Social-like responses are inducible in asocial Mexican cavefish despite the exhibition of strong repetitive behaviour

Social behaviour is a hallmark of complex animal systems; however, some species appear to have secondarily lost this social ability. In these non-social species, whether social abilities are permanently lost or suppressed is unclear. The blind cavefish Astyanax mexicanus is known to be asocial. Here, we reveal that cavefish exhibited social-like interactions in familiar environments but suppressed these interactions in stress-associated unfamiliar environments. Furthermore, the level of suppression in sociality was positively correlated with that of stereotypic repetitive behaviour, as seen in mammals. Treatment with a human antipsychotic drug targeting the dopaminergic system induced social-like interactions in cavefish, even in unfamiliar environments, while reducing repetitive behaviour. Overall, these results suggest that the antagonistic relationship between repetitive and social-like behaviours is deeply shared from teleosts through mammals.


Abstract 13
Collective behaviors, such as schooling of fishes and mass migrations of ungulates, are hallmark 14 features of a wide variety of animal species. Such phenotypic characters can be lost, either 15 through evolutionary process across generations or by certain environmental stressors within a 16 generation. Such stressors simultaneously promote stereotypic repetitive behaviors in many 17 mammals, such as those exhibited in certain ex-situ captive settings. However, in asocial species, 18 it is unclear whether social ability is permanently lost or only suppressed. It is also unclear if this 19 antagonistic regulation between repetitive and social behaviors is deeply conserved among 20 vertebrates. An evolutionary model, the Mexican tetra, Astyanax mexicanus, has a blind cave-21 dwelling morph lacking collective behaviors but exhibiting repetitive circling. In contrast, the 22 sighted surface riverine morph of A. mexicanus shows more normative behaviors. We here report 23 that social-like interactions are recoverable in blind asocial cavefish under familiar environments 24 but are suppressed under the stress-associated unfamiliar environments. In contrast, surface fish 25 revealed robust social-like behaviors in the dark, regardless of familiarity to the environment. 26 Treatment of a human antipsychotic drug also induced social-like interactions in cavefish even in 27 unfamiliar environments. In addition, the level of repetitive behavior is positively correlated with 28 that of asociality in cavefish, suggesting that the antagonistic regulation between repetitive and 29 social-like behaviors is present in teleost fish. Asocial cavefish therefore, with a deeply 30 conserved framework of behavioral regulation, are still capable of expressing social-like 31 behavior following ca. 200,000 years of adaptation to caves. 32 33

Introduction
We then analyzed social-like nearby interactions and repetitive behavior in the dark and, 79 surprisingly, revealed numerous social-like interactions in surface fish in the dark. Surface fish, 80 however, did not show repetitive turning. Furthermore, we also uncovered weak but existing 81 social-like interactions in cavefish while exhibiting significantly higher level of repetitive 82 behavior than that of surface fish. During these nearby interactions, cavefish decelerated their 83 swimming velocity, similar to surface fish. The comparison of these behaviors under familiar 84 and unfamiliar environments, indicated that cavefish significantly increased nearby interactions 85 in the familiar environment. Atypical dopamine psychiatric drug, aripiprazole, which is known to 86 increase social behaviors and reduce repetitive motions in human 31-33 , slowed swimming 87 velocity and increased nearby interaction in both surface and cavefish. Cavefish also reduced 88 repetitive turning after aripiprazole treatment. In summary, cavefish showed a robust repetitive 89 circling by largely ceding social-like nearby interactions, where this social-like interaction still 90 kept being plastic in response to the environmental cues. Together with the reported parallels in 91 pharmacological response, a set of behavioral symptoms, and gene expression profiles between 92 cavefish and human, the presented result suggests a deeply shared pathway that controls social-93 like and repetitive behaviors between teleost and mammals. 94 95 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint respect to their conspecifics. We set this distance as 6 cm for adult A. mexicanus according to (1) 142 observed distances of when two fish were first attracted to one another through 8 surface fish 143 videos in the dark (32 individuals in total, 9 min in total after back-ground subtraction, also (2) 144 histogram plots of the 'nearby bouts' across inter-pair fish distances (example of a video of each 145 surface fish and cavefish, Supplemental Fig. 1, Supplemental information). We also set a 146 minimum duration of nearby interaction as 4 sec according to the direct observation of videos 147 and histogram plots (Supplemental Fig. 2) (Materials and Methods; Supplemental information). 148 We further checked if the swimming tracks of two focal fish were nearly congruent by 149 visualizing them in each bout of the detected nearby interaction (Materials and Methods; 150 Supplemental information; Supplemental Fig. 3,4). 151 Using this nearby interaction criteria, cavefish showed low but detectable levels of 152 nearby interaction (eg. nearby interaction duration: 9.9 ± 1.7 s, N = 32 at Fig. 4h Turning bias index as a measure of repetitive circling 175 In human patients with psychiatric disorders, social tendencies seem to be suppressed in 176 those who show stereotypic repetitive motions 3,41,42 , that is, the repetitive behavior seems to 177 exclude social behavior. This antagonistic control between social and repetitive behaviors has 178 been extensively reported in mammals 2,3,43 but there are few reports from other vertebrates. In 179 cavefish we observed a highly biased one-way turning, from the ratio of the numbers of left and 180 right turnings occurring per fish every 0.33 second interval, during the 9 min of recording ( Fig.  181 3a,c,e). There was; however, no detectable left-right preference in the direction of turning among 182 individual cavefish (Fig. 3c). In contrast, surface fish showed symmetrical and balanced turning 183 tendencies (Fig. 3b,d). As with cavefish, there was no significant left or right preference in the 184 turning direction in surface fish (Fig. 3b). The biased levels and direction preference were 185 consistent for each cavefish individual over at least 10 days, indicating that biased turning is a 186 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint stable phenotype (interclass correlation: kappa = 0.92, P = 0.0016, N = 6) ( Supplementary Fig.  187 6) 44 . Using this biased turning index, we analyzed its relationship with nearby interaction.

