Social regulation of arginine vasopressin and oxytocin systems in a wild group-living fish.

The neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) are key regulators of social behaviour across vertebrates. However, much of our understanding of how these neuropeptide systems interact with social behaviour is centred around laboratory studies which fail to capture the social and physiological challenges of living in the wild. To evaluate relationships between these neuropeptide systems and social behaviour in the wild, we studied social groups of the cichlid fish Neolamprologus pulcher in Lake Tanganyika, Africa. We first used SCUBA to observe the behaviour of focal group members and then measured transcript abundance of key components of the AVP and OXT systems across different brain regions. While AVP is often associated with male-typical behaviours, we found that dominant females had higher expression of avp and its receptor (avpr1a2) in the preoptic area of the brain compared to either dominant males or subordinates of either sex. Dominant females also generally had the highest levels of leucyl-cystinyl aminopeptidase (lnpep)-which inactivates AVP and OXT-throughout the brain, potentially indicating greater overall activity (i.e., production, release, and turnover) of the AVP system in dominant females. Expression of OXT and its receptors did not differ across social ranks. However, dominant males that visited the brood chamber more often had lower preoptic expression of OXT receptor a (oxtra) suggesting a negative relationship between OXT signalling and parental care in males of this species. Overall, these results advance our understanding of the relationships between complex social behaviours and neuroendocrine systems under natural settings.

Status-dependent metabolic effects of social interactions in a group-living fish.

Social interactions can sometimes be a source of stress, but social companions can also ameliorate and buffer against stress. Stress and metabolism are closely linked, but the degree to which social companions modulate metabolic responses during stressful situations-and whether such effects differ depending on social rank-is poorly understood. To investigate this question, we studied Neolamprologus pulcher, a group-living cichlid fish endemic to Lake Tanganyika and measured the metabolic responses of dominant and subordinate individuals when they were either visible or concealed from one another. When individuals could see each other, subordinates had lower maximum metabolic rates and tended to take longer to recover following an exhaustive chase compared with dominants. In contrast, metabolic responses of dominants and subordinates did not differ when individuals could not see one another. These findings suggest that the presence of a dominant individual has negative metabolic consequences for subordinates, even in stable social groups with strong prosocial relationships.

Alternative reproductive tactics in goby fishes of the Caspian Sea.

Here we report on the reproductive morphology and histology of three Gobiidae species (the Caspian monkey goby, Neogobius pallasi; the Caspian goby, Neogobius caspius; and the round goby, Neogobius melanostomus) from the Iranian coastline of the Caspian Sea. Based on ageing, reproductive histology, and internal and external morphological measures, it appears that all three of these goby species have two types of reproductive males, a large courting, territorial, male type and a small cuckolding parasitic male type, a phenomenon known as alternative reproductive tactics (ART). Although ARTs have been reported previously for one of these species, the round goby, all reports stem from its invasive range; ARTs have never been reported before in any fish species in the Caspian Sea. In all three goby species there was a large, older male type, with a wide head, dark body colouration, and a large investment in accessory glands (AG), an organ important for female attraction and parental care. But there was also a small, light, younger male type, with a narrow head, longer urogenital papilla, and little investment in AGs. The Caspian goby were the largest of the three species, and in this species the smaller, lighter, presumably cuckolding male morph was quite rare (only about 5% of the reproductive male population). In contrast, many of the round goby and monkey goby males were the small, lighter parasitic type, making up nearly half the population of reproductive males (48% and 40%, respectively). Round goby and Caspian goby males had a prominent mesorchial gland, a fibrous sheath of pheromone-releasing connective tissue that attaches the testes to the dorsal body wall, but all the monkey goby specimens examined lacked this structure. Although ARTs are well documented across fish species and appear to be particularly common in gobies, our study provides the first evidence for ARTs in goby fishes from the Caspian Sea.

Galanin expression varies with parental care and social status in a wild cooperatively breeding fish.

