learning. It should be noted, however, that these fish did not represent outlier values in regard to previously measured variables.

thumbnail
Figure 2. Reduction in latency to escape of T fish over the 7 days of CS-US pairing

. Each point represents the mean ± SE of 24 individuals. Different letters denote statistical significance at a significant level of p<0.05 after repeated ANOVA and Bonferroni comparisons.

https://doi.org/10.1371/journal.pone.0028084.g002

Control and treatment fish did not differ significantly in the latency to escape (Fig. 3, p>0.05, Kruskall Wallis test). However, when the time between first escape and return is considered (Figure 3C) significant differences were detected (p<0.001). Fish exposed to the confinement stressor only (C2- confinement) and in combination with water off (C3-water off/confinement), escaped through the partition door and did not return to the side where the confinement frame was inserted. Fish exposed to water off only during the 7 days of training exhibited the lowest time between escaping and returning (25.2±12.09 sec) while fish exposed to water off only on day 8 after 7 days of pairing between water off and confinement showed a significantly higher time between escaping and returning (343.9±71.44 sec, p = 0.003, Dunn's comparison). The number of returns and time spent in the confinement area was also higher in C1-water off (# returns: 6.4±1.3; time spent in confinement area: 488.4±76.6 sec) as compared with T-learning (# returns: 4.9±0.9; time spent in confinement area: 378.2±61.8 sec) but not significantly different (p>0.05).

thumbnail
Figure 3. Comparison of escape behavior between T and C1-C3 fish

. Latency to escape (A), time spent in confinement area (B), time between 1st escape and 1st return to confinement area (C) and total number of returns to confinement area (D) in C1C3 (n = 6 in C1 and C2 and n =  5 in C3) and T on day 8, after 7 days of training (n = 22, 2 fish did not escape on day 8 and were not included).

https://doi.org/10.1371/journal.pone.0028084.g003

The relationship between coping styles and avoidance learning

Fish exposed to T-learning showed a pronounced individual variation in escape responses. Individuals that took less time to escape were also the individuals that took longer to return to the side of previous confinement (rs = −0.60, p = 0.009) and spent less time in the confinement area on day 8 (rs = 0.44, p = 0.039) while in addition showing the highest cortisol levels in the end of the avoidance learning test (rs = −0.44, p = 0.045), suggesting that fish escaping faster, taking longer to return and spending less time in the confinement area were more stressed even in the absence of the confinement frame.

Time to return after escaping was shown to be correlated positively to cortisol level after the net restraining stress applied on day 35 (rs = 0.60, p = 0.009, Table 1). On the contrary, individuals returning more often to the area of previous confinement (number of returns) and spending more time in that area, exhibited typical characteristics of bold individuals such as lower cortisol response after net restraining (rs = −0.48, p = 0.025,), higher feed intake after transfer to a novel environment (r = 0.44, p = 0.041), less neophobia when exposed to a novel object (r = 0.54, p = 0.01 with number of times entering 10 cm radius and r = 0.47, p = 0.029 with number of times entering 5 cm radius) and more actively trying to escape when restrained (rs = 0.58, p = 0.005).

thumbnail
Table 1. Correlation between variables indicating coping styles and fearfulness.

https://doi.org/10.1371/journal.pone.0028084.t001

Discussion

It is now generally accepted that in fish, individual variation in behaviour and physiology when exposed to environmental challenges, reflect the existence of coping styles [3], [28]. This study showed, for the first time, that Nile tilapia Oreochromis niloticus, also exhibits divergent coping styles with proactive individuals being characterized by a faster feed intake recovery after transfer into a novel environment and less neophobic when exposed to a novel object, as compared to reactive individuals. Such behavioural responses to challenges have also been described in other fish species [29][35].

In classical conditioning, repeated CS–US pairing results in the acquisition of a behavioural conditioned response (CR). In this study, behavioural conditioned response was observed after fish were exposed to the avoidance learning test. The escape behaviour differed significantly between C1-water off and the other controls and T-learning, as these fish, despite using the escape door returned very quickly to the side where the inflow water was interrupted. In C1-watter off, the use of the escape door is probably more related to exploration than to escape behaviour. Fish exposed to the US both alone or in combination with the CS, escaped to the other side of the tank and never returned during the 15 minutes of observation. Fish exposed to T-learning (pairing CS–US for 7 days followed by exposure to CS only on day 8) took longer to return to the area where the confinement frame was previously used as compared to fish exposed to water off only. Despite fish in C1-water off and T-learning were exposed to the same stimuli (water off), their behaviour differed significantly suggesting that the way the stimuli was interpreted or appraised also differed. This indicates that Nile tilapia can learn how to avoid aversive stimuli by conditioning. A previous study by [26] showed that Nile tilapia can be conditioned to display a stress response in response to conditioned stimuli. In the present study, in addition to classical conditioning, we allowed fish to escape from the aversive stimuli and the results suggest that Nile tilapia is capable of conditioned avoidance learning.

