The impact of long-term reduced access to cleaner fish on health indicators of resident client fish.

In many mutualisms, benefits in the form of food are exchanged for services such as transport or protection. In the marine cleaning mutualism, a variety of 'client' reef fishes offer 'cleaner' fish Labroides dimidiatus access to food in the form of their ectoparasites, where parasite removal supposedly protects the clients. Yet, the health benefits individual clients obtain in the long term from repeated ectoparasite removal remain relatively unknown. Here, we tested whether long-term reduced access to cleaning services alters indicators of health status such as body condition, immunity and the steroids cortisol and testosterone in four client damselfish species Pomacentrus amboinensis, Amblyglyphidodon curacao, Acanthochromis polyacanthus and Dischistodus perspicillatus To do so, we took advantage of a long-term experimental project in which several small reefs around Lizard Island (Great Barrier Reef, Australia) have been maintained cleaner-free since the year 2000, while control reefs had their cleaner presence continuously monitored. We found that the four damselfish species from reef sites without cleaners for 13 years had lower body condition than fish from reefs with cleaners. However, immunity measurements and cortisol and testosterone levels did not differ between experimental groups. Our findings suggest that clients use the energetic benefits derived from long-term access to cleaning services to selectively increase body condition, rather than altering hormonal or immune system functions.

Morphological and molecular data show no evidence of the proposed replacement of endemic Pomphorhynchus tereticollis by invasive P. laevis in salmonids in southern Germany.

Changes in parasite communities might result in new host-parasite dynamics and may threaten local fish populations. This phenomenon has been suggested for acanthocephalan parasites in the river Rhine and Danube where the species Pomphorhynchus tereticollis is becoming replaced by the Ponto-Caspian P. laevis. Developing knowledge on morphologic, genetic and behavioural differences between such species is important to follow such changes. However, disagreements on the current phylogeny of these two acanthocephalan species are producing conflicts that is affecting their correct identification. This study is offering a clearer morphological and genetic distinction between these two species. As P. tereticollis is found in rhithral tributaries of the Rhine, it was questioned whether the local salmonid populations were hosts for this species and whether P. laevis was expanding into the Rhine watershed as well. In order to test for this, brown trout, Salmo trutta, and grayling, Thymallus thymallus from South-Western Germany watersheds have been samples and screened for the occurrence of acanthocephalan parasites. For the first time, both species were confirmed to be hosts for P. tereticollis in continental Europe. P. tereticollis was found to be common, whereas P. leavis was found only at a single location in the Danube. This pattern suggest either that the expansion of P. laevis through salmonid hosts into rhithral rivers has not yet occurred, or that not yet ascertained biotic or abiotic features of rhithral rivers hinder P. laevis to spread into these areas.

The arginine-vasotocin and serotonergic systems affect interspecific social behaviour of client fish in marine cleaning mutualism.

Many species engage in mutualistic relationships with other species. The physiological mechanisms that affect the course of such social interactions are little understood. In the cleaning mutualism, cleaner fish Labroides dimidiatus do not always act cooperatively by eating ectoparasites, but sometimes cheat by taking bites of mucus from so-called "client" reef fish. The physiological mechanisms in these interspecific interactions, however, are little studied. Here, we focussed on three neuromodulator systems known to play important roles in intraspecific social behaviour of vertebrates to examine their role in clients' interspecific behaviour. We subjected the client fish Scolopsis bilineatus to ectoparasites and the exogenous manipulation of the vasotocin (AVT), isotocin (IT) and serotonin systems to test how this affects client willingness to seek cleaning and client aggression towards cleaners. We found that a single dose of AVT agonist and a selective antagonist caused clients to seek proximity to cleaners, independently of ectoparasite infection. In contrast, in a direct encounter task, the selective blocker of serotonin 5HT2A/2C receptors, Ketanserin (KET), made client reef fish more aggressive towards cleaners in the absence of cleaners' bites of mucus. IT did not yield any significant effects. Our results suggest that the AVT system plays a role in social affiliation towards an interspecific partner, while the serotonin system affects clients' acceptance of level of proximity to cleaner fish during interactions. These two systems, therefore, were apparently co-opted from intraspecific social interactions to affect the course of interspecific ones also.

Androgen responses to reproductive competition of males pursuing either fixed or plastic alternative reproductive tactics.

