Emotional responsiveness in fish from lines artificially selected for a high or low degree of laterality

Physiology & Behavior 92 (2007) 764 – 772

Emotional responsiveness in fish from lines artificially selected for a high or low degree of laterality Marco Dadda a,⁎, Eugenia Zandonà b , Angelo Bisazza a a

General Psychology Department, Via Venezia 8, 35131, University of Padova, Padova, Via Venezia 8, 35131 Padova, Italy b Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA, USA Received 16 July 2006; received in revised form 15 May 2007; accepted 4 June 2007

Abstract Evidence showing that cerebral asymmetries exist in a wide range of animals has prompted investigation into the advantages and disadvantages of brain lateralization. In the teleost fish Girardinus falcatus individuals selected for a high degree of lateralization (LAT) performed better than those fish selected for reduced lateralization (NL) in several tasks, including schooling, foraging and spatial orientation. These findings were interpreted as evidence of hemispheric specialization allowing more efficient parallel processing and thus better cognitive performance under conditions that require multitasking, but the possibility that the results may simply reflect line differences in behavioral/physiological coping styles (i.e. in their emotional responsiveness during the tests) could not be ruled out. To test the hypothesis that NL and LAT fish differ in coping style, the present study examined differences in response in these lines to a novel situation in four different conditions. NL and LAT fish did not differ in a behavioral measure of emotional response: their readiness to explore a new environment. After being isolated in a tight space they showed a similar increase in opercular beating rates, suggesting that their physiological response to an acute stressor was comparable. The overall tendency to remain close to a shoalmate after being moved to an unfamiliar place was similar in the two groups but a significant difference was found in the temporal pattern; LAT fish swam closer than NL to their mirror image in the initial stages but this difference was later reversed. NL and LAT males placed in a new, unfamiliar environment did not differ in the number of sexual acts performed but LAT males resumed sexual behavior earlier signifying that cerebral lateralization has some influence on the trade-off between predator surveillance and mating behavior. Although this study found some differences between NL and LAT lines in their response to novelty, present evidence does not seem sufficient to justify the rejection of the hypothesis that the better scores in complex tasks shown by LAT fish in previous studies were primarily due to a cognitive advantage associated with cerebral specialization © 2007 Elsevier Inc. All rights reserved. Keywords: Lateralization; Boldness; Fish; Cerebral asymmetries

1. Introduction Recent evidence demonstrating that cerebral asymmetries exist in a wide range of animals, both among vertebrates (reviewed in [1]) and invertebrates [2–4] has produced an interest into the investigation of the advantages and disadvantages of brain lateralization (see [5] for a review of current ideas). Empirical evidence on this topic has rapidly accumulated. McGrew and Marchant [6] found that among free-living chimpanzees Pan troglodytes individuals specialized in using one hand were more efficient at termite fishing than individuals not showing such a ⁎ Corresponding author. Fax: +39 30 049 8276600. E-mail address: [email protected] (M. Dadda). 0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2007.06.001

specialization [6]. Gunturkun and colleagues [7], studying pigeons Columba livia, found increased visual discrimination in strongly lateralized individuals. Rogers and collaborators [8,9] compared normally and poorly lateralized chicks Gallus gallus that were obtained by incubating eggs in the dark during the final days before hatching [10]. Chicks had to learn to discriminate between food and non-food while a model of an avian predator was moved overhead. Lateralized chicks learned faster and were more responsive to the model predator while, in the control experiment without the predator, no difference in learning ability was found. Dadda and Bisazza [11] performed a similar experiment in the teleost fish Girardinus falcatus. They used fish from lines that had been selected for high and low degrees of laterality [12,13] and compared them in a situation which required the sharing of

