Sex differences in social buffering and social contagion of alarm responses in zebrafish.

The alarm substance in fish is a pheromone released by injured individuals after a predator attack. When detected by other fish, it triggers fear/defensive responses, such as freezing and erratic movement behaviours. Such responses can also help other fish in the shoal to modulate their own behaviours: decreasing a fear response if conspecifics have not detected the alarm substance (social buffering) or triggering a fear response if conspecifics detected the alarm substance (social contagion). Response variation to these social phenomena is likely to depend on sex. Because males have higher-risk life-history strategies than females, they may respond more to social buffering where they risk not responding to a real predator attack, while females should respond more to social contagion because they only risk responding to a false alarm. Using zebrafish, we explored how the response of males and females to the presence/absence of the alarm substance is modified by the alarmed/unalarmed behaviour of an adjacent shoal of conspecifics. We found that, in social buffering, males decreased freezing more than females as expected, but in social contagion males also responded more than females by freezing at a higher intensity. Males were, therefore, more sensitive to visual information, while females responded more to the alarm substance itself. Because visual information updates faster than chemical information, males took more risks but potentially more benefits as well, because a quicker adjustment of a fear response allows to save energy to other activities. These sex differences provide insight into the modifying effect of life-history strategies on the use of social information.

Developmental Effects of Oxytocin Neurons on Social Affiliation and Processing of Social Information.

Hormones regulate behavior either through activational effects that facilitate the acute expression of specific behaviors or through organizational effects that shape the development of the nervous system thereby altering adult behavior. Much research has implicated the neuropeptide oxytocin (OXT) in acute modulation of various aspects of social behaviors across vertebrate species, and OXT signaling is associated with the developmental social deficits observed in autism spectrum disorders (ASDs); however, little is known about the role of OXT in the neurodevelopment of the social brain. We show that perturbation of OXT neurons during early zebrafish development led to a loss of dopaminergic neurons, associated with visual processing and reward, and blunted the neuronal response to social stimuli in the adult brain. Ultimately, adult fish whose OXT neurons were ablated in early life, displayed altered functional connectivity within social decision-making brain nuclei both in naive state and in response to social stimulus and became less social. We propose that OXT neurons have an organizational role, namely, to shape forebrain neuroarchitecture during development and to acquire an affiliative response toward conspecifics.SIGNIFICANCE STATEMENT Social behavior is developed over the lifetime of an organism and the neuropeptide oxytocin (OXT) modulates social behaviors across vertebrate species, and is associated with neuro-developmental social deficits such as autism. However, whether OXT plays a role in the developmental maturation of neural systems that are necessary for social behavior remains poorly explored. We show that proper behavioral and neural response to social stimuli depends on a developmental process orchestrated by OXT neurons. Animals whose OXT system is ablated in early life show blunted neuronal and behavioral responses to social stimuli as well as wide ranging disruptions in the functional connectivity of the social brain. We provide a window into the mechanisms underlying OXT-dependent developmental processes that implement adult sociality.

Pessimistic cognitive bias is associated with enhanced reproductive investment in female zebrafish.

Optimistic and pessimistic cognitive biases have been described in many animals and are related to the perceived valence of the environment. We, therefore, hypothesize that such cognitive bias can be adaptive depending on environmental conditions. In reward-rich environments, an optimistic bias would be favoured, whereas in harsh environments, a pessimistic one would thrive. Here, we empirically investigated the potential adaptive value of such bias using zebrafish as a model. We first phenotyped female zebrafish in an optimistic/pessimistic axis using a previously validated judgement bias assay. Optimistic and pessimistic females were then exposed to an unpredictable chronic stress protocol for 17 days, after which fish were euthanized and the sectional area of the different ovarian structures was quantified in both undisturbed and stressed groups. Our results show that zebrafish ovarian development responded to chronic stress, and that judgement bias impacted the relative area of the vitellogenic developmental stage, with pessimists showing higher vitellogenic areas as compared with optimists. These results suggest that pessimism maximizes reproductive investment, through increased vitellogenesis, indicating a relationship between cognitive bias and life-history organismal decisions.

Stressor controllability modulates the stress response in fish.

BACKGROUND: In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass (Dicentrarchus labrax). RESULTS: Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used. CONCLUSIONS: These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv).

Early social deprivation shapes neuronal programming of the social decision-making network in a cooperatively breeding fish.

