Early social complexity influences social behaviour but not social trajectories in a cooperatively breeding cichlid fish.

Social competence-defined as the ability to optimize social behaviour according to available social information-can be influenced by the social environment experienced in early life. In cooperatively breeding vertebrates, the current group size influences behavioural phenotypes, but it is not known whether the group size experienced in early life influences behavioural phenotypes generally or social competence specifically. We tested whether being reared in large versus small groups for the first two months of life affects social behaviours, and associated life-history traits, in the cooperatively breeding cichlid Neolamprologus pulcher between the ages of four and twelve months. As we predicted, fish raised in larger and more complex groups showed higher social competence later in life. This was shown in several ways: they exhibited more, and earlier, submissive behaviour in response to aggression from a dominant conspecific, and-in comparison to fish raised in small groups-they exhibited more flexibility in the expression of submissive behaviour. By contrast, there was no evidence that early social complexity, as captured by the group size, affects aggression or exploration behaviour nor did it influence the propensity to disperse or show helping behaviour. Our results emphasize the importance of early-life social complexity for the development of social competence.

The role of serotonin in modulating social competence in a cooperatively breeding fish.

Behavioural interactions between conspecifics rely on the appreciation of social cues, which is achieved through biochemical switching of pre-existing neurophysiological pathways. Serotonin is one of the major neurotransmitters in the central nervous system responsible for the modulation of physiological and behavioural traits, in particular social behaviour. The relative importance of serotonin in modulating optimal social responses to the available social information (i.e., social competence) is yet unknown. Here we investigate how serotonin and the serotonin 1 A receptor (5-HT1A) modulate social competence in a competitive context. In the cooperatively breeding cichlid Neolamprologus pulcher, we pharmacologically manipulated the serotonin availability and 5-HT1A activity to test their effects on social behaviours during an asymmetric contest between the owner of a defended territory containing a shelter and an intruder devoid of a territory. In this contest, the adequate response by the intruders, the focal individuals in our study, is to show submissive behaviour in order to avoid eviction from the vicinity of the shelter. While the serotonin enhancer Fluoxetine did not affect the frequency of submission towards territory owners, reducing serotonin by a low dosage of 4-Chloro-DL-phenylalanine (PCPA) increased submissive behaviour. Furthermore, threat displays towards territory owners were reduced at high dosages of Fluoxetine and also at the lowest dosage of PCPA. 5-HT1A activation increased threat displays by intruders, indicating that this receptor may not be involved in regulating social competence. We conclude that serotonin, but not its receptor 5-HT1A plays an important role in the regulation of social competence.

Stress in the social context: a behavioural and eco-evolutionary perspective.

The social environment is one of the primary sources of challenging stimuli that can induce a stress response in animals. It comprises both short-term and stable interactions among conspecifics (including unrelated individuals, mates, potential mates and kin). Social stress is of unique interest in the field of stress research because (1) the social domain is arguably the most complex and fluctuating component of an animal's environment; (2) stress is socially transmissible; and (3) stress can be buffered by social partners. Thus, social interactions can be both the cause and cure of stress. Here, we review the history of social stress research, and discuss social stressors and their effects on organisms across early life and adulthood. We also consider cross-generational effects. We discuss the physiological mechanisms underpinning social stressors and stress responses, as well as the potential adaptive value of responses to social stressors. Finally, we identify outstanding challenges in social stress research, and propose a framework for addressing these in future work.

Egg-mediated maternal effects in a cooperatively breeding cichlid fish.

