Many animals across a wide range of taxa express collective behaviors (e.g. shoaling and herding) resulting in multiple benefits, including foraging and/or antipredator effects (Pitcher and Parrish, 1993). Despite the benefits of collective behavior, this type of behavior in some species has been reduced over evolutionary time such that these species exhibit marked asociality when compared to their socially closest relatives (Pitcher and Parrish, 1993). Additionally, even among social species, this collective behavior can disappear when the animal is exposed to a stress-associated environment (Kim et al., 2016; Lewis et al., 2007). Instead, these animals may express asocial behavior and/or stereotypic repetitive behaviors, which have been particularly well characterized in mammals (e.g. ‘zoochosis’) (Lewis et al., 2007; Rose et al., 2017). Stereotypic repetitive behaviors, in contrast to collective behaviours, are generally thought of as non-functional (Lewis et al., 2007). Psychiatric conditions in humans, such as autism spectrum disorder (ASD), share similarities to atypical repetitive behavioral responses in other animals in that social activity is decreased and there is an overall increase in stereotypic repetitive behaviors, especially in stress-inducing environments (Kaplan and McCracken, 2012; Langen et al., 2011a; Runco et al., 1986). The neural mechanisms underlying the contrasting expression of social and repetitive behaviors and how the ‘choice’ between stereotypic repetitive and social modes of behavior are made remain largely unknown (Kazdoba et al., 2016; Kim et al., 2016; Langen et al., 2011b; Lewis et al., 2007). However, studies in mammals have hinted at such neural mechanisms by indicating that the dopaminergic striatal system is key for behavioral choices. For example, treatment with anti-agitation drugs (i.e. drugs to reduce repetitive behavior, for example aripiprazole and risperidone) attenuated but did not eliminate the activity of the D2-dopamine receptor-based indirect pathway in mammals (Grillner, 2018; Kim et al., 2016). Importantly, this core dopaminergic system exists in all vertebrate models from lampreys, teleosts (zebrafish), amphibians (Xenopus), and birds (chickens) to mammals (mice and humans) (Grillner and Robertson, 2016; Stephenson-Jones et al., 2011). However, among actinopterygians (ray-fin vertebrates), it is largely unknown whether an evolved asocial population had lost the ability to congregate with each other or just suppressed this activity and whether repetitive behaviors are enhanced under stress in an antagonistic manner with collective behavior.

To address these two questions, we used a suitable vertebrate model, the Mexican tetra Astyanax mexicanus, and developed a novel experimental workflow to assay collective and stereotyped behavior (Yoshizawa, 2015). A. mexicanus is emerging as a key model system for the study of diverse aspects of evolution and development, including those with relevance to human medicine, for example cataract formation, obesity, diabetes, albinism-related syndrome, and insomnia (Aspiras et al., 2015; Bilandžija et al., 2018; Bilandžija et al., 2013; Duboué et al., 2012; Duboué et al., 2011; Jaggard et al., 2017; Keene et al., 2016; Ma et al., 2014; McGaugh et al., 2014; Riddle et al., 2018; Rohner et al., 2013; Strickler et al., 2007). A. mexicanus comprises surface riverine epigean (surface fish) and cave-dwelling hypogean (cavefish) forms. Cavefish diverged from their surface-dwelling relatives 20,000~200,000 years ago (Fumey et al., 2018; Herman et al., 2018) and since then have evolved many distinct morphological and behavioral phenotypes, including eye regression/loss, pigment reduction, increased number of lateral line mechanosensors, enhanced behavior responses to vibrational stimuli, sleeplessness, hyperactivity and repetitive circling (Keene et al., 2016; Yoshizawa, 2015; Yoshizawa et al., 2018). Compared to cavefish, surface fish show typical fish phenotypes, including eyed and pigmented morphologies, no strong sensitivity to vibrational stimuli, and nocturnal sleep patterns. Cavefish have also been shown to have reduced social-like interactions, such as no detectable schooling and shoaling behavior (Kowalko et al., 2013; Patch et al., 2020; Pierre et al., 2020) or hierarchal dominance (Elipot et al., 2013). In contrast, surface fish school/shoal with cohorts and model fish (Kowalko et al., 2013) show group hierarchical dominance (Elipot et al., 2013). Importantly, the surface fish schooling behavior was concluded to be driven by visual stimuli because it diminished in the dark (Kowalko et al., 2013). In contrast, surface fish in the dark continued to show similar levels of aggression and were thought to establish a hierarchical dominance, similar to those kept in the light (Elipot et al., 2013). This motivated us to test other social-like interactions in the dark to compare non-visual-based social-like interactions between surface fish and cavefish.

Thus, we developed a sensitive method to detect social-like interactions and repetitive behavior in the dark (Figure 1). This method revealed a number of social-like interactions in surface fish in the dark. Surface fish, however, did not show repetitive turning. Furthermore, we also revealed weak but existent social-like interactions in cavefish that occurred while exhibiting a significantly higher level of repetitive behavior than that of surface fish. During these nearby interactions, cavefish decelerated their swimming speed and were likely to follow their cohorts. The comparison of these behaviors under familiar and potentially stressful, unfamiliar environments indicated that cavefish showed significantly more nearby interactions in the familiar environment. The atypical dopamine psychiatric drug aripiprazole, which is known to increase social behaviours and reduce repetitive motions in humans (Coleman et al., 2019; de Bartolomeis et al., 2015; Ghanizadeh et al., 2014), reduced swimming speed and increased nearby interactions in cavefish. Cavefish also showed reduced levels of repetitive turning after aripiprazole treatment. In summary, cavefish showed robust repetitive circling, and the degree of circling was negatively correlated with social-like nearby interactions. This cavefish interaction remained plastic and was suppressed under a potentially stressful environment. Together with the reported parallels in pharmacological responses, a set of behavioral symptoms, and gene expression profiles between cavefish and humans (Yoshizawa et al., 2018), the presented results suggest that the asocial cave population is still capable of performing social-like nearby interactions under environmental or pharmacological treatments and shares an antagonistic association between social-like and repetitive behaviors with mammals.