189
Effects of familiar and unfamiliar environments on nearby interaction and biased turnings 190 In humans, stereotypic repetitive motion is enhanced within novel unfamiliar 191 environments 45,46 . In contrast, social tendency is suppressed in these environments 45,46 . We 192 therefore tested if similar behavioral patterns could be observed in the two Astyanax morphs by 193 measuring the respective levels of repetitive turning and social-like interaction in response to 194 familiar and unfamiliar environments (Fig. 4a).

195
In the unfamiliar environment, cavefish swam longer distances (Fig. 4d, Supplemental 196 Table 1), and both surface fish and cavefish preferred to avoid the periphery of the arena, 197 compared to the familiar environment (Fig. 4b,c, Supplemental Table 1). Note that, as described 198 in the above, cavefish did not change their location preference whereas surface fish shifted from 199 the center to the periphery during the 9 min assay in the unfamiliar environment (Fig. 2b, both the duration and bout number; Supplemental Table 1). Of note, in both the familiar and 214 unfamiliar environments, surface fish showed overall significantly more nearby-interaction than 215 cavefish (two-sample Wilcoxon tests (surface fish vs cavefish), W = 415, P = 0.0006 and W = 216 503, P = 1.70 × 10 -10 in the duration of interaction, in the familiar and unfamiliar environments, 217 respectively; W = 412, P = 0.0008 and W = 501, P = 2.21 × 10 -10 in the interaction bout number, 218 in the familiar and unfamiliar environments, respectively. See Supplemental Table 1). Thus, 219 surface fish were consistently swimming closer together than cavefish. 220 It is likely that interacting fish reduce their swimming velocity to swim close to one 221 another. To test this prediction, we measured average swimming velocities: (i) during a 4 sec 222 period directly before the bout of nearby interaction, (ii) during the bout, (iii) a 4 sec period after 223 the bout, and (iv) the rest of the recording time (Supplemental Fig. 7). In the familiar 224 environment, surface fish significantly reduced their swimming velocity during the bout 225 compared to other time periods (Supplemental Fig. 7a, Supplemental Table 1). In contrast, in 226 the unfamiliar environment, surface fish didn't change swimming velocity across the recording 227 period, which significantly reduced the variation of swimming velocity (F(1,156) = 35.6, P = 228 1.50 × 10 -8 in surface fish). Cavefish did not change in their velocity profile between the familiar 229 and unfamiliar environments (F(1,294) = 1.1, P = 0.2988 in cavefish), however it is noteworthy 230 that cavefish significantly reduced their swimming velocity during the interaction bout, 231 suggesting that they can respond to their cohorts, regardless of their familiarity to the 232 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint environment (Supplemental Fig. 7a, Supplemental Table 1). Whilst, cavefish, unlike surface fish, 233 did not change their velocity profile under the unfamiliar environment; also, cavefish showed 234 much less nearby interactions in the unfamiliar environment (Supplemental Fig. 7a and Fig. 4h,i; 235 Supplemental Table 1).