Mothers can influence offspring phenotype through egg-mediated maternal effects, which can be influenced by cues mothers obtain from their environment during offspring production. Developing embryos use these components but have mechanisms to alter maternal signals. Here we aimed to understand the role of mothers and embryos in how maternal effects might shape offspring social phenotype. In the cooperatively breeding fish Neolamprologus pulcher different social phenotypes develop in large and small social groups differing in predation risk and social complexity. We manipulated the maternal social environment of N. pulcher females during egg laying by allocating them either to a small or a large social group. We compared egg mass and clutch size and the concentration of corticosteroid metabolites between social environments, and between fertilized and unfertilized eggs to investigate how embryos deal with maternal signalling. Mothers in small groups produced larger clutches but neither laid smaller eggs nor bestowed eggs differently with corticosteroids. Fertilized eggs scored lower on a principal component representing three corticosteroid metabolites, namely 11-deoxycortisol, cortisone, and 11-deoxycorticosterone. We did not detect egg-mediated maternal effects induced by the maternal social environment. We discuss that divergent social phenotypes induced by different group sizes may be triggered by own offspring experience.

Fish can infer relations between colour cues in a non-social learning task.

Transitive inference (TI) describes the ability to infer relationships between stimuli that have never been seen together before. Social cichlids can use TI in a social setting where observers assess dominance status after witnessing contests between different dyads of conspecifics. If cognitive processes are domain-general, animals should use abilities evolved in a social context also in a non-social context. Therefore, if TI is domain-general in fish, social fish should also be able to use TI in non-social tasks. Here we tested whether the cooperatively breeding cichlid Neolamprologus pulcher can infer transitive relationships between artificial stimuli in a non-social context. We used an associative learning paradigm where the fish received a food reward when correctly solving a colour discrimination task. Eleven of 12 subjects chose the predicted outcome for TI in the first test trial and five subjects performed with 100% accuracy in six successive test trials. We found no evidence that the fish solved the TI task by value transfer. Our findings show that fish also use TI in non-social tasks with artificial stimuli, thus generalizing past results reported in a social context and hinting toward a domain-general cognitive mechanism.

Stress axis programming generates long-term effects on cognitive abilities in a cooperative breeder.

The ability to flexibly adjust behaviour to social and non-social challenges is important for successfully navigating variable environments. Social competence, i.e. adaptive behavioural flexibility in the social domain, allows individuals to optimize their expression of social behaviour. Behavioural flexibility outside the social domain aids in coping with ecological challenges. However, it is unknown if social and non-social behavioural flexibility share common underlying cognitive mechanisms. Support for such shared mechanism would be provided if the same neural mechanisms in the brain affected social and non-social behavioural flexibility similarly. We used individuals of the cooperatively breeding fish Neolamprologus pulcher that had undergone early-life programming of the hypothalamic-pituitary-interrenal axis by exposure to (i) cortisol, (ii) the glucocorticoid receptor antagonist mifepristone, or (iii) control treatments, and where effects of stress-axis programming on social flexibility occurred. One year after the treatments, adults learned a colour discrimination task and subsequently, a reversal-learning task testing for behavioural flexibility. Early-life mifepristone treatment marginally enhanced learning performance, whereas cortisol treatment significantly reduced behavioural flexibility. Thus, early-life cortisol treatment reduced both social and non-social behavioural flexibility, suggesting a shared cognitive basis of behavioural flexibility. Further our findings imply that early-life stress programming affects the ability of organisms to flexibly cope with environmental stressors.

An evolutionary perspective on stress responses, damage and repair.

