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Zebrafish cooperate while inspecting predators: experimental evidence for conditional approach

Different fish species employ a conditional approach strategy during predator inspection; the risk of approaching a predator is distributed across all inspectors, but is not shared with the animals which keep its distance. Zebrafish, a highly social fish, is increasingly being used in behavioural neuroscience, but it is not known whether it displays conditional approach. In the predator inspection task, animals are observed in a tank with a refuge in one extremity, and an animated image of a predator in the other extremity, with a mirror positioned in parallel to the tank, simulating a perfectly reciprocating conspecific. In Experiment 1, animals spent more time inspecting the predator when the image was turned on, but also displayed more erratic swimming, suggesting cooperation under fear. In Experiment 2, animals spent more time inspecting predators when the mirror was parallel to the tank (“cooperating mirror”) than when the mirror was in an angle (“defecting mirror”), suggesting retaliatory behaviour; in both conditions, animals displayed more freezing and erratic swimming. These results suggest that predator inspection is associated with conditional approach, while at the same time inducing fear-like behaviour in the animal.


Introduction
When facing a predator, some fish species respond in a rather contradictory manner, by approaching instead of keeping a safe distance. Predator inspection is more frequently observed in shoaling than in lone species, with the inspection individual leaning the safety of the shoal to approach the predator. Paradoxically, inspection is both beneficial to the shoal, but increases risk to the inspecting fish for engaging with a potential threat; as a result, predator inspection is interpreted as "altruistic" or "cooperative-like". A good example is the case of the guppy Poecilia reticulata, which have been shown to use a conditional approach strategy while inspecting predators: in the presence of both predators and conspecifics, inspecting animals approach the predator solely if conspecific follows, but defects when the following conspecific also defects. Here, we attempt to determine whether the zebrafish (Danio rerio) use conditional approach strategies while inspecting predators with conspecifics. Indeed, the zebrafish is a social, shoaling species also observed to inspect predators. In the first experiment, we found that zebrafish spend more time in the inspection zone when an animated image of a predator is shown and a mirror is positioned in parallel to the tank, mimicking a perfectly reciprocating conspecific. While spending more time in the inspection zone, zebrafish also display fear-like behaviour when the animation is shown. In the second experiment, we show that zebrafish reciprocate, spending less time in the inspection zone if the mirror is in an angle (45º) with the tank. Here, the mirror image appears to defect when the inspecting animal approaches the predator, and therefore by spending less time in the inspection zone, suggests a retaliation response.
Taken together, our results demonstrate that zebrafish are also able to employ conditional approach strategies during predator inspection, being capable of cooperative-like behaviour, which could be used in models of social deficits in psychiatric disorders.