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In this study, we document that the sighted surface-dwelling form of Astyanax mexicanus is able to follow and interact with conspecifics in the dark. We also reveal that so-called ‘asocial’ cavefish retain a low but detectable level of ability to engage in social-like interactions. The social-like interaction depended on the familiarity to the environment, and interestingly, surface fish and cavefish showed opposite responses to the environments: in the familiar environment, cavefish showed more social-like nearby interactions, while surface fish showed reduced social interaction; in contrast, in the unfamiliar environment, surface fish tended to exhibit a longer duration of nearby interaction, while cavefish exhibited a reduced duration of nearby interaction. Cavefish also showed a significant negative correlation between the levels of repetitive turning behavior and social-like interactions in the familiar environment. This quantitative antagonistic relationship between repetitive behavior and social-like interactions has been reported in studies involving humans and rodents (Boulter et al., 2014; Buhr and Dugas, 2009; Hong et al., 2014), but there are few reports in teleosts. Treatment with the partial D2 receptor antagonist aripiprazole enhanced social-like interactions and suppressed repetitive turning in cavefish, which is consistent with aripiprazole treatment results in human patients (de Bartolomeis et al., 2015; Ghanizadeh et al., 2014; Nasrallah, 2008).

In terms of measuring collective behaviors, the current definition of nearby interaction is close to that of ‘attraction’ in zebrafish larvae under lighted conditions (Hinz and De Polavieja, 2017) (for a more general definition, see Lukeman et al., 2010), and we believe these two indices indicate the same interactions. However, we could not apply Hinz’s criteria to ours because they used larval fish (~24 days post-fertilization: 0.6 cm in body length; Parichy et al., 2009). Larval fish swim in a highly viscous low Reynolds number-fluid environment (Vogel, 1983) where the larval fish do not glide. Thus, the turning direction that Hinz et al. measured was sufficient to measure collective behavior. In contrast, we used young adult fish (1.5–2.0 cm), who swam in a higher Reynolds number-fluid environment and glided while swimming. Therefore, this measure of nearby duration that results from the manoeuvring efforts of the fish was more appropriate for representing the level of collective behavior than simply measuring the turning direction.

As for a potential sampling bias in this study, we equally mixed age-matched individuals from different parents. We did not exclude any outliers from our dataset except those showing atypical pre-treatment behaviors (see Materials and methods). Although our lab experienced a founder bottleneck started with approximately 80 individuals per population, we minimized potential sampling bias within our lab populations by the above procedure.

The majority of studies on shoaling and schooling behaviors of teleost fish have been conducted under environmental conditions where visual cues predominate, potentially exaggerating the importance of the visual system in these behaviours (Larsch and Baier, 2018). Observations of the maintenance of shoaling behavior in blinded fishes support the involvement of lateral line mechanosensing; however, few studies have used intact fish to test the involvement of the lateral line (Partridge and Pitcher, 1980; Pitcher, 1979; Pitcher et al., 1976). Furthermore, many shoaling tests have been conducted in somewhat physically constrained conditions (e.g. using a transparent glass divider between stimulus fish and test fish [Dreosti et al., 2015] or using a small mesh cage that constrains the swimming area of the stimulus fish [Gerlai, 2014]), where the true extent of mechanosensing in the control of shoaling behavior may be underplayed. In this study, we conducted the first systemic analysis of social-like nearby interactions in intact fish under free-swimming conditions in the dark.

In this study, we carefully set up and tested whether sighted surface fish or blind cavefish can respond to visual stimuli by measuring phototaxis and monitoring the response to moving object shade (inserted between the 850 nm infrared light source and fish arena). Our results indicated that neither fish responded to these visual stimuli at a detectable level. Although we cannot exclude the possibility that these fish may still receive low levels of visual stimuli, they do not elicit major behavioral outcomes under the current setup.

Using our method of measuring nearby interactions via idTracker-based tracking of each individual, a ‘nearby interaction’ was defined by both a threshold maximum proximity distance between two fish ( ≤ 5 cm) and a minimum threshold interaction duration ( ≥ 4 s). This method was sensitive enough to detect changes in nearby interactions in different environments (e.g. familiar and unfamiliar; Figure 3) and following drug treatment (Figure 6; Figure 2—figure supplement 4). This method is also applicable to measure a unit of collective behavior among larger groups (i.e. two fish attractions or nearby interactions among ≥5 individuals) in other animal species.

Regarding non-visual collective behavior in A. mexicanus, a quantitative trait locus (QTL) for non-visual schooling has been reported where F₂ hybrid fish generated from a pair of a surface fish and a Tinaja cavefish (a cave population that evolved independently from that of the Pachón cave; Pachón cavefish were used in this study) were used (Kowalko et al., 2013). The schooling measurement was based on how the F₂ individual responded to the school of ‘model fish’ and based on NND. The major schooling QTL is associated with vision (Kowalko et al., 2013). The QTL for non-visual schooling was detected by using only F_2, which showed negative phototaxis (i.e. had visual capacity) but exhibited large differences from non-schooling to schooling. The method presented here can improve the detection resolution in terms of nearby interactions. Regardless, it was interesting that F₂ individuals carrying homozygous cave genotypes at a marker underlying this QTL exhibited increased schooling behavior, while F₂ individuals with homozygous surface or heterozygous genotypes schooled less (Kowalko et al., 2013). This genetic study suggests two possibilities: (1) Tinaja cavefish evolved a ‘schooling allele’, which is different from the ‘nearby interaction alleles’ that Pachón cavefish have, or (2) the same allele regulates Tinaja’s schooling behavior and Pachón’s nearby interaction. Note that Tinaja’s schooling allele may simply be an appetite-driving gene since aggression or foraging against fish plastic models was not evaluated in the former study (Kowalko et al., 2013), and Tinaja cavefish show insatiable appetites (Aspiras et al., 2015) (see below for cannibalistic foraging). Based on this complexity, a crossing experiment between Tinaja and Pachón cavefish is necessary to answer these possibilities.