The reason why fish returned to the area of the tank where the confinement frame has been previously used is not clear. It should be noted that the area used for confinement was also the area used for feeding, therefore, one possibility is that the motivation to feed played a role in returning to a potentially dangerous area.

The concept of avoidance learning has been used to investigate fear in different animal species (e.g. in fish [36], [37]). The emergence of consciousness and feelings in fish has been a matter of intense scientific debate (e.g. [38][41]). Some authors [39][41] argue that this is not possible because their behaviour is simple and reflexive and they lack a neocortex. Yet, a growing body of evidence related to cognitive [42], neuroanatomic [43], [44] and emotional [36], [37], [45] aspects of fish behaviour provides strong support for the ability to feel in fish. In the present study, the observed differences in escape behaviour between fish exposed to C1-water off and T-learning suggest that these responses are not merely reflexive in nature but are associated with a subjective interpretation of the stimuli. If a reflexive response would be present one would have expected a similar behavioural response between fish exposed to the same stimulus (in our case, C1-water off and T-learning), which was not the case.

The way individual fish behaved when exposed to water off on day 8 (after 7 days of CS–US pairing) was shown to be correlated with traits indicative of coping styles. This suggests that the individual variation in how negative the CS was interpreted (negative appraisal) depends of an individuals' coping style. The link between coping styles and the subjective experience of stimuli and emotional responses has never been investigated in fish, despite studies showing that both (i.e. coping styles and emotions) are possible in fish. This study showed that fish avoiding the area of previous confinement were the fish exhibiting characteristics usually ascribed to reactive or shy individuals, such as lower feed intake recovery after transfer into a novel environment, more neophobic and higher HPI responsiveness after net restraining as compared to proactive or bold individuals. One possible explanation could be a difference in behaviour flexibility between reactive and proactive individuals, in what proactive individuals would be more flexible and therefore prone to modify learned behaviours (in this case the association between water off and the onset of confinement resulting in escaping behaviour). This explanation seems, however, unlikely as proactive individuals were shown to be less flexible in modifying learned behaviour than reactive individuals [46]. An alternative explanation is that individuals of the proactive type were less fearful when presented with a signal previously associated with an aversive stimulus, as compared to individuals of the reactive type. Fear is an important component of personality in humans [24], [47], other mammals (e.g., in dogs [48]; in rats [20], [49]) and in birds [50]. The argument for the link between coping styles and fearfulness in fish is evolutionary: fearfulness may be adaptive as it allows individuals to avoid potential threat or danger; from this view, it follows that individual variation in the threshold for when a stimuli becomes inhibitory or stimulatory, i.e. coping style, is likely to be linked with the subjective experience of that stimulus in a particular situation. Severe, chronic and/or unpredictable conditions are likely to provide reactive coping more benefits while mild, intermittent stress and/or predictable conditions are likely to favor proactive responses [51]. Therefore, emotional distress is likely an essential component of reactive coping. This study suggests that the link between coping styles/personality and the expression of emotional or affective states such as fear is an evolutionary widespread phenomenon throughout the vertebrate subphylum, including fish.

This study showed for the first time that cortisol is strongly linked to behaviours indicating fearfulness. A key question that remains to be investigated is whether the link between cortisol responsiveness and fear responses is based on a cause or effect connection. Does the fear reaction potentiate cortisol response, or does elevated cortisol exposure over time alter limbic structures in the brain that mediate fear responses [52]? Further studies are needed to unravel the time course and coordination of psychological and biological stress responses. Extensions of this study could be the investigation of the underlying brain activity in (e.g. through monoamine activity) in differential brain parts, particularly in the medial pallium, an area that is believed to be homologous of the amygdala of land vertebrates [53] and to play an important role in fear responses [54].

This study provides the first evidence that in fish, similarly to what has been found in other vertebrates, individual's coping style is predictive of how stimuli are appraised and the subsequent degree of avoidance behaviour. These results support the inclusion of emotional reactivity and appraisal as essential component of animal personality in species distributed throughout the vertebrate subphylum.

Materials and Methods

This experiment was approved by the Ethical Committee judging Animal Experiments (DEC no 2009049) of the Wageningen University, The Netherlands.