Introduction
While being widespread among animals, cooperation is an adaptive/evolutionary conundrum, as it may involve reducing one's own fitness in order to benefit another individual (Kropotkin, 1902). Different explanations have been given to this phenomenon, including kin selection (Hamilton, 1963(Hamilton, , 1964 and reciprocal altruism (Trivers, 1971). Reciprocal altruism predicts that cooperation only occurs in populations when it increases the likelihood of reciprocation at the receiving end -that is, the individual decreases its own fitness in the expectation that the other organism will act in a similar manner in a later time (Trivers, 1971). Game theoretical approaches suggest a "tit-for-tat" strategy, in which an individual cooperates in the first encounter and continues to cooperate as long as the other agent does as well (Trivers, 1971).
Experimental evidence of "tit-for-tat" has been hard to produce because the payoff of cooperating vs. defecting is known, but the benefits (and risks) cooperating are usually not known. Nonetheless, "reciprocal altruism-like" behaviour has been observed in many species (Bshary & Oliveira, 2015). An interesting example is conditional approach, a strategy used, by social fish when approaching a potential predator, leaving the shoal to obtain information on putative threat and/or try to intimidate the dangerous predator (Dugatkin, 2013). The strategy appears to function as an incentive for predator inspection, given that the risk of approaching it, is distributed between all inspectors but not shared with those that remain at a distance. Since this behaviour implies costs to the individual(s) interacting with the predator, but contributes to the shoal's fitness, it has been interpreted as cooperative-like and altruistic (Dugatkin, 1988, 1997, Dugatkin & Alfieri, 1991, 1992Pimentel et al., 2019). "Tit-for-tat" applies for conditional approach because prey may be: "nice" (i.e., inspect the predator at the first "move"), "retaliatory" (i.e., immediately head back away from the predator if the conspecific falls behind or disappears), and "forgiving" (i.e., approach the predator again if the conspecific's move of "swimming away and staying behind" is followed by "swim parallel") (Dugatkin, 1988). However, differently from tit-for-tat (Axelrod & Hamilton, 1981), conditional approach does not require that the actual payoff matrix is known (Dugatkin, 1988). Conditional approach has been shown in sticklebacks (Gasterosteus aculeatus Linnaeus 1758) (Huntingford, Lazarus, Barrie, & Webb, 1994;Milinski, 1987) and guppies (Poecilia reticulata Peter 1859) (Dugatkin, 1988, 1991, Dugatkin & Alfieri, 1991, 1992Pimentel et al., 2019), but not for zebrafish, an important model organism in the neurosciences.
Zebrafish (Danio rerio Hamilton 1822) is a model organism that is widely used in developmental biology (Parichy, 2015) and which has been introduced in the field of behavioural neuroscience and behavioural ecology (Bonan & Norton, 2015;Stewart et al., 2015). This fish is also a good model organism for biological psychiatry (Stewart et al., 2015), due to the availability of behavioural bioassays and the advantages associated with being a model organism, including low cost of acquisition and maintenance, easy handling and housing, short lifespan, and easy reproduction in the laboratory. As a highly social animal that lives in groups with well-structured social relationsincluding marginalization, dominance hierarchies, and territoriality (Spence, Gerlach, Lawrence, & Smith, 2008) -, zebrafish are also known to display predator inspection (Dugatkin et al., 2010;Pannia, Tran, Rampersad, & Gerlai, 2014); however, it is not known whether zebrafish employ conditional approach when inspecting predators.
In addition to helping to better understand complex social behaviour in this species, here we aim to introduce new behavioural techniques that can also advance research in the field of neuropsychopharmacology, including the development of drugs to treat disorders of social behaviour (Soares, Cardoso, Carvalho, & Maximino, 2018).
While the focus of this field on zebrafish social behaviour has relied mainly on shoaling, more complex behaviours, such as cooperative-like behaviour, could represent an important addition to the field. Our main aim was to investigate preliminary evidence of conditional approach in zebrafish in a predator inspection model, based in Millinski (1987) and Dugatkin (1988). In Experiment 1, a mirror was placed in parallel to a tank, and the animated image of a predator (Gangetic leaffish Nandus nandus Hamilton 1822): when the animation was turned on, animals spent more time in the inspection area than when it was turned off, suggesting that the presence of both a predator and a cooperating conspecific (mirror image) are necessary for inspection behaviour. In Experiment 2, the mirror was either placed in parallel ("cooperating mirror") or in an angle of 45º with the side of the tank which was opposite to the predator ("defecting mirror"): animals then spent less time in the inspection zone during the defecting mirror condition. Overall, animals displayed freezing and erratic swimming when the animation was visible, suggesting fear. This manuscript is the first complete report of all the studies performed to test the hypothesis that zebrafish display conditional approach during predator inspection. We report all data exclusions (if any), all manipulations, and all measures in the study.

Animals and housing
31 animals were used in each of the experiments described below; data from one animal was excluded from each experiment due to poor health after the end of the experiments, which could impact the results. Animals were bought from a commercial vendor, and arrived in the laboratory with an approximate age of 3 months (standard length = 13.2 ± 1.4 mm), and were quarantined for two weeks; the experiment began when animals had an approximate age of 4 months (standard length = 23.0 ± 3.2 mm).
Animals were kept in mixed-sex tanks during acclimation. Adult zebrafish from the wildtype strain (longfin phenotype), with an expected sex composition of 50% females weeks before experiments begun. Tanks were filled with non-chlorinated water at room temperature (28 °C) and a pH of 7.0-8.0. Lighting was provided by fluorescent lamps in a cycle of 14-10 hours (LD), according to standards of care for zebrafish (Lawrence, 2007). Water quality parameters were as follows: pH 7.0-8.0; hardness 100-150 mg/L CaCO3; dissolved oxygen 7.5-8.0 mg/L; ammonia and nitrite < 0.001 ppm. All manipulations minimized their potential suffering of animals, and followed Brazilian legislation (Conselho Nacional de Controle de Experimentação Animal -CONCEA, 2017). Animals were used for only one experiment and in a single behavioural test, to reduce interference from apparatus exposure. Experiments were approved by UEPA's IACUC under protocol 06/18.