As many busy parents will attest, caring for young often comes at the expense of having time to feed and care for oneself. Galanin is a neuropeptide that regulates food intake and modulates parental care; however, the relative importance of galanin in the regulation of feeding versus caring by parents has never been evaluated before under naturalistic settings. Here, we assessed how expression of the galanin system varied in two brain regions, the hypothalamus (which regulates feeding) and the preoptic area (which modulates social behaviours including care) in a wild cichlid fish, Neolamprologus pulcher. Females with young had higher hypothalamic expression of galanin receptor 1a, and the highest expression of galanin and galanin receptor 1a was observed in females that foraged the least. However, expression of five other feeding-related neuropeptides did not change while females were caring for young suggesting that changes in the hypothalamic galanin system may not have been directly related to changes in food intake. The preoptic galanin system was unaffected by the presence of young, but preoptic galanin expression was higher in dominant females (which are aggressive, regularly reproduce and care for young) compared to subordinate females (which are submissive, rarely reproduce but often help care for young). Additionally, preoptic galanin expression was higher in fish that performed more territory defense. Overall, our results indicate that galanin has brain-region-specific roles in modulating both parental care and social status in wild animals.

Antioxidant capacity differs across social ranks and with ascension in males of a group-living fish.

Animals that live in groups often form hierarchies in which an individual's behaviour and physiology varies based on their social rank. Occasionally, a subordinate can ascend into a dominant position and the ascending individual must make rapid behavioural and physiological adjustments to solidify their dominance. These periods of social transition and instability can be stressful and ascending individuals often incur large metabolic costs that could influence their oxidative status. Most previous investigations examining the link between oxidative status and the social environment have done so under stable social conditions and have evaluated oxidative status in a single tissue. Therefore, evaluations of how oxidative status is regulated across multiple tissues during periods of social flux would greatly enhance our understanding of the relationship between oxidative status and the social environment. Here, we assessed how antioxidant capacity in three tissues (brain, gonad, and muscle) varied among dominant, subordinate, and ascending males of the group-living cichlid fish, Neolamprologus pulcher. Antioxidant capacity in the brain and muscle of ascending males was intermediate to that of dominant (highest levels) and subordinate males (lowest levels) and correlated with differences in social and locomotor behaviours, respectively. Gonad antioxidant capacity was lower in ascending males than in dominant males. However, gonad antioxidant capacity was positively correlated with the size of ascending males' gonads suggesting that ascending males may increase gonad antioxidant capacity as they develop their gonads. Overall, our results highlight the widespread physiological consequences of social ascension and emphasize the importance of tissue-specific measures of oxidative status.

Tactic-specific antimicrobial activity suggests a parental care function for accessory glands in a marine toadfish.

Males of some species possess extra reproductive organs called accessory glands which are outgrowths of the testes or sperm duct. These organs have a well-established role in reproduction; however, they also appear to have other important functions that are less understood. Here, we investigate the function of the highly complex accessory glands of a marine toadfish, Porichthys notatus, a fish with two reproductive male types: large care-providing 'guarder' males and small non-caring 'sneaker' males. While both male types have accessory glands, guarder male accessory glands are much larger relative to their body size. We show that accessory gland fluids strongly inhibit the growth of bacterial genera associated with unhealthy eggs and have no effect on the growth of strains isolated from healthy eggs. This antibacterial effect was particularly pronounced for extracts from guarder males. Furthermore, we demonstrate that both healthy and unhealthy plainfin midshipman eggs have diverse but distinct microbial communities that differ in their composition and abundance. The highly specific inhibitory capacity of accessory gland fluid on bacteria from unhealthy eggs was robust across a wide range of ecologically relevant temperatures and salinities. Collectively, these ecological and molecular observations suggest a care function for the accessory gland mediated by antimicrobial agents.

Glucocorticoids do not promote prosociality in a wild group-living fish.

