The effect of the number and size of animated ...

PBB-72112; No of Pages 9 Pharmacology, Biochemistry and Behavior xxx (2015) xxx–xxx

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The effect of the number and size of animated conspecific images on shoaling responses of zebrafish Yohaan Fernandes a,1, Mindy Rampersad b,1, Jason Jia c, Robert Gerlai a,b,⁎ a b c

Department of Cell and System Biology, University of Toronto, Canada Department of Psychology, University of Toronto Mississauga, Canada McMaster University, Hamilton, Canada

a r t i c l e

i n f o

Available online xxxx Keywords: Zebrafish Social behavior Behavioral phenotyping Behavioral screens

a b s t r a c t The zebrafish is increasingly utilized in biomedical and psychopharmacological research aimed at modeling human brain disorders. Abnormal social behavior represents the core symptom of several neuropsychiatric and neurodevelopmental disorders. The zebrafish is a highly social species and has been proposed for modeling such disorders. Behavioral paradigms that can induce zebrafish social behavior are of importance. Here, we utilize a paradigm in which zebrafish are presented with computer animated images of conspecifics. We systematically varied the size of these images relative to the body size of the experimental fish and also investigated the potential effect of presenting different number of images in an attempt to optimize the paradigm. We report that images similar in size to the experimental fish induced a strong shoaling response (reduction of distance to the image presentation screen) both when the body size of the experimental fish was varied with the image size being held constant and when the image size was varied with the body size of the experimental fish being held constant. We also report that within the number range studied (from 1 to 8 conspecific stimulus fish), presentation of all animated shoals, but the image of a single conspecific stimulus fish, led to significant reduction of distance to the presentation screen. We conclude that the shoal image presentation paradigm induces robust social responses that are quantifiable in an automated manner, making the paradigm useful for screening of drugs and/or mutations. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Identification of mutations and drugs that influence brain function may require large scale behavioral screens especially if the target behavioral phenotype or modeled human brain disorder is complex. Social behavior and human brain disorders associated with abnormal social behavior are some of the most complex phenotypes with underlying mechanisms that are expected to involve a large number of molecular players. In the current paper, we attempt to explore the first steps towards the optimization of a simple social behavioral paradigm developed for the zebrafish, a vertebrate model organism that has been proposed to be among the most effective translational tools for large scale screening in the context of complex brain disorders (e.g. Gerlai, 2010a, 2010b). We argue that systematic analysis of methodological questions of behavior is a crucial endeavor, and the sooner the zebrafish field focuses on them, the faster we can integrate behavioral approaches

⁎ Corresponding author at: Department of Psychology, University of Toronto Mississauga, 3359 Mississauga Road North, Rm DV4023C, Mississauga, Ontario L5L 1C6, Canada. Tel.: +1 905 569 4255 (office), +1 905 569 4257 (lab); fax: +1 905 569 4326. E-mail address: [email protected] (R. Gerlai). 1 These authors contributed equally to the publication.

into multidisciplinary analysis of the biological mechanisms of vertebrate brain function and dysfunction (Gerlai, 2002, 2014b; Crabbe et al., 1999). Zebrafish are prolific (producing 200–300 eggs at each spawning), their eggs are fertilized externally, and their embryos are transparent (Kimmel, 1989). Adult fish are small (4 cm long) and cheap to maintain (Sison et al., 2006). These characteristics are among those that have made the zebrafish a favorite of developmental biologists (Vascotto et al., 1997). By now numerous forward and reverse genetic tools have been developed for this species (Grunwald and Eisen, 2002) to aid developmental biology research, but as a result the zebrafish has generated considerable interest in other subdisciplines of biology. One of these fields is behavioral neuroscience, which has seen a rapid accumulation of knowledge on zebrafish neurobiology and behavior (Gerlai, 2011; Kalueff et al., 2014). However, despite this rapid increase, the zebrafish is still considered a newcomer as our understanding of its behavioral features is rudimentary and the number of behavioral tasks with which to conduct our research is limited (Gerlai, 2012). This is a significant drawback as behavioral analysis could help us identify drugs or mutations that alter brain function and thus could aid the investigation of neurobiological mechanisms of diseases of the central nervous system (Gerlai, 2002). Briefly, the need for efficient and informative behavioral tests is clear (Sison et al., 2006).

http://dx.doi.org/10.1016/j.pbb.2015.01.011 0091-3057/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011