Experimental animals, housing and feeding

Forty-two juveniles of Nile tilapia Oreochromis niloticus with an initial body weight of 40.8±0.8 g (means±SE) were used as experimental animals. From these, 24 individuals, randomly selected, were used to characterize coping styles and avoidance learning while the remaining fish were used as controls in the avoidance learning test. All animals were obtained from a local tilapia producer (all-male, TilAqua, The Netherlands) where they had experienced common housing and feeding conditions. Upon arrival at Wageningen University, fish were group-housed in a stock tank for 15 days until the start of the experimental procedures. During this period fish were fed ad libitum with a commercial diet (2 mm floating pellets; 44% crude protein, 10% fat, 25% carbohydrates, 11.5% ash; Skretting, France) twice a day (08:00 and 16:00) by hand. The same feed was used during the experimental procedures.

During the screening for coping styles (35 days) and avoidance learning (8 days), fish were housed individually in a 40-L glass aquarium (40 cm length×30 cm width×35 cm height, 30 L water capacity, water flow rate was 4 L min−1). Tanks were part of a recirculation system operated at a water refreshment rate of 1500 L kg feed−1 d−1 [55].

Water temperature (26.5±0.1°C), pH (range between 8.6 and 8.7), conductivity (1.96±0.01 mS cm−1), TAN (0.05±0.03 mg L−1), NO2-N (0.00±0.00 mg L−1) and NO3-N (46.0±2.7 mg L−1) were checked daily. A 12 h: 12 h light: dark photoperiod was maintained with daybreak set at 7:00 h.

Coping styles

Screening for coping styles consisted of subjecting each fish to 3 subsequent tests: 1) novel environment (based on [29], [56]), 2) novel object test (based on [57]) and 3) net restraining test (based on [55]).

The novel environment test consisted of transferring individual fish to a 40-L glass aquarium and following daily feed intake recovery for 14 days. Fish (n = 24) were fed ad libitum, by hand, twice per day (08:00 and 16:00) using the same commercial feed as used during the previous 15 days. Feeding continued for a maximum of 1 h, after which the remaining pellets were collected and counted. The average feed intake of the 1st week after transfer to the novel environment was used as indicative of feed intake recovery.

Individually housed fish were kept visually isolated from one another by black plastic around tanks, except for the front side which allowed daily visual observations of the fish.

The novel object test (day 30, after onset of isolation) consisted of a sudden drop of a weighted red LEGO brick (3×3×2 cm, length×width×height) in the middle of the tank, using transparent fishing line attached to the brick to avoid visual contact between the fish and researcher. A mesh screen with squared holes (1 cm) was used on top of the aquarium to allow the determination of the number of times fish entered a 5 and 10 cm radius around the novel object. The latency to enter the 5 cm radius area was also determined using a stopwatch. Fish was considered within the 10 or 5 cm cut-offs when the head was inside that area. The observation period lasted 15 minutes after which the novel object was gently removed.

The net restraining test was conducted on day 35 and consisted of keeping each fish in an emerged net for 60 sec followed by 1 h in the respective tanks (based on [55]). While in the net, the escape behaviour of each fish was determined by counting the number of escape attempts (i.e. body displacements). Blood samples were collected 1 h after the start of net restraining. Fish were rapidly netted and placed in 0.3 g L−1 of tricaine methanesulfonate (TMS, Crescent Research Chemicals, Phoenix, Arizona, USA using 0.6 g L−1 of sodium bicarbonate as buffer). One mL of blood was collected from all fish by hypodermic syringe (containing 3 mg of Na2EDTA) from the caudal blood vessels. This procedure was finalized within 3 min after fish were caught and anaesthetized. The collected blood was placed in cooled 1.5 mL plastic tubes, mixed and centrifuged at 6000×g for 5 min at 4°C. After centrifugation plasma was collected and stored at −20°C until cortisol analysis (see below).

Avoidance learning

After being screened for coping styles each fish was exposed to an avoidance learning paradigm for 8 days (Fig. 4). Four different experimental groups of fish were established: A treatment group (T- learning, n = 24) underwent the full avoidance learning test utilising a signalled aversive stimulus (unconditioned stimulus, US). The conditioned stimulus (CS) consisted of stopping the water inflow for 30 sec (from now on water off). The US consisted of an iron frame (14 cm×35 cm) lowered into the tank until touching the dorsal fin of the fish, and then remaining there for 15 min. Additionally, 3 different control groups were established (C1- water off, C2-confinement and C3- water off/confinement). Controls were used to test the influence of CS only (C1: n = 6 fish were exposed to water off once daily during 8 days), US only (C2: n = 6 fish were exposed during 8 days to the confinement frame only, without previous signaling) and CS–US pairing (C3, n = 5, fish were exposed to CS–US pairing for 8 days, see Figure 1). C3 and T were exposed to the same procedures during 7 days of training, but on day 8, T was exposed to CS only while C3 to CS followed by US.

thumbnail
Figure 4. Schematic representation of the experimental set-up used during the avoidance learning test.