Individuals often respond to social disturbances by increasing prosociality, which can strengthen social bonds, buffer against stress, and promote overall group cohesion. Given their importance in mediating stress responses, glucocorticoids have received considerable attention as potential proximate regulators of prosocial behaviour during disturbances. However, previous investigations have largely focused on mammals and our understanding of the potential prosocial effects of glucocorticoids across vertebrates more broadly is still lacking. Here, we assessed whether experimentally elevated glucocorticoid levels (simulating endogenous cortisol responses mounted following disturbances) promote prosocial behaviours in wild groups of the cichlid fish, Neolamprologus pulcher. Using SCUBA in Lake Tanganyika, we observed how subordinate group members adjusted affiliation, helping, and submission (all forms of prosocial behaviour) following underwater injections of either cortisol or saline. Cortisol treatment reduced affiliative behaviours-but only in females-suggesting that glucocorticoids may reduce overall prosociality. Fish with elevated glucocorticoid levels did not increase performance of submission or helping behaviours. Taken together, our results do not support a role for glucocorticoids in promoting prosocial behaviour in this species and emphasize the complexity of the proximate mechanisms that underlie prosociality.

Rank- and sex-specific differences in the neuroendocrine regulation of glucocorticoids in a wild group-living fish.

Individuals that live in groups experience different challenges based on their social rank and sex. Glucocorticoids have a well-established role in coordinating responses to challenges and glucocorticoid levels often vary between ranks and sexes. However, the neuroendocrine mechanisms regulating glucocorticoid dynamics in wild groups are poorly understood, making it difficult to determine the functional consequences of differences in glucocorticoid levels. Therefore, we observed wild social groups of a cooperatively breeding fish (Neolamprologus pulcher) and evaluated how scale cortisol content (an emerging method to evaluate cortisol dynamics in fishes) and expression of glucocorticoid-related genes varied across group members. Scale cortisol was detectable in ~50% of dominant males (7/17) and females (7/15)-but not in any subordinates (0/16)-suggesting that glucocorticoid levels were higher in dominants. However, the apparent behavioural and neuroendocrine factors regulating cortisol levels varied between dominant sexes. In dominant females, higher cortisol was associated with greater rates of territory defense and increased expression of corticotropin-releasing factor in the preoptic and hypothalamic regions of the brain, but these patterns were not observed in dominant males. Additionally, transcriptional differences in the liver suggest that dominant sexes may use different mechanisms to cope with elevated cortisol levels. While dominant females appeared to reduce the relative sensitivity of their liver to cortisol (fewer corticosteroid receptor transcripts), dominant males appeared to increase hepatic cortisol breakdown (more catabolic enzyme transcripts). Overall, our results offer valuable insights on the mechanisms regulating rank- and sex-based glucocorticoid dynamics, as well as the potential functional outcomes of these differences.

Understanding fish cognition: a review and appraisal of current practices.

With over 30,000 recognized species, fishes exhibit an extraordinary variety of morphological, behavioural, and life-history traits. The field of fish cognition has grown markedly with numerous studies on fish spatial navigation, numeracy, learning, decision-making, and even theory of mind. However, most cognitive research on fishes takes place in a highly controlled laboratory environment and it can therefore be difficult to determine whether findings generalize to the ecology of wild fishes. Here, we summarize four prominent research areas in fish cognition, highlighting some of the recent advances and key findings. Next, we survey the literature, targeting these four areas, and quantify the nearly ubiquitous use of captive-bred individuals and a heavy reliance on lab-based research. We then discuss common practices that occur prior to experimentation and within experiments that could hinder our ability to make more general conclusions about fish cognition, and suggest possible solutions. By complementing ecologically relevant laboratory-based studies with in situ cognitive tests, we will gain further inroads toward unraveling how fishes learn and make decisions about food, mates, and territories.

Consequences of nest site selection vary along a tidal gradient.

