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Further evidence for the capacity of mirror self-recognition in cleaner fish and the significance of ecologically relevant marks

['Masanori Kohda', 'Laboratory Of Animal Sociology', 'Department Of Biology', 'Geosciences', 'Graduate School Of Science', 'Osaka City University', 'Osaka', 'Shumpei Sogawa', 'Alex L. Jordan', 'Department Of Collective Behaviour']

Date: 2022-02 

Abstract An animal that tries to remove a mark from its body that is only visible when looking into a mirror displays the capacity for mirror self-recognition (MSR), which has been interpreted as evidence for self-awareness. Conservative interpretations of existing data conclude that convincing evidence for MSR is currently restricted to great apes. Here, we address proposed shortcomings of a previous study on MSR in the cleaner wrasse Labroides dimidiatus, by varying preexposure to mirrors and by marking individuals with different colors. We found that (1) 14/14 new individuals scraped their throat when a brown mark had been provisioned, but only in the presence of a mirror; (2) blue and green color marks did not elicit scraping; (3) intentionally injecting the mark deeper beneath the skin reliably elicited spontaneous scraping in the absence of a mirror; (4) mirror-naive individuals injected with a brown mark scraped their throat with lower probability and/or lower frequency compared to mirror-experienced individuals; (5) in contrast to the mirror images, seeing another fish with the same marking did not induce throat scraping; and (6) moving the mirror to another location did not elicit renewed aggression in mirror-experienced individuals. Taken together, these results increase our confidence that cleaner fish indeed pass the mark test, although only if it is presented in ecologically relevant contexts. Therefore, we reiterate the conclusion of the previous study that either self-awareness in animals or the validity of the mirror test needs to be revised.

Citation: Kohda M, Sogawa S, Jordan AL, Kubo N, Awata S, Satoh S, et al. (2022) Further evidence for the capacity of mirror self-recognition in cleaner fish and the significance of ecologically relevant marks. PLoS Biol 20(2): e3001529. https://doi.org/10.1371/journal.pbio.3001529 Academic Editor: Frans B. M. de Waal, Emory University, UNITED STATES Received: April 1, 2021; Accepted: January 5, 2022; Published: February 17, 2022 Copyright: © 2022 Kohda et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was financially supported by KAKENHI Grants from JSPS to MK (nos. 17K18712, 19F19713, 19H03306 and 20K20630) and to SS (nos. 20J01170) and by the Osaka City University Strategic Research Grant 2018 and 2019 for Top Priority Researches to MK. RB is supported by the Swiss Science Foundation, Grant 310030_192673 / 1. ALJ is supported by the Max Planck Society and Deutsche Forschungsgemeinschaft Cluster of Excellence 2117 “Centre for the Advanced Study of Collective Behavior” Grant 422037984. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: GLMM, generalized linear mixed model; LMM, linear mixed model; MSR, mirror self-recognition; SEM, standard error of the mean; TL, total length; VIE, visible implant elastomer