The early social environment an animal experiences may have pervasive effects on its behaviour. The social decision-making network (SDMN), consisting of interconnected brain nuclei from the forebrain and midbrain, is involved in the regulation of behaviours during social interactions. In species with advanced sociality such as cooperative breeders, offspring are exposed to a large number and a great diversity of social interactions every day of their early life. This diverse social environment may have life-long consequences on the development of several neurophysiological systems within the SDMN, although these effects are largely unknown. We studied these life-long effects in a cooperatively breeding fish, Neolamprologus pulcher, focusing on the expression of genes involved in the monoaminergic and stress response systems in the SDMN. N. pulcher fry were raised until an age of 2 months either with their parents, subordinate helpers and same-clutch siblings (+F), or with same-clutch siblings only (-F). Analysis of the expression of glucocorticoid receptor, mineralocorticoid receptor, corticotropin releasing factor, dopamine receptors 1 and 2, serotonin transporter and DNA methyltransferase 1 genes showed that early social experiences altered the neurogenomic profile of the preoptic area. Moreover, the dopamine receptor 1 gene was up-regulated in the preoptic area of -F fish compared to +F fish. -F fish also showed up-regulation of GR1 expression in the dorsal medial telencephalon (functional equivalent to the basolateral amygdala), and in the dorsolateral telencephalon (functional equivalent to the hippocampus). Our results suggest that early social environment has life-long effects on the development of several neurophysiological systems within the SDMN.

Social stimuli increase activity of adult-born cells in the telencephalon of zebrafish (Danio rerio).

Fish have particularly high levels of adult neurogenesis, and this high neurogenic capacity may contribute to behavioural plasticity. While it is known that adult-born cells can differentiate into neurons and incorporate into neural circuits, it is unclear whether they are responsive to external stimuli and are thereby capable of contributing to behavioural change. We tested whether cells born in the telencephalon of adult zebrafish are activated by social stimuli. We marked cell birth with BrdU and, 40 days later, exposed fish to brief (15 min) visual social stimuli and assayed cellular activity through immunolocalization of phospho-S6-ribosomal protein (pS6). BrdU+/pS6+ co-labelled cells were found in six brain regions, and, in four regions [dorsal (D), dorsomedial (Dm) and dorsolateral (Dl) zones of the dorsal telencephalon and pre-optic area (POA)], the number of co-labelled cells and fraction of BrdU+ cells that labelled positive for pS6 increased during social stimulation. These results are consistent with the hypothesis that adult-born neurons play a role in regulating social behaviour.

Rising to the challenge? Inter-individual variation of the androgen response to social interactions in cichlid fish.

The Challenge Hypothesis (Wingfield et al. Am. Nat. 136, 829-846) aims to explain the complex relationship between androgens and social interactions. Despite its well acceptance in the behavioral endocrinology literature, several studies have failed to found an androgen response to staged social interactions. Possible reasons for these inconsistencies are the use of single sampling points that may miss the response peak, and the occurrence of inter-individual variability in the androgen response to social interactions. In this study we addressed these two possible confounding factors by characterizing the temporal pattern of the androgen response to social interactions in the African cichlid, Oreochromis mossambicus, and relating it to inter-individual variation in terms of the individual scope for androgen response (i.e. the difference between baseline and maximum physiological levels for each fish) and behavioral types. We found that the androgen response to territorial intrusions varies between individuals and is related to their scope for response. Individuals that have a lower scope for androgen response did not increase androgens after a territorial intrusion but were more aggressive and exploratory. In contrast males with a higher scope for response had fewer aggressive and exploratory behaviors and exhibited two peaks of KT, an early response 2-15 min after the interaction and a late response at 60-90 min post-interaction. Given that the pharmacological challenge of the Hypothalamic-Pituitary-Gonad axis only elicits the late response, we suggest that these two peaks may be regulated by different physiological mechanisms, with the early response being mediated by direct brain-gonad neural pathways. In summary, we suggest that determining the temporal pattern of the androgen response to social interactions and considering inter-individual variation may be the key to understanding the contradictory results of the Challenge Hypothesis.

Assessment of fight outcome is needed to activate socially driven transcriptional changes in the zebrafish brain.