236
We next tested whether repetitive turning is associated with the reduced social-like 237 interaction in cavefish. We did not include surface fish in this correlation analysis because 238 surface fish showed almost no variation in the repetitive turning (Fig. 4e). By fitting a 239 logarithmic curve, significant correlations between the turning bias vs. the duration, and the 240 turning bias vs. the bout number of the nearby interactions were detected in cavefish in the 241 familiar environment (R 2 = 0.118, P = 0.0308 and R 2 = 0.146, P = 0.0176, respectively; Fig.  242 5a,b). However, in the unfamiliar environment, where the nearby interactions were reduced in 243 cavefish, no significant correlation between the nearby interactions and the turning bias was 244 detected, possibly due to the significantly reduced variation in the nearby interaction (P > 0.05; 245 Fig. 5c,d).

246
In summary, an overall negative correlation between social-like nearby interaction and restricted 247 repetitive behavior was discovered in cavefish under non-stressed conditions. This relationship 248 is shared between mammals and cavefish ( 45,46 and Personal communication with Dr. Ryan Lee). 249 Particularly, the reduction of nearby interaction under the unfamiliar environment in cavefish is 250 parallel to mammals 45,46 , suggesting a possibility of shared neural processing between fish and 251 mammals in behavioral choices.

253
Involvement of dopaminergic system in the balance of the levels between nearby interaction and 254 turning bias 255 In mammals, dopaminergic inputs from the ventral tegmental area (VTA) and substantia nigra 256 pars compacta (SNpc) play an important role for behavioral choice in the cortico-striatum 257 system 3,12,13 . Within the striatum, the balance between the D1 direct-and D2 indirect-pathways is 258 crucial, from lamprey to mammals, in balancing behaviors 12,13 . An antipsychiatric drug, 259 aripiprazole, is one of two approved drugs for autism spectrum disorder (ASD) treatment by the 260 U.S. Food and Drug Administration (FDA). Aripiprazole suppresses the phasic stimulation-261 dependent activity of the indirect D2 pathway but preserves the basal tonic level of the D2 262 indirect pathway due to its partial antagonistic action on the D2 receptor in mammals 33 . 263 Aripiprazole is prescribed for the reduction of irritability, perhaps because it maintains the basal 264 inhibitory activities of the D2 pathway and reduces repetitive behaivors 33 . To address whether 265 there is a parallel response to this drug between A. mexicanus and humans, we treated surface 266 and cavefish with aripiprazole through a bath application and tested whether the treatment affects 267 social-like nearby interaction and repetitive turning in both surface fish and cavefish.

268
Consistent with our previous findings 26 , both surface fish and cavefish reduced the overall 269 swimming distance travelled under the aripiprazole treatment compared to the control or 270 pretreatment levels (P < 0.01, Fig. 6a also see the NND and IID results Fig. 6e,f).

274
In this aripiprazole treatment, we used the unfamiliar condition where the surface fish reduced 275 the variation of velocity between during and out of the bout (Supplemental Fig. 7b,c). The 276 aripiprazole treatment did not change the profile of velocity shifts during the bout (Supplemental 277  Table 1). Still, it significantly reduced the velocity in the aripiprazole treated group compared to 278 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint the control group at the post treatment (Wilcoxon test: W = 2,135, P = 1.51 × 10 -15 and W = 279 1,561, P = 9.36 × 10 -12 for surface fish and cavefish, respectively. Corrected by Bonferroni's 280 method). 281 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint

282
In this study, we document that the sighted surface-dwelling form of Astyanax mexicanus are 283 able to follow and interact with conspecifics in the dark. We also reveal that so-called 'asocial' 284 cavefish retain a low but detectable level of ability to engage in social-like interactions. The 285 current definition of nearby interaction is close to the one of 'attraction' in zebrafish larvae under 286 the lighted condition 47 , and we believe these two indexes indicate the same interactions. 287 However, since the measuring methods are distinct, we used the term 'nearby interaction' in the 288 current study. The location preference within the recording arena and social-like interaction 289 depended on the familiarity to the environment: when fish are in the familiar environment, both 290 surface and cavefish tended to explore the peripheral and cavefish showed more social-like 291 nearby interactions. In contrast, in the unfamiliar environment, surface fish tended to gather at 292 the center of the recording arena and showed less variation in the duration of nearby interaction, 293 while cavefish simply reduced nearby interaction. Cavefish also showed a significant negative 294 correlation between the levels of repetitive turning behavior and social-like interactions under the 295 familiar environment. This quantitative antagonistic control between repetitive behavior and 296 social-like interaction has also been reported in studies involving humans and rodents 43,45,46 but 297 few in teleosts. The treatment of the partial D2 receptor antagonist aripiprazole enhanced social-298 like interaction and suppressed repetitive turning in cavefish, which is consistent with 299 aripiprazole treatment results in human patients 32,33,48 .