Parents providing care must sometimes choose between rearing locations that are most favourable for offspring versus those that are most favourable for themselves. Here, we measured how both parental and offspring performance varied in nest sites distributed along an environmental gradient. The plainfin midshipman fish Porichthys notatus nests along a tidal gradient. When ascending from the subtidal to the high intertidal at low tide, both nest temperature and frequency of air exposure increase. We used one lab and two field experiments to investigate how parental nest site choices across tidal elevations are linked to the physiological costs incurred by parents and the developmental benefits accrued by offspring. Under warmer incubation conditions, simulating high intertidal nests, offspring developed faster but had higher mortality rates compared to those incubated in cooler conditions that mimicked subtidal nests. In the field, males in higher intertidal nests were more active caregivers, but their young still died at the fastest rates. Larger males claimed and retained low intertidal nests, where offspring survival and development rates were also highest. Our results suggest that males compete more intensively for nest sites in the low intertidal, where they can raise their young quickly and with lower per-offspring investments. Smaller, less-competitive males forced into higher intertidal sites nest earlier in the season and provide more active parental care, possibly to bolster brood survival under harsh environmental conditions.

Galanin and prolactin expression in relation to parental care in two sympatric cichlid species from Lake Tanganyika.

Our understanding of the hormonal mechanisms underlying parental care mainly stems from research on species with uniparental care. Far less is known about the physiological changes underlying motherhood and fatherhood in biparental caring species. Here, using two biparental caring cichlid species (Neolamprologus caudopunctatus and Neolamprologus pulcher), we explored the relative gene-expression levels of two genes implicated in the control of parental care, galanin (gal) and prolactin (prl). We investigated whole brain gene expression levels in both, male and female caring parents, as well as in non-caring individuals of both species. Caring males had higher prl and gal mRNA levels compared to caring females in both fish species. Expression of gal was highest when young were mobile and the need for parental defense was greatest and gal was lowest during the more stationary egg tending phase in N. caudopunctatus. The onset of parenthood was associated with lower expression of prl and higher expression of gal in N. pulcher, but this pattern was not observed in N. caudopunctatus. Our study demonstrates that gal gene expression is correlated with changes in parental care in two biparental cichlid species and extends both knowledge and taxonomic coverage of the possible neurogenetic mechanisms underlying parental care.

Behavioral and physiological evidence that increasing group size ameliorates the impacts of social disturbance.

Intra-group social stability is important for the long-term productivity and health of social organisms. We evaluated the effect of group size on group stability in the face of repeated social perturbations using a cooperatively breeding fish, Neolamprologus pulcher In a laboratory study, we compared both the social and physiological responses of individuals from small versus large groups to the repeated removal and replacement of the most dominant group member (the breeder male), either with a new male (treatment condition) or with the same male (control condition). Individuals living in large groups were overall more resistant to instability but were seemingly slower to recover from perturbation. Members of small groups were more vulnerable to instability but recovered faster. Breeder females in smaller groups also showed greater physiological preparedness for instability following social perturbations. In sum, we discover both behavioral and physiological evidence that living in larger groups helps to dampen the impacts of social instability in this system.

Social buffering of stress in a group-living fish.

Living in groups affords individuals many benefits, including the opportunity to reduce stress. In mammals, such 'social buffering' of stress is mediated by affiliative relationships and production of the neuropeptide oxytocin, but whether these mechanisms facilitate social buffering across vertebrates remains an open question. Therefore, we evaluated whether the social environment influenced the behavioural and physiological recovery from an acute stressor in a group-living cichlid, Neolamprologus pulcher. Individual fish that recovered with their social group displayed lower cortisol levels than individuals that recovered alone. This social buffering of the stress response was associated with a tendency towards lower transcript abundance of arginine vasotocin and isotocin in the preoptic area of the brain, suggesting reduced neural activation of the stress axis. Individuals that recovered with their social group quickly resumed normal behaviour but received fewer affiliative acts following the stressor. Further experiments revealed similar cortisol levels between individuals that recovered in visual contact with their own social group and those in visual contact with a novel but non-aggressive social group. Collectively, our results suggest that affiliation and familiarity per se do not mediate social buffering in this group-living cichlid, and the behavioural and physiological mechanisms responsible for social buffering may vary across vertebrates.