Group living animals must be able to express different behavior profiles depending on their social status. Therefore, the same genotype may translate into different behavioral phenotypes through socially driven differential gene expression. However, how social information is translated into a neurogenomic response and what are the specific cues in a social interaction that signal a change in social status are questions that have remained unanswered. Here, we show for the first time, to our knowledge, that the switch between status-specific neurogenomic states relies on the assessment of fight outcome rather than just on self- or opponent-only assessment of fighting ability. For this purpose, we manipulated the perception of fight outcome in male zebrafish and measured its impact on the brain transcriptome using a zebrafish whole genome gene chip. Males fought either a real opponent, and a winner and a loser were identified, or their own image on a mirror, in which case, despite expressing aggressive behavior, males did not experience either a victory or a defeat. Massive changes in the brain transcriptome were observed in real opponent fighters, with losers displaying both a higher number of differentially expressed genes and of coexpressed gene modules than winners. In contrast, mirror fighters expressed a neurogenomic state similar to that of noninteracting fish. The genes that responded to fight outcome included immediate early genes and genes involved in neuroplasticity and epigenetic modifications. These results indicate that, even in cognitively simple organisms such as zebrafish, neurogenomic responses underlying changes in social status rely on mutual assessment of fighting ability.

Temporal variation in brain transcriptome is associated with the expression of female mimicry as a sequential male alternative reproductive tactic in fish.

Distinct patterns of gene expression often underlie intra- and intersexual differences, and the study of this set of coregulated genes is essential to understand the emergence of complex behavioural phenotypes. Here, we describe the development of a de novo transcriptome and brain gene expression profiles of wild-caught peacock blenny, Salaria pavo, an intertidal fish with sex-role reversal in courtship behaviour (i.e., females are the courting sex) and sequential alternative reproductive tactics in males (i.e., larger and older nest-holder males and smaller and younger sneaker males occur). Sneakers mimic both female's courtship behaviour and nuptial coloration to get access to nests and sneak fertilizations, and later in life transition into nest-holder males. Thus, this species offers the unique opportunity to study how the regulation of gene expression can contribute to intersex phenotypes and to the sequential expression of male and female behavioural phenotypes by the same individual. We found that at the whole brain level, expression of the sneaker tactic was paralleled by broader and divergent gene expression when compared to either females or nest-holder males, which were more similar between themselves. When looking at sex-biased transcripts, sneaker males are intersex rather than being either nest-holder or female-like, and their transcriptome is simultaneously demasculinized for nest-holder-biased transcripts and feminized for female-biased transcripts. These results indicate that evolutionary changes in reproductive plasticity can be achieved through regulation of gene expression, and in particular by varying the magnitude of expression of sex-biased genes, throughout the lifetime of the same individual.

Androgen response to social competition in a shoaling fish.

Androgens respond to social challenges and this response has been interpreted as a way for males to adjust androgen-dependent behavior to social context. However, the androgen responsiveness to social challenges varies across species and a conceptual framework has been developed to explain this variation according to differences in the mating system and parental care type, which determines the regimen of challenges males are exposed to, and concomitantly the scope (defined as the difference between the physiological maximum and the baseline levels) of response to a social challenge. However, this framework has been focused on territorial species and no clear predictions have been made to gregarious species (e.g. shoaling fish), which although tolerating same-sex individuals may also exhibit intra-sexual competition. In this paper we extend the scope of this conceptual framework to shoaling fish by studying the endocrine response of zebrafish (Danio rerio) to social challenges. Male zebrafish exposed to real opponent agonistic interactions exhibited an increase in androgen levels (11-ketotestosterone both in Winners and Losers and testosterone in Losers). This response was absent in Mirror-fighters, that expressed similar levels of aggressive behavior to those of winners, suggesting that this response is not a mere reflex of heightened aggressive motivation. Cortisol levels were also measured and indicated an activation of the hypothalamic-pituitary-interrenal axis in Winners of real opponent fighters, but not Losers or in Mirror-fighters. These results confirm that gregarious species also exhibit an endocrine response to an acute social challenge.

Agonistic interactions elicit rapid changes in brain nonapeptide levels in zebrafish.