Fish exposed to avoidance learning (T-learning, n = 24) were trained for 7 days to associate water off (CS) with the onset of a confinement stress (US) followed by exposure to CS only on day 8. Fish in C1-water off (n = 6) were exposed to the CS only, i.e. water off during 8 days; Fish in C2- confinement (n = 6) were exposed to the US only, i.e., confinement during 8 days without previous signaling by stopping the water inflow; Fish in C3-water off/confinement (n = 5) were exposed to CS–US pairing for 8 days. During the 7 days of training the latency to escape was determined. On day 8 in addition to the escape behaviour measures also blood was collected (15 minutes after the start of the US or CS) for cortisol measurements.

https://doi.org/10.1371/journal.pone.0028084.g004

Each tank was divided in 2 partitions using a PVC divider containing an escape door (half circle, 8 cm diameter) that was opened upon CS presentation. Fish were trained to associate US with CS for 7 days (1 training per day). The latency to escape (i.e. to swim to the side with no confinement frame) was determined daily. In addition to the latency to escape, at this step also the time taken between the first escape and the first return, the total number of returns and the total time spent in the (previous) confinement area, were registered. These behaviours were used as a measure of the degree of responsiveness to a frightening stimulus (based on [36]). After 15 min of observation on day 8 (during this time fish could choose whether and when to return to the previous confinement area), fish were netted and rapidly killed by severing the spinal cord just behind the head. Afterwards, blood (for cortisol analysis) were immediately collected. Blood was processed as described earlier.

Control fish were sampled (for blood), 15 minutes after the start of the US or CS. Fish used in C1C3 and T were all exposed to the experimental conditions prior to the start of the avoidance learning test (however in C1–C3 no coping styles data were collected).

Analysis of cortisol

Plasma cortisol levels were measured with a commercially available competitive binding Coat-A-Count® Cortisol kit (SIEMENS Medical Solutions Diagnostics, Los Angeles, CA, USA) adapted from [58]. Briefly, 50 µl of each sample to be assayed was transferred into an Ab-Coated tube and 1 ml of 125I Cortisol added. The tubes were then incubated for 45 min at 37°C in a water bath. The contents of all tubes were decanted, and allowed to drain for 5 min before being readonagammacounter (2470 WIZARD2TM, PerkinElmerTM, Inc., Zaventem, Belgium) for 1 min. A calibration curve was constructed on logit-log graph paper and used to convert results from percent binding cortisol to concentration (ng ml−1). The Coat-A-Count cortisol antiserum cross-reacts 100% with cortisol, 11.4% with 11-deoxycortisol, 0.98% with cortisone, 0.94% ith corticosterone and 0.02% with progesterone.

Data analysis

Statistical analyses were performed using SPSS 16.0 for windows. Relationships between variables were investigated using Spearman correlation. To determine whether latency to escape changed over the learning period, a repeated ANOVA (n = 24) was used followed by Bonferroni comparisons. The value of 1000 sec was used when fish did not escape during the 15 minutes observation period. Kruskal Wallis test and Dunn's post-hoc comparison were used to compare the escape behaviour (homogeneity of variances could not be obtained even after data transformation) between controls and treatments. Statistical significance was taken at p<0.05.

Acknowledgments

We thank Menno ter veld, Aart Hutten, Wian Nusselder and Gonçalo Santos for technical support and sampling.

Author Contributions

Conceived and designed the experiments: CM PS LC EH OO JS. Performed the experiments: CM. Analyzed the data: CM EH OO JS. Contributed reagents/materials/analysis tools: CM BC. Wrote the paper: CM EH OO JS.