Parental care and neuropeptide dynamics in a cichlid fish Neolamprologus caudopunctatus.

The hypothalamic neuropeptides arginine vasopressin (AVP) and oxytocin (OT) modulate social behavior across a wide variety of species. However, the role of arginine vasotocin (AVT) and isotocin (IT, the teleost homologs of AVP and OT) in regulating biparental care especially in the context of monogamy is not well studied. Here, using high-performance liquid chromatography (HPLC), we investigated how bioactive whole brain AVT and IT neuropeptide levels vary in relation to the phase of the breeding cycle and sex, in a monogamous biparental cichlid fish, Neolamprologus caudopunctatus. Since non-caring individuals of this species readily cannibalize eggs, but caring parents never do, we further investigated whether there might be changes in AVT/IT whole brain levels that correspond to the transition from a non-breeding, egg cannibal to an egg caring parent. We found that AVT levels were higher in females than in males and that AVT levels were highest when the need to defend the young was greatest. Breeding pairs that had a strong pair-bond and a higher frequency of nest care had the highest levels of AVT, whereas individuals that spent little time close to their breeding partner, displayed aggression towards their partner and neglected their nest duties (signs of a weak pair bond), had lower whole brain AVT levels. Isotocin (IT) levels did not differ between sexes and we did not detect any variation across the breeding cycle, with pair-bonding scores or with parental behavior. Our results show that whole brain AVT levels are linked to the breeding cycle, nest maintenance and pair-bonding score in this species. Furthermore, our study highlights species and sex-specific nonapeptides patterns in bi-parental caring fish and contributes to the increasing knowledge on neuroendocrinological mechanisms underlying parental care.

Sperm maturation and male tactic-specific differences in ejaculates in the plainfin midshipman fish Porichthys notatus.

Using the plainfin midshipman fish Porichthys notatus, a species with alternative reproductive tactics (ARTs), we investigated how sperm maturation shapes sperm competitive abilities. We compared sperm performance and morphology before and after final sperm maturation by sampling sperm from the testes and stripped ejaculates of guarders and sneakers. In accordance with sperm competition risk theory, ejaculates from sneaker males had three times as much sperm as ejaculates from guarder males and sneaker males produced faster swimming sperm than guarder males, but this was only the case after final sperm maturation had occurred. Additionally, fully mature sperm found in ejaculates had larger heads and midpieces than sperm found in the testes. These results emphasize the important role played by non-sperm components of an ejaculate in mediating sperm performance and potentially also morphology.

Direct and indirect effects of chemical contaminants on the behaviour, ecology and evolution of wildlife.

Chemical contaminants (e.g. metals, pesticides, pharmaceuticals) are changing ecosystems via effects on wildlife. Indeed, recent work explicitly performed under environmentally realistic conditions reveals that chemical contaminants can have both direct and indirect effects at multiple levels of organization by influencing animal behaviour. Altered behaviour reflects multiple physiological changes and links individual- to population-level processes, thereby representing a sensitive tool for holistically assessing impacts of environmentally relevant contaminant concentrations. Here, we show that even if direct effects of contaminants on behavioural responses are reasonably well documented, there are significant knowledge gaps in understanding both the plasticity (i.e. individual variation) and evolution of contaminant-induced behavioural changes. We explore implications of multi-level processes by developing a conceptual framework that integrates direct and indirect effects on behaviour under environmentally realistic contexts. Our framework illustrates how sublethal behavioural effects of contaminants can be both negative and positive, varying dynamically within the same individuals and populations. This is because linkages within communities will act indirectly to alter and even magnify contaminant-induced effects. Given the increasing pressure on wildlife and ecosystems from chemical pollution, we argue there is a need to incorporate existing knowledge in ecology and evolution to improve ecological hazard and risk assessments.

Stress axis regulation during social ascension in a group-living cichlid fish.