The teleost fish nonapeptides, arginine vasotocin (AVT) and isotocin (IT), have been implicated in the regulation of social behavior. These peptides are expected to be involved in acute and transient changes in social context, in order to be efficient in modulating the expression of social behavior according to changes in the social environment. Here we tested the hypothesis that short-term social interactions are related to changes in the level of both nonapeptides across different brain regions. For this purpose we exposed male zebrafish to two types of social interactions: (1) real opponent interactions, from which a Winner and a Loser emerged; and (2) mirror-elicited interactions, that produced individuals that did not experience a change in social status despite expressing similar levels of aggressive behavior to those of participants in real-opponent fights. Non-interacting individuals were used as a reference group. Each social phenotype (i.e. Winners, Losers, Mirror-fighters) presented a specific brain profile of nonapeptides when compared to the reference group. Moreover, the comparison between the different social phenotypes allowed to address the specific aspects of the interaction (e.g. assessment of opponent aggressive behavior vs. self-assessment of expressed aggressive behavior) that are linked with neuropeptide responses. Overall, agonistic interactions seem to be more associated with the changes in brain AVT than IT, which highlights the preferential role of AVT in the regulation of aggressive behavior already described for other species.

Social odors conveying dominance and reproductive information induce rapid physiological and neuromolecular changes in a cichlid fish.

BACKGROUND: Social plasticity is a pervasive feature of animal behavior. Animals adjust the expression of their social behavior to the daily changes in social life and to transitions between life-history stages, and this ability has an impact in their Darwinian fitness. This behavioral plasticity may be achieved either by rewiring or by biochemically switching nodes of the neural network underlying social behavior in response to perceived social information. Independent of the proximate mechanisms, at the neuromolecular level social plasticity relies on the regulation of gene expression, such that different neurogenomic states emerge in response to different social stimuli and the switches between states are orchestrated by signaling pathways that interface the social environment and the genotype. Here, we test this hypothesis by characterizing the changes in the brain profile of gene expression in response to social odors in the Mozambique Tilapia, Oreochromis mossambicus. This species has a rich repertoire of social behaviors during which both visual and chemical information are conveyed to conspecifics. Specifically, dominant males increase their urination frequency during agonist encounters and during courtship to convey chemical information reflecting their dominance status. RESULTS: We recorded electro-olfactograms to test the extent to which the olfactory epithelium can discriminate between olfactory information from dominant and subordinate males as well as from pre- and post-spawning females. We then performed a genome-scale gene expression analysis of the olfactory bulb and the olfactory cortex homolog in order to identify the neuromolecular systems involved in processing these social stimuli. CONCLUSIONS: Our results show that different olfactory stimuli from conspecifics' have a major impact in the brain transcriptome, with different chemical social cues eliciting specific patterns of gene expression in the brain. These results confirm the role of rapid changes in gene expression in the brain as a genomic mechanism underlying behavioral plasticity and reinforce the idea of an extensive transcriptional plasticity of cichlid genomes, especially in response to rapid changes in their social environment.

Social interactions elicit rapid shifts in functional connectivity in the social decision-making network of zebrafish.

According to the social decision-making (SDM) network hypothesis, SDM is encoded in a network of forebrain and midbrain structures in a distributed and dynamic fashion, such that the expression of a given social behaviour is better reflected by the overall profile of activation across the different loci rather than by the activity of a single node. This proposal has the implicit assumption that SDM relies on integration across brain regions, rather than on regional specialization. Here we tested the occurrence of functional localization and of functional connectivity in the SDM network. For this purpose we used zebrafish to map different social behaviour states into patterns of neuronal activity, as indicated by the expression of the immediate early genes c-fos and egr-1, across the SDM network. The results did not support functional localization, as some loci had similar patterns of activity associated with different social behaviour states, and showed socially driven changes in functional connectivity. Thus, this study provides functional support to the SDM network hypothesis and suggests that the neural context in which a given node of the network is operating (i.e. the state of its interconnected areas) is central to its functional relevance.

Neurogenomic mechanisms of social plasticity.

Group-living animals must adjust the expression of their social behaviour to changes in their social environment and to transitions between life-history stages, and this social plasticity can be seen as an adaptive trait that can be under positive selection when changes in the environment outpace the rate of genetic evolutionary change. Here, we propose a conceptual framework for understanding the neuromolecular mechanisms of social plasticity. According to this framework, social plasticity is achieved by rewiring or by biochemically switching nodes of a neural network underlying social behaviour in response to perceived social information. Therefore, at the molecular level, it depends on the social regulation of gene expression, so that different genomic and epigenetic states of this brain network correspond to different behavioural states, and the switches between states are orchestrated by signalling pathways that interface the social environment and the genotype. Different types of social plasticity can be recognized based on the observed patterns of inter- versus intra-individual occurrence, time scale and reversibility. It is proposed that these different types of social plasticity rely on different proximate mechanisms at the physiological, neural and genomic level.