Variation in stress responses has been investigated in relation to environmental factors, species ecology, life history and fitness. Moreover, mechanistic studies have unravelled molecular mechanisms of how acute and chronic stress responses cause physiological impacts ('damage'), and how this damage can be repaired. However, it is not yet understood how the fitness effects of damage and repair influence stress response evolution. Here we study the evolution of hormone levels as a function of stressor occurrence, damage and the efficiency of repair. We hypothesise that the evolution of stress responses depends on the fitness consequences of damage and the ability to repair that damage. To obtain some general insights, we model a simplified scenario in which an organism repeatedly encounters a stressor with a certain frequency and predictability (temporal autocorrelation). The organism can defend itself by mounting a stress response (elevated hormone level), but this causes damage that takes time to repair. We identify optimal strategies in this scenario and then investigate how those strategies respond to acute and chronic exposures to the stressor. We find that for higher repair rates, baseline and peak hormone levels are higher. This typically means that the organism experiences higher levels of damage, which it can afford because that damage is repaired more quickly, but for very high repair rates the damage does not build up. With increasing predictability of the stressor, stress responses are sustained for longer, because the animal expects the stressor to persist, and thus damage builds up. This can result in very high (and potentially fatal) levels of damage when organisms are exposed to chronic stressors to which they are not evolutionarily adapted. Overall, our results highlight that at least three factors need to be considered jointly to advance our understanding of how stress physiology has evolved: (i) temporal dynamics of stressor occurrence; (ii) relative mortality risk imposed by the stressor itself versus damage caused by the stress response; and (iii) the efficiency of repair mechanisms.

Predator-induced maternal effects determine adaptive antipredator behaviors via egg composition.

In high-risk environments with frequent predator encounters, efficient antipredator behavior is key to survival. Parental effects are a powerful mechanism to prepare offspring for coping with such environments, yet clear evidence for adaptive parental effects on offspring antipredator behaviors is missing. Rapid escape reflexes, or "C-start reflexes," are a key adaptation in fish and amphibians to escape predator strikes. We hypothesized that mothers living in high-risk environments might induce faster C-start reflexes in offspring by modifying egg composition. Here, we show that offspring of the cichlid fish Neolamprologus pulcher developed faster C-start reflexes and were more risk averse if their parents had been exposed to cues of their most dangerous natural predator during egg production. This effect was mediated by differences in egg composition. Eggs of predator-exposed mothers were heavier with higher net protein content, and the resulting offspring were heavier and had lower igf-1 gene expression than control offspring shortly after hatching. Thus, changes in egg composition can relay multiple putative pathways by which mothers can influence adaptive antipredator behaviors such as faster escape reflexes.

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.

The serotonin 1A receptor modulates the social behaviour within groups of a cooperatively-breeding cichlid.

The neurotransmitter serotonin (5-HT) reduces aggressive behaviour in a number of vertebrates, and the 5-HT1A receptor is known to be involved in this regulation. However, the role of this receptor in the modulation of sociopositive behaviour remains largely unknown. Here we investigated the role of the 5-HT1A receptor in the regulation of aggressive, submissive and affiliative behaviour in the cooperatively-breeding cichlid Neolamprologus pulcher. In two experiments, we performed intramuscular injections of a 5-HT1A agonist (8-OH-DPAT) and antagonist (Way-100635) followed by recordings of social behaviour of injected fish within their social groups. We determined the concentrations and post-injection times when the drugs had the greatest effect on social behaviour. We recorded spontaneous social behaviour in both experiments. In the second experiment we also recorded behaviour after social groups received a territorial challenge by live presentations of either conspecifics or egg predators. The 5-HT1A agonist caused an increase in aggression and a decrease in submission and affiliation, whereas the antagonist had the opposite effects. Thus, the 5-HT1A receptor plays an important regulatory role not only for aggressive but also sociopositive behaviour.

Early social experience has life-long effects on baseline but not stress-induced cortisol levels in a cooperatively breeding fish.

In cooperatively breeding cichlid fish, the early social environment has lifelong effects on the offspring's behaviour, life-history trajectories and brain gene expression. Here, we asked whether the presence or absence of parents and subordinate helpers during early life also shapes fluctuating levels of cortisol, the major stress hormone in the cichlid Neolamprologus pulcher. To non-invasively characterize baseline and stress-induced cortisol levels, we adapted the 'static' holding-water method often used to collect waterborne steroid hormones in aquatic organisms by including a flow-through system allowing for repeated sampling without handling of the experimental subjects. We used 8-year-old N. pulcher either raised with (+F) or without (-F) parents and helpers in early life. We found that N. pulcher have a peak of their circadian cortisol cycle in the early morning, and that they habituated to the experimental procedure after four days. Therefore, we sampled the experimental fish in the afternoon after four days of habituation. -F fish had significantly lower baseline cortisol levels, whereas stress-induced cortisol levels did not differ between treatments. Thus, we show that the early social environment has life-long effects on aspects of the physiological stress system of the Hypothalamic-Pituitary-Interrenal (HPI) axis. We discuss how these differences in physiological state may have contributed to the specialization in different social and life-history trajectories of this species.