References

  1. 1. Romero LM, Dickens MJ, Cyr NE (2009) The reactive scope model - A new model integrating homeostasis, allostasis, and stress. Horm Behav 55: 375–389.LM RomeroMJ DickensNE Cyr2009The reactive scope model - A new model integrating homeostasis, allostasis, and stress.Horm Behav55375389
  2. 2. Koolhaas JM, Korte SM, De Boer SF, Van Der Vegt BJ, Van Reenen CG, et al. (1999) Coping styles in animals: current status in behavior and stress-physiology. Neurosci Biobehav Rev 23: 925–935.JM KoolhaasSM KorteSF De BoerBJ Van Der VegtCG Van Reenen1999Coping styles in animals: current status in behavior and stress-physiology.Neurosci Biobehav Rev23925935
  3. 3. Øverli Ø, Sørensen C, Pulman KG, Pottinger TG, Korzan W, et al. (2007) Evolutionary background for stress-coping styles: Relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates. Neurosci Biobehav Rev 31: 396–412.Ø. ØverliC. SørensenKG PulmanTG PottingerW. Korzan2007Evolutionary background for stress-coping styles: Relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates.Neurosci Biobehav Rev31396412
  4. 4. Coppens CM, de Boer SF, Koolhaas JM (2010) Coping styles and behavioural flexibility: towards underlying mechanisms. Phil. Trans. R. Soc. B. 365: 4021–4028.CM CoppensSF de BoerJM Koolhaas2010Coping styles and behavioural flexibility: towards underlying mechanisms. Phil. Trans. R. Soc. B.36540214028
  5. 5. Bell AM (2007) Evolutionary Biology: Animal personalities. Nature 447: 539–540.AM Bell2007Evolutionary Biology: Animal personalities.Nature447539540
  6. 6. Sih A, Bell AM, Johnson JC, Ziemba RE (2004) Behavioral syndromes: an integrative overview. Q Rev Biol 79: 241–277.A. SihAM BellJC JohnsonRE Ziemba2004Behavioral syndromes: an integrative overview.Q Rev Biol79241277
  7. 7. Réale D, Reader S, Sol D, McDougall PT, Dingemanse NJ (2007) Integrating animal temperament within ecology and evolution. Biol Rev 82: 291–318.D. RéaleS. ReaderD. SolPT McDougallNJ Dingemanse2007Integrating animal temperament within ecology and evolution.Biol Rev82291318
  8. 8. Korte SM, Koolhaas JM, Wingfield JC, McEwen BS (2005) The Darwinian concept of stress: benefits of allostasis and costs of allostatic load and the trade-offs in health and disease. Neurosci Biobehav Rev 29: 3–38.SM KorteJM KoolhaasJC WingfieldBS McEwen2005The Darwinian concept of stress: benefits of allostasis and costs of allostatic load and the trade-offs in health and disease.Neurosci Biobehav Rev29338
  9. 9. Pottinger TG, Carrick TR (1999) Modification of the plasma cortisol response to stress in rainbow trout by selective breeding. Gen Comp Endocr 116: 122–132.TG PottingerTR Carrick1999Modification of the plasma cortisol response to stress in rainbow trout by selective breeding.Gen Comp Endocr116122132
  10. 10. Van Oers K, Drent PJ, De Goede P, Van Noordwijk AJ (2004) Realized heritability and repeatability of risk-taking behaviour in relation to avian personalities. Proc R Soc B 271: 65–73.K. Van OersPJ DrentP. De GoedeAJ Van Noordwijk2004Realized heritability and repeatability of risk-taking behaviour in relation to avian personalities.Proc R Soc B2716573
  11. 11. Frijda NH (1986) The emotions. Cambridge University Press, London. 56. NH Frijda1986The emotions.Cambridge University Press, London. 56
  12. 12. Panksepp J (2005) Affective consciousness: core emotional feelings in animals and humans. Conscious Cogn 14: 30–80.J. Panksepp2005Affective consciousness: core emotional feelings in animals and humans.Conscious Cogn143080
  13. 13. Chandroo KP, Yue S, Moccia RD (2004) An evaluation of current perspectives on consciousness and pain in fishes. Fish Fish 5: 281–295.KP ChandrooS. YueRD Moccia2004An evaluation of current perspectives on consciousness and pain in fishes.Fish Fish5281295
  14. 14. Izard CE (1991) The psychology of emotions New York: Plenum. 451 p.CE Izard1991The psychology of emotions New York: Plenum451
  15. 15. Budaev SV (1997) Personality in the guppy (Poecilia reticulata): a correlational study of exploratory behavior and social tendency. J Comp Psychol 111: 399–411.SV Budaev1997Personality in the guppy (Poecilia reticulata): a correlational study of exploratory behavior and social tendency.J Comp Psychol111399411