Based on our results of pharmacological ablation of the lateral line, the nearby interaction of surface fish in the dark is likely regulated by lateral line mechanosensors (Figure 2—figure supplement 4). However, this result does not mean that the involvement of other sensory systems, such as tactile and olfactory systems, can be excluded (Timmermann et al., 2004). Further tests are needed to address which of these sensory systems plays the crucial role in regulating nearby interactions in fish in the dark.

In the dark, surface fish changed their swimming direction based on the presence of other individuals, as if they sensed and were attracted to others, similar to what has been observed in zebrafish larvae under lighted conditions (Figure 2—figure supplement 2; Hinz and De Polavieja, 2017). However, surface fish failed to form a clear cluster, that is a ‘shoal’, in the dark. Instead, surface fish individually responded to one another. We believe that the failure to form a shoaling cluster was based on the lack of visual information (see Videos 1 and 4 under light and dark conditions, respectively), yet surface fish maintained their collective tendency in the dark by using lateral line sensing.

The nearby interactions detected in this study did not appear to be related to foraging activities or aggression (i.e. two fish come close while expressing aggression or cannibalistic foraging [Elipot et al., 2013]). We categorized the nearby activities into five classes of activity and visualized them to show the frequent role changes of leaders and followers within single bouts in the same fish pair (Figure 2—figure supplement 2C and E). This role change is rarely observed in aggressive encounters (Elipot et al., 2013). In addition, cavefish slowed during the nearby-interaction bout compared with other periods of time, which contradicts the speed shift observed during aggression—fish thrust during aggression (Elipot et al., 2013). Finally, we carefully assessed damage to the fins and scales, and although these tissues are good indicators of aggressive encounters, noticeable damage was not detected. Consistent with this observation, both surface fish and cavefish rarely showed startle responses after touching one another. We therefore conclude that A. mexicanus rarely attacked one another in our assay system and that the majority of detected nearby interactions can be characterized as shoaling-like activities.

The degree and type of nearby interactions in the dark that were recorded in this study appear relevant within the context of each morph’s natural habitat. Surface fish have a known nocturnal predator, prawn fish (Wilson et al., 2004), and adult surface fish form a tighter shoaling cluster at night (personal observation in the laboratory). These facts imply that shoaling can be advantageous by simply lowering individual risk among cohorts, the so-called ‘attack dilution effect’, and/or by increasing sensitivity in detecting potential predators by the collective effect of more sensors of multiple individuals (Pitcher and Parrish, 1993). In cavefish, a higher level of nearby interactions detected in the familiar laboratory setting was also observed in their natural habitat, Cueva de El Pachón, Tamaulipas, Mexico (2019, personal observation). While they did not form a recognizable shoal, cavefish responded to conspecifics and tended to follow each other. This social-like tendency could merely be a residual of regressive evolution to a troglobitic niche following their divergence from surface riverine ancestors (Langecker et al., 1991; Yoshizawa et al., 2012). Alternatively, this weak social-like tendency could remain advantageous for finding mates, locating food or evading potential dangers within their cave environment.

The young adult surface fish used in this study had reached an appropriate developmental stage to express the shoaling behavior, and all of their behaviors were assessed only in the day. Since the shoaling motivation was seen at night with very limited visual inputs (see above), it is important to investigate shoaling capacity at night in future studies.

In the unfamiliar setting, the cavefish reduced the social-like interaction and kept high-level of repetitive turning, suggesting that cavefish lost the strategy to use social-like interactions to confront unfamiliar environmental inputs, which surface fish have.

Restricted repetitive behaviors are observed in mammals kept in restricted/isolated environments and in humans with stereotypic movement disorders, including ASD (Lewis et al., 2007; Rose et al., 2017). This motor disorder has been considered non-functional. However, since these stereotypic behaviors are seen in many vertebrate species as well as in typically developing human infants, there may be an advantage to repetitive behaviours. To understand the behavioural function of repetitive motion, an emerging hypothesis—predictive processing—was introduced in cognitive neuroscience (Palmer et al., 2017). In this framework, to develop an internal model for the habitat ( = environment), the central nervous system is continuously attempting to minimize prediction errors, that is, differences between the environmental signals sampled by an animal’s sensory systems and that animal’s internal model of the world within the brain. The brain is assumed to have two strategies to minimize differences between sensory input and the internal model when novel stimuli are encountered: (i) to use a better fitting model among many existing internal models or (ii) to locomote back to a ‘no-novel stimulus environment’ to sample the sensory inputs that had already been implemented in the current internal model (Friston, 2010). Stereotypic repetitive circling seems to be a result of choosing the latter strategy by sampling predictable sensory inputs (strong lateral line inputs on one side of the body). This method can reduce the prediction errors between the sensory inputs and those predictions from the internal model (e.g. left turning provides the strong right lateral line signals in cavefish). In contrast, surface fish responded to unfamiliar stimuli and did not increase repetitive turning, perhaps by selecting a better fitting model among the available internal models (i.e. strategy (i)). Therefore, based on this predictive processing framework, surface fish and cavefish apparently have different strategies. Both of these changes could minimize the errors between the sensory inputs and the predicted inputs associated with the internal model. Given that surface fish appear to acclimatize to novel environments much quicker than cavefish measured by foraging latency (personal observation), this suggests that surface fish may store many internal models that are proxies of the current environment. Having many internal models is thought to better highlight new salient stimuli in a changing environment (i.e. a good fitting-internal model yields better attention to the new object). The difference in the richness of internal models between surface fish and cavefish is addressable by examining relationships between a number of different environmental setups and how individuals can accurately navigate to reach food without panicking/repetitive motion.

