Parasite infection rather than tactile stimulation is the proximate cause of cleaning behaviour in reef fish.

Cleaning behaviour is a popular example of non-kin cooperation. However, quantitative support for this is generally sparse and the alternative, that cleaners are parasitic, has also been proposed. Although the behaviour involves some of the most complex and highly developed interspecific communication signals known, the proximate causal factors for why clients seek cleaners are controversial. However, this information is essential to understanding the evolution of cleaning. I tested whether clients seek cleaners in response to parasite infection or whether clients seek cleaners for tactile stimulation regardless of parasite load. Parasite loads on client fish were manipulated and clients exposed to cleaner fish and control fish behind glass. I found that parasitized client fish spent more time than unparasitized fish next to a cleaner fish. In addition, parasitized clients spent more time next to cleaners than next to control fish, whereas unparasitized fish were not attracted to cleaners. This study shows, I believe for the first time, which is somewhat surprising, that parasite infection alone causes clients to seek cleaning by cleaners and provides insight into how this behaviour evolved.     

Degrees of honesty: cleaning by the redlip cleaner wrasse Labroides rubrolabiatus

Cleaning symbioses among coral reef fishes are highly variable. Cleanerfishes vary in how much they cooperate with (i.e. remove only ectoparasites) or cheat (i.e. bite healthy tissue, scales or mucus) on their fish clients. As a result, clients use various strategies to enforce cooperation by cleaners (e.g. punishment or partner choice), and cleaners use tactile stimulation to manipulate cheated client behaviour. We provide the first detailed observations of cleaning behaviour of the redlip cleaner wrasse Labroides rubrolabiatus and ask where interactions with this cleanerfish lie on the continuum of cleanerfish honesty, client control, and cleanerfish manipulation. Ninety per cent of redlip cleaner wrasses took jolt-inducing cheating bites from their clients, but they did so at a very low rate (~ 2 jolts per 100 s inspection). Retaliatory chases by clients were uncommon. Three-quarters (30 of 40) of cleaner wrasses used tactile stimulation on their clients, but rarely did so to reconcile with cheated clients. Instead, the majority (70%) of tactile stimulation events targeted a passing client that then stopped for inspection. The relationship between redlip cleaner wrasses and their clients appears to be less conflictual than those documented in other Labroides cleanerfishes. Future studies should test whether this low level of conflict is consistent across space and time and is underpinned by a preference for ectoparasites over other client-gleaned items. As an active cleaner that appears to take few cheating bites from their clients, L. rubrolabiatus has the potential to be as important a driver of fish health and community structure on coral reefs as its better-known relatives.

     

Cleaner fish use tactile dancing behavior as a preconflict management strategy

The most commonly asked question about cooperative interactions is how they are maintained when cheating is theoretically more profitable. In cleaning interactions, where cleaners remove parasites from apparently cooperating clients, the classical question asked is why cleaner fish can clean piscivorous client fish without being eaten, a problem Trivers used to explain reciprocal altruism. Trivers suggested that predators refrain from eating cleaners only when the repeated removal of parasites by a particular cleaner results in a greater benefit than eating the cleaner. Although several theoretical models have examined cheating behavior in clients, no empirical tests have been done (but see Darcy ). It has been observed that cleaners are susceptible to predation. Thus, cleaners should have evolved strategies to avoid conflict or being eaten. In primates, conflicts are often resolved with conflict or preconflict management behavior. Here, I show that cleaner fish tactically stimulate clients while swimming in an oscillating "dancing" manner (tactile dancing) more when exposed to hungry piscivorous clients than satiated ones, regardless of the client's parasite load. Tactile dancing thus may function as a preconflict management strategy that enables cleaner fish to avoid conflict with potentially "dangerous" clients.     