  16. 16. Budaev SV, Zhuikov AY (1998) Avoidance learning and personality in the guppy Poecilia reticulata. J Comp Psychol 112: 92–94.SV BudaevAY Zhuikov1998Avoidance learning and personality in the guppy Poecilia reticulata.J Comp Psychol1129294
  17. 17. Brown C, Jones F, Braithwaite V (2005) In situ examination of boldness-shyness traits in the tropical poeciliid, Brachyraphis episcopi. Anim Behav 70: 1003–1009.C. BrownF. JonesV. Braithwaite2005In situ examination of boldness-shyness traits in the tropical poeciliid, Brachyraphis episcopi.Anim Behav7010031009
  18. 18. Dadda M, Zandonà E, Bisazza A (2007) Emotional responsiveness in fish from lines artifivially selected for a high and low degree of laterality. Physiol Behav 92: 764–772.M. DaddaE. ZandonàA. Bisazza2007Emotional responsiveness in fish from lines artifivially selected for a high and low degree of laterality.Physiol Behav92764772
  19. 19. Thomas RJ, King TA, Forshaw HE, Marples NM, Speed MP, et al. (2010) The response of fish to novel prey: evidence that dietary conservatism is not restricted to birds. Behav Ecol 21: 669–675.RJ ThomasTA KingHE ForshawNM MarplesMP Speed2010The response of fish to novel prey: evidence that dietary conservatism is not restricted to birds.Behav Ecol21669675
  20. 20. Steimer T, la Fleur S, Schulz P (1997) Neuroendocrine correlates of emotional reactivity and coping in male rates from the Roman high (RHA/Verh). And low (RLA/Verh)- Avoidance lines. Behav Genet 27: 503–512.T. SteimerS. la FleurP. Schulz1997Neuroendocrine correlates of emotional reactivity and coping in male rates from the Roman high (RHA/Verh). And low (RLA/Verh)- Avoidance lines.Behav Genet27503512
  21. 21. Tong EMW (2010) Personality influences in appraisal-emotion relationships: the role of neuroticism. J Personality 78: 393–417.EMW Tong2010Personality influences in appraisal-emotion relationships: the role of neuroticism.J Personality78393417
  22. 22. Whittle S, Allen NB, Lubman DI, Yücel M (2006) The neurobiological basis of temperament: Towards a better understanding of psychopathology. Neurosci Biobehav Rev 30: 511-525. S. WhittleNB AllenDI LubmanM. Yücel2006The neurobiological basis of temperament: Towards a better understanding of psychopathology.Neurosci Biobehav Rev 30511-525
  23. 23. Rothbart MK, Jones LB (1998) Temperament, self-regulation, and education. School Psychol Rev 27: 479–491.MK RothbartLB Jones1998Temperament, self-regulation, and education.School Psychol Rev27479491
  24. 24. McCrae RR, Costa PT (1997) Personality trait structure as a human universal. Am Psychol 5: 509–516.RR McCraeCosta PT Jr1997Personality trait structure as a human universal.Am Psychol5509516
  25. 25. Shin LM, Liberzon I (2010) The neurocircuitry of fear, stress, and anxiety disorders. Neuropsychopharmacology 35: 169–191.LM ShinI. Liberzon2010The neurocircuitry of fear, stress, and anxiety disorders.Neuropsychopharmacology35169191
  26. 26. Moreira PSA, Volpato GL (2004) Conditioning of stress in Nile tilapia. J Fish Biol 64: 961–969.PSA MoreiraGL Volpato2004Conditioning of stress in Nile tilapia.J Fish Biol64961969
  27. 27. Barreto RE, Volpato GL (2007) Evaluating feeding as unconditioned stimulus for conditioning of an endocrine effect in Nile tilapia. Physiol Behav 92: 867–872.RE BarretoGL Volpato2007Evaluating feeding as unconditioned stimulus for conditioning of an endocrine effect in Nile tilapia.Physiol Behav92867872
  28. 28. Schjolden J, Winberg S (2007) Genetically determined variation in stress responsiveness in rainbow trout: behaviour and neurobiology. Brain Behav Evol 70: 227–238.J. SchjoldenS. Winberg2007Genetically determined variation in stress responsiveness in rainbow trout: behaviour and neurobiology.Brain Behav Evol70227238
  29. 29. Øverli Ø, Pottinger TG, Carrick TR, Øverli E, Winberg S (2002) Differences in behaviour between rainbow trout selected for high- and low-stress responsiveness. J Exp Biol 205: 391–395.Ø. ØverliTG PottingerTR CarrickE. ØverliS. Winberg2002Differences in behaviour between rainbow trout selected for high- and low-stress responsiveness.J Exp Biol205391395
  30. 30. Øverli Ø, Winberg S, Pottinger TG (2005) Behavioral and neuroendocrine correlates of selection for stress responsiveness in rainbow trout — a review. Integr Comp Biol 45: 463–474.Ø. ØverliS. WinbergTG Pottinger2005Behavioral and neuroendocrine correlates of selection for stress responsiveness in rainbow trout — a review.Integr Comp Biol45463474
