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Individual differences in cortisol levels and behaviour of Senegalese sole (Solea senegalensis) juveniles: Evidence for coping styles
Applied Animal Behaviour Science 124 (2010) 75–81
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Individual differences in cortisol levels and behaviour of Senegalese sole (Solea senegalensis) juveniles: Evidence for coping styles Patrı´cia Isabel Mota Silva a,c,*, Catarina I.M. Martins a, Sofia Engrola a, Giovanna Marino b, Øyvind Øverli c, Luis E.C. Conceic¸a˜o a a
Centro de Cieˆncias do Mar (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Institute for Environmental Protection and Research (ISPRA), Via di Casalotti 300, I-00166 Rome, Italy c Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 14 January 2010 Available online 12 February 2010
Individual variation in stress physiology and behaviour has been previously reported in several fish species. As seen in other vertebrates, existence of stress coping styles seems to be reflected by the presence of individual variation. Aggressive behaviour, amongst others, is one of the most commonly used parameters to characterize coping styles. However, not all fish species exhibit aggressive behaviour, such as the flatfish Senegalese sole, Solea senegalensis (Kaup, 1858). Therefore, the goal of this study was to determine the magnitude of individual variation in behavioural parameters other than aggression (feeding motivation and activity during stress) as well as in growth and stress response in Senegalese sole. The relationship between these variables was investigated to determine whether they could be used as indicators of coping styles. Thirty-six juvenile fish (9.9 2.2 g) were individually housed for 73 days. Feeding motivation, measured as the time (in s) taken by each fish to react to feed, was determined on days 10, 17, 24 and 31. Blood samples for plasma cortisol were collected on days 51 and 71 for determination of undisturbed and stress levels, respectively. The stress test consisted of holding each fish individually in a net, outside the water, for 3 min. Duration of escape attempts, i.e. the time taken by each fish to stop struggling (in an attempt to escape) in the net, was quantified. The results showed a pronounced individual variation in both control (CV = 54%) and acute stress (CV = 71%) cortisol levels. Senegalese sole also exhibited high coefficient of variation in the behavioural parameters: 75% in feeding latency and 96% in duration of escape attempts. Growth (RGR = 1.17 0.38) showed to be the parameter with lower variation of only 32% and was not correlated with any of the measured parameters. A significant correlation between undisturbed cortisol levels and duration of escape attempts was found. Undisturbed cortisol levels (8.08 4.36 ng/ml) were negatively correlated with duration of escape attempts (P = 0.009, rs = 0.503). Correlations between plasma cortisol levels after stress (398.45 282.67 ng/ml) and the behavioural parameters were not found. The observed individual variation in behaviour and stress physiology as well as their relationship suggests the existence of coping styles in Senegalese sole where proactive individuals exhibit shorter feeding latency, higher duration of escape attempts and lower undisturbed cortisol levels than passive individuals. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Individual variation Personality Feeding motivation Activity Stress response Flatfish
1. Introduction * Corresponding author at: Centro de Cieˆncias do Mar (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. Tel.: +351 916489641. E-mail address: [email protected] (P.I.M. Silva). 0168-1591/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.applanim.2010.01.008
Individual variation in growth, stress response and behaviour has been described in several fish species (Jobling and Reinsnes, 1986; Jobling and Koskela, 1996;
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Carter et al., 1998; Carter and Bransden, 2001; Martins et al., 2005, 2006; Øverli et al., 2006). Until recently, this variation was considered to be a consequence of the establishment of social hierarchies, with dominant fish exhibiting superior growth and lower response to stress. However, recent studies demonstrate that individual variation is not simply a consequence of social hierarchies but also of inherent (i.e. genetic) factors (Øverli et al., 2002; Martins et al., 2005; Schjolden and Winberg, 2007). Several studies showed that, in the absence of social hierarchies, fish still exhibit a large individual variation in growth, stress response and behaviour and that this variation is not only consistent over time (e.g. Martins et al., 2005; van de Nieuwegiessen et al., 2008) but also across generations (Pottinger and Carrick, 1999). In fact, like in mammals, fish seem to express differential coping styles or personalities, i.e. ‘‘coherent set of behavioural and physiological stress responses which are consistent over time and which are characteristic to a certain group of individuals’’ (Koolhaas et al., 1999). Coping styles include two major types, the proactive (active coping or fight-flight or bold personalities) and reactive (passive coping, or conservation-withdrawal or shy personalities) responses (Koolhaas et al., 1999). On one hand, high levels of aggression, territorial control, active avoidance of a negative stimulus and other behavioural responses that suggest active efforts to offset a negative stimulus characterize a proactive response. On the other hand, low levels of aggression and avoidance of a negative stimulus characterize behaviourally a reactive response (Koolhaas et al., 1999; Øverli et al., 2007). Individual differences in behaviour are associated with differences in the physiological stress response. Studies in rainbow trout demonstrate that stress responsiveness in terms of confinement induce changes in plasma cortisol which are negatively correlated with aggressive behaviour (Øverli et al., 2004). This finding supports that proactive coping responses are related with low secretion of cortisol in plasma under stress conditions while passive coping responses are related with an enhanced secretion. Differences in behaviour have been shown not to be just associated to physiological stress response but also with cortisol levels under undisturbed conditions. Øverli et al. (2007) showed that fish that obtained a high total feeding score, i.e. fish that resume feeding quicker after being isolated, showed high aggression (related to proactive coping styles) and low levels of plasma cortisol under undisturbed conditions. These findings suggest that individuals with low levels of undisturbed plasma cortisol display proactive coping styles. In fish, coping styles have been described in rainbow trout (Øverli et al., 2002, 2006), brown trout (Kristiansen, 1999; Brelin et al., 2005), sticklebacks (Ward et al., 2004), fighting fish (Verbeek et al., 2008), halibut (Kristiansen and Ferno¨, 2007) and in African catfish (Martins et al., 2006; van de Nieuwegiessen et al., 2008). The understanding of coping styles in fish is of major importance, not only under an evolutionary perspective but also under practical situations as in aquaculture. Coping styles can be a helpful model in understanding individual adaptive capacity and vulnerability to stress-related disease