Punishment and partner switching cause cooperative behaviour in a cleaning mutualism

What are the mechanisms that prevent partners from cheating in potentially cooperative interactions between unrelated individuals? The cleaner fish Labroides dimidiatus and client reef fish both benefit from an interaction as long as the cleaner eats ectoparasites. However, the cleaner fish prefers some client mucus, which constitutes cheating. Field observations suggested that clients control such cheating by using punishment (chasing the cleaner) or by switching partners (fleeing from the cleaner). Here, we tested experimentally whether such client behaviours result in cooperative cleaner fish. Cleaners were allowed to feed from Plexiglas plates containing prawn items and fish flake items. A lever attached to the plates allowed us to mimic the behaviours of clients. As cleaners showed a strong preference for prawn over flakes, we taught them that eating their preferred food would cause the plate to either chase them or to flee, while feeding on flakes had no negative consequences. We found a significant shift in cleaner fish foraging behaviour towards flake feeding after six learning trials. As punishment and terminating an interaction resulted in the cleaners feeding against their preferences in our experiment, we propose that the same behaviours in clients improve the service quality of cleaners under natural conditions.     

A decrease in the abundance and strategic sophistication of cleaner fish after environmental perturbations

Coral reef ecosystems are declining worldwide and under foreseeable threat due to climate change, resulting in significant changes in reef communities. It is unknown, however, how such community changes impact interspecific interactions. Recent extreme weather events affecting the Great Barrier Reef, that is, consecutive cyclones and the 2016 El Niño event, allowed us to explore potential consequences in the mutualistic interactions involving cleaner fish Labroides dimidiatus (hereafter “cleaner”). After the perturbations, cleaner densities were reduced by 80%, disproportionally compared to the variety of reef fish clients from which cleaners remove ectoparasites. Consequently, shifts in supply and demand yielded an increase in the clients’ demand for cleaning. Therefore, clients became less selective toward cleaners, whereas cleaners were able to choose from a multitude of partners. In parallel, we found a significant decline in the ability of cleaners to manage their reputation and to learn to prioritize ephemeral food sources to maximize food intake in laboratory experiments. In other words, cleaners failed to display the previously documented strategic sophistication that made this species a prime example for fish intelligence. In conclusion, low population densities may cause various effects on individual behavior, and as a consequence, interspecific interactions. At the same time, our data suggest that a recovery of population densities would cause a recovery of previously described interaction patterns and cleaner strategic sophistication within the lifetime of individuals.     

Asymmetric cheating opportunities and partner control in a cleaner fish mutualism

How can cooperation persist if, for one partner, cheating is more profitable than cooperation in each round, while the other partner has no option to cheat? Our laboratory experiments suggest that such a situation exists between the cleaner fish Labroides dimidiatus and its nonpredatory client reef fish species, which actively seek cleaners to have their ectoparasites removed. Clients Ctenochaetus striatus regularly jolted in response to cleaner mouth contact, and these jolts were not linked to the removal of parasites. In addition, cleaners did not search for parasites but fed on mucus when exposed to anaesthetized clients, which could not control the cleaners' behaviour. Field data showed that clients often terminated an interaction immediately after a jolt. Client species with access to only one cleaning station, owing to their small territories or home ranges, terminated interactions mainly by chasing cleaners while clients with access to two or more cleaning stations mainly swam away. Thus, the chasing of cleaners appeared to be a form of punishment, imposing costs on the cleaner at the client's (momentary) expense. Chasing yields future benefits, as jolts were on average less frequent during interactions between cleaners and individuals that had terminated their previous interaction by aggressive chasing.     

Caribbean Cleaning Gobies Prefer Client Ectoparasites Over Mucus

If cooperation often involves investment, then what specific conditions prevent selection from acting on cheaters that do not invest? The mutualism between the Indo‐Pacific cleaner wrasse Labroides dimidiatus and its reef fish clients has been a model system to study conflicts of interest and their resolution. These cleaners prefer client mucus over ectoparasites – that is, they prefer to cheat – but punishment and partner switching by clients enforce cooperative behaviour by cleaners. By contrast, clients of Caribbean cleaning gobies (Elacatinus spp.) do not to use punishment or partner switching. Here, we test the hypothesis that the behavioural differences between these two cleaner fish systems are caused by differences in cleaner foraging preferences. In foraging choice experiments, we offered broadstripe cleaning gobies Elacatinus prochilos client‐derived parasitic isopods, client mucus and a control food item. The cleaning gobies significantly preferred ectoparasites over mucus or the control item, which contrasts with cleaner wrasses. We propose that the low level of cleaner–client conflict arising from cleaning goby foraging preferences explains the observed lack of strategic partner control behaviour in the clients of cleaning gobies.     