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attention between two simultaneous tasks: prey capture and predator vigilance. Food-deprived individuals entered a compartment adjacent to the home tank to capture live brine shrimps either in the presence or absence of a live predator situated at some distance. With the predator visible, lateralized fish were twice as fast at catching shrimps than non-lateralized fish, while no difference in capture rate was recorded when the predator was absent and subjects were not required to share attention between vigilance and prey capture. Moreover, in a situation requiring the sharing of attention between retrieving food items scattered on the surface and avoiding unsolicited male mating attempts, lateralized females were significantly more efficient than non-lateralized females in retrieving food while no difference was found in control experiments in which the male was absent and subjects were not required to share attention between the two tasks [14]. Evidence for differences between these selected lines were also found in other contexts. Schools of lateralized fish (G. falcatus) observed in a novel environment showed significantly more cohesion and coordination than schools of nonlateralized fish. Moreover, in schools composed of both lateralized and non-lateralized fish, the latter were more often at the periphery of the school while lateralized fish occupied the center, a position normally safer and energetically less expensive [15]). Lateralized fish also proved to be better than nonlateralized fish at using features or geometric cues to re-orient themselves in a small environment [16]. It was suggested that the superiority of lateralized individuals demonstrated in these studies reflects their greater efficiency of neural computation [9,14]. In particular, specialization of cognitive function could enable separate and parallel processing to take place in the two hemispheres [8], thus allowing lateralized individuals to perform better under conditions that imposed a high cognitive load (e.g., multitasking). However, none of the above-mentioned studies could exclude that lateralized and non-lateralized individuals also differing in other traits, for example the way they respond behaviorally and physiologically to a novel situation. It is therefore possible that the difference in performance on cognitive tasks was due to a difference in “emotional reaction” to the test situation rather than to lateralization per se. Indeed, recent research has shown that individuals within a population may display distinct physiological and behavioral profiles [17,18]. These suites of correlated traits correspond to distinct coping styles, also referred to as animal personalities or behavioral syndromes [19,20]. One dimension, the boldness– shyness continuum in relation to anti-predator behavior, has been extensively studied and appears to have a genetic basis [21,22]. Coping styles influence behavior in a number of contexts (feeding, aggression, mating, dispersal, etc.) and often correspond to distinct hormonal profiles [23,24]. The neuroendocrine basis of coping styles is just beginning to be understood. In the rainbow trout Oncorhynchus mykiss, for example, plasma cortisol response to stress is under genetic control and can be artificially selected for [25]. Fish with low post-stress cortisol values tended to become dominant over high responding individuals and were more rapid to resume normal locomotory behavior and start feeding after being socially isolated [26,27]. Coping styles in this species have been shown

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to affect learning with bold trout requiring fewer trials in a conditioning task [28]. In the laterality studies just mentioned, it is possible that differences in coping styles have arisen as a side effect of the procedure employed to obtain lateralized and non-lateralized animals. For example random drift and correlated selection, i.e. genetic changes in traits not directly subjected to selection, are very common in artificial selection experiments [29]. In selected lines of G. falcatus genetic changes in emotionality may thus have occurred due to bottlenecks or as a consequence of using the reaction to a model predator to score individual laterality [12,13]. Differences in personality might also be directly associated with a different organization of cerebral functions. The two hemispheres of the brain play different roles in emotion, attention, perceptual processing and control of motor responses (reviewed in [1]) and some authors have suggested that differences in hemispheric dominance can lead to individuals that differ in cognitive styles and emotional responsiveness ([8], Andrew 2005, commentary to [5]). In this study, we subjected fish from lines selected for high and low degree of behavioral lateralization to four tests aimed at determining differences in coping styles. In the first experiment we tested the readiness of a fish to explore a new environment. Two further experiments measured, respectively, the tendency of a female to leave a shoal, and the tendency of males to resume mating behavior after being transferred to a new environment. In the last experiment we measured respiratory rate, a correlated response to the stress reaction when fish were moved to an unfamiliar place. 2. Methods 2.1. Subjects We used subjects from three stocks of fish (RD, LD and NL) that differed in laterality and that were obtained through selective breeding [12,13]. From 1997 to 2001 we artificially selected for left turning, right turning or no turning preference using a detour test [30] that scores the direction taken by a fish when facing a barrier behind which a model predator is visible. Directionally selected lines showed significant differences in their responses when compared with a control line. The line selected for no turning preferences also showed a significant response to selection and after a few generations it was composed of a great majority of fish that were non-lateralized at the individual level [13]. In 2001 we established three stocks of fish from the fifth and sixth generations of this selection experiment. Stocks have been maintained until the present in the following way: at approximately monthly intervals, the sub-adult progeny of each stock are measured in the detour tests. Individuals that satisfy requirements are admitted into the breeding tanks to produce the next generations. Requirements are that the fish turn 80% of the time or more to the left for LD stock, 80% or more to the right for RD stock and 50% in each direction for NL stock. To reduce inbreeding, fish from an unselected population chosen with the same criteria are added to the stocks at a proportion of about 5%.