(Koolhaas et al., 1999). Furthermore, when fish are cultured in intensive husbandry systems, characterized by high stocking densities and fixed feeding locations, it is expected that reactive fish gain little feed over time and grow weakly which will result in loss of production and compromised welfare (Huntingford and Adams, 2005). Understanding this individuality can be used to approach remedial measures, such as selection programs and alteration of husbandry systems to optimize fish farming. Senegalese sole, Solea senegalensis is a flatfish with high economic and commercial potential for aquaculture and is being exploited in some southern European countries, such as Portugal and Spain (Dinis et al., 1999; Imsland et al., 2003). Despite the commercial importance of Senegalese sole very little is known on its individual variation in growth, stress response and behaviour. Araga˜o et al. (2008) reported a coefficient variation (CV) in growth between 24 and 29% in juvenile Senegalese sole. However, it is not known whether this variation in growth is related to the existence of coping styles. In sole, which is a nonaggressive species (Salas-Leiton et al., 2008) it may be more difficult to assess coping styles, as one of the methods used is to determine the outcome of fights. Also measuring individual feed intake (or residual feed intake) that has been shown to predict coping styles in African catfish (Martins et al., 2005) seems to be more difficult than in other fish species as Senegalese sole present a sluggish feeding behaviour with a swallow-and-spit behaviour often being observed (Dinis et al., 2000). Therefore, the use of behavioural observations (others than aggression and feed intake) to predict coping styles in Senegalese sole may be especially interesting. The aim of this study was to determine individual variation in growth, feeding behaviour and stress response in Senegalese sole and whether such individual differences and their relationships could be used as predictor of coping styles in a non-aggressive species. 2. Material and methods 2.1. Fish, housing and feeding The experiment was carried out at the LEOA Research Facility (University of Algarve, Faro, Portugal) using a closed seawater system (temperature: 19.46 2.15 8C (mean SD); salinity: 29.59 1.26 ppt) with a photoperiod of 12 h light:12 h dark. This experiment lasted for 73 days. Thirty-six Senegalese sole S. senegalensis juveniles with an initial weight of 9.9 2.2 g were obtained from INRBIPIMAR Pilot Station (Olha˜o, Portugal). All fish were kept in 21 l flat-bottom beige fiberglass tanks, 0.21 m2 area, with a water flow rate of 73.5 l h 1. Each tank was divided in three equal compartments (0.07 m2 of area) using plastic nets with 1 cm2 holes covered with sponge to allow continuous water flow rate inside the tank. Fish were housed individually in each compartment, without having physical and visual contact with each other. Tanks were daily cleaned and water parameters daily measured. Fish were fed every day three times a day (11:00, 15:00, and 17:00). The commercial diet Migas 4 (2.0 mm) (A. Coelho e Castro, Po´voa de Varzim, Portugal) used had 53% protein, 11% lipid, 15% carbohydrate,
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according to the manufacturer’s data. Diets were hydrated to allow feed to sink quickly. Fish were initially fed with 2.6% of biomass and this quantity was daily adjusted based on visual inspection of the feed remaining, so that fish were fed to satiation. Water quality parameters were maintained according to the species. 2.2. Experimental procedures Before the start of the experiment fish were held in Ramalhete facilities at the University of Algarve (Faro, Portugal) on a semi-closed system at a rearing density of 0.5 kg/m2 and fed ad libitum. Fish were individually weighted (to 0.001 g) at the start and end of the experiment. Feeding motivation was measured as the feeding latency (s) defined as the time taken by each fish to start swimming towards the feed. This behaviour was measured directly using a stopwatch for the 3 meals on days 10, 17, 24 and 31. Day 10 was used as the first day to measure feeding motivation because it was thought it would represent the feeding motivation of fish already adapted to isolation. Previous studies with African catfish showed that a period of 15 days was sufficient as an adaptation period to isolation (Martins et al., 2005). Afterwards measurements were repeated once per week for an extra 3-week period to allow the determination of how consistent feeding motivation was. Individual feed intake was not measured since this specie presents a swallow-and-spit and sluggish feeding behaviour that could be translated into erroneous results. Blood samples for determination of control (i.e. undisturbed) plasma cortisol were collected on day 51. On day 71 all fish were subjected to an acute stress by holding each fish individually in a net outside the water for 3 min (Araga˜o et al., 2008). Afterwards fish were returned to their holding tanks and after 30 min blood samples were taken (based on Ruane et al., 2001). Fish were fasted for 24 h prior to blood sampling. Prior to blood sampling fish were anaesthetized with 2-phenoxyethanol (Sigma, Madrid, Spain; 200 ppm). Blood was withdrawn from the caudal vein using heparinised syringes. This procedure was finalized within 3 min after fish were anaesthetized. For 5 fish, this procedure took longer than the 3 min and the respective blood samples were not considered for further analysis. The collected blood was centrifuged at 1500 g for 2 min at room temperature and plasma was stored at 25 8C for further cortisol analyses. In four samples, the resulting plasma from centrifugation was reddish and was not considered for analysis. This resulted in a total of 27 blood samples for cortisol determination. The time between blood samplings was long since 15 days are usually used to allow the recovery of the fish after blood sampling (van Zutphen et al., 2001). During the acute stress induction, the time taken by each fish to stop moving was measured using a stopwatch. This behaviour was defined as duration of escape attempts (s). At the end of the experiment fish were kept alive for further experimental work.
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2.3. Cortisol analysis Plasma cortisol was measured in 25 ml samples using commercially available solid phase 125Iodine RIA measuring the total amount of hormone in unextracted serum (Coat-A-Count Cortisol1, D.P.C. Los Angeles, CA). The sensitivity of the assay was 2 ng ml 1 and the intra-assay coefficients of variation were 4.7 and 6.4 (n = 3), respectively. Data was obtained using a gamma counter (Kontron Analytical MDA 312) and analyzed using RIA software. 2.4. Data analysis All results are expressed as means standard deviation (SD). Growth, expressed as relative growth rate (RGR, %/day), was calculated, between final and initial body weight, using the formula: (eg 1) 100 with g = [(ln final weight ln initial weight)/time] (Ricker, 1958). Coefficient of variation (CV, %) was calculated as follows: [(standard deviation/mean) 100]. Pearson correlation was used to verify the consistency of individual feeding latency on different days. The feeding latency of an individual was calculated as the average of the 3 meals measured on days 17, 24 and 31 (day 10 was excluded as it failed on consistency test). Differences in feeding latency over time were tested using repeated measured design. Mauchly’s test was used to check the sphericity assumption and the Bonferroni test for making pairwise comparisons (Field, 2000). A paired t-test (dependent samples) was used to verify whether the netting and air exposure induced a stress response in Senegalese sole. The relationship between feeding latency (log transformed), undisturbed cortisol levels (log x + 1 transformed) and cortisol levels obtained after an acute stress was investigated using Pearson correlation. The duration of escape attempts and growth rate failed to achieve normality even after transformation and their relationship with other variables was investigated using Spearman correlation. Statistical analyses were performed using SPSS (version 15.0) for windows. Statistical significance was taken at P < 0.05. 3. Results 3.1. Individual variation in behaviour, plasma cortisol and growth Duration of escape attempts (CV = 96%), feeding latency (CV = 75%) and the cortisol levels obtained after an acute stress (CV = 71%) showed the highest individual variation (Table 1). Feeding latency decreased significantly over time (repeated measured ANOVA, F1,35 P < 0.001, Fig. 1). On day 10, feeding latency was significantly higher than all the other sampling days (Bonferroni pairwise comparisons, P < 0.001) and not correlated with other days, which suggested an adaptation period. Therefore, day 10 was not considered in further analyses. The netting and exposure of Senegalese sole to air during 3 min resulted in a significant increase of plasma cortisol levels (paired t-test, t(26) = 7.369, P < 0.001).