Choosy reef fish select cleaner fish that provide high-quality service

Reef fish that actively visit cleaner fish to have parasites and dead or infected tissue removed face two potential problems: they might have to wait while cleaners inspect other clients, and cleaners might feed on healthy body tissue, a behaviour that is referred to as cheating. Individuals of some ‘client’ species have large home ranges, which cover several cleaning stations, while others have small territories or home ranges with access to only one cleaning station. The former can thus choose between cleaners, while the latter cannot. We investigated whether clients with large home ranges change cleaning partners to outplay cleaners against each other to achieve (1) priority of access over clients with no choice at cleaning stations and (2) control over cheating by cleaners. We followed individuals of longnosed parrotfish, Hipposcarus harid, for up to 120 min in their natural environment and noted their interactions with cleaner wrasses, Labroides dimidiatus. Individuals were likely to return to the same cleaning station if the previous interaction had ended without conflict but changed cleaners for the next inspection if they had been either cheated or ignored, at least if the time between two consecutive visits was short. The overall attractiveness of a cleaning station seemed to be largely independent of service quality, which appeared to be similar at all stations. This is the first empirical evidence that the option to change partners is used as a control mechanism to stabilize cooperative behaviour.     

Experimental evidence that partner choice is a driving force in the payoff distribution among cooperators or mutualists: the cleaner fish case

Supply and demand largely determine the price of goods on human markets. It has been proposed that in animals, similar forces influence the payoff distribution between trading partners in sexual selection, intraspecific cooperation and interspecific mutualism. Here we present the first experimental evidence supporting biological market theory in a study on cleaner fish, Labroides dimidiatus . Cleaners interact with two classes of clients: choosy client species with access to several cleaners usually do not queue for service and do not return if ignored, while resident client species with access to only one cleaning station do queue or return. We used plexiglas plates with equal amounts of food to simulate these behaviours of the two client classes. Cleaners soon inspected 'choosy' plates before 'resident' plates. This supports previous field observations that suggest that client species with access to several cleaners exert choice to receive better (immediate) service.     

Cleaner wrasse prefer client mucus: support for partner control mechanisms in cleaning interactions

Recent studies on cleaning behaviour suggest that there are conflicts between cleaners and their clients over what cleaners eat. The diet of cleaners usually contains ectoparasites and some client tissue. It is unclear, however, whether cleaners prefer client tissue over ectoparasites or whether they include client tissue in their diet only when searching for parasites alone is not profitable. To distinguish between these two hypotheses, we trained cleaner fish Labroides dimidiatus to feed from plates and offered them client mucus from the parrotfish Chlorurus sordidus, parasitic monogenean flatworms, parasitic gnathiid isopods and boiled flour glue as a control. We found that cleaners ate more mucus and monogeneans than gnathiids, with gnathiids eaten slightly more often than the control substance. Because gnathiids are the most abundant ectoparasites, our results suggest a potential for conflict between cleaners and clients over what the cleaner should eat, and support studies emphasizing the importance of partner control in keeping cleaning interactions mutualistic.     