Social Status and Arginine Vasotocin Neuronal Phenotypes in a Cichlid Fish.

The nonapeptide arginine vasotocin (AVT) and its mammalian homologue arginine vasopressin play a key role in the regulation of social behaviour across vertebrates. In teleost fishes, three AVT neuronal populations have been described in the preoptic area (POA): the parvocellular (pPOA), the magnocellular (mPOA) and the gigantocellular (gPOA). Neurons from each of these areas project both to the pituitary and to other brain regions, where AVT is supposed to regulate neural circuits underlying social behaviour. However, in the fish species studied so far, there is considerable variation in which AVT neuronal populations are involved in behavioural modulation and in the direction of the effect. In this study, the association between AVT neuronal phenotypes and social status was investigated in the Mozambique tilapia (Oreochromis mossambicus). This species is an African female mouth-brooding cichlid fish in which males form breeding aggregations in which dominant males establish territories and subordinate males to act as floaters. With respect to sex differences in AVT neuronal phenotypes, females have a larger number of AVT neurons in the pPOA and mPOA. Within males, AVT appeared associated with social subordination, as indicated by the larger cell body areas of AVT neurons in mPOA and gPOA nuclei of non-territorial males. There were also positive correlations between submissive behaviour and the soma size of AVT cells in all three nuclei and AVT cell number in the mPOA. In summary, the results provide evidence for an involvement of AVT in the modulation of social behaviour in tilapia, but it was not possible to identify specific roles for specific AVT neuronal populations. The results presented here also contrast with those previously published for another cichlid species with a similar mating system, which highlights the species-specific nature of the pattern of association between AVT and social behaviour even within the same taxonomic family.

Brain levels of nonapeptides in four labrid fish species with different levels of mutualistic behavior.

There is strong evidence that brain nonapeptides are implicated as modulators of a wide array of social and reproductive behaviors in fishes. However, the question remains, as to whether there is a link between the distribution of active nonapeptides across brain regions and fishes specific behavioral phenotypes. To explore this link we compared the nonapeptides' profile across the brains of fishes representing different degrees of mutualistic behavior (here: cleaning behavior). Herein we studied the quantitative distribution of both nonapeptides, arginine vasotocin (AVT) and isotocin (IT), in the brains of four species of fish belonging to the family Labridae: two are obligatory cleaners throughout their entire life (Labroides dimidiatus and Labroides bicolor), one species is a facultative cleaner (Labropsis australis; juveniles are cleaners and adults are corallivorous), and one is a non-cleaner species, corallivorous throughout its entire life (Labrichthys unilineatus). The biologically available AVT and IT concentrations were measured simultaneously in distinct brain macro-areas: forebrain, optic tectum, cerebellum and brain stem, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We showed that the levels of both AVT and IT varied significantly across species, as measured in the whole brain or in the specific macro-areas. Significantly higher AVT concentrations in the cerebellum which were found in the obligate cleaners seemed to be related to expression of mutualistic behavior. On the other hand, the higher levels of brain IT in the non-cleaner L. unilineatus suggested that these might be linked to the development of sexual dimorphism, which occurs only in this non-cleaner species.

Oestradiol and prostaglandin F2α regulate sexual displays in females of a sex-role reversed fish.

The mechanisms regulating sexual behaviours in female vertebrates are still poorly understood, mainly because in most species sexual displays in females are more subtle and less frequent than displays in males. In a sex-role reversed population of a teleost fish, the peacock blenny Salaria pavo, an external fertilizer, females are the courting sex and their sexual displays are conspicuous and unambiguous. We took advantage of this to investigate the role of ovarian-synthesized hormones in the induction of sexual displays in females. In particular, the effects of the sex steroids oestradiol (E2) and testosterone (T) and of the prostaglandin F2α (PGF2α) were tested. Females were ovariectomized and their sexual behaviour tested 7 days (sex steroids and PGF2α) and 14 days (sex steroids) after ovariectomy by presenting females to an established nesting male. Ovariectomy reduced the expression of sexual behaviours, although a significant proportion of females still courted the male 14 days after the ovary removal. Administration of PGF2α to ovariectomized females recovered the frequency of approaches to the male's nest and of courtship displays towards the nesting male. However, E2 also had a positive effect on sexual behaviour, particularly on the frequency of approaches to the male's nest. T administration failed to recover sexual behaviours in ovariectomized females. These results suggest that the increase in E2 levels postulated to occur during the breeding season facilitates female mate-searching and assessment behaviours, whereas PGF2α acts as a short-latency endogenous signal informing the brain that oocytes are mature and ready to be spawned. In the light of these results, the classical view for female fishes, that sex steroids maintain sexual behaviour in internal fertilizers and that prostaglandins activate spawning behaviours in external fertilizers, needs to be reviewed.