301
The majority of studies on the shoaling and schooling behaviors of teleost fish have been 302 conducted under environmental conditions where visual cues predominate, potentially 303 exaggerating the importance of the visual system in these behaviors 49 . Observations of the 304 maintenance of shoaling behavior in blinded fishes supports the involvement of lateral line 305 mechanosensing in shoaling in addition to the visual sensing in some teleost species, yet, few 306 studies used intact fish to test the involvement of the lateral line 50-52 . Furthermore, many 307 shoaling tests were conducted in somewhat physically constrained conditions (e.g. using a 308 transparent glass divider between stimulus fish and test fish 53 or using a small mesh-cage 309 constraining the swimming area of stimulus fish 34 ), where the true extent of mechanosensing in 310 controlling shoaling behavior may be underplayed. In this study, we conducted the first systemic 311 analysis of social-like nearby-interactions in intact fish under free-swimming conditions in the 312 dark. 313 Our method, measuring nearby interactions via idTracker-based tracking of each 314 individual, a 'nearby interaction' is defined by both a threshold maximum proximity distance 315 between two fish (≦ 6 cm) and a minimum threshold interaction duration (≧ 4 s). This method is 316 sensitive enough to detect changes in nearby interaction under different environments (familiar 317 and unfamiliar; Fig. 4) and drug treatments ( Fig. 6 and Supplemental Fig. 5). This method is also 318 applicable to measure a unit of collective behavior among larger groups (i.e., two fish attraction 319 or nearby interaction among ≧ 5 individuals) in other animal species. 320 As for non-visual collective behavior in A. mexicanus, a quantitative trait locus (QTL) for non-321 visual schooling has been reported where F2 hybrid fish generated from a pair of a surface fish 322 and a Tinaja cavefish (an independently evolved cave population from that of Pachón cave; 323 Pachón cavefish were used in this study) were used 27 . The QTL for non-visual schooling was 324 detected by using only F2 which showed negative phototaxis (i.e., having visual capacity) but 325 exhibited a wide variation from non-schooling to schooling. Interestingly, F2 individuals carrying 326 homozygous cave genotypes at a marker underlying this QTL increased in schooling behavior, 327 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint while F2 with homozygous surface or heterozygous genotypes less schooled 27 . This genetic study 328 suggests that (1) Tinaja cavefish evolved a 'schooling gene' independently from Pachón cavefish 329 and surface fish, or (2) the cavefish collective behavior is regulated by different molecular 330 mechanism from that of surface fish if Tinaja and Pachón cavefish share the 'schooling gene' 331 cave allele. According to our environmental and pharmacological treatments that indicated the 332 reduced but existing social-like response in Pachón cavefish, we expect that the former scenario 333 (1: Tinaja has a distinct allele for schooling) is likely the case. Crossing experiment between 334 Tinaja and Pachón cavefish is necessary to answer this question. 335 From our results of pharmacological ablation of the lateral line, the nearby interaction of 336 surface fish in the dark is likely regulated by the lateral line mechanosensors (Supplemental Fig.  337 5). However, this result does not mean that the involvement of other sensory systems, such as 338 tactile and olfactory, can be excluded 54 . Further tests are needed to address which of these 339 sensory systems plays the more crucial role in regulating nearby interaction in fish in the dark. 340 In the dark, surface fish changed their swimming direction according to the other 341 individuals, as if they sensed and were attracted to others, similar to what has been observed in 342 zebrafish larvae in lighted conditions (Supplemental Fig. 3) 47 . However, surface fish failed to 343 form a clear cluster like 'shoal' in the dark. Instead, surface fish responded to one another, 344 individually. We believe that the failure to form a shoaling cluster is based on the lack of the 345 visual information (see also Supplemental Movie 1 under the light), yet, surface fish keep their 346 collective tendency in the dark by using lateral line sensing. 347 The nearby interactions detected in this study do not seem to be related to foraging 348 activities or aggression (i.e., two fish come close during expressing aggression or cannibalistic 349 foraging 30 ). We carefully assessed damage of the fins and scales, and despite these tissues being 350 good indicators of aggressive encounters, noticeable damage was not detected. Consistent with 351 this observation, both surface fish and cavefish rarely showed startle responses after touching 352 one another. We therefore conclude that A. mexicanus rarely attacked one another in our assay 353 system, and that the majority of detected nearby interaction can be characterized as shoaling-like 354 activities.