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In this study we compared two groups, non-lateralized fish (NL) and lateralized fish (LAT). All fish were adults that had satisfied the laterality criterion appropriate for their stock. All NL fish came from the NL stock and therefore had the same laterality score (50% left and 50% right turns). Due to their limited availability, the group of lateralized fish (LAT) was composed of both LD and RD fish. In each experiment we checked for statistical differences between LD and RD lateralized fish and if no significant difference was found, they were pooled together for comparison with NL fish. Fish were maintained in small heterosexual groups (10–15 fish) of the same laterality and kept in 70-L glass aquaria with abundant vegetation (Ceratophillum sp.) and artificial lighting (16:8 h LD); water temperature was maintained at 25 ± 2 °C and all fish were fed dry fish food and nauplii of Artemia salina twice a day. 2.2. Experiment 1: exploratory tendency in an unfamiliar environment This experiment tested the readiness of a fish to explore a new environment after emerging from a small box, a method that has proved to be useful in investigations of both inter-and intraspecific boldness differences in fish [31,32]. We used a modification of the method to study the boldness of eight populations of the poeciliid Brachyraphis episcopi. The apparatus consisted of a black plastic square box (15 × 15 × 12 cm) provided with a trapdoor (14.5 × 12 cm) that could be lifted via a nylon thread connected to a pulley system allowing the fish to leave the box and explore the new environment. The box was placed in the center of a white plastic circle (42 cm Ø) and positioned inside a square-shaped glass aquarium (60 × 60 × 40 cm), with a 3 cmlayer of aquarium grit on the bottom, filled with 10 cm of water and lit by two 18 W-neon lights. Above the apparatus, at a 2.50 mdistance, a video camera was positioned to record the experimental sessions. The apparatus was entirely surrounded by black curtains. Twenty LAT (10 RD, 10 LD) and 20 NL adult females were used for this experiment. To reduce handling time prior to the test and to allow repeated measures of the same individual, 24 h before the test, each subject was taken from its home aquarium and placed in a separate glass tank (40 × 20 × 35 cm) with 3 smaller females from the stock population. The day of the experiment the subject was put in a small cylinder (6 cm Ø) to be carried to the apparatus. We gently inserted the fish from the cylinder into the removable upper side of the box. After a 5 min acclimatization period the door was slowly lifted allowing the fish to leave the box and the behavior of the fish was recorded for 4 min. Each subject was observed twice a day, once in the morning and once in the afternoon (with an approximate 3 h interval between tests), for three consecutive days, with a total of 6 observation sessions per fish. After each session the fish was returned to a separate glass tank. Analysing the video recordings, we measured the time taken to emerge from the box after we had opened the trapdoor and the time taken to cross the white circle. Following [31] we defined the boldness score as the time taken for the fish's snout to emerge from the box and a “hesitancy score” was defined as the time the fish took to cross

the white circle minus the time it took to emerge from the box. If the fish still had not emerged after 4 min we terminated the trial and allotted the fish a ceiling value of 240 s. Four subjects (1 RD, 1 LD and 2 NL) were tested each day in random order. 2.3. Experiment 2: shoal tendency in a novel environment Aggregation in shoals is a common response of fish to a potential hazard [33,34]. Placed in a novel tank containing a mirror, fish of a large number of species show a strong tendency to swim tightly parallel to the mirror as if they considered their mirror image as a shoalmate [35]. In this test the subjects were placed singly in a novel environment surrounded by mirrors and we measured the tendency of the subject to separate from a virtual shoalmate. Subjects were placed in an apparatus (Fig. 1) that consisted of a glass aquarium (60 × 60 × 45 cm) with eight mirrors (27 × 37 cm) placed around the aquarium's walls to create an octagonal shape. The bottom of the aquarium was white with a green line painted at a 1 cm distance from the mirrors. In this way it was possible to determine with precision when the fish swam at a distance of more than 1 cm from the mirror. The apparatus was filled with 10 cm of water and lit by two 18 Wneon lights. A hollow transparent cylinder (10 cm Ø, 15 cm height) was placed in the middle of the apparatus and a video camera was positioned above the apparatus at a distance of 2.5 m. The whole apparatus was surrounded by black curtains. Twenty LAT (10 RD, 10 LD) and 14 NL adult females were used for this experiment. To reduce handling time prior the test, 24 h before the test, each subject was taken from its home aquarium and placed in a separate glass (20 × 40 × 30 cm) tanks with 3 smaller females from the stock population. On the day of the experiment, a fish was placed inside the transparent cylinder for two minutes after which the cylinder was lifted using a nylon thread. Every subject was observed for 10 min. Observing the video recordings, we could measure the frequency of separation from the mirror (defined as a distance of more than one centimeter from the mirror), the duration of the