Social instability promotes hormone-behavior associated patterns in a cichlid fish.

Androgens are known to respond to social challenges and to control the expression of social behavior and reproductive traits, such as gonadal maturation and sperm production, expression of secondary sex characters and reproductive behaviors. According to the challenge hypothesis variation in androgen levels above a breeding baseline should be explained by the regime of social challenges faced by the individual considering the trade-offs of androgens with other traits (e.g. parental care). One prediction that can be derived from the challenge hypothesis is that androgen levels should increase in response to social instability. Moreover, considering that a tighter association of relevant traits is expected in periods of environmental instability, we also predict that in unstable environments the degree of correlations among different behaviors should increase and hormones and behavior should be associated. These predictions were tested in a polygamous cichlid fish (Mozambique tilapia, Oreochromis mossambicus) with exclusive maternal care. Social instability was produced by swapping dominant males among groups. Stable treatment consisted in removing and placing back dominant males in the same group, in order to control for handling stress. Cortisol levels were also measured to monitor stress levels involved in the procedure and their relation to the androgen patterns and behavior. As predicted androgen levels increased in males in response to the establishment of a social hierarchy and presence of receptive females. However, there were no further differential increases in androgen levels over the social manipulation phase between social stable and social unstable groups. As predicted behaviors were significantly more correlated among themselves in the unstable than in the stable treatment and an associated hormone-behavior pattern was only observed in the unstable treatment.

Cortisol mediates cleaner wrasse switch from cooperation to cheating and tactical deception.

Recent empirical research, mostly done on humans, recognizes that individuals' physiological state affects levels of cooperation. An individual's internal state may affect the payoffs of behavioural alternatives, which in turn could influence the decision to either cooperate or to defect. However, little is known about the physiology underlying condition dependent cooperation. Here, we demonstrate that shifts in cortisol levels affect levels of cooperation in wild cleaner wrasse Labroides dimidiatus. These cleaners cooperate by removing ectoparasites from visiting 'client' reef fishes but prefer to eat client mucus, which constitutes cheating. We exogenously administrated one of three different compounds to adults, that is, (a) cortisol, (b) glucocorticoid receptor antagonist mifepristone RU486 or (c) sham (saline), and observed their cleaning behaviour during the following 45min. The effects of cortisol match an earlier observational study that first described the existence of "cheating" cleaners: such cleaners provide small clients with more tactile stimulation with their pectoral and pelvic fins, a behaviour that attracts larger clients that are then bitten to obtain mucus. Blocking glucocorticoid receptors led to more tactile stimulation to large clients. As energy demands and associated cortisol concentration level shifts affect cleaner wrasse behavioural patterns, cortisol potentially offers a general mechanism for condition dependent cooperation in vertebrates.

Castration affects reproductive but not aggressive behavior in a cichlid fish.

Gonads are the main source of sex steroids, which have been implicated in the regulation of sexually differentiated behavior, such as reproductive and aggressive displays. In the Mozambique tilapia (Oreochromis mossambicus) territorial males have higher androgen levels than non-territorials, express reproductive behavior and use a urine-borne pheromone to signal their social status towards conspecifics. Here we investigated the effects of gonadectomy on the circulating levels of androgens and cortisol, and on the expression of aggressive and reproductive behavior (nest building, courtship behavior, and nuptial coloration). Males were either castrated, urine bladder damaged, or sham-operated and visually exposed to a group of females during 8 consecutive days and subsequently to a male on day 9. The urine bladder damaged treatment was included in the experimental design because a full castration procedure in this species causes quite often damage to the urine bladder. Gonadectomy lowers dramatically the circulating levels of androgens measured at 4 and 8days post-castration and abolishes the expression of nest building, courtship behavior and nuptial coloration, but has no effect on the expression of aggressive behavior. These results confirm the gonads as the main source of androgens in this species and show that androgens are necessary for the expression of reproductive behaviors. However, the expression of aggressive behavior seems to be decoupled from gonadal steroids, namely androgens, suggesting the action of independent central mechanisms.