Sample size determination
Sample sizes were determined based on results regarding conditional approach in guppies (Pimentel et al., 2018). Time on the inspection zone was chosen as the primary endpoint, and calculations for sample sizes are valid only for that endpoint.
Calculations were based on Rosner's (2016) method for comparing two means, and assumed α = 0.05 and power 80% on a two-tailed analysis. Based on these calculations, 15 animals were used in each group in both experiments. Animals were randomly drawn from the tank immediately before testing, and the order with which they were allocated to different conditions was randomized via generation of random numbers using the randomization tool in http://www.randomization.com/. Blinding was not possible, due to the positioning of the camera.

Experimental apparatus
The experimental apparatus was based on that described by Pimentel et al.
(2019) for guppies ( Fig. 1), consisting in a glass tank with 40.6 cm x 18 cm x 25 cm (l X w X h). The bottom of the tank was divided in 10 equally-sized quadrants; the quadrant that was farther from the side in which the stimulus was presented was numbered "section 1" (refuge zone), and an artificial plant was placed there to serve as refuge. Parallel to the quadrant that was farther from section 1, a computer screen (Samsung T20c310lb, 20″, LED screen, nominal brightness 20 cd/m³) allowed the presentation of animated images. These animations were produced on LibreOffice Presentation (Pimentel et al., 2019), and consisted of a moving image of a leaffish (Nandus nandus), a sympatric predator for zebrafish (Engeszer, Patterson, Rao, & Parichy, 2007;Parichy, 2015). Parallel to one of the longer walls of the tank (Fig. 1B), a mirror, with the length of 40.6 cm, was positioned. The mirror, therefore, occupied the whole length of the experimental apparatus and, as a consequence, created an image of a second zebrafish swimming in parallel to the focal animal. In Experiment 2, the mirror was either parallel to the tank (Cooperating mirror) or forming and angle of 45º with the tank (Defecting mirror).

General design
Animals were tested individually and only once. Before beginning a trial, animals were individually transferred to the experimental tank, behaviour was filmed for 5 min while swimming freely. During this acclimation period, the screen was turned off. Animals were randomly allocated to groups, using the random number generator at https://randomization.com, to reduce allocation bias. Moreover, the order with which individuals from each group were tested was also randomized. Each trial took 15 min, and consisted in an acclimation stage (min. 0-5), immediately after the animal was introduced in the tank and during which the video was turned off, followed by the stimulation stage (min. 6-15), during which the video was turned on. Trials were recorded in digital format by a camera (Sony DCR-DVD610) mounted above the tank.
Given the position of the camera in relation to the mirror, it was not possible to blind video transcription to treatment. Digital videos were later viewed, and behaviour was manually recorded using XPlo-Rat (http:///scotty.ffclrp.usp.br). The following variables were recorded: • Freezing: Time spent immobile, except for eye and opercular movements; • Erratic swimming: Number of events of zig-zagging, fast swimming episodes.

Experiment 1
Experiment 1 was a conceptual replication of experiments in guppies (Pimentel et al., 2019), and attempted to understand whether approach and defect behaviours were potential responses to the specific predator inspection context, or whether the animal was simply responding to the conspecific (i.e., following its own mirror image). In the "Predator" condition, the animation was shown after 5 minutes of acclimation, as described in "General design", above; in the "No-predator" condition, no animation was shown.

Experiment 2
Experiment 2 was a conceptual replication of studies on cooperation vs. defection made on guppies (Dugatkin, 1988) and sticklebacks (Milinski, 1987). The rationale for the experiment is that, for two interacting fish approaching a predator, defecting (staying behind) is beneficial because the defecting individual decreases its risk but increases its payoff (i.e., information gathered) by watching the fate of the other fish. Conditional approach predicts that inspecting fish should immediately retaliate defecting conspecifics by also defecting (Bshary & Oliveira, 2015;Dugatkin, 1988;Pimentel et al., 2019). We attempt to simulate this situation by using two mirror conditions, a "cooperating" mirror (parallel to the tank) and a "defecting" mirror (in an angle of 45º to the tank); in this last situation, as the fish approached the predator, its mirror image would appear to turn away.

Statistical analysis
Data for time on the inspection, avoidance, and refuge zones were assessed using independent samples t-tests. Bayes Factors were also calculated, using the

Open science practices
Experiments were not formally pre-registered. Datapackages and analysis scripts can be found on GitHub (https://github.com/lanec-unifesspa/zebrafishTFT/ ). Preprint versions of this paper have been submitted to bioRxiv.