355
The degree and type of nearby interaction in the dark recorded in this study appears 356 relevant within the context of each morph's natural habitat. Surface fish have a known nocturnal 357 predator, prawn fish 55 , and the adult surface fish form a tighter shoaling cluster at night (personal 358 observation in the lab). These facts imply that the shoaling can be advantageous by simply 359 lowering individual risk among cohorts, so called 'attack dilution effect', and/or increased 360 sensitivity in detecting potential predators by collective-effect of more sensors of multiple 361 individuals 1 . In cavefish, the higher level of nearby interactions and lower repetitive behaviors 362 observed in the familiar lab setting were also observed in their natural habitat, Cueva de El 363 Pachón, Tamaulipas, Mexico (2019, personal observation). Whilst they did not form a 364 recognizable shoal, cavefish responded to conspecifics and tended to follow each other. This 365 social-like tendency could be merely a residual of regressive evolution to a troglobitic niche 366 following their divergence from surface riverine ancestors 56,57 . Alternatively, this weak social-367 like tendency could remain advantageous in mate-finding, food location or evasion responses to 368 potential dangers within their cave environment. 369 The young adult surface fish used in this study had reached an appropriate developmental stage 370 to express the shoaling behavior and all of their behaviors were assayed only in the day. Since 371 the shoaling motivation was seen in the night with very limited visual inputs (see above), it is 372 important to investigate the shoaling capacity in the night in future studies. 373 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint In the unfamiliar setting, the cavefish did not enhance the social-like interaction and rather 374 increased repetitive turning, suggesting that cavefish lost a strategy to use the social-like 375 interaction to confront the unfamiliar environmental inputs.

377
Restricted repetitive behaviors are observed in stereotypic movement disorder, autism spectrum 378 disorder in humans, and also observed in mammals kept in a restricted/isolated environments 2,4 . 379 This motor disorder has been thought of as non-functional. However, since these stereotypic 380 behaviors are seen in many vertebrate species as well as normally developing human infants, 381 there may be an advantage to repetitive behaviors. Karl Friston proposed the Free-Energy 382 Principle that explains how the brain processes a given stimulus 58 . In this principle, the brain or 383 the central nervous system is continuously attempting to minimize prediction errors (differences 384 between the environmental signals sampled by an animal's sensory systems, and animal's 385 internal model of the world within the brain). The brain has two strategies to minimize 386 differences between sensory input and the internal model when novel stimuli are encountered: (i) 387 to update the internal model, or (ii) to locomote back to a 'no-novel stimulus environment', to 388 sample the sensory inputs that are already implemented in the current internal model 58 .

389
Stereotypic repetitive circling seems a result of choosing the latter strategy by sampling 390 predictable sensory inputs (strong lateral-line inputs on the one side of the body, and much less 391 inputs from the other side of the body). This method can reduce the prediction errors between the 392 sensory inputs and those predictions from the internal model (e.g., left turning provides the 393 strong right lateral line signals in cavefish). In contrast, surface fish responded to the unfamiliar 394 stimuli by avoiding the peripheral area and homogenizing the nearby interaction duration, yet, 395 did not increase repetitive turning. Therefore, surface fish and cavefish have apparently different 396 strategies to minimize the errors between the sensory inputs and the predicted inputs by the 397 internal model. Given that surface fish appear to acclimatize to novel environments much 398 quicker than cavefish, this suggests that surface fish can update their internal model more 399 frequently than cavefish. This possibility should be tested in A. mexicanus in the near future.