Animals living in groups often form social hierarchies, with characteristic behaviours and physiologies associated with rank. However, when social opportunities arise and a subordinate ascends into a dominant position, quick adjustments are necessary to secure this position. Such periods of social transition are typically associated with elevated glucocorticoid production, but the precise regulation of the stress axis during these occasions is not well understood. Using the group-living cichlid, Neolamprologus pulcher, the effects of social ascension on the stress axis were assessed. Ascenders rapidly filled experimentally created vacancies, adopting a dominant behavioural phenotype within 72 h-elevating aggression, activity, and workload, while receiving high rates of affiliative behaviours from their group members. Despite assuming behavioural dominance within their groups, ascenders displayed higher cortisol levels than dominants three days post-ascension. Additionally, compared to subordinates, ascenders had increased transcript abundance of steroidogenic acute regulatory protein (star) and cytochrome p450 side-chain cleavage enzyme (p450scc) in the head kidney, indicating activation of the stress axis. Cortisol levels were lowest in ascenders that displayed low rates of aggression, potentially reflecting the reestablishment of social stability in these groups. Increased transcript abundance of both glucocorticoid receptors (gr1 and gr2) in the brain's preoptic area (POA) of ascenders compared to dominants suggested an enhanced capacity for cortisol regulation via negative feedback. Our results reveal a regulatory cascade of behavioural and physiological interactions and highlight the importance of investigating the underlying mechanisms regulating the stress axis.

Metabolic Costs of Exposure to Wastewater Effluent Lead to Compensatory Adjustments in Respiratory Physiology in Bluegill Sunfish.

Municipal wastewater effluent is a major source of aquatic pollution and has potential to impact cellular energy metabolism. However, it is poorly understood whether wastewater exposure impacts whole-animal metabolism and whether this can be accommodated with adjustments in respiratory physiology. We caged bluegill sunfish (Lepomis macrochirus) for 21 days at two sites downstream (either 50 or 830 m) from a wastewater treatment plant (WWTP). Survival was reduced in fish caged at both downstream sites compared to an uncontaminated reference site. Standard rates of O2 consumption increased in fish at contaminated sites, reflecting a metabolic cost of wastewater exposure. Several physiological adjustments accompanied this metabolic cost, including an expansion of the gill surface area available for gas exchange (reduced interlamellar cell mass), a decreased blood-O2 affinity (which likely facilitates O2 unloading at respiring tissues), increased respiratory capacities for oxidative phosphorylation in isolated liver mitochondria (supported by increased succinate dehydrogenase, but not citrate synthase, activity), and decreased mitochondrial emission of reactive oxygen species (ROS). We conclude that exposure to wastewater effluent invokes a metabolic cost that leads to compensatory respiratory improvements in O2 uptake, delivery, and utilization.

Growth and otolith morphology vary with alternative reproductive tactics and contaminant exposure in the round goby Neogobius melanostomus.

Round goby Neogobius melanostomus sagittal (saccular) otolith morphology was compared between males of the two alternative reproductive tactics (termed guarder and sneaker males) and between males captured from sites of high or low contamination. Otolith size increased with fish size and also displayed an ontogenetic shift in shape, becoming relatively taller as otoliths grew in size. Despite a considerable overlap in age between males adopting the two reproductive tactics, size-at-age measurements revealed that guarder males are significantly larger than sneakers at any given age and that they invest more into somatic growth than sneaker males. Controlling for body size, sneaker males possessed heavier sagittal otoliths than guarder males. Subtle otolith shape differences were also found between the two male tactics and between sites of high and low contaminant exposure. Sneaker males had relatively shorter otoliths with more pronounced notching than guarder males. Fish captured at sites of high contamination had otoliths showing slower growth rates in relation to body size and their shapes had more pronounced caudal points and ventral protrusions when compared with fish captured at sites of low contamination. The results are discussed in relation to life-history tradeoffs between the male tactics in terms of reproductive and somatic investment as well as the putative metabolic costs of exposure to contaminants. Overall, this study reveals that male alternative reproductive tactics and environmental contaminants can have small, yet measurable, effects on otolith morphology and these factors should be accounted for in future research when possible.