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Recently, a simple behavioral test paradigm utilizing the natural tendency of the zebrafish to form groups (shoals) has started to be utilized (Saverino and Gerlai, 2008; Abaid et al., 2012). Shoaling behavior (Pitcher, 1983) of zebrafish can be observed in nature (Spence et al., 2008; Engeszer et al., 2007a, 2007b) and can also be easily induced in the laboratory (Saverino and Gerlai, 2008; Miller and Gerlai, 2011a). When a single zebrafish sees a group of conspecifics it will approach and attempt to join the group (Gerlai et al., 2000). Most of our understanding about shoaling in zebrafish has come from studies utilizing live shoals (Maaswinkel et al., 2013; Pagnussat et al., 2013; Green et al., 2012; Miller et al., 2012; Miller and Gerlai, 2007, 2008, 2011a, 2011b, 2012). While these studies have increased our ability to investigate dynamic features of shoaling behavior, the need for a simple and efficient social behavior test paradigm continues to exist (Miller and Gerlai, 2011a). Recently, such a test paradigm has been developed. An alternative to measuring social behavior in live shoals is to provide computer generated conspecific images and measure the response of a single experimental subject to this stimulus (Saverino and Gerlai, 2008). Measuring the response of a single subject makes identification of mutants in mutagenesis studies easier and using computers to deliver the stimulus also has advantages (Sison et al., 2006; Gerlai, 2010a, 2010b). For example, one can precisely control the parameters of the stimulus, including numerous visual features, and the timing and location of its presentation. Computerized stimulus delivery eliminates experimental error variation inherent to studies using live shoals and also reduces the amount of experimenter interference. Furthermore, computerization of the stimulus presentation allows for the running of multiple trials simultaneously (Fernandes and Gerlai, 2009), characteristics that can significantly increase throughput to a level required for large scale mutation and/or drug screens (Gerlai, 2010a, 2010b). Importantly, in a recent study the responses of zebrafish to computer animated conspecific images were found statistically indistinguishable from those induced by presentation of video-recordings of live stimulus fish, of live fish placed outside of the experimental tank and of live stimulus fish placed inside the experimental tank separated from the experimental fish by a perforated transparent Plexiglas barrier (Qin et al., 2014). These results suggest that induction of robust shoaling responses does not necessarily require live stimulus fish or even three dimensional appearance of movement of the presented stimuli. Computerized delivery of social stimuli and the analysis of shoaling responses to these stimuli have been successfully employed in a variety of studies investigating a range of complex questions including the neurochemical correlates of social behavior (Gerlai, 2014a, 2014b; Saif et al., 2013; Scerbina et al., 2012), the effect of embryonic alcohol exposure (Fernandes and Gerlai, 2009), and the effects of acute and/or chronic alcohol exposure (Gerlai et al., 2009). Given the potentially broad applicability of the shoaling paradigm, it is important to find optimal parameters of shoal image presentation, a method that would induce the most robust, maximal shoaling response in zebrafish. A robust shoaling paradigm may be utilized to investigate the biological (genetic) underpinnings of this complex behavior, and would also allow one to analyze pharmacological agents that modify this response. Furthermore, it would allow one to examine functional changes in the brain induced in zebrafish models of human brain disorders associated with abnormal social behavior (Gerlai, 2012). However, systematic analysis of the parameters of stimulus fish image presentation has not been conducted for zebrafish. The present study represents the first step in this optimization process. Here, we examine the potential effect of the size of the animated conspecific images and also the number of the presented images on the strength of the shoaling response in zebrafish. Body size may be important in the context of dominance and mating competition. Larger fish are usually more dominant and can compete for food and mates more successfully (Huntingford et al., 1990). Body size may also be relevant from the perspective of the oddity effect (Landeau and Terborgh, 1986). Fish that stand out among other shoal members may be more