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Table 1 Means, coefficient of variation (CV), minimum (min.) and maximum (max.) of feeding behaviour (feeding latency), duration of escape attempts, plasma cortisol (control and after an acute stress) and growth of Senegalese sole housed individually. Parameter Behaviour Feeding latency (s) Duration of escape attempts (s) Plasma cortisol Control (ng/ml) Acute stress (ng/ml) Growth (%/day)
Mean SD
CV (%)
Min.
Max.
38.30 28.62 7.20 6.91
74.73 95.98
7.33 1.00
117.00 27.00
8.08 4.36 398.45 282.67
54.01 70.94
0.80 21.00
17.60 949.00
1.17 0.38
32.72
0.28
1.71
tion in plasma cortisol levels before (54%) and after an acute stress (71%) was applied. Undisturbed cortisol levels obtained in this study (8.08 4.36 ng/ml) differed from those reported by Araga˜o et al. (2008; approx. 2 ng/ml) in Senegalese sole groups. However, differences could be due to different cortisol analytical methods as well to individual housing condition, which can act as a stressful situation in fish (Øverli et al., 2002). Nevertheless, it must be noticed that in Senegalese sole the response to an acute stress in both individually (present study) and grouped housed conditions (Araga˜o et al., 2008; Costas et al., 2008) has a considerable Fig. 1. Feeding latency over time for Senegalese sole juveniles housed individually. Values are means (+SD). Different letters indicates significant difference (n = 36).
3.2. Correlations There was a trend towards a significant positive correlation between undisturbed plasma cortisol levels and feeding latency (P = 0.0778, r2 = 0.119, Fig. 2A). The undisturbed plasma cortisol levels were significantly correlated with duration of escape attempts (P = 0.009, rs = 0.503, Fig. 2B): individuals with lower undisturbed cortisol levels seemed more motivated to eat and were more responsive towards an acute stressor. No correlation was found between plasma cortisol levels obtained after an acute stress and the behaviour parameters (P > 0.05). Feeding latency and duration of escape attempts were also not correlated (P > 0.05). Growth was not correlated with any of the measured parameters (P > 0.05). 4. Discussion This study reports for the first time individual differences in behaviour, stress response and growth of S. senegalensis, a flatfish with increasing interest for the aquaculture sector. Over the past years, there has been an increased interest on studying individual differences in behaviour and stress physiology and their relationships, as these seem to reflect the existence of coping styles in fish (Øverli et al., 2007). Individual variation in cortisol responsiveness (as one of the most common methods to assess the stress response in fish, Wendelaar Bonga, 1997; Ruane et al., 2001; Martins et al., 2006) has also been addressed in this study. Senegalese sole exhibited a pronounced individual varia-
Fig. 2. Relationship between individual differences in behaviour (feeding latency and duration of escape attempts) and cortisol levels obtained in isolated and undisturbed Senegalese sole juveniles. (A) Relationship between feeding latency and undisturbed plasma cortisol and (B) Relationship between duration of escape attempts and undisturbed plasma cortisol (Pearson r2 and P values).
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variation, up to 73%. Other studies with African catfish and sea bass (Dicentrarchus labrax) have also reported comparable individual variation in both undisturbed levels of plasma cortisol (Martins et al., 2006; Marino et al., 2001), and poststressor levels (Martins et al., 2006). In terms of behaviour the present study reports pronounced individual variation in feeding and duration of escape attempts (75 and 96%, respectively). The variation obtained in the duration of escape attempts is comparable with the variation of another variable that also reflects locomotory activity during stress reported by Øverli et al. (2006) in female rainbow trout that had an individual variation in locomotor activity during confinement test of approximately 46%. Also in the same study pronounced individual variation was observed in feeding score, another way of measuring feeding motivation. The observed individual variation in behaviour and stress physiology suggests differences in coping styles in Senegalese sole. This is supported by the type of relationships found between plasma cortisol, feeding motivation and duration of escape attempts. The relationship found between undisturbed plasma cortisol levels and duration of escape attempts was never reported in fish. In pigs, the number of escape attempts when individuals are exposed to a backtest has been shown to reflect the existence of coping styles (Bolhuis et al., 2004). The number of escape attempts was shown to be related with baseline cortisol levels obtained in stall-housing gilts (Geverink et al., 2003). These authors found a relationship similar to the relationship found in this study: individuals with lower escape attempts exhibit higher baseline cortisol levels. However, this relationship was not observed when gilts were grouped housed. If this lack of relationship would also apply in grouped housed fish is still to be determined. Nevertheless, lower escape attempts suggest a passive coping style, which has been associated with higher basal corticosteroid concentrations (Korte et al., 1997). The trend found between undisturbed plasma cortisol and feeding motivation in sole juveniles is similar to the relationship reported by Øverli et al. (2007) for rainbow trout. Individuals with a higher feeding score had lower undisturbed cortisol levels until a certain level after which the relationship was inverted however, in the present study a linear trend line yielded a better fit than the curvilinear approach. Øverli et al. (2006, 2007) reported a relationship between feeding motivation and activity during acute confinement stress. In the present study a relationship between feeding motivation and duration of escape attempts (movement during stress) was not found. Nevertheless it must be noticed that in the studies by Øverli et al. feeding motivation was measured immediately after transfer to isolation (reflecting a behaviour of adaptation to isolation) and not after the end of an adaptation period, which was the case in the present study. In combination, the results suggest that individuals with low undisturbed cortisol levels, a short feeding latency and active avoidance after stress are proactive individuals. On the contrary, individuals that present high-undisturbed cortisol levels, higher feeding latency and short duration of escape attempts seem to be passive individuals. However, no correlation was found between post-stress cortisol