Towards an Evolutionary Theory of Stress Responses.

All organisms have a stress response system to cope with environmental threats, yet its precise form varies hugely within and across individuals, populations, and species. While the physiological mechanisms are increasingly understood, how stress responses have evolved remains elusive. Here, we show that important insights can be gained from models that incorporate physiological mechanisms within an evolutionary optimality analysis (the 'evo-mecho' approach). Our approach reveals environmental predictability and physiological constraints as key factors shaping stress response evolution, generating testable predictions about variation across species and contexts. We call for an integrated research programme combining theory, experimental evolution, and comparative analysis to advance scientific understanding of how this core physiological system has evolved.

Early social and ecological experience triggers divergent reproductive investment strategies in a cooperative breeder.

Unlike eusocial systems, which are characterized by reproductive division of labour, cooperative breeders were predicted not to exhibit any reproductive specialization early in life. Nevertheless, also cooperative breeders face a major life-history decision between dispersal and independent breeding vs staying as helper on the natal territory, which might affect their reproductive strategies. In the cooperatively-breeding cichlid Neolamprologus pulcher early-life social and predator experiences induce two behavioural types differing in later-life social and dispersal behaviour. We performed a long-term breeding experiment to test whether the two early-life behavioural types differ in their reproductive investment. We found that the early-dispersing type laid fewer and smaller eggs, and thus invested overall less in reproduction, compared to the philopatric type. Thus N. pulcher had specialised already shortly after birth for a dispersal and reproductive strategy, which is in sharp contrast to the proposition that reproductively totipotent cooperative breeders should avoid reproductive specialization before adulthood.

Transcriptomic signatures of social experience during early development in a highly social cichlid fish.

The social environment encountered early during development can temporarily or permanently influence life history decisions and behaviour of individuals and correspondingly shape molecular pathways. In the highly social cichlid fish Neolamprologus pulcher, deprivation of brood care permanently affects social behaviour and alters the expression of stress axis genes in juveniles and adults. It is unclear when gene expression patterns change during early life depending on social experience, and which genes are involved. We compared brain gene expression of N. pulcher at two time points during the social experience phase when juveniles were reared either with or without brood care, and one time point shortly afterwards. We compared (a) whole transcriptomes and (b) expression of 79 genes related to stress regulation, in order to define a neurogenomic state of stress for each fish. At developmental day 75, that is, after the social experience phase, 43 genes were down-regulated in fish having experienced social deprivation, while two genes involved in learning and memory and in post-translational modifications of proteins (PTM), respectively, were up-regulated. Down-regulated genes were mainly associated with immunity, PTM and brain function. In contrast, during the experience phase no genes were differentially expressed when assessing the whole transcriptome. When focusing on the neurogenomic state associated with the stress response, we found that individuals from the two social treatments differed in how their brain gene expression profiles changed over developmental stages. Our results indicate that the early social environment influences the transcriptional activation in fish brains, both during and after an early social experience, possibly affecting plasticity, immune system function and stress axis regulation.

Simulating more realistic predation threat using attack playbacks.