Fig. 1. Schematic representation of the apparatus used in experiment 2 to measure individual shoaling tendency in a novel environment. Each subject was placed in the transparent cylinder in the center of the apparatus. After 2 min the fish was released and its shoaling behavior in relation to its mirror image was recorded for 10 min. The number of separations from the mirror (more than one centimeter) was taken as the measure of shoaling tendency.

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separation and the swimming direction (clockwise or counterclockwise). 2.4. Experiment 3: male mating behavior in a novel environment Tradeoffs between mating and vigilance against predators have been demonstrated in many species [36]. Among poeciliids, there is a wide interspecific variation in their readiness to resume mating behavior when moved to a novel potentially hazardous environment [37] and intra-specific differences in readiness to mate following a perceived threat have been reported in some species [38–40]. In this experiment we measured the number of sexual acts and the latency to initiate mating behaviour in lateralized and non-lateralized males after they had been moved to a novel environment. The apparatus used for the experiment consisted of a glass aquarium (68 × 68 × 38 cm) with a green plastic bottom (47 × 47 cm). Four females matched for dimensions (3.8– 4.2 cm), coming from the stock population, were kept in the apparatus for at least 3 days before the test started. The entire apparatus was lit by two 18 W-neon lights. A video camera was mounted 1 m above the apparatus. Twenty-three LAT (11 RD, 12 LD) and 18 NL adult females were used for this experiment. Subjects were dip-netted, placed in a little plastic opaque container (8 × 6 cm) moved to the experimental room and gently inserted into the apparatus. Fish were video recorded for 40 min and from the video clip we determined the total number of male mating attempts and the latency to the first and fifth mating attempt. 2.5. Experiment 4: operculum opening frequency In fish, ventilatory frequency is a good indicator of the physiological stress reaction and many studies have shown that ventilation rates readily respond to frightening stimuli such as alarm substances or predator models [41–43]. In this test, we measured the opening frequency of the operculum after fish had been moved to an unfamiliar place. The experimental apparatus consisted of a small plastic bin (12 × 12 × 8 cm) with a rectangular section (6.5 × 2 × 3.5 cm) inside made of green plastic. The apparatus was funnel-shaped so that after the reduction of the water level (to a 5 cm limit) by opening a small tap, the fish could gently enter a rectangular section (6.5 × 2 cm) of the apparatus. Following [43], we constrained fish in a small space to avoid the confounding effect of elevation of metabolic rate following escape bursts. A digital camera was mounted 15 cm above the apparatus. The apparatus was lit by two 18 W-neon lights and surrounded with black curtains. Ten LAT (5 RD, 5 LD) and 10 NL adult females were used for this experiment. To standardize and reduce handling time prior to the test and to allow repeated measures of the same individual, 24 h before the test, the subjects of the experiment were moved singly from the common tank to an identical aquarium containing only three smaller individuals. On the day of the experiment, the subject was rapidly dip-netted and carried to the apparatus. We gently placed the fish into the apparatus,