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levels and feeding motivation or duration of escape attempts. Such relationship has been reported in other studies suggesting coping styles in fish. Animals that showed reduced locomotion during stress test and high motivation towards feed exhibited lower post-stress cortisol levels (Øverli et al., 2006). These authors used a different stress test in which fish were kept in a small, immersed box. Such test may have allowed individuals to express their coping strategy. In the stress test used in our study fish were kept in a net outside the water, which may have limited the individual’s ability to cope. Feeding motivation, cortisol levels obtained before and after stress and the duration of escape attempts obtained during the stress test were collected at different time points. Obtaining and correlating variables at different time points may seem questionable. However, both feeding motivation and cortisol levels have been shown to be consistent over time in several fish species (Pottinger and Carrick, 1999; Tort et al., 2001; Martins et al., 2005; Schjolden et al., 2005) making the difference in sampling times less relevant. Nevertheless, it is important to understand how consistent these parameters are for Senegal sole and subsequently evaluate the possible implications of correlating variable collected at different time points. The consistency of the individual differences found in the present study should be further studied. As defined by Koolhaas et al. (1999), copying styles are defined as ‘‘coherent set of behavioural and physiological stress responses which are consistent over time. . .’’. Therefore one need to understand whether the individual variation in stress physiology and behaviour as well as the relationships found in this study are consistent over time and genetically linked. If these individual differences prove to be genetically based then selection programs can be adopted. It has been already demonstrated in strains of rainbow trout selected for consistently high (HR) or low (LR) responses to stress that there is a heritable association between increased cortisol production, anxiety-like behaviour, brain monoaminergic function and altered cognitive function (Pottinger and Carrick, 1999, 2001; Øverli et al., 2001; Moreira et al., 2004). A recent study even show that yolk-sac larvae originating from the HR strain were more sensitive to environmental stressors than LR yolk-sac larvae, suggesting that coping styles are inherent since social experience or variable access to food resources could not modify behavioural strategy in this case (Ho¨glund et al., 2008). Most of the previous studies on individual differences are related to individual differences in growth since the consequences of individual variation in growth for the fish farming industry are extensive. One of the main reasons for these studies was because until recently, growth variation was thought to be the result of social interactions resulting in that dominant fish consumed more feed and therefore grew faster than subordinates. However, if the presence of social interactions would be the main reason for growth variation it would be expected that in the absence of social interactions (such as in the case of individual housing) this variation would be reduced. This seems not be the case in the present study. Senegalese sole housed individually
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exhibited an individual variation in growth of 33%, suggesting that these individual differences may be inherent. Similar results were observed by Martins et al. (2005) that reported a variation in growth of 53% in African catfish Clarias gariepinus individually housed. Studies performed with Senegalese sole housed in groups reported an individual variation in growth ranging from 21% till 29% (Araga˜o et al., 2008; Costas et al., 2008) showing values in fact somewhat smaller than the values obtained in this study using individual housing. This suggests that in Senegalese sole the presence of social interactions does not explain the presence of individual differences in growth. Growth variation was observed in other flatfish both individually housed such as Greenback flounder (CV 19%, Carter and Bransden, 2001) and in groups such as Atlantic halibut (CV approx. 21%, Kristiansen et al., 2004). Furthermore, to which extend coping styles differ in performance traits such as feed efficiency as shown in Martins et al. (2005, 2006) and van de Nieuwegiessen et al. (2008) would be an interesting point to study within the context of aquaculture. In fact, the link between performance traits and coping strategies is largely unknown for aquaculture species. This is mainly due to experimental protocols that are limited in time and despite allowing a characterization of individual behaviour and stress response do not allow a proper characterization of performance traits that need longer experimental periods. 5. Conclusion In conclusion, this study quantifies for the first time the individual variation in growth, undisturbed and poststress cortisol levels, feeding behaviour and activity during acute stress in Senegalese sole. The relationships between these variables support the existence of coping styles. Behaviour traits like feeding latency and duration of escape attempts are easier, faster and cheaper to measure and were shown to predict undisturbed cortisol levels. When linked with performance traits, these behaviour traits, together with physiological measurements, may be of interest as a discriminatory tool in genetic selection programs. Acknowledgments We wish to thank H. Teixeira for practical assistance. This study benefited from funding by Project PROMAR SP5.P117/03 (programme INTERREG III A, co-funded by FEDER, European Commission), SFRH/BPD/49051/2008 and SFRH/BD/44103/2008 from ‘‘Fundac¸a˜o para a Cieˆncia e Tecnologia’’ (Portugal). References Araga˜o, C., Corte-Real, J., Costas, B., Dinis, M.T., Conceic¸a˜o, L.E.C., 2008. Stress response and changes in amino acid requirements in Senegalese sole (Solea senegalensis Kaup 1858). Amino Acids 34, 143– 148. Bolhuis, J.E., Schouten, W.G.P., Leeuw, J.A.D., Scharama, J.W., Wiegant, V.M., 2004. Individual coping characteristics, rearing conditions and behavioural flexibility in pigs. Behav. Brain Res. 152 (2), 351–360.
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Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness. Brain Behav. Evol. 57, 214–224. Øverli, Ø., Pottinger, T.G., Carrick, T.R., Øverli, E., Winberg, S., 2002. Differences in behaviour between rainbow trout selected for highand low-stress responsiveness. J. Exp. Biol. 205, 391–395. Øverli, Ø., Korzan, W.J., Ho¨glund, E., Winberg, S., Bollig, H., Watt, M., Forster, G.S., Barton, B.A., Øverli, E., Renner, K.J., Summers, C.H., 2004.
P.I.M. Silva et al. / Applied Animal Behaviour Science 124 (2010) 75–81 Stress coping style predicts aggression and social dominance in rainbow trout. Horm. Behav. 45, 235–241. Øverli, Ø., Sørensen, C., Nilsson, G.E., 2006. Behavioural indicators of stress-coping style in rainbow trout: do males and females react differently to novelty? Physiol. Behav. 87, 506–512. Øverli, Ø., Sørensen, C., Pulman, K.G.T., Pottinger, T.G., Korzan, W., Summers, C.H., Nilsson, G.E., 2007. Evolutionary background for stresscoping styles: relationships between physiological, behavioural, and cognitive traits in non-mammalian vertebrates. Neurosci. Biobehav. Rev. 31, 396–412. Pottinger, T.G., Carrick, T.R., 1999. Modification of the plasma cortisol response to stress in rainbow trout by selective breeding. Gen. Comp. Endocr. 116, 122–132. Pottinger, T.G., Carrick, T.R., 2001. Stress responsiveness affects dominant–subordinate relationships in rainbow trout. Horm. Behav. 40, 419–427. Ricker, W.E., 1958. Handbook of computations for biological statistics of fish populations. In: Bull. Fish Res. Board Can. 119, Fisheries Research Board of Canada Publications, Ottawa, pp. 1–300. Ruane, N.M., Huisman, E.A., Komen, J., 2001. Plasma cortisol and metabolite level profiles in two isogenic strains of common carp during confinement. J. Fish Biol. 59, 1–12. ˜ avate, J.P., 2008. Growth, Salas-Leiton, E., Anguis, V., Manchado, M., Can feeding and oxygen consumption of Senegalese sole (Solea senegalensis) juveniles stocked at different densities. Aquaculture 285, 84–89.