Use of virtual proxies of live animals are rapidly gaining ground in studies of animal behaviour. Such proxies help to reduce the number of live experimental animals needed to stimulate the behaviour of experimental individuals and to increase standardisation. However, using too simplistic proxies may fail to induce a desired effect and/or lead to quick habituation. For instance, in a predation context, prey often employ multimodal cues to detect predators or use specific aspects of predator behaviour to assess threat. In a live interaction, predator and prey often show behaviours directed towards each other, which are absent in virtual proxies. Here we compared the effectiveness of chemical and visual predator cues in the cooperatively breeding cichlid Neolamprologus pulcher, a species in which predation pressure has been the evolutionary driver of its sociality. We created playbacks of predators simulating an attack and tested their effectiveness in comparison to a playback showing regular activity and to a live predator. We further compared the effectiveness of predator odour and conspecific skin extracts on behaviours directed towards a predator playback. Regular playbacks of calmly swimming predators were less effective than live predators in stimulating a focal individual's aggression and attention. However, playbacks mimicking an attacking predator induced responses much like a live predator. Chemical cues did not affect predator directed behaviour.

Heritabilities, social environment effects and genetic correlations of social behaviours in a cooperatively breeding vertebrate.

Social animals interact frequently with conspecifics, and their behaviour is influenced by social context, environmental cues and the behaviours of interaction partners, allowing for adaptive, flexible adjustments to social encounters. This flexibility can be limited by part of the behavioural variation being genetically determined. Furthermore, behaviours can be genetically correlated, potentially constraining independent evolution. Understanding social behaviour thus requires carefully disentangling genetic, environmental, maternal and social sources of variations as well as the correlation structure between behaviours. Here, we assessed heritability, maternal, common environment and social effects of eight social behaviours in Neolamprologus pulcher, a cooperatively breeding cichlid. We bred wild-caught fish in a paternal half-sibling design and scored ability to defend a resource against conspecifics, to integrate into a group and the propensity to help defending the group territory ("helping behaviour"). We assessed genetic, social and phenotypic correlations within clusters of behaviours predicted to be functionally related, namely "competition," "aggression," "aggression-sociability," "integration" and "integration-help." Helping behaviour and two affiliative behaviours were heritable, whereas there was little evidence for a genetic basis in all other traits. Phenotypic social effects explained part of the variation in a sociable and a submissive behaviour, but there were no maternal or common environment effects. Genetic and phenotypic correlation within clusters was mostly positive. A group's social environment influenced covariances of social behaviours. Genetic correlations were similar in magnitude but usually exceeding the phenotypic ones, indicating that conclusions about the evolution of social behaviours in this species could be provisionally drawn from phenotypic data in cases where data for genetic analyses are unobtainable.

Early-life manipulation of cortisol and its receptor alters stress axis programming and social competence.

In many vertebrate species, early social experience generates long-term effects on later life social behaviour. These effects are accompanied by persistent modifications in the expression of genes implicated in the stress axis. It is unknown, however, whether stress axis programming can affect the development of social competence, and if so, by which mechanism(s). Here, we used pharmacological manipulations to persistently reprogramme the hypothalamic-pituitary-interrenal axis of juvenile cooperatively breeding cichlids, Neolamprologus pulcher. During the first two months of life, juveniles were repeatedly treated with cortisol, mifepristone or control treatments. Three months after the last manipulation, we tested for treatment effects on (i) social competence, (ii) the expression of genes coding for corticotropin-releasing factor ( crf), glucocorticoid receptor ( gr1) and mineralocorticoid receptor ( mr) in the telencephalon and hypothalamus and (iii) cortisol levels. Social competence in a social challenge was reduced in cortisol-treated juveniles, which is in accordance with previous work applying early-life manipulations using different social experiences. During early life, both cortisol and mifepristone treatments induced a persistent downregulation of crf and upregulation of mr in the telencephalon. We suggest that these persistent changes in stress gene expression may represent an effective physiological mechanism for coping with stress. This article is part of the theme issue 'Developing differences: early-life effects and evolutionary medicine'.

Divergent brain gene expression profiles between alternative behavioural helper types in a cooperative breeder.