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the water level was reduced in 40 s and its behavior was observed for 5 min. We recorded opercular opening rates of the subject in two separate sessions, one in the morning and one in the afternoon, separated by a 3-hour period. Four subjects (1 RD, 1 LD, 2 NL) were tested daily. 3. Statistical methods We tested data for normality and homogeneity of variance, and where necessary, appropriate transformation of the data was performed. All probabilities are two-tailed. Statistics were done using SPSS 11.5.1. 4. Results 4.1. Experiment 1: exploratory tendency in an unfamiliar environment Boldness and hesitancy score were analyzed with repeated measures ANOVA; the two daily observation sessions (morning and afternoon) and the three days of the experiment were the “within-subjects” factors while the type of laterality was the “between-subjects” factor. The two groups of lateralized subjects (RD and LD) did not differ statistically for “boldness score” (F(1, 18) = 0.034, p = 0.856, power = .054). We found a significant effect of the three test days (F(2, 36) = 3.518, p = 0.040, power = .619) (from a latency of 55.40 ± 8.04 s in the first day to a latency of 33.2 ± 6.26 s in the third day) and a marginally non-significant effect of the two sessions (F(1, 18) = 4.245, p = 0.054, power = .496). No interactions were statistically significant. RD and LD were therefore pooled together and considered as a single group (LAT) for subsequent analyses. There were no statistically significant differences between LAT and NL subjects in latency times (F(1, 34) = 0.028, p = 0.869, power = .053; Fig. 2). Latency to exit the box increased significantly between the two observation periods (F(2, 68) = 10.317, p b 0.001, power= .984), while it significantly decreased after the first day (F(1, 34) = 8.459, p =0.006, power = .807). When considering the “hesitancy score” we found no significant difference between the two groups of lateralized

Fig. 2. Latency to emerge from the box for LAT and NL subjects in three consecutive days. Each day, fish were subjected to two tests: one in the morning and one in the afternoon (mean ± standard error).

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subjects RD and LD (F(1, 18) = 2.636, p = 0.122, power = .336) and no other factors or interactions were significant. RD and LD were therefore pooled together. When compared, LAT and NL subjects revealed no statistically significant differences (F(1, 34) = 0.614, p = 0.439, power = .119). No other factors or interactions were significant. 4.2. Experiment 2: shoal tendency in a novel environment The number of separations from the mirror was computed separately for each minute. Data were analysed using repeated measures ANOVA; the minute of the test was the “within subjects” factor while laterality type was the “between subjects” factor. The number of separations from the mirror per minute was the dependent variable. When we compared RD and LD fish, there was no significant effect due to direction of laterality (F(1, 18) = 1.365, p = 0.258, power = .198) or minute of test (F(9, 162) = 1.678, p = 0.098, power = .756) and no significant interaction. RD and LD were therefore pooled together and considered as a single group (LAT) for subsequent analysis. When LAT and NL subjects were compared (Fig. 3), we found no significant effect of lateralization (F(1, 32) = 1.864, p = 0.182, power = .263) or of minute of the test (F(9, 288) = 1.400, p = 0.188, power = .672; linear trend (F(1, 32) = 4.712, p = 0.037) while the interaction (minute × lateralization) was significant (F(9, 288) = 3.103, p = 0.001, power = .975). NL subjects show a significantly greater frequency of separation from the mirror than LAT subjects in the first minute (t(32) = 2.659, p = 0.012) whereas, the reverse occurred in the fourth (t(32) = 2.406, p = 0.022), and ninth minute (t(32) = 2.247, p = 0.032). No significant difference was found when the 10 min of test were considered (t(32) = 0.936, p = 0.356). In this experiment we did the normality criterion for the single minutes was not meet even after logtransformation of the data. To confirm the above results we performed a non-parametric analysis (Mann–Whitney U test) on each minute of the test. RD and LD fish did not differ in any of the minutes of the test or in total time (p N 0.1 in all tests). NL subjects show a greater frequency of separation from the mirror in the first minute (p = 0.017) whereas, the reverse occurred in the fourth (p = 0.023), and ninth minute (p = 0.037). No difference was found in the remaining minutes or when the 10 min of test were considered. (p N 0.1 in all tests).

Fig. 3. Tendency to leave a shoal companion in LAT and NL subjects measured as the number of times fish separate from the mirror in a minute (mean ± standard error).