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Schjolden, J., Stoskhus, A., Winberg, S., 2005. Does individual variation in stress responses and agonistic behavior reflect divergent stress coping strategies in juvenile rainbow trout? Physiol Biochem. Zool. 78, 715–723. Schjolden, J., Winberg, S., 2007. Genetically determined variation in stress responsiveness in rainbow trout: behaviour and neurobiology. Brain Behav. Evol. 70, 227–238. Tort, L., Montero, D., Robaina, L., Ferna´ndez-Palacios, H., Izquierdo, M.S., 2001. Consistency of stress response to repeated handling in the gilthead sea bream Sparus aurata Linnaeus, 1758. Aquacult. Res. 32, 593–598. van de Nieuwegiessen, P.G., Schrama, J.W., Verreth, J.A.J., 2008. A note on alarm cues in juvenile African catfish. Clarias gariepinus Burchell: indications for opposing behavioural strategies. Appl. Anim. Behav. Sci. 113, 270–275. van Zutphen, L.F.M., Baumans, V., Beynen, A.C., 2001. In: van Zutphen, L.F.M., Baumans, V., Beynen, A.C. (Eds.), Principles of Laboratory Animal Science. Elsevier Publications, Amsterdam, p. 41. Verbeek, P., Iwamoto, T., Murakami, N., 2008. Variable stress-responsiveness in wild type and domesticated fighting fish. Physiol. Behav. 93, 83–88. Ward, A.J.W., Thomas, P., Hart, P.J.B., Krause, J., 2004. Correlates of boldness in three-spined sticklebacks (Gasterosteus aculeatus). Behav. Ecol. Sociobiol. 55, 561–568. Wendelaar Bonga, S.E., 1997. The stress response in fish. Physiol. Rev. 77, 591–625.
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Applied Animal Behaviour Science journal homepage: www.elsevier.com/locate/applanim
Individual differences in cortisol levels and behaviour of Senegalese sole (Solea senegalensis) juveniles: Evidence for coping styles Patrı´cia Isabel Mota Silva a,c,*, Catarina I.M. Martins a, Sofia Engrola a, Giovanna Marino b, Øyvind Øverli c, Luis E.C. Conceic¸a˜o a a
Centro de Cieˆncias do Mar (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Institute for Environmental Protection and Research (ISPRA), Via di Casalotti 300, I-00166 Rome, Italy c Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 14 January 2010 Available online 12 February 2010
Individual variation in stress physiology and behaviour has been previously reported in several fish species. As seen in other vertebrates, existence of stress coping styles seems to be reflected by the presence of individual variation. Aggressive behaviour, amongst others, is one of the most commonly used parameters to characterize coping styles. However, not all fish species exhibit aggressive behaviour, such as the flatfish Senegalese sole, Solea senegalensis (Kaup, 1858). Therefore, the goal of this study was to determine the magnitude of individual variation in behavioural parameters other than aggression (feeding motivation and activity during stress) as well as in growth and stress response in Senegalese sole. The relationship between these variables was investigated to determine whether they could be used as indicators of coping styles. Thirty-six juvenile fish (9.9 2.2 g) were individually housed for 73 days. Feeding motivation, measured as the time (in s) taken by each fish to react to feed, was determined on days 10, 17, 24 and 31. Blood samples for plasma cortisol were collected on days 51 and 71 for determination of undisturbed and stress levels, respectively. The stress test consisted of holding each fish individually in a net, outside the water, for 3 min. Duration of escape attempts, i.e. the time taken by each fish to stop struggling (in an attempt to escape) in the net, was quantified. The results showed a pronounced individual variation in both control (CV = 54%) and acute stress (CV = 71%) cortisol levels. Senegalese sole also exhibited high coefficient of variation in the behavioural parameters: 75% in feeding latency and 96% in duration of escape attempts. Growth (RGR = 1.17 0.38) showed to be the parameter with lower variation of only 32% and was not correlated with any of the measured parameters. A significant correlation between undisturbed cortisol levels and duration of escape attempts was found. Undisturbed cortisol levels (8.08 4.36 ng/ml) were negatively correlated with duration of escape attempts (P = 0.009, rs = 0.503). Correlations between plasma cortisol levels after stress (398.45 282.67 ng/ml) and the behavioural parameters were not found. The observed individual variation in behaviour and stress physiology as well as their relationship suggests the existence of coping styles in Senegalese sole where proactive individuals exhibit shorter feeding latency, higher duration of escape attempts and lower undisturbed cortisol levels than passive individuals. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Individual variation Personality Feeding motivation Activity Stress response Flatfish
1. Introduction * Corresponding author at: Centro de Cieˆncias do Mar (CCMar), Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. Tel.: +351 916489641. E-mail address: [email protected] (P.I.M. Silva). 0168-1591/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.applanim.2010.01.008
Individual variation in growth, stress response and behaviour has been described in several fish species (Jobling and Reinsnes, 1986; Jobling and Koskela, 1996;
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Carter et al., 1998; Carter and Bransden, 2001; Martins et al., 2005, 2006; Øverli et al., 2006). Until recently, this variation was considered to be a consequence of the establishment of social hierarchies, with dominant fish exhibiting superior growth and lower response to stress. However, recent studies demonstrate that individual variation is not simply a consequence of social hierarchies but also of inherent (i.e. genetic) factors (Øverli et al., 2002; Martins et al., 2005; Schjolden and Winberg, 2007). Several studies showed that, in the absence of social hierarchies, fish still exhibit a large individual variation in growth, stress response and behaviour and that this variation is not only consistent over time (e.g. Martins et al., 2005; van de Nieuwegiessen et al., 2008) but also across generations (Pottinger and Carrick, 1999). In fact, like in mammals, fish seem to express differential coping styles or personalities, i.e. ‘‘coherent set of behavioural and physiological stress responses which are consistent over time and which are characteristic to a certain group of individuals’’ (Koolhaas et al., 1999). Coping styles include two major types, the proactive (active coping or fight-flight or bold personalities) and reactive (passive coping, or conservation-withdrawal or shy personalities) responses (Koolhaas et al., 1999). On one hand, high levels of aggression, territorial control, active avoidance of a negative stimulus and other behavioural responses that suggest active efforts to offset a negative stimulus characterize a proactive response. On the other hand, low levels of aggression and avoidance of a negative stimulus characterize behaviourally a reactive response (Koolhaas et al., 1999; Øverli et al., 2007). Individual differences in behaviour are associated with differences in the physiological stress response. Studies in rainbow trout demonstrate that stress responsiveness in terms of confinement induce changes in plasma cortisol which are negatively correlated with aggressive behaviour (Øverli et al., 2004). This finding supports that proactive coping responses are related with low secretion of cortisol in plasma under