Juveniles of the cooperatively breeding cichlid fish Neolamprologus pulcher either consistently provide help in form of alloparental egg care ("cleaners") or consistently abstain from helping ("noncleaners"). These phenotypes are not based on heritable genetic differences. Instead, they arise during ontogeny, which should lead to differences in brain structure or physiology, a currently untested prediction. We compared brain gene expression profiles of cleaners and noncleaners in two experimental conditions, a helping opportunity and a control condition. We aimed to identify (a) expression differences between cleaners and noncleaners in the control, (b) changes in gene expression induced by the opportunity and (c) differences in plasticity of gene expression between cleaners and noncleaners. Control cleaners and noncleaners differed in the expression of a single gene, irx2, which regulates neural differentiation. During the opportunity, cleaners and noncleaners had three upregulated genes in common, which were implicated in neuroplasticity, hormonal signalling and cell proliferation. Thus, the stimulus in the opportunity was sufficiently salient. Cleaners also showed higher expression of seven additional genes that were unique to the opportunity. One of these cleaner-specific genes is implicated in neuropeptide metabolism, indicating that this process is associated with cleaning performance. This suggests that the two types employed different pathways to integrate social information, preparing them for accelerated reaction to future opportunities. Interestingly, three developmental genes were downregulated between the control and the opportunity in cleaners only. Our results indicate that the two behavioural types responded differently to the helping opportunity and that only cleaners responded by downregulating developmental genes.

Brain activation patterns following a cooperation opportunity in a highly social cichlid fish.

In highly social species, individuals frequently face opportunities to cooperate. The molecular and neural mechanisms that integrate internal and external information prior to cooperative responses are not well understood. Using expression levels of egr-1, a genomic marker of neural activity, we quantified the neural response to an alloparental-care opportunity in a cooperatively breeding fish, a component of cooperative behaviour, across brain regions and time. In this species, alloparental care and submission are considered alternative strategies to appease dominants. We therefore investigated whether brood care and defence as well as submissive displays were associated with egr-1 expression. Finally, we predicted potential targets of the egr-1 transcription factor in the cichlid genome. This target prediction suggested that egr-1 regulates the expression of transcription factors involved in nervous system development, which could be implicated in social memory formation associated with cooperation. Egr-1 expression levels differed between test and control individuals and across time. Compared to a control, individuals experiencing the cooperation opportunity expressed less egr-1 in two brain regions, the cerebellum and the telencephalon. This down-regulation was independent of their behavioural reaction, i.e. whether they cooperated or not. However, within the subset of test individuals, egr-1 expression increased as a function of the amount of submissive behaviours, but not of cooperative behaviours, in the hypothalamus and potentially the telencephalon. These regions host structures that play a role in social decision-making; suggesting that egr-1 might be a suitable proxy for neural activation due to the social interaction component of the cooperation opportunity, rather than the actual alloparental care component.

Life-History Multistability Caused by Size-Dependent Mortality.

Body size is a key determinant of mortality risk. In natural populations, a broad range of relationships are observed between body size and mortality, including positive and negative correlations. Previous evolutionary modeling has shown that negatively size-dependent mortality can result in life-history bistability, with early maturation at small size and late maturation at large size representing alternative fitness optima. Here we present a general analysis of conditions under which such life-history bistabilities can occur, reporting the following findings. First, alternative fitness optima can be found for any arbitrarily chosen forms of mortality functions, including functions according to which mortality smoothly declines with size. Second, while bistabilities occur more readily under negatively size-dependent mortality, our analysis reveals that they can also emerge under positively size-dependent mortality, a feature missed in earlier work. Third, any sharp drop of mortality with size facilitates bistability. Fourth, if the mortality regime involves more than one such sharp drop, multistable life histories can occur, with alternative fitness optima straddling each of the drops. Paradoxically, our findings imply that, fifth, a species-poor predator community capable of creating a rugged mortality regime is conducive to evolutionary multistability, which could act as a stepping stone toward prey life-history diversification, whereas a species-rich predator community that results in a smoothly varying mortality regime may prevent diversification through this pathway.