Alternative reproductive tactics (ARTs), which can be plastic or fixed for life, may be characterized by distinct hormonal profiles. The relative plasticity hypothesis predicts flexible androgen regulation for adult males pursuing plastic tactics, but a less flexible regulation for males using a fixed tactic throughout life. Furthermore, androgen profiles may respond to changes in the social environment, as predicted by the social reciprocity models of hormone/behaviour interactions. The cichlid fish Lamprologus callipterus provides a rare opportunity to study the roles of androgens for male ARTs within a single species, because fixed and plastic ARTs coexist. We experimentally exposed males to competitors pursuing either the same or different tactics to test predictions of the relative plasticity and the social reciprocity models. Androgen profiles of different male types partly comply with predictions derived from the relative plasticity hypothesis: males of the plastic bourgeois/sneaker male trajectory showed different 11-ketotestosterone (11-KT) levels when pursuing either bourgeois or parasitic sneaker male behaviours. Surprisingly, males pursuing the fixed dwarf male tactic showed the highest free and conjugated 11-KT and testosterone (T) levels. Our experimental social challenges significantly affected the free 11-KT levels of bourgeois males, but the androgen responses did not differ between challenges involving different types of competitors. Furthermore, the free T-responses of the bourgeois males correlated with their aggressive behaviour exhibited against competitors. Our results provide new insights into the endocrine responsiveness of fixed and plastic ARTs, confirming and refuting some predictions of both the relative plasticity and the social reciprocity models.

Dear Enemies Elicit Lower Androgen Responses to Territorial Challenges than Unfamiliar Intruders in a Cichlid Fish.

In many territorial species androgen hormones are known to increase in response to territorial intrusions as a way to adjust the expression of androgen-dependent behaviour to social challenges. The dear enemy effect has also been described in territorial species and posits that resident individuals show a more aggressive response to intrusions by strangers than by other territorial neighbours. Therefore, we hypothesized that the dear enemy effect may also modulate the androgen response to a territorial intrusion. Here we tested this hypothesis in male cichlid fish (Mozambique tilapia, Oreochromis mossambicus) using a paradigm of four repeated territorial intrusions, either by the same neighbour or by four different unfamiliar intruders. Neighbour intruders elicited lower aggression and a weaker androgen response than strangers on the first intrusion of the experiment. With repeated intrusions, the agonistic behaviour of the resident males against familiar intruders was similar to that displayed towards strangers. By the fourth intrusion the androgen response was significantly reduced and there was no longer a difference between the responses to the two types of intruders. These results suggest that the dear enemy effect modulates the androgen response to territorial intrusions and that repeated intrusions lead to a habituation of the androgen response.

Intra- and interspecific social challenges modulate the levels of an androgen precursor in a seasonally territorial tropical damselfish.

Studies on different vertebrate groups have provided evidence that androgen levels in males increase after competitive social interactions during the breeding season, as postulated by the Challenge Hypothesis. However, social modulation of androgen levels may vary with latitude and may differ between species holding seasonal versus year-round territories. Here, we tested the Challenge Hypothesis on a seasonal tropical damselfish, Abudefduf sexfasciatus, where males temporarily defend territory and eggs against both intra- and interspecific individuals. Carrying out simulated territorial intrusions (STIs) in the laboratory, we document for the first time a consistent increase in the plasma level of the androgen precursor 11-ketoandrostenedione (11KA) in fish confronted to either intra- or interspecific challenges. Collecting samples in the field also revealed higher 11KA levels in fish facing frequent territorial interactions than in non-territorial individuals. Levels of 11-ketotestosterone (11KT) were high in territorial males in the field, but were not incremented after simulated territorial intrusions in the laboratory. Plasma levels of cortisol and testosterone were not affected by challenges but were different in wild and captive specimens. Although the endocrine responses to STIs did not differ between intra- and interspecific challenges, agonistic displays expressed by resident fish were more intense towards intraspecific intruders. Taken together, our study emphasizes the need to incorporate androgen precursor concentrations to advance our understanding on the physiology of territorial interactions.

Physiological plasticity to water flow habitat in the damselfish, Acanthochromis polyacanthus: linking phenotype to performance.