stress conditions while passive coping responses are related with an enhanced secretion. Differences in behaviour have been shown not to be just associated to physiological stress response but also with cortisol levels under undisturbed conditions. Øverli et al. (2007) showed that fish that obtained a high total feeding score, i.e. fish that resume feeding quicker after being isolated, showed high aggression (related to proactive coping styles) and low levels of plasma cortisol under undisturbed conditions. These findings suggest that individuals with low levels of undisturbed plasma cortisol display proactive coping styles. In fish, coping styles have been described in rainbow trout (Øverli et al., 2002, 2006), brown trout (Kristiansen, 1999; Brelin et al., 2005), sticklebacks (Ward et al., 2004), fighting fish (Verbeek et al., 2008), halibut (Kristiansen and Ferno¨, 2007) and in African catfish (Martins et al., 2006; van de Nieuwegiessen et al., 2008). The understanding of coping styles in fish is of major importance, not only under an evolutionary perspective but also under practical situations as in aquaculture. Coping styles can be a helpful model in understanding individual adaptive capacity and vulnerability to stress-related disease
(Koolhaas et al., 1999). Furthermore, when fish are cultured in intensive husbandry systems, characterized by high stocking densities and fixed feeding locations, it is expected that reactive fish gain little feed over time and grow weakly which will result in loss of production and compromised welfare (Huntingford and Adams, 2005). Understanding this individuality can be used to approach remedial measures, such as selection programs and alteration of husbandry systems to optimize fish farming. Senegalese sole, Solea senegalensis is a flatfish with high economic and commercial potential for aquaculture and is being exploited in some southern European countries, such as Portugal and Spain (Dinis et al., 1999; Imsland et al., 2003). Despite the commercial importance of Senegalese sole very little is known on its individual variation in growth, stress response and behaviour. Araga˜o et al. (2008) reported a coefficient variation (CV) in growth between 24 and 29% in juvenile Senegalese sole. However, it is not known whether this variation in growth is related to the existence of coping styles. In sole, which is a nonaggressive species (Salas-Leiton et al., 2008) it may be more difficult to assess coping styles, as one of the methods used is to determine the outcome of fights. Also measuring individual feed intake (or residual feed intake) that has been shown to predict coping styles in African catfish (Martins et al., 2005) seems to be more difficult than in other fish species as Senegalese sole present a sluggish feeding behaviour with a swallow-and-spit behaviour often being observed (Dinis et al., 2000). Therefore, the use of behavioural observations (others than aggression and feed intake) to predict coping styles in Senegalese sole may be especially interesting. The aim of this study was to determine individual variation in growth, feeding behaviour and stress response in Senegalese sole and whether such individual differences and their relationships could be used as predictor of coping styles in a non-aggressive species. 2. Material and methods 2.1. Fish, housing and feeding The experiment was carried out at the LEOA Research Facility (University of Algarve, Faro, Portugal) using a closed seawater system (temperature: 19.46 2.15 8C (mean SD); salinity: 29.59 1.26 ppt) with a photoperiod of 12 h light:12 h dark. This experiment lasted for 73 days. Thirty-six Senegalese sole S. senegalensis juveniles with an initial weight of 9.9 2.2 g were obtained from INRBIPIMAR Pilot Station (Olha˜o, Portugal). All fish were kept in 21 l flat-bottom beige fiberglass tanks, 0.21 m2 area, with a water flow rate of 73.5 l h 1. Each tank was divided in three equal compartments (0.07 m2 of area) using plastic nets with 1 cm2 holes covered with sponge to allow continuous water flow rate inside the tank. Fish were housed individually in each compartment, without having physical and visual contact with each other. Tanks were daily cleaned and water parameters daily measured. Fish were fed every day three times a day (11:00, 15:00, and 17:00). The commercial diet Migas 4 (2.0 mm) (A. Coelho e Castro, Po´voa de Varzim, Portugal) used had 53% protein, 11% lipid, 15% carbohydrate,
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according to the manufacturer’s data. Diets were hydrated to allow feed to sink quickly. Fish were initially fed with 2.6% of biomass and this quantity was daily adjusted based on visual inspection of the feed remaining, so that fish were fed to satiation. Water quality parameters were maintained according to the species. 2.2. Experimental procedures Before the start of the experiment fish were held in Ramalhete facilities at the University of Algarve (Faro, Portugal) on a semi-closed system at a rearing density of 0.5 kg/m2 and fed ad libitum. Fish were individually weighted (to 0.001 g) at the start and end of the experiment. Feeding motivation was measured as the feeding latency (s) defined as the time taken by each fish to start swimming towards the feed. This behaviour was measured directly using a stopwatch for the 3 meals on days 10, 17, 24 and 31. Day 10 was used as the first day to measure feeding motivation because it was thought it would represent the feeding motivation of fish already adapted to isolation. Previous studies with African catfish showed that a period of 15 days was sufficient as an adaptation period to isolation (Martins et al., 2005). Afterwards measurements were repeated once per week for an extra 3-week period to allow the determination of how consistent feeding motivation was. Individual feed intake was not measured since this specie presents a swallow-and-spit and sluggish feeding behaviour that could be translated into erroneous results. Blood samples for determination of control (i.e. undisturbed) plasma cortisol were collected on day 51. On day 71 all fish were subjected to an acute stress by holding each fish individually in a net outside the water for 3 min (Araga˜o et al., 2008). Afterwards fish were returned to their holding tanks and after 30 min blood samples were taken (based on Ruane et al., 2001). Fish were fasted for 24 h prior to blood sampling. Prior to blood sampling fish were anaesthetized with 2-phenoxyethanol (Sigma, Madrid, Spain; 200 ppm). Blood was withdrawn from the caudal vein using heparinised syringes. This procedure was finalized within 3 min after fish were anaesthetized. For 5 fish, this procedure took longer than the 3 min and the respective blood samples were not considered for further analysis. The collected blood was centrifuged at 1500 g for 2 min at room temperature and plasma was stored at 25 8C for further cortisol analyses. In four samples, the resulting plasma from centrifugation was reddish and was not considered for analysis. This resulted in a total of 27 blood samples for cortisol determination. The time between blood samplings was long since 15 days are usually used to allow the recovery of the fish after blood sampling (van Zutphen et al., 2001). During the acute stress induction, the time taken by each fish to stop moving was measured using a stopwatch. This behaviour was defined as duration of escape attempts (s). At the end of the experiment fish were kept alive for further experimental work.