Geographic Variation in the Behaviour of the Cleaner Fish Labroides dimidiatus (Labridae)

Geographical variation in the outcome of interspecific interactions has a range of proximate ecological causes. For instance, cleaning interactions between coral reef fishes can result in benefits for both the cleaner and its clients. However, because both parties can cheat and because the rewards of cheating may depend on the local abundance of ectoparasites on clients, the interaction might range from exploitative to mutualistic. In a comparative analysis of behavioural measures of the association between the cleaner fish Labroides dimidiatus and all its client species, we compared cleaning interactions between two sites on the Great Barrier Reef that differ with respect to mean ectoparasite abundance. At Heron Island, where client fish consistently harbour fewer ectoparasites, client species that tended to pose for cleaners were more likely to receive feeding bites by cleaners than client species that did not pose for cleaners. This was not the case at Lizard Island, where ectoparasites are significantly more abundant. Client fish generally spent more time posing for cleaners at Lizard Island than their conspecifics at Heron Island. However, fish at Heron Island were inspected longer on average by cleaners than conspecifics at Lizard Island, and they incurred more bites and swipes at their sides per unit time from cleaners. These and other differences between the two sites suggest that the local availability of ectoparasites as a food source for cleaners may determine whether clients will seek cleaning, and whether cleaners will feed on parasites or attempt to feed on client mucus. The results suggest that cleaning symbiosis is a mosaic of different outcomes driven by geographical differences in the benefits for both participants.     

Size and stripes: how fish clients recognize cleaners

Little is known of how individuals find each other in interspecific mutualisms involving free-living partners. We tested the importance of two factors, namely body size and the presence of a lateral body stripe, in the recognition of cleanerfish by their fish clients. Clients on an Indonesian reef flat readily approached wooden models of the bluestreak cleaner wrasse, Labroides dimidiatus, which varied in size and stripe characteristics. The composition of the clientele of models was not significantly different from that of natural cleaning stations, suggesting that fish visiting the models were seeking to be cleaned. Normal-sized models of cleaner wrasses attracted significantly more clients, which showed more intense interest and stayed with the models for significantly longer, than super-sized models. For normal-sized models, the number of clients increased as the length of the cleaner's lateral stripe increased (from 0, to 44, 67 and 100% of body length). However, there was no effect of stripe length on client numbers for super-sized models. Client interest also did not vary with stripe length for models of either size. Small body size and the presence of a lateral stripe therefore appear to be long-distance signals that their bearer may be a cleaner, but after initial attraction, client interest is maintained by other cues. Alternative short-distance signals may include colour, other visual signals such as cleanerfish dances, or physical contact between cleaner and client.     

Short‐Term Variation in the Level of Cooperation in the Cleaner Wrasse Labroides dimidiatus: Implications for the Role of Potential Stressors

There is a wealth of game theoretical approaches to the evolution and maintenance of cooperation between unrelated individuals and accumulating empirical tests of these models. This contrasts strongly with our lack of knowledge on proximate causes of cooperative behaviour. Marine cleaning mutualism has been used as a model system to address functional aspects of conflict resolution: client reef fish benefit from cleaning interactions through parasite removal, but cleaner fish Labroides dimidiatus prefer client mucus. Hence, feeding against their preference represents cooperative behaviour in cleaners. Cleaners regularly cheat non‐predatory clients while they rarely cheat predatory clients. Here, we asked how precisely cleaners can adjust service quality from one interaction to the next. We found that non‐predatory clients receive a better service if the previous client was a predator than if the previous client was a non‐predator. In a related laboratory experiment, a hand‐net used as a stressor resulted in cleaners feeding more against their preference in subsequent interactions. The combination of the cleaners’ behaviour in the two studies shows that the cleaners’ service quality for a given client species is not fixed, but it can be manipulated. The results suggest that short‐term stress is one factor that causes cleaners to increase their levels of cooperation, a hypothesis that is amenable to further experiments manipulating the endocrine system.     

Does client size affect cleaner fish choice of client? An empirical test using client fish models

To determine whether the choice of client fishes in the cleaner fish Labroides dimidiatus was influenced by client size, cleaner fish were given a choice of equal amount of food spread on large and small client redfin butterflyfish Chaetodon trifasciatus models. All large models received bites from cleaners compared to 27% for small models. Seventy‐nine per cent of cleaners took their first bite from the large fish model. The results suggest that client size may affect cleaner fish choice.     