Finally, we considered the time spent by each subject at a distance greater than 1 cm from the mirror. RD and LD subjects did not differ in total time spent far from the mirror (F(1, 18) = 1.506, p = 0.236, power = .213) and there was no effect of minute (F(9, 162) = 1.108, p = 0.360, power = .536) or significant interaction. There is no significant difference between LAT and NL (F(1, 32) = 1.306, p = 0.262, power = .198). Time spent away from the mirror tended to decrease with time (F(9, 288) =2.062, p = 0.033, power = .864; linear trend F(1, 32) = 3.359; p = 0.076) and the interaction was not significant. No significant difference was found when the 10 min of test were considered (t(32) = 1.727, p = 0.094). Mann–Whitney analysis confirmed a lack of significant difference between RD and LD fish and between NL and LAT (p b 0.05). In total, LD subjects swam clockwise for 67 ± 0.11% of the time (one sample T test, t(9) = 4.782, p = 0.001), while RD fish swam clockwise for 39 ± 0.15% of the time (t(9) = 2.160, p = 0.059). The difference between LD and RD subjects was highly significant (t(18) = 4.537, p b 0.001). NL fish showed no preference in their swimming direction (52% ± 0.14; t(13) = 0.495, p = 0.629). 4.3. Experiment 3: male mating behavior in a novel environment Subjects from the RD and LD groups did not differ in the number of mating attempts during the test (RD 32.66 ± 7.66; LD 44.18 ± 17.41, two sample T test t(21) = 2.021, p = 0.064). RD fish showed a latency of 10.61 ± 5.52 min to their first mating attempt and LD fish a latency of 9.67 ± 5.51 min. The difference between these groups was not significant (two-sample T-test, t(21)= 0.404, p = 0.690). No significant difference was found between the groups in the latency to the fifth mating attempt (LD 12.38 ± 4.82; RD 13.64 ± 4.92; t(21) = 0.629, p = 0.536). RD and LD were therefore pooled together and considered as a single group (LAT) for subsequent analysis. LAT and NL fish did not differ in the number of mating attempts (LAT 38.17 ± 14.20; NL 32.94 ± 11.50, two sample T test t(39) = 1.269, p = 0.212). However LAT subjects had shorter latencies than NL both to the first (LAT 10.16 ± 5.41; NL 15.20 ± 3.84; t(39) = 3.337, p = 0.002) and to the fifth mating (LAT 13.04± 4.80; NL 17.86 ± 3.79; t(39) = 3.486, p = 0.001). 4.4. Experiment 4: operculum opening frequency Average opercular opening frequency was computed separately for each minute. Data were analyzed with repeated measures ANOVA with the two observation sessions (morning or afternoon) and the minute of the test as “within-subjects” factors and the type of laterality was the “between-subjects” factor. Since the respiratory rate has been correlated with the dimensions of the fish [31], for these analyses, the length of the subjects was considered to be a covariate. No significant difference was found between the two groups of lateralized (RD and LD) subjects (F(1, 8) = 0.998, p = 0.344, power = .144). There was no statistically significant difference between the morning and afternoon observation sessions (F(1, 8) = 0.039, p = 0.848, power = .054). No other factors or interactions were

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Fig. 4. Operculum opening frequency (mean openings per second ± standard error) in LAT and NL subjects exposed to the stress of confinement in a small place.

significant. RD and LD were therefore pooled together and considered as a single group (LAT) for subsequent analysis. LAT and NL fish (Fig. 4) did not differ in their opercular opening rate (F(1, 20) = 0.503, p = 0.486, power = .175). There was no statistically significant difference between the two observation sessions (F(1, 20) = 1.540, p = 0.229, power = .219). No other factors or interactions were significant. 5. Discussion A series of recent studies have compared lateralized and nonlateralized fish in several biologically relevant situations. The former showed superior performance in several tasks including schooling, spatial orientation and prey capture [11,15,16]. These results were interpreted as evidence that lateralization of cognitive functions improves neural-processing, especially under conditions that imposed a high cognitive load, by enabling separate and parallel processing to take place in the two cerebral hemispheres [5,8]. The aim of the present study was to test the alternative hypothesis that differences in performance between lateralized and non-lateralized fish might reflect the different emotionality displayed by these two groups of fish during laboratory tests. In the first experiment we measured the readiness of a fish to emerge from a shelter and explore a novel environment, a test that had been used as a measure of boldness in fish both at both inter-and intra-specific level [44,31,32]. In this experiment there were two significant effects: the latency in exiting from the plastic box decreased during the three days of tests and fish were significantly slower in exiting during the second daily session compared to the first one. We found, however, no significant differences between fish selected for high laterality and fish selected for low laterality as regards the latency to explore an unfamiliar environment. The hypothesis that lateralized and non-lateralized fish did not basically differ in their response to a new environment was confirmed by the results of experiment 4 in which we quantified their opercular beating rate, a more direct measure of physiological response to a stressor. Change in respiratory rate is a common vertebrate response to a perceived threatening situation and is thought to be in preparation to physical responses such as fighting or