Previous studies have indicated that there is an antagonistic relationship between repetitive and social behavior (Kim et al., 2016; Lewis et al., 2007). For example, social impairment and repetitive behavior are thought to be linked together as revealed by results following oxytocin infusion (reviewed in Kim et al., 2016). In contrast, in a study of ketogenic diet treatment in individuals with autism, social behavior in some treated patients had recovered to normative levels, while repetitive behaviour was not significantly attenuated (Lee et al., 2018). This result suggested that repetitive behavior can be independent of social impairment. The mechanisms that controls switching between social and repetitive behaviors is still largely unknown. Many studies of repetitive behavior and social behavior have focused on the cortico-striatal system, where many patients who show repetitive behavior (including those with Gilles de la Tourette syndrome, obsessive–compulsive disorder, ASDs, Parkinson’s disease, and Huntington’s disease) have abnormal functional and/or histological features diagnosed by PET and fMRI (Langen et al., 2011a). Importantly, this striatal system, particularly its dopaminergic system, has also been suggested to drive social behavior, where the dopamine signal from the VTA stimulates D1-dopamine receptor-positive striatal neurons in the nucleus accumbens, driving social behaviour in mice (Gunaydin et al., 2014; Kim et al., 2016). Among vertebrates, the striatum has been proposed as the conserved bridging centre. The striatum integrates (1) higher levels of sensory inputs from the cerebral cortex/dorsal pallium and (2) information-evaluating dopaminergic input from the substantia nigra par compacta (SNc). The dopaminergic striatal pathway is largely conserved among vertebrate species. This includes the direct and indirect pathways, each of which is associated with a specific type of dopamine receptor—D1 and D2 receptors, respectively. The major function of this striatal dopaminergic system is thought to be to select behavioral modules (e.g. mating, foraging, and aggression modules) based on given sensory inputs and internal states (Grillner, 2018; Grillner and Robertson, 2016). For example, a fish may ignore or follow mechanical signals from cohorts based on the fish’s internal state.

In addition to the striatal behavior switching centre, many other brain regions are known to induce social/social-like behaviors. For example, from teleosts to mammals, the best neurochemically conserved social decision-making network has been reported to be the preoptic area (POA based on an analysis of 10 gene products across 12 brain decision-making processing regions in 88 species; dopaminergic neurons also exist in this region) (O’Connell and Hofmann, 2012; O’Connell and Hofmann, 2011).

Accordingly, bath treatment with a partial D2-dopamine receptor antagonist, aripiprazole, showed a parallel response between cavefish and humans, suggesting a potential shared pathway that controls social-like and repetitive behaviors in teleosts and mammals and possibly involves the striatum, POA, and/or other dopaminergic processing nuclei (Grillner, 2018; Kim et al., 2016; O’Connell and Hofmann, 2011). Future studies will address whether the antagonistic relationship between social-like and repetitive behaviors is based on the switch of behavior modules in the striatal system or whether repetitive motions simply fragment social-like interactions, although the individual may continue to have the motivation to congregate.

It will be exciting to examine the evolutionary and neurophysiological changes in the dopaminergic system between cavefish and surface fish and how these changes are associated with the integration of an internal model, which the predictive-processing theory proposes, in the future.

All data generated and analyzed during this study are included in the supplementary Source Data file. Program scripts/codes are available in the public data depository (https://doi.org/10.5281/zenodo.5122894). All raw video data are available upon request. Sample video files are available at (https://doi.org/10.5281/zenodo.5122894).

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[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Social-like responses are inducible in the asocial Mexican cavefish despite the exhibition of strong repetitive behavior" for consideration by eLife.

Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Guest Editor and a Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Hans Hofmann (Reviewer #3).

Our decision has been reached after consultation between the reviewers and editors. Although the work is of interest, we regret to inform you that we have concluded that the findings at this stage are too preliminary for further consideration at eLife. That said, we would be willing re-consider a new submission at a later stage, if you were able to add the missing controls and address the other points raised. Please note that resubmission does not guarantee re-evaluation, let alone eventual acceptance.

Specifically, although you present a significant body of data, which supports many of your conclusions, there has been substantial concern about the lack of appropriate controls in order to rule out alternative explanations of the results. Furthermore, there have been questions about how some of the behaviours were defined and the interpretation of the relationship between social repetitive behaviours. Given that the behaviours described are influenced by speed and tendency to occupy central positions in fish, there have been concerns to what extend these behaviours would be representative of social behaviour, rather than kinematic differences in behaviour that are affected by a drug treatment already known to reduce movement speed.

Please note that we aim to publish articles with a single round of revision that under normal circumstances can be accomplished within two months. This means that articles that have potential, but in our judgment would need extensive additional work, will not be considered for revision. We do not intend any criticism of the quality of the data or the rigor of the science.

Reviewer #1:

Social-like responses are inducible in the asocial Mexican cavefish despite the exhibition of strong repetitive behavior by Iwashita and Yoshizawa examines social behavior in cavefish and their surface counterparts. While multiple previously published studies found a loss of schooling and shoaling behavior in cavefish compared to their highly social surface counterparts, as well as an essential role for vision in schooling in surface fish, through careful and thorough analysis, the authors overcome some of the limitations of previous studies to reveal that surface fish do indeed have social interactions in the dark, and that social interactions, while reduced, are indeed present in cavefish. Further, they find that in cavefish, the environment plays a role in these social interactions: these interactions increase in frequency and duration in a familiar environment. Further, they examine turning and find that both biased turning and social interactions are altered by a drug targeting the dopaminergic system in cavefish. Together, this study presents an interesting approach to examining social behavior in a comparative context, using novel methods in this context which allow these authors to uncover aspects of social behavior in a natural population of fish that were previously unknown.