The relationships among animal form, function and performance are complex, and vary across environments. Therefore, it can be difficult to identify morphological and/or physiological traits responsible for enhancing performance in a given habitat. In fishes, differences in swimming performance across water flow gradients are related to morphological variation among and within species. However, physiological traits related to performance have been less well studied. We experimentally reared juvenile damselfish, Acanthochromis polyacanthus, under different water flow regimes to test 1) whether aspects of swimming physiology and morphology show plastic responses to water flow, 2) whether trait divergence correlates with swimming performance and 3) whether flow environment relates to performance differences observed in wild fish. We found that maximum metabolic rate, aerobic scope and blood haematocrit were higher in wave-reared fish compared to fish reared in low water flow. However, pectoral fin shape, which tends to correlate with sustained swimming performance, did not differ between rearing treatments or collection sites. Maximum metabolic rate was the best overall predictor of individual swimming performance; fin shape and fish total length were 3.3 and 3.7 times less likely than maximum metabolic rate to explain differences in critical swimming speed. Performance differences induced in fish reared in different flow environments were less pronounced than in wild fish but similar in direction. Our results suggest that exposure to water motion induces plastic physiological changes which enhance swimming performance in A. polyacanthus. Thus, functional relationships between fish morphology and performance across flow habitats should also consider differences in physiology.

Intra- and interspecific challenges modulate cortisol but not androgen levels in a year-round territorial damselfish.

Interactions between individuals of different species are commonplace in animal communities. Some behaviors displayed during these interspecific social interactions may be very similar to those displayed during intraspecific social interactions. However, whether functional analogies between intra- and interspecific behaviors translate at the proximate level into an overlap in their underlying endocrine mechanisms remains largely unknown. Because steroids both mediate social behaviors and respond to them, we approached this question by comparing the behavioral and steroid response of free-living dusky gregories (Stegastes nigricans) to standardized territorial intrusions (sTI) of either conspecific or heterospecific food competitors. Stegastes nigricans is a year-round territorial fish that 'cultivates' the algae on which it feeds and is highly aggressive to both intra- and interspecific intruders. Behavioral differences between intra- and interspecific aggressive responses to sTI were marginal, and sTI tests caused an increase in cortisol levels that was positively related with the levels of aggression. In contrast, androgen levels did not increase in response to sTI, yet they showed a positive relationship with agonistic behavior. These results parallel a pattern that was first described for year-round territorial bird species. Furthermore, they suggest that changes in endocrine-hormone levels during territoriality might be independent of the species that induces the territorial response.

Immune activation is inversely related to, but does not cause variation in androgen levels in a cichlid fish species.

Studies on birds and mammals indicate that sexual traits may signal superior health because active immunity, like inflammatory responses to infections, is suppressive to the production of androgens that facilitate the expression of these traits. Here we test this possible pathway for honest signaling in a teleost species, Sarotherodon galilaeus, by activating the immune system with sheep red blood cells (SRBC), which is a non-pathogenic T- and B-cell stimulating antigen. Two weeks after the start of treatment adult males injected with SRBC showed a significant increase in antibody production in comparison with control males. The variation in specific antibody production was negatively related with variation in both testosterone and 11-ketotestosterone levels. This suggests that investment in immune protection is incompatible with increased activity of the hypothalamic-pituitary-gonadal axis. However, opposite to our expectation no difference in androgen levels was found between placebo and SRBC treatment suggesting that immune activation did not cause androgen suppression in our studied species.

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.

Tactile stimulation lowers stress in fish.

In humans, physical stimulation, such as massage therapy, reduces stress and has demonstrable health benefits. Grooming in primates may have similar effects but it remains unclear whether the positive effects are due to physical contact or to its social value. Here we show that physical stimulation reduces stress in a coral reef fish, the surgeonfish Ctenochaetus striatus. These fish regularly visit cleaner wrasses Labroides dimidiatus to have ectoparasites removed. The cleanerfish influences client decisions by physically touching the surgeonfish with its pectoral and pelvic fins, a behaviour known as tactile stimulation. We simulated this behaviour by exposing surgeonfish to mechanically moving cleanerfish models. Surgeonfish had significantly lower levels of cortisol when stimulated by moving models compared with controls with access to stationary models. Our results show that physical contact alone, without a social aspect, is enough to produce fitness-enhancing benefits, a situation so far only demonstrated in humans.

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.

Experience modulates both aromatase activity and the sensitivity of agonistic behaviour to testosterone in black-headed gulls.