401
Previous studies have indicated that there is an antagonistic relationship between repetitive and 402 social behavior 2,3 . For example, social impairment and repetitive behavior are thought to be 403 linked together, as revealed by the results of oxytocin infusion (reviewed in 3 ). In contrast, in an 404 autism study of the ketogenic diet treatment, some of treated patients recovered their social 405 behavior to the normative level, while repetitive behavior was not significantly attenuated 59 . This 406 result suggests that the repetitive behavior can be independent from social impairment. 407 Therefore, the mechanism which controls switching between social and repetitive behaviors is 408 still largely unknown. Many studies of repetitive behavior and social behavior have focused on 409 the cortex-striatal system, where many patients who show repetitive behavior (including Gilles 410 de la Tourette syndrome, obsessive-compulsive disorder, autism spectrum disorders, Parkinson's 411 disease, and Huntington's disease) have abnormal functional and/or histological features 412 diagnosed by PET and fMRI 9 . Importantly, this striatal system, particularly its dopaminergic 413 system, is also suggested to drive social behavior, where the dopamine signal from the ventral 414 tegmental area (VTA) stimulates the D1-dopamine receptor positive-striatal neurons in the 415 nucleus accumbens, driving social behavior in mouse 3,60 . Among vertebrates, the striatum was 416 proposed as the conserved bridging center. Located at the basal ganglia, the striatum integrates 417 (1) higher-level of sensory inputs from the cerebral cortex/dorsal pallium and (2) the 418 information-evaluating dopaminergic input from substantia nigra par compacta (SNc). 419 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint The dopaminergic striatal pathway is largely conserved among vertebrate species. This includes 420 the direct and indirect pathways, each associated with a specific type of dopamine receptor-D1 421 and D2 receptors, respectively. The major function of this striatal dopaminergic system is 422 thought to be to select behavioral modules (eg. mating, foraging module, and aggression 423 modules) according to given sensory inputs and internal states 12 . For example, a fish may ignore 424 or follow to the mechanical signals from cohorts according to the fish's internal state. The 425 treatment of a partial D2-dopamine receptor antagonist, aripiprazole, showed a parallel response 426 between cavefish and human, suggesting a potential shared pathway that controls social-like and 427 repetitive behaviors between teleost and mammals 3,11 . It will be exciting to address the 428 evolutionary and neurophysiological changes in the striatal dopaminergic system between 429 cavefish and surface fish, and how it is associated to the integration of internal model, which 430 Friston proposed, in the future. 431 432 Conclusion 433 In the current study, surface fish still expressed strong social-like interaction in the darkness. We 434 also report that cavefish retain a lower level of the ability for the social-like interactions through 435 200,000 years of evolution in the dark. Both surface fish and cavefish were capable of 436 responding to familiar and unfamiliar environmental stimuli, and changed social-like interaction 437 accordingly. This was surprising given: (1) surface fish were thought to be unable to express 438 social-like collective behavior in the dark 14,61 , and (2) cavefish were thought to be asocial in both 439 light and dark environments 14,61 . Cavefish also showed a quantitatively negative correlation 440 between repetitive turning and nearby interaction, paralleling results documented in humans for 441 the negative interactions between stereotyped behavior and sociality 45,46 , which is, as far as we 442 know, the first time that this quantitative pattern has been shown in teleost species. The 443 behavioral shifts caused by the treatment of a partial D2 receptor antagonist also overlapped 444 between cavefish and mammals 32,33,48 . The relevance of these findings reveals cavefish as an 445 evolutionary model for the choice of social and repetitive behavioral modules, as well as a new 446 translational biomedical system for the balance between simple repetitive and complex 447 behaviors. 448 449 Acknowledgement 450 We thank to C. Balaan for constructive comments on the insight of social-like interactions in 451 cavefish. We also thank to A. Hudson for English edit and significant improvement in the logical Hawaii Community Foundation (18CON-90818).