The authors present a significant body of data, which supports many of their conclusions.

1. Additional information regarding how nearby interactions are defined would be helpful, along with an analysis to determine that these interactions are not simply occurring by chance, but rather signify social interactions.

2. Whether there is a relationship between turning bias (suggested here as a repetitive behavior) and social suppression is still unclear, and alternative explanations for the correlation between these behaviors in cavefish, should be discussed.

3. The authors do a great job of presenting many detailed analyses and explanations of how they performed these analyses. However, as structured, the amount of analysis presented in supplemental figures makes the manuscript difficult to follow in places. For example, there are six supplemental figures associated with Figure 3. However, many of these are unrelated to Figure 3, and there is a whole section of the paper from Lines 137-179 which only discusses these supplemental figures. Thus, some of these should be made into main figures for the paper. Similarly, there are 2 Supplementary files which describe methods that should go into main text.

4. The measurements of nearby interactions is crucial to the interpretations of the paper, and very interesting. However, I was a bit confused about how the authors chose their cutoffs for nearby interactions (for example, why was 4 seconds a better cut off than 3 seconds). Further, I was a little confused about whether some of these social interactions could occur by random chance. It seems like the authors could test this, as they have already performed "random" location permutation tests – are these close interactions overrepresented relative to what you would see if you analyzed these synthesized random videos? If this is the case, this would greatly strengthen their arguments.

5. The argument that there is a tradeoff between repetitive behavior and social behavior was weak – Couldn't these two traits just both be influenced by the pharmacological agent?

Reviewer #2:

The authors have presented a study that offers insight into a potentially fascinating question of social evolution. They examine two populations of Astyanax fish derived from surface or cave dwelling progenitors, comparing their behaviour in the absence of light shorter than 850nm (which they term 'dark', although this is not correct). Based on a previous finding that certain behaviours are reduced in the dark in this species, the authors sought to test whether other behaviours, which they term social-like interactions, are also diminished in the dark. They report 'numerous social-like' behaviours also occur in the dark in surface fish, and claim that treatment with a human anti-psychotic drug slowed velocity and restored some behaviours.

Although the aims are commendable, the experiment is poorly controlled and suffers from some major flaws. The first is in the usage and definitions of social behaviours. Most of the patterns of behaviours reported are associated with tendency to move towards or away from the centre of the tank. This is a crucial aspect as the 'social' behaviours reported by the authors are directly influenced by the tendency to mill in the centre of the tank, which has subsequent motion effects on turning frequency and inter-individual distance. Secondly, and most problematic for the interpretations of the authors is that although the authors claim there is no change in behaviour under their infra-red lighting conditions, the light sources they use emit at 850nm, which is within a known sensitivity range for many teleost species (Sensitivity Differences in Fish Offer Near-Infrared Vision as an Adaptable Evolutionary Trait Shcherbakov et al). Even other Astyanax studies use much longer wavelength light (e.g. 970nm). The most parsimonious explanation for almost all results is therefore that the surface fish can still see, and in fact the potentially strong side-welling light may have considerable consequences for their behaviour. The authors attempt to address this by presenting a visual stimulus (Video 4), but this stimulus is presented from above the water, whereas the light is side-welling and from below the water level. As such, it is not clear that this overhead stimulus would be visible in low-intensity, side-welling light in the assumed visible spectrum. Given the possibility that fish can see 850nm light, and the very poor controls conducted in this experiment to test for this, I simply do not accept the author's conclusion that the visual system plays no role in the behaviours described here. This problem is exacerbated in the main interpretations of the paper, which are the comparisons of swimming behaviour between cave and surface fish. A source of side-welling light would induce avoidance behaviour in sighted fish, and therefore also reduce the thigmotaxis commonly observed in this species. The finding that surface fish maintain more central positions in the tank is most likely a consequence of this avoidance, and without proper controls this remains the most parsimonious interpretation. That this effect was not observed in the cave fish, which certainly cannot see, provides more evidence for this interpretation. Because there is the potential that blind fish showed normal thigmotactic and exploratory behaviour of the entire tank, whereas surface fish were more centrally located in the tank (potentially because they avoided areas of strong side-welling light), then all subsequent analyses of inter-individual distance are questionable, since they are all impacted by the fact that surface fish are more central in the IR condition.

The relevant control would be to measure the tendency to avoid tank edges with natural light that is side-welling, but as can be seen in Video 3, the side-welling light is (surprisingly) removed with barriers, so no comparison can be made.

Unfortunately, I find this paper lacks the relevant controls that would be required to support the authors' interpretations.

Reviewer #3:

In the manuscript "Social-like responses are inducible in the asocial Mexican cavefish despite the exhibition of strong repetitive behavior," Iwashita and Yoshizawa examine to which extent the supposedly "asocial" blind cavefish, Astyanax mexicanus, exhibits "social-like interactions" depending on the familiarity of the environment. The authors show that such interactions exist, possibly regulated by the dopaminergic system.

Overall, this is a well-executed study that brings to bear advanced tracking technology to identify subtle behavioral phenotypes. In that sense it achieves its goals. To which extent those phenotypes have any adaptive value and whether the mechanisms underlying repetitive behavior might be shared between teleosts and mammals remains to be seen. In any case, the study should be of interest to readers in several fields.

(1) In Results, every experiment is introduced with a reference to similar human phenomena. I think it is fine to make this connection in the Introduction and Discussion, but it seems forced in the Results.

2) The authors may want to pay close attention to their use of terminology. It appears that words like "social," "collective," and "cooperative" are used interchangeably, where they clearly mean different things.

3) In the Discussion, the authors call on Friston's so-called "Free-Energy Principle" to interpret their results, which seems quite unnecessary. Also note that Friston's supposed principle rests on circular reasoning and cannot be experimentally falsified. I consider it unscientific.