In young black-headed gulls (Larus ridibundus), exposure to testosterone increases the sensitivity of agonistic behaviour to a subsequent exposure to this hormone. The aim of this paper is twofold: to analyze whether social experience, gained during testosterone exposure, mediates this increase in hormonal sensitivity (priming), and whether this in turn is mediated by an increase in central aromatase activity. To this end, we performed three experiments. In the first juvenile gulls were exposed to two consecutive treatments with testosterone (T1 and T2), with more than a week interval in between. During T1, half of the birds were housed in social isolation (Iso) and the other half in groups (Soc). All birds were re-housed in a new social situation during the second treatment. The increase in social behaviour during T2 was significantly more rapid in Soc than Iso birds. In experiment 2 we show that 17beta-estradiol treatment facilitates the behaviour measured in experiment 1. In experiment 3 we used a set-up comparable with that of experiment 1, but birds were sacrificed early in the T2 period. Aromatase activity in the preoptic area and the hypothalamus was measured using the tritiated water releasing method. In some parts of the preoptic area and hypothalamus aromatase activity was higher in Soc birds relative to Iso birds. The results indicate that social experience can modulate the increase of social behaviour to testosterone via modulation of aromatase activity and independently of actual hormone levels.

Aggressive behaviour and energy metabolism in a cichlid fish, Oreochromis mossambicus.

We have investigated the effect of mirror-elicited agonistic behaviour on oxygen consumption in the Mozambique tilapia, Oreochromis mossambicus (Cichlidae). Males exposed to their mirror image showed higher frequencies of both lateral display and tail-beating and escalated aggression more frequently than males exposed to a transparent glass that was used as a control for the presence of a novel object in the tank. This aggressive response was correlated with an increase in oxygen consumption. Overt aggression was highly correlated with display behaviour and with locomotor activity. Bivariate analyses showed high correlation (explaining about 64% of variation) between overt aggression, locomotor activity and metabolic rates. Weakly positive bivariate correlations between displays and metabolic rates turned spurious after partialling out aggression. The data suggest that energetic costs only emerge late during the conflict, when animals escalate their aggressive behaviour.

Alternative male reproductive tactics and the immunocompetence handicap in the Azorean rock-pool blenny, Parablennius parvicornis.

In the Azorean rock-pool blenny (Parablennius parvicornis) reproductively active males display alternative morphotypes, which differ in the expression of secondary sexual characters (SSC). Males expressing SSC, the M+ morphotype, have high androgen levels and compete for crevices that will be visited by females to spawn. M+ males holding nests court females and care for the eggs. Males with low expression of SSC, the M- morphotype, have low levels of androgens and reproduce by stealing fertilizations from the M+ males. Based on the hypothesis that androgens are immunosuppressive, we expected these morphotypes to differ in immunocompetence. To test this hypothesis, we conducted a field study in which we collected repeated blood samples to monitor leukocyte populations (blood smears), and to measure the primary antibody response of males that were experimentally challenged with a foreign non-pathogenic antigen (sheep red blood cells). Circulating levels of 11-ketotestosterone and testosterone were higher in M+ males than in M- males. Neither granulocyte nor thrombocyte counts did covariate with androgens or male tactic. In contrast, lymphocyte counts and humoral antibody response were negatively correlated with body size, and as expected, both were lower in M+ than in M- males. Interestingly, in M+ males androgen levels decreased after immunization, and this was less in nest-holder males than in M+ males that were floating around in the pools. Within each morphotype we found no relationship between androgens and immunocompetence. The latter result is not supportive for androgen regulated immunosuppression in M+ males. A possible alternative is enhancement of immunity in M- males. These males had relatively high levels of injuries in comparison with M+ males. High immunity might be a consequence of high infection rate because of such injuries.

Intra-sexual variation in male reproduction in teleost fish: a comparative approach.

The occurrence of intra-sexual variation in reproduction is a widespread phenomenon in teleosts. One such form of variation consists in the occurrence of alternative male types: males that invest resources in mate attraction and males that exploit the investment of the former males, by trying to sneak fertilizations during spawning. These alternative reproductive tactics can be classified according to their plasticity during the life span of the individuals (i.e., fixed vs. sequential vs. reversible). Furthermore, the differences between morphs within a given species may involve a set of different traits, including reproductive behavior, the differentiation of male morphological traits, and the patterns of gonad tissue allocation and the differentiation of gonadal accessory glands. In this paper, we review the available data on four species exhibiting different types of intra-sexual plasticity in reproduction that have been studied in our lab. The data on the proximate mechanisms, androgens and forebrain arginine-vasotocin (AVT), underlying these alternative tactics suggest that between-morph differences in androgen levels, especially in 11-ketotestosterone, are especially present in species where the alternative male types have evolved morphological traits that are tactic-specific (i.e., sexual polymorphisms) and that differences in AVT appear to be related to between-morph differences in the expression of courtship behavior. Therefore, this comparative approach leads us to propose that the different endocrine systems are involved in the differentiation of different sets of traits that make up alternative phenotypes, and that the differentiation of alternative tactics is not controlled by a single endocrine system (e.g., androgens).