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2.3. Cortisol analysis Plasma cortisol was measured in 25 ml samples using commercially available solid phase 125Iodine RIA measuring the total amount of hormone in unextracted serum (Coat-A-Count Cortisol1, D.P.C. Los Angeles, CA). The sensitivity of the assay was 2 ng ml 1 and the intra-assay coefficients of variation were 4.7 and 6.4 (n = 3), respectively. Data was obtained using a gamma counter (Kontron Analytical MDA 312) and analyzed using RIA software. 2.4. Data analysis All results are expressed as means standard deviation (SD). Growth, expressed as relative growth rate (RGR, %/day), was calculated, between final and initial body weight, using the formula: (eg 1) 100 with g = [(ln final weight ln initial weight)/time] (Ricker, 1958). Coefficient of variation (CV, %) was calculated as follows: [(standard deviation/mean) 100]. Pearson correlation was used to verify the consistency of individual feeding latency on different days. The feeding latency of an individual was calculated as the average of the 3 meals measured on days 17, 24 and 31 (day 10 was excluded as it failed on consistency test). Differences in feeding latency over time were tested using repeated measured design. Mauchly’s test was used to check the sphericity assumption and the Bonferroni test for making pairwise comparisons (Field, 2000). A paired t-test (dependent samples) was used to verify whether the netting and air exposure induced a stress response in Senegalese sole. The relationship between feeding latency (log transformed), undisturbed cortisol levels (log x + 1 transformed) and cortisol levels obtained after an acute stress was investigated using Pearson correlation. The duration of escape attempts and growth rate failed to achieve normality even after transformation and their relationship with other variables was investigated using Spearman correlation. Statistical analyses were performed using SPSS (version 15.0) for windows. Statistical significance was taken at P < 0.05. 3. Results 3.1. Individual variation in behaviour, plasma cortisol and growth Duration of escape attempts (CV = 96%), feeding latency (CV = 75%) and the cortisol levels obtained after an acute stress (CV = 71%) showed the highest individual variation (Table 1). Feeding latency decreased significantly over time (repeated measured ANOVA, F1,35 P < 0.001, Fig. 1). On day 10, feeding latency was significantly higher than all the other sampling days (Bonferroni pairwise comparisons, P < 0.001) and not correlated with other days, which suggested an adaptation period. Therefore, day 10 was not considered in further analyses. The netting and exposure of Senegalese sole to air during 3 min resulted in a significant increase of plasma cortisol levels (paired t-test, t(26) = 7.369, P < 0.001).
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Table 1 Means, coefficient of variation (CV), minimum (min.) and maximum (max.) of feeding behaviour (feeding latency), duration of escape attempts, plasma cortisol (control and after an acute stress) and growth of Senegalese sole housed individually. Parameter Behaviour Feeding latency (s) Duration of escape attempts (s) Plasma cortisol Control (ng/ml) Acute stress (ng/ml) Growth (%/day)
Mean SD
CV (%)
Min.
Max.
38.30 28.62 7.20 6.91
74.73 95.98
7.33 1.00
117.00 27.00
8.08 4.36 398.45 282.67
54.01 70.94
0.80 21.00
17.60 949.00
1.17 0.38
32.72
0.28
1.71
tion in plasma cortisol levels before (54%) and after an acute stress (71%) was applied. Undisturbed cortisol levels obtained in this study (8.08 4.36 ng/ml) differed from those reported by Araga˜o et al. (2008; approx. 2 ng/ml) in Senegalese sole groups. However, differences could be due to different cortisol analytical methods as well to individual housing condition, which can act as a stressful situation in fish (Øverli et al., 2002). Nevertheless, it must be noticed that in Senegalese sole the response to an acute stress in both individually (present study) and grouped housed conditions (Araga˜o et al., 2008; Costas et al., 2008) has a considerable Fig. 1. Feeding latency over time for Senegalese sole juveniles housed individually. Values are means (+SD). Different letters indicates significant difference (n = 36).
3.2. Correlations There was a trend towards a significant positive correlation between undisturbed plasma cortisol levels and feeding latency (P = 0.0778, r2 = 0.119, Fig. 2A). The undisturbed plasma cortisol levels were significantly correlated with duration of escape attempts (P = 0.009, rs = 0.503, Fig. 2B): individuals with lower undisturbed cortisol levels seemed more motivated to eat and were more responsive towards an acute stressor. No correlation was found between plasma cortisol levels obtained after an acute stress and the behaviour parameters (P > 0.05). Feeding latency and duration of escape attempts were also not correlated (P > 0.05). Growth was not correlated with any of the measured parameters (P > 0.05). 4. Discussion This study reports for the first time individual differences in behaviour, stress response and growth of S. senegalensis, a flatfish with increasing interest for the aquaculture sector. Over the past years, there has been an increased interest on studying individual differences in behaviour and stress physiology and their relationships, as these seem to reflect the existence of coping styles in fish (Øverli et al., 2007). Individual variation in cortisol responsiveness (as one of the most common methods to assess the stress response in fish, Wendelaar Bonga, 1997; Ruane et al., 2001; Martins et al., 2006) has also been addressed in this study. Senegalese sole exhibited a pronounced individual varia-
Fig. 2. Relationship between individual differences in behaviour (feeding latency and duration of escape attempts) and cortisol levels obtained in isolated and undisturbed Senegalese sole juveniles. (A) Relationship between feeding latency and undisturbed plasma cortisol and (B) Relationship between duration of escape attempts and undisturbed plasma cortisol (Pearson r2 and P values).