easily detected by predators and this may be a disadvantage especially when the unique individual is larger than the rest of the shoal members. Thus, the size of the experimental fish relative to that of the stimulus fish may matter and the relative size difference may influence the strength of the shoaling response in the experimental fish. We predict that due to the conflict between the effect of body size in the context of mating versus predation risk (larger fish are more successful at mating but may be predated upon with higher probability), the experimental fish should show the strongest shoaling response towards conspecific images whose body size is similar to that of the experimental fish. In addition, we also investigated whether the number of stimulus fish presented may alter the strength of the shoaling response. Larger shoals may allow better detection of predators by the shoal members (more eyes see more), but they may also lead to increased competition for resources such as food, and may also result in increased interference or stress among the shoal members. Zebrafish have been observed to form shoals in nature ranging in numerical size from only a few (2–3) to several hundred members per shoal (Spence et al., 2008). In several other fish species, larger, i.e. more numerous, shoals were shown to be preferred to smaller ones, at least when the number of shoal members tested ranged between 1 and 10 stimulus fish (Gómez-Laplaza and Gerlai, 2011a, 2011b, 2012, 2013a, 2013b; Piffer et al., 2013, 2012; Agrillo and Bisazza, 2014; Agrillo et al., 2012; Bisazza et al., 2014). Based on these results, we predict that zebrafish will show less strong shoaling responses towards shoals that contain fewer stimulus fish at least in the small number range studied in the current experiment. To test the validity of the above predictions we systematically varied (a) the size of the experimental zebrafish while presenting stimulus fish of a constant size; (b) the size of the stimulus fish image while keeping the size of the experimental fish constant and (c) the number of stimulus fish images of a size identical to that of the experimental fish. As a measure of the strength of the shoaling response, we quantified the distance of the experimental zebrafish to the computer screen presenting the different stimuli. 2. Methods 2.1. Animals and housing Adult sexually mature zebrafish (Danio rerio) between the ages of 12 and 18 months of the AB strain bred in our facility (University of Toronto Mississauga Vivarium, Mississauga, ON, Canada) were used in the current study. Progenitors of this population were obtained from the Zebrafish International Resource Center (ZIRC, Eugene, OR, USA). The experimental fish were sixth generation from the original founders. All fish were held in social groups in their home tanks, 3 liter plastic tanks that were part of a high density system rack (Aquaneering Inc., San Diego CA) that had a multistage filtration system equipped with mechanical (sponge), biological (fluidized glass bed), chemical (activated carbon) filtration and a UV light-based sterilizing unit as described before (e.g. Jia et al., 2014). The housing density was 10–13 fish per 3 liter tank. The water on the rack (system water) was identical to the water used in all experiments and was obtained using reverse osmosis purification. To obtain the desired water chemistry (300 microSiemens conductivity and neutral pH), the reverse osmosis purified water was supplemented by sea salt (Instant Ocean, Big Al's Aquarium Warehouse, Mississauga, Ontario, Canada). 2.2. Behavioral apparatus The experimental setup was a 37-liter glass aquarium (50 × 25 × 30 cm, length × width × height) that had a flat LCD computer screen (17 inch Samsung SyncMaster 732N monitor) placed flush on its left and right side (Fig. 1). Each monitor was connected to a Dell Vostro 1510 Laptop running a custom made software application (Saverino

Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011

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Fig. 1. The experimental setup (A) and an example of the stimulus shoal presented (B). Note the two computer monitors (stimulus presentation screens) placed flush against the two side walls of the experimental tank. These monitors could present animated (moving) images of conspecifics (stimulus) for predetermined periods of time. For each experimental fish, the side of presentation was constant but the presentation side varied across the experimental subjects. The size or the number of images shown was systematically varied across the three experiments as described in the Methods. For further details on the image presentation parameters experimental procedures and design see Methods. (Modified from Jia et al., 2014; Pather and Gerlai, 2009).

and Gerlai, 2008; Qin et al., 2014) that allowed the presentation of animated fish images on one or the other side. Although images of stimulus fish were presented against a black background using a computer monitor on a single side (see below), the illumination level in the test tank was homogeneous due to the strong light provided by a 15 W fluorescent light-tube placed directly above the tank. The back and the bottom of the tank was coated with white corrugated plastic sheets to increase contrast and to reduce glare and reflections for videotracking analysis. Two identical experimental setups were used in parallel. The behavior of experimental fish was recorded onto the hard drive of 2 video-cameras (JVC GZ-MG37u and GZ-MG50) and the digital recordings were transferred to the hard drive of a desktop computer (Dell, Dimension 8400) and later replayed and analyzed using the Ethovision Color Pro Videotracking software (Version 8.5XT, Noldus Info Tech, Wageningen, the Netherlands). 2.3. Behavioral test procedure Experimental zebrafish were placed in the test aquarium singly, and 30 s later we started a 23-minute long recording session. During particular intervals of the recording session, depending on the experimental condition, the single experimental subject was presented with five individually moving animated images of zebrafish (Fig. 1), which were 3.2 cm (in Experiment 1), or varied in size (in Experiment 2). In Experiment 3, the image presentation was identical to that of Experiments 1 and 2, but here the size of the images matched the experimental fish and the number of these images was systematically varied as described below. In all experiments the images of zebrafish were moving in

realistic manner following a horizontal path with slight vertical adjustments with a speed similar to that of live zebrafish (ranging between 1.5 and 4 cm/s). The variation in vertical movement and horizontal speed followed a stochastic pattern as determined by the software application developed in house (see e.g. Qin et al., 2014). Each experimental fish received the image presentation on only one side, i.e. either on the left or the right side, but the image presentation side varied randomly across the subjects. In all experiments, the animated images were of high-resolution photographs of a single adult female zebrafish of the AB strain, exhibiting the wild type striped pattern, coloration and wild type short fin phenotype. The photograph showed the fish from the side in a swimming position, i.e. with half erect fins. The rationale for the choice of this stimulus has been detailed elsewhere (Gerlai et al., 2009; Fernandes and Gerlai, 2009). In experiment 1, the experimental subjects themselves varied in size, while the size of the animated zebrafish images remained constant at 3.2 cm. Experimental fish were measured from head to tail in order to determine body length, and were subsequently divided into three groups based on these measurements. Fish that were up to 2.9 cm were considered Small (smaller than the size of the stimulus fish image); fish that were between 3.0 and 3.5 cm were considered to be Medium (approximately matching the size of the stimulus fish image), and fish larger than 3.5 were considered to be Large (larger than the size of the stimulus fish image). In experiment 2, the size of the experimental fish tested was kept constant (3.2 cm ± 0.2 cm), but the size of the animated zebrafish image (stimulus fish) varied (the sizes employed were 2, 2.5, 3.2, 4 and 4.5 cm, respectively). In experiment 3, the size of the experimental subjects was constant

Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011

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(3.2 cm ± 0.2 cm) and the size of the animated zebrafish images also remained constant at 3.2 cm (matching the size of the experimental fish), but the number of animated stimulus fish per shoal varied (1, 3, 5 or 8 animated conspecific images were shown respectively). The sequence of stimulus presentation was as follows: 10-minute no-stimulus period (acclimation period), 10-minute stimulus (images of conspecifics) presentation period followed by a 3-minute no stimulus interval. In each experiment, experimental fish received only one behavioral session with a particular condition, a between subject experimental design, and the order of different stimulus presentations randomly varied across treatment conditions. Last, the quantification of behavioral responses was completed blind and the codes for the treatment conditions were broken only after the statistical analyses were completed.

In case of significant effects, post hoc Tukey Honestly Significant Difference (HSD) multiple comparison test and/or paired t-tests were conducted as appropriate. In addition, we also examined whether the change of distance from the stimulus screen induced by the presentation of the shoal stimulus was significantly below random chance. For this, we compared the reduction of distance value to 0 cm (random chance) using one-tailed, one sample t-tests with Bonferroni correction. For all our experiments the effect of sex and the interaction between sex and the other independent variables were found highly non-significant and therefore the above analyses were repeated with sexes pooled. Statistical findings are presented only for this latter set of analyses. Significance was accepted when the probability of the null hypothesis was less than 5% (p b 0.05). 3. Results

2.4. Quantification of behavior and statistical analysis The digital video files (AVI format) were later replayed and analyzed using Ethovision as described in detail by Gerlai et al. (2009), an automated method of behavior quantification. We quantified the distance between the experimental fish and the stimulus presentation computer screen and express this measure as averages for 1-minute intervals of the recording session. We regard this distance as a measure of the strength of the subject's preference for the conspecifics images, i.e. the strength of the shoaling response with smaller distances indicating stronger preference. We analyzed our results using SPSS (version 14.1) for the PC. In experiment 1, repeated measure variance analysis (ANOVA) was performed to investigate the effect of interval (23, 1-minute intervals, the repeated measure factor), sex (between subject factor) and Fish size (between subject factor with 3 levels (Small, Medium and Large)), and the interaction between these factors. In experiment 2, repeated measure ANOVA was employed to investigate the effect of interval (23, 1-minute intervals, the repeated measure factor), sex (between subject factor) and Image size (between subject factor with 5 levels (2, 2.5, 3.2, 4.0, and 4.5)), and the interaction between these factors. In experiment 3, repeated measure ANOVA was used to investigate the effect of interval (23, 1-minute intervals, the repeated measure factor), sex (between subject factor) and Shoal size (between subject factor with 4 levels (1,3,5 and 8)). A univariate ANOVA was also performed for measures derived from the interval data (for details see Results).

When we controlled the size of the experimental fish while holding the stimulus fish size constant, we found experimental fish with a body length that was smaller or equal to the size of the stimulus fish to exhibit a strong shoaling response (Fig. 2). However, experimental fish that were larger in size than the stimulus fish showed a diminished shoaling response (Fig. 2), observations that were confirmed by our statistical analyses. To homogenize variances we performed a logarithm scale transformation and conducted a repeated measures ANOVA using the transformed data set. This analysis revealed a significant Interval effect (F(22, 1100) = 4.926, p b 0.0001), which was in accordance with the observed robust stimulus presentation induced reduction of distance to the stimulus screen. We also found a significant effect of Fishsize (F(1, 50) = 3.698, p b 0.05), confirming experimental fish of different sizes responded to the stimulus fish differently. The Interval × Fish-size Interaction was found non-significant (F(44, 1100) = 1.034, p N 0.05). Because ANOVA has been found insensitive to detect the significance of interaction terms (Wahlsten, 1990), and because multiple range post hoc statistical tests such as the Tukey HSD test are inappropriate for repeated measures design we performed the following follow-up analysis. We averaged the pre-stimulus and stimulus period data and subtracted the former from the latter to obtain what we call the stimulus-induced reduction of distance (Fig. 3). Variance analysis of this data confirmed Fish-size to have a significant effect (F(2, 50) = 4.48, p = 0.016). Tukey's HSD post hoc test revealed a significant (p b 0.05) difference between the large fish and small fish, found the