4) The discussion of possible striatal mechanisms underlying the observed phenomena seems premature. The dopaminergic system was manipulated via systemic application of a drug (in aquarium water), so no inferences can be made about where the drug may have acted.

5) It is not clear that this study presents any evidence for the existence of "a deeply shared pathway that controls social-like and repetitive behaviors between teleost and mammals." The results presented here may well be consistent with this idea. However, to show this would require a phylogenetic comparative analysis, which was not the goal of the present study.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Social-like responses are inducible in asocial Mexican cavefish despite the exhibition of strong repetitive behaviour" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Guest Reviewing Editor and Christian Rutz as the Senior Editor. The reviewers have opted to remain anonymous.

While we are in principle happy to publish your article, please carefully address the remaining comments from Reviewer #2 in a final revision. Furthermore, please note that eLife has recently adopted the STRANGE framework, to help improve reporting standards and reproducibility in animal behaviour research. In your final revision, please consider scope for sampling biases and potential limitations to the generalisability of your findings, adding subject attribute data and discussion text as appropriate:

Reviewer #1:

The authors have done a good job with this revision. Their responses to the Reviewers' comments are thorough and convincing, and the revised manuscript reads well and is considerably strengthened. I think this will be a nice contribution.

Reviewer #2:

The authors addressed the majority of my concerns, and I was impressed by the extensive revisions and additional experiments and analysis they performed, and felt that these strengthened the manuscript.

1. The revised manuscript does an excellent job of explaining how nearby interactions were defined and why. However, this raised an additional question: The authors defined nearby interactions by identifying differences between simulated groups and actual groups of fish. The authors use IID (distance between each fish and every other fish in the group) to determine these cutoffs. However, the interactions are between only two fish, so it seems like NND would be the appropriate metric to use to determine what should be a nearby interaction, and this appears to be what the authors used in the last version of the manuscript. I recommend using pairwise interactions rather than group interactions to define what will ultimately be an analysis of interactions between pairs of fish.

2. The authors redoing experiments using IR light diffused across the tank resulted in many of the same conclusions as the previous manuscript. However, there were some differences in the reported results using this new method (notably, a lack of difference between cavefish and surface fish in NND and IID, differences in effects of the environment on position in the arena, etc.). Do the authors believe that the surface fish could see in the original set of assays but could not in the second, or do they have another explanation for these differences in results when the lights are moved to a different location?

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

[…] Specifically, although you present a significant body of data, which supports many of your conclusions, there has been substantial concern about the lack of appropriate controls in order to rule out alternative explanations of the results. Furthermore, there have been questions about how some of the behaviours were defined and the interpretation of the relationship between social repetitive behaviours. Given that the behaviours described are influenced by speed and tendency to occupy central positions in fish, there have been concerns to what extend these behaviours would be representative of social behaviour, rather than kinematic differences in behaviour that are affected by a drug treatment already known to reduce movement speed.

We took these concerns seriously and responded to all these points. Our responses are described in the cover letter. Briefly, we found that fish significantly slowed their swimming speed during social-like interactions (Figure 2G and H; Figure 3H and I; Figure 6E and F) and showed role switches between 'leader' and 'follower' during each interaction event (Figure 2—figure supplement 2). We further showed that these social-like events occurred in both peripheral and central areas of the recording arena (Figure 2—figure supplements 1 and 3) and showed no visual responsivity in the current setup. These results provide robust support that this detected behaviour represents an ‘affinity to each other’ and not unidirectional aggression or a result of avoiding peripheral areas.

Please note that we aim to publish articles with a single round of revision that under normal circumstances can be accomplished within two months. This means that articles that have potential, but in our judgment would need extensive additional work, will not be considered for revision. We do not intend any criticism of the quality of the data or the rigor of the science.

Reviewer #1:

[…] 1. Additional information regarding how nearby interactions are defined would be helpful, along with an analysis to determine that these interactions are not simply occurring by chance, but rather signify social interactions.

In this revision, we further described the detection method of the social-like interactions. Briefly, we filtered the nearby-interaction events by distance (below 5 cm) and duration (more than 4 s) based on the comparisons between actual and simulated data. The actual data were from 18 groups of social surface fish. The simulated data were generated by randomly sampling four individual fish’s X-Y coordinates (four fish per group in our experiment). Using these X-Y coordinates, we recalculated the inter-individual distances among each of the 1,280 simulated four-fish groups. The ranges of distance (≤ 3-6 cm) and duration (≥ 3-8 s) showed differences between the actual and simulated data. We chose a conservative set of cut-off values (i.e., ≤ 5 cm and ≥ 4 s). We also carefully classified the types of nearby interactions into the following categories: leading, dispersing, and approaching (Figure 2—figure supplement 2). As expected for reciprocal interactions, we observed role switches between leaders and followers within a single nearby-interaction bout event and between bouts within the same individual pair (Figure 2—figure supplement 2). This interaction can be categorized as fish in the “attraction” zone defined by Lukeman et al. (Lukeman et al., 2010). However, the fish could not form a collective cluster, probably due to the lack of long-distance sensing (visual sensing) in the dark. In fact, the surface fish could form a cluster under lighted conditions (Figure 2—figure supplement 3). We discussed these points in the Discussion section from the paragraph “Using our method of measuring nearby interactions via idTracker-based tracking…” (pg. 18) to the paragraph “In the dark, surface fish changed their swimming direction based on…” (pg. 20).

2. Whether there is a relationship between turning bias (suggested here as a repetitive behavior) and social suppression is still unclear, and alternative explanations for the correlation between these behaviors in cavefish, should be discussed.

We interpreted the reviewer’s point to mean that we cannot assume mutual exclusivity between turning bias and social-like interaction. We added a paragraph discussing the potential mechanisms of this antagonistic relationship (pg. 24). Briefly, turning bias or repetitive behaviour reduced the probability of social-like interactions, which could be due to a switch in the behaviour module in the striatal system or a simple fragmentation of social-like interaction events even though the individual may have the motivation to engage in social-like activities.