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fleeing [45,46]. Respiratory rate is a non-invasive measure of the stress response in fish and it has been shown to correlate with plasma corticosteroids levels under standard conditions [47,48]. Several species were shown to respond to frightening stimuli with increased ventilation rates [41–43] and recently, Brown and colleagues [31] observed significant differences in opercular beating rates between fish from populations with high and low levels of predation pressure when fish were experimentally confined in a small space. Our data on respiratory rate provide no evidence that lateralized and non lateralized fish differ in their response to an acute stress situation. However, some caution should be exercised in drawing conclusions from this experiment since the sample size was smaller than the other experiments and statistical power of the tests is very low. We found some differences between lateralized and nonlateralized fish in experiments 2 and 3. In experiment 2 we measured the tendency to shoal with a companion, a common behavioral response to predation risk in fish [33,34]. Ward and colleagues [49] showed that individual stickleback which resume feeding rapidly after a simulated predatory attack (bold fish) were also competitively dominant and displayed low shoaling tendencies. In our study we used a modification of the standard shoaling tests, the mirror test [35,50,51] that allows the shoaling tendency of single individuals to be measured. On the whole, LAT and NL fish did not differ in their tendencies to depart from the mirror. However differences in timing were found between the two groups. Compared with non-lateralized subjects, lateralized fish showed a greater tendency to stay close their mirror image in the first two minute of the test, but the reverse was observed in the remaining minutes of the test. When moved to an unfamiliar place, males from lateralized and non-lateralized stocks did not differ in the total number of mating attempts they performed. However we found a significant difference between lateralized and non-lateralized fish in the latency to the first mating attempt, with NL males nearly 50% slower than LAT males in resuming sexual activity. Mating activities often enhance conspicuousness and reduce vigilance, thus increasing exposure to predation [36,52,53] Male propensity to exhibit mating behavior under a perceived threat is strongly influenced by coping style [38–40]. Results of this experiment therefore suggest that NL and LAT fish may differ widely with regard to a trade-off between mating and antipredator behaviour. The differences in female shoaling tendency and in male sexual readiness when fish were placed in an unfamiliar environment might indicate that a genetic differentiation in emotionality had occurred during the artificial selection process. There are two possible ways in which selection for laterality might have generated differences in coping style among selected lines. The first is that personality differences are a correlated trait of selection for different degrees and direction of behavioral lateralization. Since the two cerebral hemispheres play different roles in the control of emotion, attention, perception and motor responses some authors have suggested that individual differences in hemispheric dominance may lead to variation in cognitive styles and emotional responsiveness ([8]; Andrew