Biting cleaner fish use altruism to deceive image-scoring client reef fish

Humans are more likely to help those who they have observed helping others previously. Individuals may thus benefit from being altruistic without direct reciprocity of recipients but due to gains in 'image' and associated indirect reciprocity. I suggest, however, that image-scoring individuals may be exploitable by cheaters if pay-offs vary between interactions. I illustrate this point with data on cleaner-client reef fish interactions. I show the following: (i) there is strong variation between cleaners with respect to cheating of clients (i.e. feeding on client tissue instead of parasites); (ii) clients approach cleaners, that they observe cooperating with their current client and avoid cleaners that they observe cheating; (iii) cleaners that cheat frequently are avoided more frequently than more cooperative cleaners (iv) cleaners that cheat frequently behave altruistically towards their smallest client species; (v) altruistic acts are followed by exploitative interactions. Thus, it appears that cleaners indeed have an image score, which selects for cooperative cleaners. However, cheating cleaners use altruism in potentially low-pay-off interactions to deceive and attract image-scoring clients that will be exploited.     

Does access to the bluestreak cleaner wrasse Labroides dimidiatus affect indicators of stress and health in resident reef fishes in the Red Sea?

Interactions between the bluestreak cleaner wrasse Labroides dimidiatus and its client reef fish are a textbook example of interspecific mutualism. The fact that clients actively visit cleaners and invite inspection, together with evidence that cleaners eat many client ectoparasites per day, indeed strongly suggests a mutualistic relationship. What remains unknown is how parasite removal affects the physiology of clients and thereby their body condition, health, and immune function. Here we addressed these issues in a field study in Ras Mohammed National Park, Egypt. In our study area, small reef patches are inter-spaced with areas of sandy substrate, thereby preventing many species (i.e., residents, including cleaner wrasses) from travelling between the reef patches. This habitat structure leads to a mosaic of resident clients with and without access to bluestreak cleaner wrasses, further referred to as “cleaner access”, on which we focused our study. We found that residents with cleaner access had higher body condition than residents without cleaner access. However, indicators of stress like variation in cortisol levels corrected for handling time and various immune parameters were apparently unaffected by cleaner access. In fact antibody responses were significantly higher in fishes without cleaner access. This suggests that cleaner access decreases the need for active immunity and that this releases resources that might be allocated to other functions such as somatic growth and reproduction.     

Arginine vasotocin regulation of interspecific cooperative behaviour in a cleaner fish

In an interspecific cooperative context, individuals must be prepared to tolerate close interactive proximity to other species but also need to be able to respond to relevant social stimuli in the most appropriate manner. The neuropeptides vasopressin and oxytocin and their non-mammalian homologues have been implicated in the evolution of sociality and in the regulation of social behaviour across vertebrates. However, little is known about the underlying physiological mechanisms of interspecific cooperative interactions. In interspecific cleaning mutualisms, interactions functionally resemble most intraspecific social interactions. Here we provide the first empirical evidence that arginine vasotocin (AVT), a non-mammalian homologue of arginine vasopressin (AVP), plays a critical role as moderator of interspecific behaviour in the best studied and ubiquitous marine cleaning mutualism involving the Indo-Pacific bluestreak cleaner wrasse Labroides dimidiatus. Exogenous administration of AVT caused a substantial decrease of most interspecific cleaning activities, without similarly affecting the expression of conspecific directed behaviour, which suggests a differential effect of AVT on cleaning behaviour and not a general effect on social behaviour. Furthermore, the AVP-V1a receptor antagonist (manning compound) induced a higher likelihood for cleaners to engage in cleaning interactions and also to increase their levels of dishonesty towards clients. The present findings extend the knowledge of neuropeptide effects on social interactions beyond the study of their influence on conspecific social behaviour. Our evidence demonstrates that AVT pathways might play a pivotal role in the regulation of interspecific cooperative behaviour and conspecific social behaviour among stabilized pairs of cleaner fish. Moreover, our results suggest that the role of AVT as a neurochemical regulator of social behaviour may have been co-opted in the evolution of cooperative behaviour in an interspecific context, a hypothesis that is amenable to further testing on the potential direct central mechanism involved.     