Introduction Passing the mark test, in which subject animals touch or scrape a mark placed on their body in a location that can only be indirectly viewed in mirror, demonstrates the capacity for mirror self-recognition (MSR), which has been interpreted as evidence for self-awareness (e.g., [1–7]). Variations of this test have been applied to many species of vertebrates. Most often, the results are clearly negative, including studies on lesser apes, monkeys, pig, dog, cat, panda, crows, and parrots (e.g., [1–2,5,8–16]). However, a small number of socially intelligent species including elephant, dolphin, horse, magpie, and a crow have been argued to have passed the test (e.g., [17–23]), although this interpretation has been criticized [7,13]. As a consequence, only the evidence on chimpanzees, and to a lesser degree on orangutans, has so far obtained unequivocal approval as evidence for MSR and hence self-awareness [7]. Furthermore, there is disagreement on whether there are intermediate levels of self-awareness [24,25] or whether self-awareness may represent a cognitive discontinuity [7,8]. Morgan’s canon states that simpler explanations must be excluded in order to more complex cognitive interpretations being acceptable, and adhering to this logic suggests that we must acknowledge the concerns regarding evidence for MSR in nonapes raised by Gallup and Anderson [7]. The authors propose the following criteria that need to be fulfilled in combination to conclude that a species passed the mark test: (i) repeated studies (ideally by different laboratories) showing positive results; (ii) linked to the first point, a reasonable number of individuals should pass the test; and (iii) additional experiments should exclude alternative explanations for mirror-related actions. These additional experiments can be fairly simple but telling. For example, 2 rhesus macaques that had shown habituation to a permanent mirror (without showing evidence for MSR) started to behave aggressively again after the mirror was simply moved to another side of the cage, clearly showing that they did not recognize themselves [26]. Also, individuals naive to mirrors should not pass the mark test spontaneously, as was the case for the 2 mirror-naive chimpanzees in the original study, which did not inspect their mark within the first 30 minutes of exposure [1]. Additionally, marked individuals that see another marked individual rather than their own mirror image should not attempt to remove the mark. Finally, both Gallup and Anderson [7] as well as de Waal [24] emphasize that the interpretation of results becomes less clear if the mark is not just painted on the skin but attached below the skin. This is because the physical sensation of the mark (or head implant for physiological studies), together with seeing the mark in the mirror, may trigger mark-related behavior and then also inspection of other body parts, as, for example, in rhesus macaques [27], a species that otherwise fails the mark test. Thus, it was argued that the monkeys may have learned contingencies rather than recognizing themselves in the mirror [24]. Note, however, that this interpretation differs from the one by the authors, who propose that the salience of the mark triggered closer inspection and, as a consequence, MSR [27]. Marking procedures are also a crucial element of the debate triggered by recent results on the behavior of cleaner fish Labroides dimidiatus when exposed to a mirror [25]. Cleaner wrasse obtain their food by eating parasites and mucus of the surface of other fish, so-called clients [28], and Gnathiid isopods are their main food [29]. These small crustaceans appear as small dark dots on clients. Kohda and colleagues. [25] used this feature of cleaner fish ecology to mark subjects in a salient way, i.e., by injecting a brown elastomer marking on the throat such that the marking was only visible when subjects swam upward in front of a mirror. In this previous experiment, brown marking, but not invisible elastomer implants, caused 3 out of 4 mirror-experienced cleaners to scrape their throats several times after swimming in front of the mirror, but not when a mirror was absent [25]. Despite these results, a number of criticisms by de Waal [24] and Gallup and Anderson [7] potentially apply, including the possibility that the elastomer marks also produced some physical sensation, akin to the head implant in rhesus macaques. Here, we provide results on various follow-up experiments designed to challenge the interpretation by Kohda and colleagues [25] that cleaners pass the mark test. The first aim was to test whether the earlier results can be reproduced by a new experimenter with a larger sample size. Furthermore, we tested whether the brown color of the marking was crucial for the throat-scraping responses. If cleaners are only responding to ecologically relevant markings, then blue or green marks would not elicit throat scraping. In order to obtain further information on the role of cleaners feeling the mark, we injected the mark deeper in some cleaners in a further experiment. If such marking caused irritation such that cleaners scraped their throat in the absence of a mirror, but did not do so under normal (shallow) marking procedures, it would suggest that the standard marking is not comparable with a skull implant for electrophysiological experiments in rhesus macaques, but that deeper marking may be. Furthermore, we marked mirror-naive individuals and exposed them to a mirror. Based on the 2 data points on chimpanzees [1], we expected that these subjects should show no or at least less throat scraping during the 120-minute exposure. Introducing a new experimental paradigm, we also marked mirror-experienced cleaner pairs that could see each other through a transparent barrier. If seeing any cleaner with a brown mark on the throat would somehow remind subjects of the mark on their own throat, then we would expect throat scraping in this experiment as well. Our final experiment involved changing the position of the mirror once cleaners had stopped to aggress their own reflection. If moving the mirror would lead to renewed aggression, this result would emphasize the importance of learned spatial contingencies as opposed to self-recognition. Before passing mark test, animals progress through Stages 1 to 3 (chimpanzee, dolphin, elephant, and cleaner fish [1,17,18,25]). The first stage involves social interactions including aggression (animals mistake a reflection for another conspecific), the second stage involves repetitive atypical behavior against mirror reflection, by which animals are thought to check the contingency in movements between their own body and the mirror reflection (i.e., testing whether the reflection is self or not), and the final stage when animals perform self-directed behavior using the mirror (understanding the reflection to be self). These processes are considered to be a basic evidence for MSR [1,17], and in this study, we also describe these behaviors in cleaner fish.