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2005 Commentary to [5]). A link between cerebral asymmetries and emotional response has indeed been recognized in studies of human lateralization [54–56] and recent results suggest this possibility for animals too. In nonhuman primates the perception and the expression of emotions is lateralized [57] and in two species, the chimpanzee and the marmoset, right-handers were found to show more explorative behavior than left-handers [58,59]. Recently, a possible covariation of laterality and boldness has been reported among natural populations of the poeciliid fish Brachyraphis episcopi ([18,31]. Differences in the latency to emerge into a new compartment were observed in the zebrafish Danio rerio, between wild type fish and fish bearing the fsi mutation that causes situs inversus, but also affects behavioral lateralization [60]. The second possibility is that changes in personality have occurred as a side effect of the selection procedure. To score and select fish with different behavioral lateralization, we repeatedly measured the direction of escape reaction of individual G. falcatus when a realistic model predator was encountered [12,13]. It is conceivable that this selection procedure might also act upon allele frequency of genes that control boldness in antipredator behavior. There is a third possible explanation which may account for the differences between LAT and NL without necessarily assuming that differences in personality had occurred. If the hypothesis suggested by Rogers [8] is correct, we would expect lateralized individuals to be more efficient in situations of high cognitive load. A fish shoaling in a new environment must perform several concurrent cognitive tasks, i.e. monitoring the new environment for predator presence, taking continuous track of the position and changes of direction of the shoal mates, maintaing cohesiveness and alignment with the other shoal mates [15,61]. A similar high cognitive load is expected to occur for Girardinus males engaged in sexual behaviour in a novel place. They need to monitor the surroundings for competitors presence or predatory attacks, take track of the female's movements and responses, co-ordinate perceptual and motor skills to insert their copulatory organ in the female genital opening [62]. Individuals with a more efficient management of attention resources (lateralized fish in Rogers' hypothesis) may be able to shift earlier from the sole scanning of the new location to the simultaneous performance of two or more tasks required by the test. In addition, lateralized fish, thanks to their better ability to run simultaneous cognitive tasks without interference may be in general more “self-confident” after being displaced to a novel situation. Among primates, left and right-handed individuals have been found to differ in coping style [58,59]. By contrast, we found little evidence in this study that the two lateralized types of fish, LD (left detour) and RD (right detour), differed in their emotional response. It is necessary however, to note that the sample size in these comparisons was usually rather small and the power of the statistical tests was consequently very low. An exception to the general resemblance of fish from the two lateralized stocks was found in the direction of swimming in the mirror test, with LD subjects swimming predominantly clockwise (thus keeping the virtual companion on their left

side) and RD fish swimming predominantly counter-clockwise. This finding is in agreement with previous studies indicating that LD and RD fish have a similar but completely mirrorreversed organization of cerebral functions, while NL fish tend to have a bilateral representation of most cognitive functions [30,63,64]. There is also confirmation of the complementary use of the two eye-system found in other species [51]: fish that prefer to observe a shoal mate with one eye (experiment 2 of this study) also prefer to look at a predator with the contra-lateral eye (detour test and [11]). In summary, in this study we found limited evidence that lateralized and non-lateralized fish differ in their emotional response. Two tests, one measuring the readiness of a fish in emerging into an unfamiliar area and one of the respiratory rate after an acute stress, demonstrated no difference in performance linked to the degree of cerebral lateralization but partial differences were found in two other tests measuring a behavioural response after placement in a novel area: the latency to male mating behaviour and schooling tendency in females. We therefore cannot presently exclude the possibility that lines of fish selected for different degrees of lateralization also differ in their shyness/boldness dimension or in other aspects related to coping style although the observed differences might also be explained by different capabilities in the management of attention resources under conditions that impose a high cognitive load. In one recent study that compared cognitive abilities of the same fish (LAT and NL) used here [11], subjects had to enter a compartment adjacent to the home tank to capture live brine shrimps either in the presence or in the absence of a live predator situated at some distance. With a predator present, LAT fish were found to be almost twice as fast as NL fish in catching their prey. In another study, we found that schools of lateralized fishes moving in a novel environment showed significantly more cohesion and coordination than schools of non-lateralized fish. Following Rogers' suggestion [8], we hypothesized that these difference derived from a cognitive advantage conferred by cerebral specialization to lateralized individuals. Differences between lateralized and non-lateralized fish found in the present study do not seem sufficient to explain the difference in performance of schooling or of prey capture, in terms of being entirely due to a variation in the shyness/boldness dimension or in other aspects related to coping style. Thus, we cannot presently reject the hypothesis that the better performance of lateralized individuals in complex tasks is due to an augmented efficiency in neural-processing associated with cerebral specialization. This study highlighted some of the difficulties in evaluating intra-specific variation in copying styles when the analysis is restricted to behavioral data. The differences in the reaction to a novel situation seen here could have arisen either from variation in emotionality, i.e. a different response of NL and LAT fish to a potentially harmful situation, or from variation in cognition, e.g. a greater capacity for parallel processing in lateralized fish. In mice, chicks, tits, trouts and quails, coping style was found to be associated with response of neuro-endocrine system to stressors [24,25,65–67]. Future studies providing a more direct measure of the physiological response to stress, such as variation of

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