The Meaning of Jolts by Fish Clients of Cleaning Gobies

Cooperative interactions offer the inherent possibility of cheating by each of the interacting partners. A key challenge to behavioural observers is to recognize these conflicts, and find means to measure reliably cheating in natural interactions. Cleanerfish Labroides dimidiatus cheat by taking scales and mucus from their fish clients and such dishonest cleaning has been previously recognized in the form of whole‐body jolts by clients in response to cleaner mouth contact. In this study, we test whether jolts may be a general client response to cheating by cleaners. We experimentally varied the ectoparasite loads of yellowtail damselfish (Microspathodon chrysurus), a common client of the cleaning goby Elacantinus evelynae, and compared the rates of jolts on parasitized and deparasitized clients. As predicted if jolts represent cleaner cheating, deparasitized clients jolted more often than parasitized clients, and overall jolt rates increased over time as client parasite load was presumably reduced by cleaning activity. Yellowtail damselfish in the wild jolted significantly less frequently than those in captivity, which is consistent with a loss of ectoparasites during capture. Our results suggest that jolts by clients of cleaning gobies are not related to the removal of ectoparasites. Client jolts may therefore be a generally accurate measure of cheating by cleanerfish.     

The cost of cleanerfish mimics to their models

In aggressive mimicry, a 'predatory' species resembles a model that is harmless or beneficial to a third species, the 'dupe'. Perhaps the most extraordinary case of aggressive mimicry occurs in Indo‐Pacific cleaning symbioses, where cleaner wrasses (the models) remove ectoparasites from larger fish clients. Several species of fangblennies mimic cleaners in behaviour and coloration. Instead of removing ectoparasites, however, fangblennies tear off fins, skin and scales from unsuspecting clients (the dupes). There is some debate over the extent to which cleanerfish mimics are really mimics because in some populations, the contribution of fish tissue to fangblenny diet is limited. In this study, I examine the impact of the resemblance between bluestriped fangblennies ( Plagiotremus rhinorhynchus ) and its putative model, the juvenile bluestreak cleaner wrasse ( Labroides dimidiatus ), on the model's cleaning activity to test the theoretical prediction that mimics should decrease the fitness of their models. I show that the presence of a bluestripe fangblenny in the vicinity of cleaner wrasses results in significantly lower client visit rates and inspection times compared to cleaners without a fangblenny nearby, and discuss why cleaner wrasses tolerate mimics near cleaning stations.     

Treatment with the glucocorticoid antagonist RU486 reduces cooperative cleaning visits of a common reef fish, the lined bristletooth

Cooperation often involves a conflict of interest. This is particularly true in situations where one individual seeks out a service but cannot properly control the quality of the service given by the partner who would gain from defecting. An example is cleaning mutualism involving the bluestreak cleaner wrasse (Labroides dimidiatus) and its reef-fish 'clients'. These cleaners may reduce the stress experienced by their clients by removing parasites; however they occasionally cheat clients (i.e. defect) by eating mucus and other living tissues. Here we present experimental support for the hypothesis that stress responses increase the motivation for clients to seek out such risky asymmetric interactions. We manipulated the stress response by blocking glucocorticoid receptors with the antagonist RU486 in a species that is a regular visitor of cleaner fish, the lined bristletooth (Ctenochaetus striatus). Field observations 1 week after treatment with RU486 showed that antagonist treatment led to a reduction in cleaning duration compared to control treatment. This was not explained by a general effect on client behavior as intraspecific social behavior appeared unaffected. We propose that antagonist treatment reduced stress responses to the presence of ectoparasites, which in turn reduced the client's perception of benefits from seeking out cleaning interactions. The results demonstrate a hitherto overlooked variable role of stress and stress responses on cooperative behavior.