Results
In Experiment 1, a significant difference was found for time spent in inspection zone ( Fig. 2A) while the RM-ANOVA was not significant for the interaction effect, the higher Bayes Factor suggests that the interaction model is preferred against all models.
In Experiment 2, a significant difference was found for time spent in inspection zone (Fig. 3A) Moreover, no significant differences were found for time in the refuge zone ( Fig. 3C

Discussion
In experiment 1, we found that the animal spends more time in the inspection zone than in other zones when the predator stimulus is present, suggesting that the presence of a predator in the concomitant presence of a conspecific induces predator inspection, while simultaneously observing that the individual has a fear response in the form of increased erratic swimming and freezing behaviour. In experiment 2 we observed that the animal spends more time in inspection in the cooperating mirror condition than in the defecting mirror condition, and in both conditions we observed that it had fear-like behaviour. The results of both experiments suggest that zebrafish exhibits conditional approach behaviour.
In the present experiments, animals behaved towards conspecifics by approaching the mirror, and towards the predator by keeping a distance and freezing and/or displaying erratic swimming; however, when both stimuli are present, the animal emits predator inspection, entering the inspection zone and orienting towards the predator image. While it has been previously shown that visual contact with conspecifics decrease fear responses in zebrafish (Faustino et al., 2017), the presence of the conspecific image did not inhibit fear in the present experiment, as the animal still displays freezing and erratic swimming. We reported similar effects on guppies (Pimentel et al., 2019).
In the cooperating mirror condition, zebrafish spent more time inspecting the predator than fish in the defecting mirror condition, suggesting that zebrafish employs a "conditional approach" strategy. Similarly to "tit-for-tat", in conditional approach the individual approaches the predator in the first movement, and subsequently approaches only if the second fish (the mirror image) swims along, retreating if the conspecific also retreated (Dugatkin, 1988). In guppies, these dynamics have been interpreted as indicative of "nice" behaviour (the first movement of the game is cooperative), "retaliation" (individuals cease inspection if co-inspectors move far enough), and "forgiveness" (a fish individual resumes inspection after retaliating only if the inspector's next step is to cooperate) (Dugatkin, 1997). Results from our Experiment 2 suggest that the animal retaliates after defection (i.e., reduces inspection in the defecting mirror condition), while results from Experiment 1 suggest "niceness" and "forgiveness".
Zebrafish were thus shown to be able to change their strategy from avoiding to approaching predators when conspecifics are present. If in visual contact with a conspecific (their own reflection), they proceed to spend more time inspecting predator (cooperation mirror condition), a situation that significantly changed in the defecting mirror condition, as their company was absent. This looks like a typical "conditional approach" situation: conditionally, the individual swims towards the predator in the first movement, but subsequent close inspection only occurs if the second fish (the mirror image) swims along, retreating immediately if the accompanying conspecific also abandons (Dugatkin, 1988). This is an indication of the social conditions necessary the evolution of predator approach behaviour as a cooperative strategy in zebrafish, as in guppies and sticklebacks (Dugatkin & Alfieri, 1991;Huntingford et al., 1994;Edenbrow et al., 2017): strong social bonds between conspecifics that enable their first bold move (cooperative behaviour) which can confer benefits to those inspecting and to the shoal (Bshary & Oliveira, 2015). However, if facing defecting shoalmates, individuals may retaliate, ceasing inspection but are able to "forgive" if baseline conditions are restored. Our findings allow us to include zebrafish in the restrict group of teleost fish species capable of complex decision making, responding to the quality of social bonds by being able to change strategy as conditions change.
Zebrafish has been previously shown to display predator inspection, at low levels, without the presence of conspecifics (Dugatkin et al., 2010;Pannia et al., 2014).
Our results are the first demonstration that predator inspection is related to cooperativelike behaviour in zebrafish, even when the animal displays fear-like behaviour towards the predator. These results suggest that the social behaviour of zebrafish is more complex than previously thought, and can be further exploited in the study of the neural bases of altruism in an interesting model organism.   difference is plotted on a floating axes on the right as a bootstrap sampling distribution. The mean difference is depicted as a dot; the 95% confidence interval is indicated by the ends of the vertical error bar. In (D-E), the raw data is plotted on the upper axes; each paired set of observations is connected by a line. In (D'-E'), the paired mean differences for comparisons between groups are shown in a Cumming estimation plot, with each paired mean difference plotted as a bootstrap sampling distribution of Cohen's d. Mean differences are depicted as dots; the 95% confidence interval is indicated by the ends of the vertical error bar.