Fig. 2. Zebrafish whose size is smaller than (first graph) or similar to (second graph) the size of conspecific images show robust shoaling responses and swim closer to the stimulus screen upon and during the presentation of the images. Means + SEM are shown. The designations ‘Small’ (total length up to 2.9 cm, n = 20), ‘Medium’ (total length between 3.0 and 3.5 cm, n = 18), and ‘Large’ (total length above 3.5 cm, n = 15) indicate the size of the experimental fish relative to the size of the stimulus fish image (3.2 cm) presented. The distances for 1 minute intervals of stimulus presentation are shown by grey symbols whereas the distances for 1 minute intervals of no-stimulus periods are shown by black symbols. The dashed horizontal line represents random chance, the midpoint of the tank. Note the robust reduction of distance to the stimulus screen upon and during stimulus presentation for the Small and Medium sized experimental zebrafish and the almost complete lack of this shoaling response in the experimental fish whose body size was larger than the size of the images (Large). For further details of methods and statistical results see Methods and Results.

Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011

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Fig. 3. The reduction of distance in response to stimulus presentation (average distance during stimulus presentation minus the average distance before the stimulus presentation) is significantly affected by the relative size of the experimental fish. Means + SEM are shown. The designations ‘Small’ (total length up to 2.9 cm, n = 20), ‘Medium’ (total length between 3.0 and 3.5 cm, n = 18), and ‘Large’ (total length above 3.5 cm, n = 15) indicate the size of the experimental fish relative to the size of the stimulus fish image (3.2 cm) presented. Note that Small and Medium sized experimental zebrafish reduced their distance significantly compared to random chance whereas the apparent reduction of distance in Large experimental fish was statistically indistinguishable from random chance. For further details of methods and statistical results see Methods and Results.

difference between large and medium fish close to but not reaching significance (p = 0.07), and found the difference between medium and small fish non-significant (p N N 0.05). Next we examined whether the reduction of distance to the stimulus screen in response to the presentation of the shoal stimulus was different from random chance, i.e. 0 cm reduction. One-tailed one sample t-tests with Bonferroni correction showed that when the experimental fish were smaller than (small size, t = − 4.419, df = 19, p b 0.001) or similar (medium size, t = − 5.550, df = 17, p b 0.001) to the size of the conspecific images the experimental fish swam significantly closer to the images (significant reduction of distance) than what would be expected in case of random chance. However, the reduction of distance seen in experimental fish that were larger than the conspecific images did not significantly differ from random chance (large size, t = − 1.690, df = 14, p N 0.05). The size differences between the experimental fish may be the result of differential growth rate and may also represent differences in dominance status among these fish. The size of the fish may correlate with the developmental stage of the fish. Both dominance status and developmental stage may influence shoaling decisions. Thus, to examine the effect of relative size difference between experimental and stimulus fish, and to remove the potential confounding effects of developmental stage and/or dominance status, we performed the second experiment in which the size of the experimental fish was held constant and the size of the stimulus fish was systematically varied and controlled. The results shown on Fig. 4 suggest that experimental fish responded differently depending upon the size of the stimulus fish. The 3.2 cm long stimulus fish (identical in size to the test fish) induced the strongest shoaling response (largest reduction of distance to the stimulus screen upon stimulus presentation) and the smallest stimulus fish induced only diminished responses if at all. ANOVA confirmed a significant effect of Interval (F(22, 1892) = 15.686, p b 0.001), and also found the Interval × Image-size Interaction significant (F(88, 1892) = 2.209, p b 0.001). The main effect of Image-size was found non-significant (F(4, 86) = 1.415, p N 0.05). Similar to the analysis performed for the data obtained in the first experiment, we calculated the reduction of distance upon stimulus