  31. 31. Kristiansen TS, Fernö A (2007) Individual behaviour and growth of halibut (Hippoglossus hippoglossus L.) fed sinking and floating feed: Evidence of different coping styles. Appl Anim Behav Sci 104: 236–250.TS KristiansenA. Fernö2007Individual behaviour and growth of halibut (Hippoglossus hippoglossus L.) fed sinking and floating feed: Evidence of different coping styles.Appl Anim Behav Sci104236250
  32. 32. Silva PIM, Martins CIM, Engrola S, Marino G, Øverli Ø, et al. (2010) Individual differences in cortisol levels and behaviour of Senegalese sole (Solea senegalensis) juveniles: evidence for coping styles. Appl Anim Behav Sci 124: 75–81.PIM SilvaCIM MartinsS. EngrolaG. MarinoØ. Øverli2010Individual differences in cortisol levels and behaviour of Senegalese sole (Solea senegalensis) juveniles: evidence for coping styles.Appl Anim Behav Sci1247581
  33. 33. Martins CIM, Castanheira MF, Engrola S, Costas B, Conceição LEC (2011) Individual differences in metabolism predict coping styles in fish. Appl Anim Behav Sci 130: 135–143.CIM MartinsMF CastanheiraS. EngrolaB. CostasLEC Conceição2011Individual differences in metabolism predict coping styles in fish.Appl Anim Behav Sci130135143
  34. 34. Dadda M, Domenichini A, Piffer L, Argenton F, Bisazza A (2010) Early differences in epithalamic left-right asymmetry influence lateralization and personality of adult zebrafish. Behav Brain Res 206: 208-15. M. DaddaA. DomenichiniL. PifferF. ArgentonA. Bisazza2010Early differences in epithalamic left-right asymmetry influence lateralization and personality of adult zebrafish.Behav Brain Res 206208-15
  35. 35. MacKenzie S, Ribas L, Pilarczyk M, Capdevila DM, Kadri S, et al. (2009) Screening for coping style increases the power of gene expression studies. PLoS ONE 4: e5314.S. MacKenzieL. RibasM. PilarczykDM CapdevilaS. Kadri2009Screening for coping style increases the power of gene expression studies.PLoS ONE4e5314
  36. 36. Yue S, Moccia RD, Duncan IJH (2004) Investigating fear in domestic rainbow trout, Oncorhynchus mykiss, using an avoidance learning task. Appl Anim Behav Sci 87: 343–54.S. YueRD MocciaIJH Duncan2004Investigating fear in domestic rainbow trout, Oncorhynchus mykiss, using an avoidance learning task.Appl Anim Behav Sci8734354
  37. 37. Yue S, Duncan IJH, Moccia RD (2008) Investigating fear in rainbow trout (Oncorhynchus mykiss) using the conditioned-suppression paradigm. J Appl Anim Welf Sci 11: 14–27.S. YueIJH DuncanRD Moccia2008Investigating fear in rainbow trout (Oncorhynchus mykiss) using the conditioned-suppression paradigm.J Appl Anim Welf Sci111427
  38. 38. Chandroo KP, Duncan IJH, Moccia RD (2004) Can fish suffer?: perspectives on sentience, pain, fear and stress. Appl Anim Behav Sci 86: 225–250.KP ChandrooIJH DuncanRD Moccia2004Can fish suffer?: perspectives on sentience, pain, fear and stress.Appl Anim Behav Sci86225250
  39. 39. Rose JD (2002) The neurobehavioral nature of fishes and the question of awareness and pain. Rev Fish Sci 10: 1-38: JD Rose2002The neurobehavioral nature of fishes and the question of awareness and pain.Rev Fish Sci 10:1-38
  40. 40. Rose JD (2007) Anthropomorphism and 'mental welfare' of fishes. Dis Aquat Organ 75: 139–154.JD Rose2007Anthropomorphism and 'mental welfare' of fishes.Dis Aquat Organ75139154
  41. 41. Cabanac M, Cabanac AJ, Parent A (2009) The emergence of consciousness in phylogeny. Behav Brain Res 198: 267–272.M. CabanacAJ CabanacA. Parent2009The emergence of consciousness in phylogeny.Behav Brain Res198267272
  42. 42. Braithwaite VA (2006) Cognitive ability in fish. Fish Physiol 24: 1–37.VA Braithwaite2006Cognitive ability in fish.Fish Physiol24137
  43. 43. Portavella M, Vargas JP (2005) Emotional and spatial learning in goldfish is dependent on different telencephalic pallial systems. Eur J Neurosci 21: 2800–2806.M. PortavellaJP Vargas2005Emotional and spatial learning in goldfish is dependent on different telencephalic pallial systems.Eur J Neurosci2128002806
  44. 44. Rodriguez F, Broglio C, Duran E, Gomez A, Salas C (2006) Neural mechanisms of learning in teleost fish. In: Brown C, Laland K, Krause J, editors. Fish Cognition and Behaviour. Oxford: Blackwell Publishing. pp. 243–277.F. RodriguezC. BroglioE. DuranA. GomezC. Salas2006Neural mechanisms of learning in teleost fish.C. BrownK. LalandJ. KrauseFish Cognition and BehaviourOxfordBlackwell Publishing243277