General discussion There is a current controversy regarding the interpretation of results from the mirror task, reignited by recent results on cleaner fish [7,24,25]. Our aim was to present cleaner fish to a variety of largely new experiments aimed at challenging the interpretation by Kohda and colleagues [25] that cleaner fish show self-recognition. We welcome a general discussion on these new results. As we see it, the additional experiments largely support the notion that cleaner fish indeed show self-recognition in the mirror task. We have greatly increased sample size, showing that throat scraping is a general behavior of cleaners when marked and exposed to a mirror. Subjects need to see on their mirror image rather than the mark on another individual to scrape their own throat. Furthermore, cleaners recognize the individual in the mirror rather than having learned that a fish at a certain location should no longer be confronted. In each of these experiments, cleaners could have behaved in ways that would have invalidated the conclusion by Kohda and colleagues [25] that cleaners pass the mirror test, but they never did. We acknowledge that the cleaners’ behavior in each single additional experiment can probably be explained without invoking MSR. However, the combined accumulated evidence should be more difficult to be dismissed than the previous study. A remaining potential shortcoming of the current study is that the mark was injected rather than painted. We do not see how this can be changed in fish. We showed that a deeper injection causes scraping frequencies that are independent of the presence/absence of a mirror. Furthermore, the results from the 2 experiments with marked fish suggest that the visual input must be the mirror image rather than a marked conspecific. Both results make it less likely that the standard marking procedure causes a visual sensory feedback loop during mirror exposure, as proposed by de Waal [24]. At the minimum, cleaner fish are able to learn that only the mirror image provides contingencies for self and use this knowledge to scrape a body part with an apparent parasite attached when spotted in the mirror. Mirror-naive cleaners scraped their throat less frequently than mirror-experienced individuals or even not at all. Thus, one could argue that the results from this experiment qualitatively fit evidence for cleaner self-recognition. On the other hand, the timing with which several mirror-naive cleaners started using the mirror reflection to scrape their throat was early. Thus, the data could currently be used as evidence both for or against MSR [7]. To solve the puzzle fairly, we need intensively observe behaviors of nonmarked naive fish in initial hour of mirror presentation. We consider any strong conclusions of the strange timing of this fish premature, as we also lack quantitative data on other species. In his classic study, Gallup [1] had only tested 2 naive chimpanzees for 30 minutes, and no similar data have been collected in other studies as far as we are aware. One important conclusion from our study is that cleaners only respond to markings of apparently high ecological relevance. We therefore encourage colleagues to think hard about which marks could be relevant for their study species in order to increase the likelihood of responses [36]. Only particularly curious and/or playful species may inspect any marking, regardless of its ecological relevance. Fish are generally not known for curiosity and playfulness (but see [37]), making ecological relevance of the mark a potentially imperative prerequisite. This also implies that we cannot expect a fish showing inspection behavior of otherwise invisible body parts when in front of a mirror, and fish would not be able to touch these parts anyway. Our subjects were all wild caught as adults and hence had plenty of experience searching and eating small crustacean ectoparasites prior to our experiments. Unfortunately, the life cycle of cleaner wrasse cannot be completed in the laboratory, preventing experiments on parasite-naive individuals to test whether the behavior of current subjects was a response to an innate or acquired stimulus. Independently of the answer to that question, cleaners have to perceive the mirror image as relevant for self, including parts they had never seen without a mirror. In conclusion, we propose that the validity and/or the conclusions from the mirror task need further investigation. Given the negative results on a great variety of large-brained endotherm vertebrates, the positive results for cleaner fish present a puzzle. High ecological relevance of the stimulus, in combination with experience with the stimulus, may potentially be an important part of the answer. Familiarity with all components of the task may greatly enhance the probability that subjects are able to combine the available information to form new insights. We note that cleaner fish show evidence for a variety of unexpected advanced cognitive abilities. For example, cleaner wrasse use predators as social tools against aggressive clients [38] and can generalize across predatory species in learning experiments mimicking the social tool use scenario [39]. Furthermore, cleaners apparently use configurational learning to give priority to ephemeral clients over more permanently accessible ones [40–42]. Finally, cleaners can incorporate what other cleaners can or cannot see [43], a supposedly key building block of a theory of mind [44]. Cleaner wrasse have an average brain to body ratio for a labrid fish [45], making it likely that their 2,000 interactions with client reef fish provide such abundant learning opportunities that cleaners eventually reach more advanced insights within the narrow ecologically relevant context. With this perspective, more species may be found to show evidence for MSR if the task can be made ecologically relevant to them. The main open question in our view is how MSR relates to self-awareness. We cannot provide an answer. Nevertheless, we agree with de Waal [24] that self-awareness is not necessarily an all or nothing. Indeed, any moving animal must have a basic notion of self, i.e., the size and shape of its body, in order to avoid bumping into obstacles [46]. In contrast, recognizing oneself in a mirror does not necessarily imply the presence of other, supposedly advanced cognitive processes. Children recognize themselves in a mirror long before they pass the Sally–Anne test for conscious attribution of intentions and beliefs to other individuals [47,48]. Conversely, specific brain damage prevents MSR without impairing theory of mind in adult humans [4]. Similarly, MSR may need to be combined with mental time traveling abilities to grasp the concept of death. Given the available evidence, we conclude that the degree of self-awareness may well differ between species and in ways that are independent of performance in the mirror test.

Supporting information S1 Data. Raw data of the data represented in this manuscript. https://doi.org/10.1371/journal.pbio.3001529.s001 (XLSX)

Acknowledgments We are grateful to the members of the Laboratory of Animal Sociology, Osaka City University for their fruitful discussion.

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