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variation, up to 73%. Other studies with African catfish and sea bass (Dicentrarchus labrax) have also reported comparable individual variation in both undisturbed levels of plasma cortisol (Martins et al., 2006; Marino et al., 2001), and poststressor levels (Martins et al., 2006). In terms of behaviour the present study reports pronounced individual variation in feeding and duration of escape attempts (75 and 96%, respectively). The variation obtained in the duration of escape attempts is comparable with the variation of another variable that also reflects locomotory activity during stress reported by Øverli et al. (2006) in female rainbow trout that had an individual variation in locomotor activity during confinement test of approximately 46%. Also in the same study pronounced individual variation was observed in feeding score, another way of measuring feeding motivation. The observed individual variation in behaviour and stress physiology suggests differences in coping styles in Senegalese sole. This is supported by the type of relationships found between plasma cortisol, feeding motivation and duration of escape attempts. The relationship found between undisturbed plasma cortisol levels and duration of escape attempts was never reported in fish. In pigs, the number of escape attempts when individuals are exposed to a backtest has been shown to reflect the existence of coping styles (Bolhuis et al., 2004). The number of escape attempts was shown to be related with baseline cortisol levels obtained in stall-housing gilts (Geverink et al., 2003). These authors found a relationship similar to the relationship found in this study: individuals with lower escape attempts exhibit higher baseline cortisol levels. However, this relationship was not observed when gilts were grouped housed. If this lack of relationship would also apply in grouped housed fish is still to be determined. Nevertheless, lower escape attempts suggest a passive coping style, which has been associated with higher basal corticosteroid concentrations (Korte et al., 1997). The trend found between undisturbed plasma cortisol and feeding motivation in sole juveniles is similar to the relationship reported by Øverli et al. (2007) for rainbow trout. Individuals with a higher feeding score had lower undisturbed cortisol levels until a certain level after which the relationship was inverted however, in the present study a linear trend line yielded a better fit than the curvilinear approach. Øverli et al. (2006, 2007) reported a relationship between feeding motivation and activity during acute confinement stress. In the present study a relationship between feeding motivation and duration of escape attempts (movement during stress) was not found. Nevertheless it must be noticed that in the studies by Øverli et al. feeding motivation was measured immediately after transfer to isolation (reflecting a behaviour of adaptation to isolation) and not after the end of an adaptation period, which was the case in the present study. In combination, the results suggest that individuals with low undisturbed cortisol levels, a short feeding latency and active avoidance after stress are proactive individuals. On the contrary, individuals that present high-undisturbed cortisol levels, higher feeding latency and short duration of escape attempts seem to be passive individuals. However, no correlation was found between post-stress cortisol
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levels and feeding motivation or duration of escape attempts. Such relationship has been reported in other studies suggesting coping styles in fish. Animals that showed reduced locomotion during stress test and high motivation towards feed exhibited lower post-stress cortisol levels (Øverli et al., 2006). These authors used a different stress test in which fish were kept in a small, immersed box. Such test may have allowed individuals to express their coping strategy. In the stress test used in our study fish were kept in a net outside the water, which may have limited the individual’s ability to cope. Feeding motivation, cortisol levels obtained before and after stress and the duration of escape attempts obtained during the stress test were collected at different time points. Obtaining and correlating variables at different time points may seem questionable. However, both feeding motivation and cortisol levels have been shown to be consistent over time in several fish species (Pottinger and Carrick, 1999; Tort et al., 2001; Martins et al., 2005; Schjolden et al., 2005) making the difference in sampling times less relevant. Nevertheless, it is important to understand how consistent these parameters are for Senegal sole and subsequently evaluate the possible implications of correlating variable collected at different time points. The consistency of the individual differences found in the present study should be further studied. As defined by Koolhaas et al. (1999), copying styles are defined as ‘‘coherent set of behavioural and physiological stress responses which are consistent over time. . .’’. Therefore one need to understand whether the individual variation in stress physiology and behaviour as well as the relationships found in this study are consistent over time and genetically linked. If these individual differences prove to be genetically based then selection programs can be adopted. It has been already demonstrated in strains of rainbow trout selected for consistently high (HR) or low (LR) responses to stress that there is a heritable association between increased cortisol production, anxiety-like behaviour, brain monoaminergic function and altered cognitive function (Pottinger and Carrick, 1999, 2001; Øverli et al., 2001; Moreira et al., 2004). A recent study even show that yolk-sac larvae originating from the HR strain were more sensitive to environmental stressors than LR yolk-sac larvae, suggesting that coping styles are inherent since social experience or variable access to food resources could not modify behavioural strategy in this case (Ho¨glund et al., 2008). Most of the previous studies on individual differences are related to individual differences in growth since the consequences of individual variation in growth for the fish farming industry are extensive. One of the main reasons for these studies was because until recently, growth variation was thought to be the result of social interactions resulting in that dominant fish consumed more feed and therefore grew faster than subordinates. However, if the presence of social interactions would be the main reason for growth variation it would be expected that in the absence of social interactions (such as in the case of individual housing) this variation would be reduced. This seems not be the case in the present study. Senegalese sole housed individually
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exhibited an individual variation in growth of 33%, suggesting that these individual differences may be inherent. Similar results were observed by Martins et al. (2005) that reported a variation in growth of 53% in African catfish Clarias gariepinus individually housed. Studies performed with Senegalese sole housed in groups reported an individual variation in growth ranging from 21% till 29% (Araga˜o et al., 2008; Costas et al., 2008) showing values in fact somewhat smaller than the values obtained in this study using individual housing. This suggests that in Senegalese sole the presence of social interactions does not explain the presence of individual differences in growth. Growth variation was observed in other flatfish both individually housed such as Greenback flounder (CV 19%, Carter and Bransden, 2001) and in groups such as Atlantic halibut (CV approx. 21%, Kristiansen et al., 2004). Furthermore, to which extend coping styles differ in performance traits such as feed efficiency as shown in Martins et al. (2005, 2006) and van de Nieuwegiessen et al. (2008) would be an interesting point to study within the context of aquaculture. In fact, the link between performance traits and coping strategies is largely unknown for aquaculture species. This is mainly due to experimental protocols that are limited in time and despite allowing a characterization of individual behaviour and stress response do not allow a proper characterization of performance traits that need longer experimental periods. 5. Conclusion In conclusion, this study quantifies for the first time the individual variation in growth, undisturbed and poststress cortisol levels, feeding behaviour and activity during acute stress in Senegalese sole. The relationships between these variables support the existence of coping styles. Behaviour traits like feeding latency and duration of escape attempts are easier, faster and cheaper to measure and were shown to predict undisturbed cortisol levels. When linked with performance traits, these behaviour traits, together with physiological measurements, may be of interest as a discriminatory tool in genetic selection programs. Acknowledgments We wish to thank H. Teixeira for practical assistance. This study benefited from funding by Project PROMAR SP5.P117/03 (programme INTERREG III A, co-funded by FEDER, European Commission), SFRH/BPD/49051/2008 and SFRH/BD/44103/2008 from ‘‘Fundac¸a˜o para a Cieˆncia e Tecnologia’’ (Portugal). References Araga˜o, C., Corte-Real, J., Costas, B., Dinis, M.T., Conceic¸a˜o, L.E.C., 2008. Stress response and changes in amino acid requirements in Senegalese sole (Solea senegalensis Kaup 1858). Amino Acids 34, 143– 148. Bolhuis, J.E., Schouten, W.G.P., Leeuw, J.A.D., Scharama, J.W., Wiegant, V.M., 2004. Individual coping characteristics, rearing conditions and behavioural flexibility in pigs. Behav. Brain Res. 152 (2), 351–360.
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