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presentation by averaging the pre-stimulus period data, the stimulus period data and then subtracting the former from the latter (Fig. 5). Variance analysis of this data set confirmed a significant Image-size effect (F(4,86) = 5.50, p = 0.001). Tukey's HSD post hoc test revealed that experimental fish that were shown the 3.2 cm long images reduced their distance to the image presentation screen significantly (p b 0.001) more than fish that were shown the 2.0 cm long or the 2.5 cm long stimulus fish, while other differences were non-significant (p N 0.05). Next we examined whether the apparent reduction of distance induced by the stimulus presentation is indeed different from random chance (0 cm reduction). One-sample one tailed t-tests showed that all stimuli when presented, except the smallest (2 cm long) conspecific images (t = − 0.731, df = 18, p N 0.05), significantly (t N |− 2.933|, df ≥ 15, p b 0.05) reduced the distance of the experimental fish to the stimulus screen. Given that in both experiments 1 and 2 we have found that stimulus fish images of size similar to that of the experimental fish induce a robust shoaling response, in the analysis of the potential effect of the number of stimulus fish we used images of size identical to that of the experimental fish. In this experiment, the experimental fish were presented with shoals of different numerical sizes. The results shown on Fig. 6 suggest that experimental fish in all four shoal size conditions responded with a reduction of distance to the stimulus screen upon stimulus presentation. In accordance with this observation, ANOVA also confirmed a significant Interval effect (F(22, 1474), p b 0.01), but found the main effect of shoal-size (F(3, 67) = 0.355, p N 0.05) and the Interval × shoal-size interaction (F(66, 1474) = 0.979, p N 0.05) nonsignificant. Subsequent analysis of the reduction of distance upon stimulus presentation (Fig. 7) also found no significant effect of shoal-size (F(3, 67) = 0.86, p N 0.05) and thus no differences in the reduction of distance between any of the four stimulus treatment groups. Next we examined whether the apparently robust change of distance to stimulus screen we observed in all stimulus groups was indeed below random chance. One-sample one tailed t-tests with Bonferroni correction found that experimental fish of all groups, except those exposed to a single conspecific image reduced their distance significantly below random chance (t N |−2.76|, df N 16, p b 0.05) and even the reduction of distance seen in fish exposed to the single conspecific image was bordering significance as compared to random chance (t = − 2.395, df = 17, p = 0.056). 4. Discussion Social behavior is a complex and highly important phenomenon worthy of investigation in its own right (Krause et al., 2013). Given the highly social nature of the zebrafish, this species may be particularly relevant for the analysis of social behavior, especially group forming (Miller and Gerlai, 2011a). Abnormalities associated with social behavior accompany a number of human CNS disorders, and for some of these disorders, e.g. the autism spectrum disorders and schizophrenia, these abnormalities represent defining features of the disease (Shapiro and Hertzig, 1991; Brady, 1984). Model organisms may be successfully employed for the analysis of such human disorders. The zebrafish, due to its evolutionarily conserved features demonstrated at multiple levels of its biological organization, but particularly due to the high nucleotide sequence homology found between zebrafish and mammalian genes, has been considered to be an excellent translationally relevant model organism for human diseases, including brain disorders (Kalueff et al., 2014). Thus, analysis of social behavior of zebrafish may have relevance both from a basic science as well as from clinical perspectives (Gerlai, 2014a, 2014b). The shoal stimulus paradigm employed in the current study has been successfully utilized in numerous recent investigations. For example, it allowed the experimenters to analyze the effect of alcohol on the vertebrate brain both when this substance was administered during embryonic development (Fernandes and Gerlai, 2009) or when

Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011

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Fig. 4. Medium sized (3.2 cm long) experimental zebrafish show the strongest reduction of distance (shoaling response) towards animated images of conspecifics whose size matched or was bigger than their own. Means + SEM are shown. The length (“body size”) of the animated conspecific image is indicated above the graphs. Sample sizes for groups exposed to the differently sized images were as follows: n(2.0 cm) = 19, n (2.5 cm) = 19, n(3.2 cm) = 16, n(4.0 cm) = 18, and n(4.5 cm) = 19. The distances for 1 minute intervals of stimulus presentation are shown by grey symbols whereas the distances for 1 minute intervals of no-stimulus periods are shown by black symbols. The dashed horizontal line represents random chance, the midpoint of the tank. Note the robust reduction of distance to the stimulus screen upon and during stimulus presentation for the 3.2 cm, 4.0 cm and 4.5 cm long animated conspecific images. Note the diminished response induced by the images whose length was smaller than that of the experimental fish's, i.e. the 2.0 cm and 2.5 cm long images. For further details of methods and statistical results see Methods and Results.

Fig. 5. The reduction of distance induced by animated conspecific image presentation is dependent upon the size of the shown images. Means + SEM are shown. The length (“body size”) of the animated conspecific image is indicated above the graphs. Sample sizes for groups exposed to the differently sized images were as follows: n(2.0 cm) = 19, n (2.5 cm) = 19, n(3.2 cm) = 16, n(4.0 cm) = 18, and n(4.5 cm) = 19. Note that experimental fish that were shown the 3.2 cm long animated images (same length as their own) reduced their distance in response to this stimulation significantly more than those experimental fish that were shown the smaller sized images (2.0 cm and 2.5 cm long conspecific images). Also note that all images except the smallest (2.0 cm long) induced a reduction of distance from the stimulus screen that was significantly different from random chance. For further details of methods and statistical results see Methods and Results.