3. The authors do a great job of presenting many detailed analyses and explanations of how they performed these analyses. However, as structured, the amount of analysis presented in supplemental figures makes the manuscript difficult to follow in places. For example, there are six supplemental figures associated with Figure 3. However, many of these are unrelated to Figure 3, and there is a whole section of the paper from Lines 137-179 which only discusses these supplemental figures. Thus, some of these should be made into main figures for the paper. Similarly, there are 2 Supplementary files which describe methods that should go into main text.

Thank you very much for this constructive suggestion, and we fully agree with the reviewer’s point. We incorporated these supplemental data into the main figure (Figure 2) and simplified the logical flow.

4. The measurements of nearby interactions is crucial to the interpretations of the paper, and very interesting. However, I was a bit confused about how the authors chose their cutoffs for nearby interactions (for example, why was 4 seconds a better cut off than 3 seconds). Further, I was a little confused about whether some of these social interactions could occur by random chance. It seems like the authors could test this, as they have already performed "random" location permutation tests – are these close interactions overrepresented relative to what you would see if you analyzed these synthesized random videos? If this is the case, this would greatly strengthen their arguments.

Again, thank you very much for this significant advice. We introduced new paragraphs in the main body describing how we determined the cut-off parameters based on the comparisons between the actual and simulated randomly sampled data. The filtered nearby interactions (≤ 5 cm and ≥ 4 s) showed very similar behavioural modes—approaches, leading and following within the same bout—as in the lighted conditions with surface fish.

5. The argument that there is a tradeoff between repetitive behavior and social behavior was weak – Couldn't these two traits just both be influenced by the pharmacological agent?

We addressed this point above.

Reviewer #2:

[…] Although the aims are commendable, the experiment is poorly controlled and suffers from some major flaws. The first is in the usage and definitions of social behaviours. Most of the patterns of behaviours reported are associated with tendency to move towards or away from the centre of the tank. This is a crucial aspect as the 'social' behaviours reported by the authors are directly influenced by the tendency to mill in the centre of the tank, which has subsequent motion effects on turning frequency and inter-individual distance. Secondly, and most problematic for the interpretations of the authors is that although the authors claim there is no change in behaviour under their infra-red lighting conditions, the light sources they use emit at 850nm, which is within a known sensitivity range for many teleost species (Sensitivity Differences in Fish Offer Near-Infrared Vision as an Adaptable Evolutionary Trait Shcherbakov et al). Even other Astyanax studies use much longer wavelength light (e.g. 970nm). The most parsimonious explanation for almost all results is therefore that the surface fish can still see, and in fact the potentially strong side-welling light may have considerable consequences for their behaviour. The authors attempt to address this by presenting a visual stimulus (Video 4), but this stimulus is presented from above the water, whereas the light is side-welling and from below the water level. As such, it is not clear that this overhead stimulus would be visible in low-intensity, side-welling light in the assumed visible spectrum. Given the possibility that fish can see 850nm light, and the very poor controls conducted in this experiment to test for this, I simply do not accept the author's conclusion that the visual system plays no role in the behaviours described here.

Thank you for pointing out these critical aspects. The first point, that is, that fish congregated in the centre to avoid ‘visible’ illumination from the side, regardless of their affinity for the others was rejected by looking at the swimming trajectories of the fish. The nearby interactions occurred in the centre and at the edges. Accordingly, we improved the visualization method for the fish trajectories (e.g., Figure 2—figure supplements 1 and 3).

Astyanax studies using 970-nm illumination are from one lateral line expert group, i.e., Dr. Sheryl Coombs laboratory. Additionally, thank you for sharing the reference regarding near-IR sensitivities in multiple teleost species. We re-designed the assay system and doublechecked that the sighted and non-sighted fish did not change their behaviour under our 850-nm illumination (Figure 1—figure supplement 1; Video 3). Considering a published report by Parry et al., where Astyanax retina microspectrophotometry showed no detectable absorption above 700 nm (Parry et al., 2003), we conclude that the current setup is dark based on Astyanax light sensitivity.

This problem is exacerbated in the main interpretations of the paper, which are the comparisons of swimming behaviour between cave and surface fish. A source of side-welling light would induce avoidance behaviour in sighted fish, and therefore also reduce the thigmotaxis commonly observed in this species. The finding that surface fish maintain more central positions in the tank is most likely a consequence of this avoidance, and without proper controls this remains the most parsimonious interpretation. That this effect was not observed in the cave fish, which certainly cannot see, provides more evidence for this interpretation. Because there is the potential that blind fish showed normal thigmotactic and exploratory behaviour of the entire tank, whereas surface fish were more centrally located in the tank (potentially because they avoided areas of strong side-welling light), then all subsequent analyses of inter-individual distance are questionable, since they are all impacted by the fact that surface fish are more central in the IR condition.

The relevant control would be to measure the tendency to avoid tank edges with natural light that is side-welling, but as can be seen in Video 3, the side-welling light is (surprisingly) removed with barriers, so no comparison can be made.

Unfortunately, I find this paper lacks the relevant controls that would be required to support the authors' interpretations.

Based on the comments by Reviewer 2, we systematically replicated all the experiments with a background LED light with a light diffuser. In this setting, surface fish and cavefish did not show a preference for the centre of the arena, did not show a preference for the illuminated or non-illuminated areas, and did not respond to the moving shade between the light source and fish tank. These facts supported the notion that surface fish did not respond to 850-nm light. In our original side-light setting, LEDs were naked without diffusers; thus, surface fish may have been able to sense the infrared light-LED itself or thermal energy. With the reported result of Astyanax retina’s microspectrophotometry (sensitive less than 700 nm; Parry et al., 2003), we conclude that our 850-nm infrared light did not affect the behaviours of either sighted surface fish or non-sighted cavefish.