The hormonal control of begging and early aggressive behavior: experiments in black-headed gull chicks.

The hormonal control of begging and sibling competition is largely unknown, but recent evidence suggests a role for steroid hormones. We tested the influence of the aromatizable androgen testosterone (T), the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT), and 17beta-estradiol (E) on both begging behavior and aggressive behavior in black-headed gull chicks (Larus ridibundus). Chicks of this species have a conspicuous begging display, while their frequently performed early aggressive behavior is facilitated by testosterone and important for territorial defense. Hormone treatment was applied by implants between days 6 and 16 after hatching. Behavior was tested by means of standard stimulus tests. The results were validated in a second experiment under semi-natural conditions. Begging was suppressed by T and DHT and not affected by E. Aggressive Pecking was strongly facilitated by T. The erect threat posture, characteristic for older chicks, was facilitated by T, DHT, and E and the nest-oriented threat display, typical for young chicks, only by T and DHT. Growth was suppressed in the T group. The results indicate that androgen production, needed for territorial defense, has costs in terms of a suppression of begging and growth. It is discussed to what extent older chicks may avoid these costs by converting testosterone to estrogen and why pre-natal and post-natal exposure to androgens differ in their effect on begging behavior.

Endocrine correlates of intra-specific variation in the mating system of the St. Peter's fish (Sarotherodon galilaeus).

The Challenge Hypothesis postulates that androgen levels are a function of the social environment in which the individual is living. Thus, it is predicted that in polygynous males that engage in social interactions, androgen levels should be higher than in monogamous animals that engage in parental care. In this study, we tested this hypothesis at the intra-specific level using a teleost species, Sarotherodon galilaeus, which exhibits a wide variation in its mating system. Experimental groups of individually marked fish were formed in large ponds with different operational sex-ratios (OSR) to study the effects of partner availability on blood plasma levels of sex steroids [11-ketotestosterone (11-KT), testosterone (T), and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)] and gonadosomatic index (GSI). Polygyny mostly occurred in the female biased OSR groups. 17,20beta-P and gonadosomatic index did not differ among OSR groups. However, 11-KT was high in male biased OSR and positively correlated with aggressive challenges, thereby supporting the central postulate of the Challenge Hypothesis. The results of T were the inverse of those of 11-KT, probably because 11-KT is metabolized from T. 11-KT levels of polygynous males did not differ neither from those of monogamous males, nor from those of males that participated in parental care. These results do not support the expected relationships between polygyny, parental care, and androgen levels. The differences from expectations for 11-KT may be related to the fact that in S. galilaeus, the mating and the parenting phase are not clearly separated and thus, males may still fight and court while they are brooding.

Social stimuli, testosterone, and aggression in gull chicks: support for the challenge hypothesis.

We tested the challenge hypothesis for the hormonal regulation of aggression in chicks of the black-headed gull, Larus ridibundus. Chicks of this species are highly aggressive toward conspecifics, but never to peers that hatched from the same clutch (modal clutch size is three). Therefore, in the first experiment small families were housed together in large groups (challenged condition) and compared to families kept isolated (nonchallenged condition). As expected, in the challenged condition during the initial stage of territory establishment basal levels of testosterone (T) were clearly higher than those in the nonchallenged condition. In the second experiment we tested the effect of a short social challenge on short-term T-fluctuations. The design was based on an earlier experiment, showing that after temporary T-treatment chicks become very sensitive to social challenges while having low basal T-levels. We now show that these social challenges induce brief elevations in plasma T-levels. These peaks are similar to those in previously untreated chicks but untreated chicks do not respond with aggression to a challenge. Therefore, we conclude that the initial exposure to elevated T-levels increases the sensitivity to brief changes in T induced by social challenges. In this way exposure to T, that may be detrimental for development, is minimized while birds remain able to defend territories. This is the first report showing that the challenge hypothesis as established for adult birds, is also applicable for aggressive behavior in young birds outside the sexual context. Furthermore we suggest that a phase of priming with T is necessary to obtain the high behavioral responsiveness to a challenge.