458
Data availability 459 The video datasets generated during and/or analyzed during the current study are available at 460 university's shared server and will be deposited to Zenodo. All program scripts used in this study 461 is attached in this submission (DEMO_code.zip) and will be deposited in GitHub, shortly. 462 463 464 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. overnight (~16 hrs). In the next morning, group fish behaviors in the 37.85L tank was recorded 514 as described in the "Recording group fish behavior" section. 515 516 Tracking 517 The first minute of each original video was discarded since this time interval contains the 518 moment that four fish were released from their home container and agitating the water surface, 519 disrupting observation. Before tracking each fish's position by idTracker software 35 , the 520 background visual artefacts were subtracted by a custom made ImageJ macro script. Briefly, if a 521 video was recorded with white background, each frame image was adjusted by the "Add" 522 operation of the 'Image Calculator' function with an inverted median z-stacked image of 4,000 523 frames calculated by 'Z Project...' and 'Invert' functions of ImageJ 64 . If a video has a black 524 background, each frame image was adjusted by "Subtract" operation of the 'Image Calculater' 525 with a median z-stacked image of 4,000 frames calculated with 'Z Project…' function of ImageJ 526 (no 'Invert'). Then, the frame images' contrasts were adjusted according to each video. Each 527 processed video was saved as the AVI format by using the jpeg compression in ImageJ. Using 528 these background-subtracted videos, the X-Y coordinate of each fish was extracted by tracking 529 each fish's ID under the ID-detection algorithm of idTracker 35 . Parameters of idTracker were 530 usually set as Number of individuals as 4, Intensity threshold as 0.75-0.89, Minimum size as 30-531 50, and "Select region by Polygon" function, and check the "Remove background" option. We 532 aimed for a successful tracking frame ratio of above 92 % by changing these parameters. Even 533 with a high ratio of successful tracking frames (e.g., 99%), the outputs of idTracker can have 534 errors in IDs by switching the IDs between different fish. These errors were manually surveyed 535 frame-by-frame by using idPlayer, an accompanied software of idTracker 35 . The corrected IDs 536 were re-assigned by a custom python script. If a fish had periods without ID during the analyzing 537 videos, the estimated X-Y coordinates were calculated in those durations by averaging velocity 538 and swimming direction between the X-Y coordinates before and after the missing ID period. 539 Tracking results were used for the repetitive turning and nearby interaction analyses (see below). 540 541 Biased repetitive turning analyses 542 Changes of the angles in swimming directions in every 5 frame window (i.e., every 0.33 s) were 543 calculated as radian. When the X-Y coordinates of the three-time points (t0, t1, and t2) were as 544 (x0, y0), (x1, y1) and (x2, y2), the swimming angle ө between two vectors (x1-x0, y1-y0) and 545 (x2-x1, y2-y1) is: ө=arctan(((x1-x2)*(y0-y1)-(y1-y2)*(x0-x1)) / ((x1-x2)* (x0-x1) + (y1-y2)* 546 (y0-y1)) (the cross product of two vectors). The numbers of frames that have positive radian 547 values (N1), representing anticlockwise turns, and negative radian values (N2), representing 548 clockwise turns, were totaled. The repetitive turning index was then calculated as Log 10 549 (N1/N2). As fish did not show any preference in the direction of turning (Fig. 3b,c, and 550 Supplemental Fig. 6), the repetitive turning index was expressed as the absolute values of Log 10 551 (N1/N2). 552 553 Recording of single fish repetitive behavior 554 Repetitive behavior assays were carried out on single fish, each individual fish being, in turn, 555 transferred from the home container to a 25.5 x 38.0 cm Pyrex Grass Baking Dish filled with ~4 556 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint cm depth of water by using a fine brine shrimp net. Two of these dish arenas were lit by a 557 custom-made infrared light box from underneath (a 58.4 × 41.2 × 15.2 cm opaque storage box 558 equipped with infrared LED strip SMD3528 850nm strip: LightingWill). Videos for this 559 behavioral assay were recorded with the same camera setup as the familiarity experiment, except 560 that a c-mount zoom lens (macro 1.8/12.5-75mm C-mount zoom lens, Toyo Lens) was fitted.

561
A repeat behavioral assay using the same fish was recorded 10 days after the first. To identify 562 individuals, each fish was labeled with a physical implanted dye (Visible Implant Elastomer 563 (VIE) tags, Northwest Marine Technology, Inc. Anacortes, WA, USA) whilst anesthetized in a 564 bath treatment of the ice-cold 0.2 mg/mL of MS-222 (MillliporeSigma, St. Louis, MO), directly 565 after the first recording. After 10 days, the fish behavior was recorded using the same procedure. 566 567 Social-like nearby interaction analysis 568 The inter-individual distances (IID) and nearest-neighbor distances (NND) were calculated 569 according to Partridge et al 65 . For IID, the average of the distances between the four fish being 570 assayed (6 distances) was calculated per video frame. Then, the mean and standard error of 571 means (s.e.m.) were calculated by using one 4-fish group as N = 1 (for example, N = 12 in each 572 of the surface fish and cavefish experimental groups in Fig. 2e). For NND, the average of 4 573 nearest distances between a focal fish and the nearest individual per video frame, then the mean 574 and s.e.m. were calculated as in IID.

575
The permutation test of NND and IID was performed by random selection of one individual from 576 each group (one of four fish) and forming a fake 4-fish group, where none of each individual 577 physically swam in the same arena. We then calculated nearest neighbor distances and inter-578 individual distances among these 4 fish by using their 9 min X-Y coordinates (> 8,600 frames). 579 The fake 4-fish group were randomly and exclusively formed 1,000 times in each surface fish 580 and cavefish.