Reviewer #3:

[…] (1) In Results, every experiment is introduced with a reference to similar human phenomena. I think it is fine to make this connection in the Introduction and Discussion, but it seems forced in the Results.

Thank you for the suggestion. We removed most human-related statements from the Results section with the exception of the section on aripiprazole treatment. Aripiprazole treatment is justifiable only in relation to its use in humans as a medical treatment, due to its complex pharmacological profile and the history of its development.

2) The authors may want to pay close attention to their use of terminology. It appears that words like "social," "collective," and "cooperative" are used interchangeably, where they clearly mean different things.

This is a great suggestion. In the current revision, we avoided using “cooperative.” Additionally, we carefully chose the words “social-like” and “collective” based on the context.

3) In the Discussion, the authors call on Friston's so-called "Free-Energy Principle" to interpret their results, which seems quite unnecessary. Also note that Friston's supposed principle rests on circular reasoning and cannot be experimentally falsified. I consider it unscientific.

We removed reference to the “free-energy principle’; however, predictive coding is a well-accepted theory. We discussed a possible neural mechanism underlying biased turning based on predictive coding and a possible experimental approach to assessing the theory on pg. 22-23.

4) The discussion of possible striatal mechanisms underlying the observed phenomena seems premature. The dopaminergic system was manipulated via systemic application of a drug (in aquarium water), so no inferences can be made about where the drug may have acted.

We have expanded our discussion by taking into account the systemic effect of drug treatment. Now the sentence reads “Accordingly, bath treatment with a partial D2-dopamine receptor antagonist, aripiprazole, showed a parallel response between cavefish and humans, suggesting a potential shared pathway that controls social-like and repetitive behaviours in teleosts and mammals and possibly involves the striatum, POA, and/or other dopaminergic processing nuclei (Grillner, 2018; Kim et al., 2016; O’Connell and Hofmann, 2011)” (pg. 24).

5) It is not clear that this study presents any evidence for the existence of "a deeply shared pathway that controls social-like and repetitive behaviors between teleost and mammals." The results presented here may well be consistent with this idea. However, to show this would require a phylogenetic comparative analysis, which was not the goal of the present study.

We agree with the reviewer and eliminated phylogeny-related statements. This statement now reads “the presented results suggest that the asocial cave population is still capable of performing social-like nearby interactions under environmental or pharmacological treatments and shares an antagonistic association between social-like and repetitive behaviours with mammals.” (pg. 6).

[Editors’ note: what follows is the authors’ response to the second round of review.]

Reviewer #2:

The authors addressed the majority of my concerns, and I was impressed by the extensive revisions and additional experiments and analysis they performed, and felt that these strengthened the manuscript.

1. The revised manuscript does an excellent job of explaining how nearby interactions were defined and why. However, this raised an additional question: The authors defined nearby interactions by identifying differences between simulated groups and actual groups of fish. The authors use IID (distance between each fish and every other fish in the group) to determine these cutoffs. However, the interactions are between only two fish, so it seems like NND would be the appropriate metric to use to determine what should be a nearby interaction, and this appears to be what the authors used in the last version of the manuscript. I recommend using pairwise interactions rather than group interactions to define what will ultimately be an analysis of interactions between pairs of fish.

Thank you for addressing this critical point with thoughtful comments. We strategically chose IID (average distance between each pair of fish) instead of NND (nearest neighbour distance) because we intended to measure nearby interactions (one-by-one) while two, three, or four fish were interacting with each other. This allowed us to count the social-like events that we carefully investigated when the filtered social-like events matched what we observed in fish behaviours in each recorded video. For example, suppose fish A and fish B are interacting with each other, and fish C passes by fish A more closely than it passes by fish B. With the cut-off based on IID, the fish A-fish B interaction is continuously counted while fish C passes by fish A because this cut-off is based on the average distance of each pair of fish (Figure 2—figure supplement 1A). The fish A-fish C interaction may not be counted because of a shorter duration than the cut-off period. In contrast, by using the NND cut-off, three events are counted separately: (1) the fish A-fish B interaction before fish C passes by, (2) the fish A-fish C interaction, and (3) the fish A-fish B interaction after fish C passes by (note, NND is shorter than IID). This may exclude the fish A-fish B interaction since each of durations (1) and (3), not the sum of (1) and (3), can be shorter than the duration cut-off.

Again, we are reporting nearby interaction durations/bouts of fish pairs within a cluster of fish (i.e., shorter distances than the IID-based cut off). The NND-based cut-off fragments the nearby interaction bout and reduces the S/N ratio (the passing-by events are noise).

To clarify this point, we added the sentence "IID instead of NND was strategically chosen because we were interested in interactions not only between two fish (NND) but also among three or four fish (IID) that were closer than expected by chance” to the Results section on pg 8.

2. The authors redoing experiments using IR light diffused across the tank resulted in many of the same conclusions as the previous manuscript. However, there were some differences in the reported results using this new method (notably, a lack of difference between cavefish and surface fish in NND and IID, differences in effects of the environment on position in the arena, etc.). Do the authors believe that the surface fish could see in the original set of assays but could not in the second, or do they have another explanation for these differences in results when the lights are moved to a different location?

Whether the fish could "see or not see" is an important point. We made this point only in response to Reviewer 2 in our resubmission cover letter because it is not necessary for the reader to know about our original side-light setting. What we wrote in our response was, "In our original side-light setting, LEDs were naked without diffusers; thus, surface fish may have been able to sense the infrared light-LED itself or thermal energy."

We hope this answers your question.

Author details

1. Motoko Iwashita

   School of Life Sciences, the University of Hawai‘i at Manoa, Honolulu, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6653-6823

2. Masato Yoshizawa

   School of Life Sciences, the University of Hawai‘i at Manoa, Honolulu, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - review and editing

   For correspondence

   yoshizaw@hawaii.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8455-8252

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