  45. 45. Sneddon L (2007) Assessing pain perception in fish from physiology to behaviour. Comp Biochem Physiol A 146: S78-S78: L. Sneddon2007Assessing pain perception in fish from physiology to behaviour.Comp Biochem Physiol A 146:S78-S78
  46. 46. Ruiz-Gomez MDL, Huntingford FA, Øverli Ø, Thörnqvist P-O, Höglund E (2011) Response to environmental change in rainbow trout selected for divergent stress coping styles. Physiol Behav 102: 317–322.MDL Ruiz-GomezFA HuntingfordØ. ØverliP-O ThörnqvistE. Höglund2011Response to environmental change in rainbow trout selected for divergent stress coping styles.Physiol Behav102317322
  47. 47. Pineles SL, Vogt DS, Orr SP (2009) Personality and fear responses during conditioning: Beyond extraversion. Pers Indiv Differ 46: 48–53.SL PinelesDS VogtSP Orr2009Personality and fear responses during conditioning: Beyond extraversion.Pers Indiv Differ464853
  48. 48. Svartberg K, Forkman B (2002) Personality traits in the domestic dog (Canis familiaris). Appl Anim Behav Sci 79: 133-155: K. SvartbergB. Forkman2002Personality traits in the domestic dog (Canis familiaris).Appl Anim Behav Sci 79:133-155
  49. 49. Steimer T, Driscoll P (2003) Divergent stress responses and coping styles in psychogenetically selected Roman high-(RHA) and low-(RLA) avoidance rats: Behavioural, neuroendocrine and developmental aspects. Stress 6: 87–100.T. SteimerP. Driscoll2003Divergent stress responses and coping styles in psychogenetically selected Roman high-(RHA) and low-(RLA) avoidance rats: Behavioural, neuroendocrine and developmental aspects.Stress687100
  50. 50. Garamszegi LZ, Eens M, Török J (2008) Birds reveal their personality when singing. PLOS One 3: e2647.LZ GaramszegiM. EensJ. Török2008Birds reveal their personality when singing.PLOS One3e2647
  51. 51. Wingfield JC (2003) Control of behavioural strategies for capricious environments. Anim Behav 66: 807–815.JC Wingfield2003Control of behavioural strategies for capricious environments.Anim Behav66807815
  52. 52. Schulkin J, Morgan MA, Rosen JB (2005) A neuroendocrine mechanism for sustaining fear. Trends Neurosci 28: 629–635.J. SchulkinMA MorganJB Rosen2005A neuroendocrine mechanism for sustaining fear.Trends Neurosci28629635
  53. 53. Portavella M, Torres B, Salas C (2004) Avoidance response in goldfish: emotional and temporal involvement of medial and lateral telencephalic pallium. J Neurosci 24: 2335–2342.M. PortavellaB. TorresC. Salas2004Avoidance response in goldfish: emotional and temporal involvement of medial and lateral telencephalic pallium.J Neurosci2423352342
  54. 54. LeDoux J (2003) The emotional brain, fear, and the amygdala. Cell Mol Neurobiol 23: 727–738.J. LeDoux2003The emotional brain, fear, and the amygdala.Cell Mol Neurobiol23727738
  55. 55. Martins CIM, Ochola D, Ende S, Eding E, Verreth JAJ (2009) Is growth retardation present in Nile tilapia Oreochromis niloticus cultured in low water exchange recirculating aquaculture systems? Aquaculture 298: 43–50.CIM MartinsD. OcholaS. EndeE. EdingJAJ Verreth2009Is growth retardation present in Nile tilapia Oreochromis niloticus cultured in low water exchange recirculating aquaculture systems?Aquaculture2984350
  56. 56. Øverli Ø, Sørensen C, Nilsson GE (2006) Behavioral indicators of stress-coping style in rainbow trout: Do males and females react differently to novelty? Physiol Behav 87: 506–512.Ø. ØverliC. SørensenGE Nilsson2006Behavioral indicators of stress-coping style in rainbow trout: Do males and females react differently to novelty?Physiol Behav87506512
  57. 57. Frost AJ, Winrow-Giffen A, Ashley PJ, Sneddon LU (2009) Plasticity in animal personality traits: does prior experience alter the degree of boldness? Proc R Soc B 274: 333–339.AJ FrostA. Winrow-GiffenPJ AshleyLU Sneddon2009Plasticity in animal personality traits: does prior experience alter the degree of boldness?Proc R Soc B274333339
  58. 58. Irwin S, Kenny AP, O'Halloran J, Fitzgerald RD, Duggan PF (1999) Adaptation and validation of a radioimmunoassay kit for measuring plasma cortisol in turbot. Comp Biochem Physiol C 124: 27–31.S. IrwinAP KennyJ. O'HalloranRD FitzgeraldPF Duggan1999Adaptation and validation of a radioimmunoassay kit for measuring plasma cortisol in turbot.Comp Biochem Physiol C1242731