582
A 'nearby interaction bout' was defined by a minimum interaction duration threshold of four 583 seconds and a maximum physical distance threshold of 6 cm distance, between the two fish. As a 584 note, the definition of nearby interaction is close to the one of 'attraction' by Hinz et al.,where 585 attraction was expressed as the probability that the focal turns to the side where the other fish is 586 located 47 . The current nearby interaction is to measure the preference to stay or follow one 587 another without considering the turning direction. We use a term 'nearby interaction' in this 588 study to avoid confusion. 589 Nearby interactions were extracted from each fish's X-Y coordinates by a custom-made Matlab 590 script. The bout duration was adjusted by subtracting the passing-by duration (PbDur) (see 591 following and Supplemental Information, Supplemental Fig. 2). The PbDur is defined as the 592 average duration of a fish passing-by another fish where these fish do not express the nearby 593 interaction. The purpose of subtracting the PbDur from the bout durations is to remove the period 594 from the nearby interaction events that resulted from just physical proximity rather than the 595 actual social-like interaction. PbDur was calculated as: PbDur = 6 cm /V, where V is an average 596 velocity of four fish in the same group during 9 min or 5 min periods (Supplemental Information; 597 Supplemental Fig. 2). If V is low, PbDur becomes longer; therefore, it excludes 'by-chance' 598 events where two fish may remain within 6 cm of one another while not expressing attraction 599 toward one another. The swimming velocities of 'during the bout,' '4 seconds before bout 600 commencement,' and '4sec directly following the bout' were also extracted and calculated using 601 a custom made Matlab script. 602 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020.  . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. a Schematics of experimental procedure. Fish were acclimated in the home tray for more than 4 828 days and were transferred to the recording tank in this unfamiliar environment. After the 829 recording, fish were return to the home tray and kept for more than 4 days. Then, fish were 830 returned to the recording tank and were acclimated to the recording tank for 4 days. After the 831 acclimation, the IR LED array was set to illuminate fish from the side, and fish movements were 832 recorded under this familiar(ized) environment. b a diagram explains the Position index. c Plots 833 for Positional preference of the fish for region 1. Three bars in the bean pods represent 25 834 (bottom bar), median (middle) and 75 (top) percentiles of data. The generalized linear mixed-835 effects model indicated that environment (Env: familiar vs unfamiliar) and the interaction of Env 836 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made was significantly increased in the unfamiliar environment in cavefish. e, f, g Turning bias index 838 (e), Nearest neighbor distance (NND) (f), and Interindividual distance (IID) (g) showed no 839 significant change between the familiar and unfamiliar environments but differed between SF 840 and CF. h, i The duration (h) and the bout number of nearby interactions (i) showed the 841 significant difference in Pop, and the significant interaction in Env × Pop via generalized linear 842 mixed-effects model (Supplemental Table 1). Post-hoc test indicated the significant reduction of 843 nearby interaction in the unfamiliar environment comparing with the familiar environment. SF: 844 surface fish, CF: cavefish. **: P < 0.01, ***: P < 0.001. All statistics scores are available in 845 Supplemental Table 1. 846 847 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint 862 Figure 6. The partial agonist for the D2 receptor, aripiprazole, enhanced nearby 863 interactions and reduced turning bias in cavefish.

864
The pretreatment behaviors were recorded more than four days before the aripiprazole treatment.

865
Aripiprazole and the control DMSO were treated for ~16 hours before the posttreatment 866 recording under the unfamiliar environment. a Total swimming distances before (blank) and 867 after (red shaded) the treatment, between the aripiprazole and control treatments, and between 868 surface fish and cavefish. Both surface fish and cavefish significantly reduced swimming 869 distance after the aripiprazole treatment (N = 12 for each of surface fish and cavefish). b 870 Turning bias index indicated that cavefish significantly reduced biased turning after the 871 aripiprazole treatment (N = 12 for each of surface fish and cavefish). c, d The duration (c) and 872 the bout number of nearby interactions (d) showed the significant difference in Pop (surface fish 873 and cavefish), and the significant interaction in PrePost (pre and post treatments) × Exp (control 874 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint and aripiprazole treatments) × Pop via generalized linear mixed-effects model (Supplemental 875  Table 1). Post-hoc test indicated the significant increase of nearby interaction in the aripiprazole 876 treated groups of surface fish and cavefish. N =12 for each of surface fish and cavefish. e, f 877 Nearest neighbor distance (NND) (e), and Interindividual distance (IID) (f) showed no detectable 878 change in cavefish but were significantly shortened in surface fish under the aripiprazole 879 treatment. N =12 for each of surface fish and cavefish. 880 All statistics scores are available in Supplemental Table 1  881  882 . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted August 20, 2020. ; https://doi.org/10.1101/2020.08.20.258640 doi: bioRxiv preprint