it was delivered acutely or chronically for the adult fish (Gerlai et al., 2009). It also allowed the investigation of associative learning as well as memory in zebrafish (Pather and Gerlai, 2009; Jia et al., 2014). One reason for the success of this shoaling paradigm is that it builds upon a natural tendency of zebrafish to form groups. Consequently, the fish do not have to be trained or forced to perform the appropriate response, and shoaling is efficiently induced upon even the first presentation of conspecifics or their images (Saverino and Gerlai, 2008; Engeszer et al., 2007b). Importantly, the shoaling response has been found to be not only robust and easy to induce, but also quite stable over time and/or in response to repeated administration of the shoaling stimulus (Miller and Gerlai, 2007, 2012). Although successfully employed, systematic analysis of the parameters of the shoaling stimulus has not been performed despite existing evidence suggesting that zebrafish are not indifferent to features of the conspecific images presented (Saverino and Gerlai, 2008). In the current study, we have started the systematic analysis of certain features of the shoaling stimulus. First we have manipulated the size of the conspecific images relative to the size of the experimental subjects. Intuitively, and based upon indirect evidence obtained with other fish species (Gómez-Laplaza and Gerlai, 2011b; Agrillo et al., 2012 and references therein), investigators tended to use zebrafish images of a size identical to that of their experimental fish. However, according to our knowledge, our study is the first that actually demonstrated that similarity between the size of the stimulus and the experimental fish

Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011

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Fig. 6. The reduction of distance to the stimulus screen induced by animated conspecific images was not significantly affected by the number of animated images presented. Means + SEM are shown. The number of the animated conspecific images (numerical shoal size) presented is indicated above the graphs. Sample sizes for groups exposed to the differently shoal sizes were as follows: n(1) = 18, n (3) = 18, n(5) = 17, and n(8) = 18. The distances for 1 minute intervals of stimulus presentation are shown by grey symbols whereas the distances for 1 minute intervals of no-stimulus periods are shown by black symbols. The dashed horizontal line represents random chance, the midpoint of the tank. For further details of methods and statistical results see Methods and Results.

leads to the strongest shoaling response in the latter. We addressed the question of relative body size in two experiments. In the first one we held the size of the conspecific image constant and varied the size of the experimental fish and in the second experiment we did the opposite, we held the size of the experimental fish constant but varied the size of the conspecific images. In both experiments, we found that when the size of the stimulus and experimental fish was similar, the experimental fish showed strong shoaling. Images whose size was bigger compared to the experimental fish also induced an appreciable shoaling response. However, experimental fish exhibited reduced shoaling or absence of the response towards images that were smaller in size compared to their own. The consistent nature of findings in these two experiments implies that the differential effect of relative image size is not dependent upon the absolute size, and thus the developmental stage or age or dominance status, of the experimental fish, at least not within the size range studied here. In summary, the findings of

Fig. 7. Reduction of distance induced by numerically different shoal sizes (different number of animated images of conspecifics). Means + SEM are shown. The number of the animated conspecific images (numerical shoal size) presented is indicated below the X-axis. Sample sizes for groups exposed to the differently shoal sizes were as follows: n(1) = 18, n (3) = 18, n(5) = 17, and n(8) = 18. Note that although the effect of numerical shoal size was found non-significant, the stimulus shoals with 3, 5 and 8 images induced a reduction of distance to stimulus screen in the experimental fish that was significantly different random chance whereas the “shoal” with a single image did not. For further details of methods and statistical results see Methods and Results.

the first two experiments presented here validate the wide-spread use of stimulus fish whose size matches that of the test fish. These results also allow us to speculate about the possible function of shoaling and its adaptive nature, at least in the context of the shoaling paradigm we employed. Joining a group of fish that are larger in body size than the individual that is attempting to join the group should confer no advantages for this individual from the perspective of competition for mates or for food. Smaller fish are expected to lose in such competitions (Huntingford et al., 1990 and references therein). However, consider that all birds of prey and most of the piscivorous fish that are sympatric with zebrafish can easily capture and swallow fully-grown adult zebrafish and much larger fish too. These predators are likely to focus their attention on larger, and not smaller, zebrafish for foraging economy reasons. Thus being smaller in a group of larger shoalmates may confer adaptive advantages for zebrafish from the perspective of predator avoidance. The experimental paradigm employed a novel tank that is expected to induce mild fear and/or anxiety in the experimental subject (Stewart et al., 2012; Gerlai, 2010b). Consequently, we propose that the shoaling response induced in this paradigm is primarily driven by antipredatory motivation, a speculation that is in accordance with finding experimental fish to prefer similarly sized or relatively larger images as reported in the current study. This speculation is also in line with prior findings demonstrating the effect of fear inducing stimuli leading to strengthening of shoaling responses in zebrafish (Speedie and Gerlai, 2008). Furthermore, in addition to answering the question of what the adaptive function of shoaling may be, this speculation may have translational (predictive) relevance too. If our speculation is correct, we predict that compounds and drugs with anxiolytic or anxiogenic properties may reduce or enhance, respectively, the strength of shoaling, and that the shoaling paradigm as employed here with zebrafish may enable the investigator to test the efficacy of pharmaceutical agents in this context as well. Contrary to our expectations, systematic modification of the number of animated images presented did not alter the strength of the shoaling response in the experimental zebrafish. At least within the range from 1 to 8 images, we found all shoal stimuli to induce a shoaling response that was statistically indistinguishable across the four different stimulus groups, although the shoaling response was found marginal towards the single conspecific image presented. Previously, we found the interaction between two zebrafish (one experimental and one stimulus fish image) to be associated with aggression, i.e. agonistic behavior, and not shoaling (Gerlai et al., 2000). When the experimental fish encountered a single stimulus fish the experimental fish often exhibited aggressive behavioral responses including lateral display, undulating movements and attempts to bite the stimulus fish, whereas when the

Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011

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experimental fish encountered a group of stimulus fish, no such aggressive behaviors were exhibited (Gerlai et al., 2000). Interestingly, the dose-dependent changes induced by acute alcohol, a drug of abuse known to alter both aggression as well as social behaviors, also differed in these two domains of behavior in zebrafish (Gerlai et al., 2000). Thus, we propose that the motivational drive behind the reduction of distance to the conspecific image is dependent upon how many images are shown: it is likely to be aggression when a single image is presented while it is likely to be shoaling when multiple images are shown. Thus, it is also likely that the test paradigm that presents one stimulus fish versus multiple stimulus fish will tap into different neurobiological processes. The question of why we could not find differential effects of conspecific image presentations with the studied 3, 5 or 8 images cannot yet be answered. We only examined the effect of different number of stimulus images within this small number range (between 1 and 8), and it is possible that zebrafish could show a stronger response to a shoal containing more than 8 members (or images). It is also possible that zebrafish can distinguish and show a preference for the numerically larger shoal even within the employed small number range. For example, when two numerically different shoals were contrasted, i.e. one presented on the left and the other on the right side of the experimental tank simultaneously, a range of fish species were found to show a preference for the numerically larger shoal even in the number range employed in our current study (Gómez-Laplaza and Gerlai, 2011b and references therein). Thus, it is possible that zebrafish also have similar shoal size estimation abilities and could exhibit a preference for the larger shoal in binary choice tasks, a question that will be investigated in the future. Although numerous empirical and methodological questions remain unanswered, we argue that the current shoal-image paradigm will have an important role in the characterization of social behavior and the analysis of functional changes in the brain of zebrafish. It is a simple method that induces a robust and consistent shoaling response when multiple images of conspecifics matched in size to the experimental fish are shown. Yet, it taps into a complex brain function that may be dependent upon a large number of mechanisms. This behavioral paradigm may be employed in large scale mutation and drug screening experiments as a first pass screening tool, because both image presentation and behavioral response quantification are automated and do not require the constant presence of an experimenter. Last, it may be employed for a variety of purposes, including the analysis of social behavior (Gerlai, 2014a, 2014b), fear and anxiety (Gerlai, 2010b), as well as learning and memory (Jia et al., 2014; Pather and Gerlai, 2009). Acknowledgments Supported by NSERC (grant #311637) to RG. References Abaid N, Spinello C, Laut J, Porfiri M. Zebrafish (Danio rerio) responds to images animated by mathematical models of animal grouping. Behav Brain Res 2012;232: 406–10. Agrillo C, Bisazza A. Spontaneous versus trained numerical abilities. A comparison between the two main tools to study numerical competence in non-human animals. J Neurosci Methods 2014;234C:82–91. Agrillo C, Piffer L, Bisazza A, Butterworth B. Evidence for two numerical systems that are similar in humans and guppies. PLoS One 2012;7(2):e31923. http://dx.doi.org/10. 1371/journal.pone.0031923. Bisazza A, Butterworth B, Piffer L, Bahrami B, Petrazzini ME, Agrillo C. Collective enhancement of numerical acuity by meritocratic leadership in fish. Sci Rep 2014;4:4560. http://dx.doi.org/10.1038/srep04560. Brady JP. Social skills training for psychiatric patients. I: Concepts, methods, and clinical results. Am J Psychiatry 1984;141:333–40. Crabbe JC, Wahlsten D, Dudek BC. Genetics of mouse behavior: interactions with laboratory environment. Science 1999;284:1670–2. Engeszer RE, Patterson LB, Rao AA, Parichy DM. Zebrafish in the wild: a review of natural history and new notes from the field. Zebrafish 2007a;4:21–40. Engeszer RE, Barbiano LA, Ryan MJ, Parichy DM. Timing and plasticity of shoaling behaviour in the zebrafish, Danio rerio. Anim Behav 2007b;74:1269–75. Fernandes Y, Gerlai R. Long-term behavioral changes in response to early developmental exposure to ethanol in zebrafish. Alcohol Clin Exp Res 2009;33:601–9. Gerlai R. Phenomics: fiction or the future? Trends Neurosci 2002;25:506–9.

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Please cite this article as: Fernandes Y, et al, The effect of the number and size of animated conspecific images on shoaling responses of zebrafish, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.01.011