Oxidative stress in the brain may contribute to the onset and progression of mental health disorders. Markers of oxidative stress in serum or blood plasma have been used to predict cognitive decline or identify subjects at risk of developing neurodegenerative disease. However, plasma markers of oxidative stress are rarely directly linked to patterns of oxidative stress in the brain in the same study. Here, we assessed whether peripheral markers of oxidative stress were positively linked to oxidative stress measurements from several macrodissected brain divisions in a highly social cichlid fish, Astatotilapia burtoni. We generated composite measures of brain oxidative stress marker variation by performing principal component analysis on brain markers including oxidative DNA damage (8-OHdG), total antioxidant capacity (TAC), and glutathione levels measured across four brain divisions in dominant and subordinate males. We found that plasma oxidative damage, measured as derivatives of reactive oxygen metabolites (d-ROMs), was not significantly associated with the first two principal components representing brain oxidative stress marker patterns. The link between plasma total antioxidant capacity and brain oxidative stress patterns (PC1 and PC2) varied between dominant and subordinate individuals, but within these social states there were no significant associations between plasma TAC and brain oxidative stress marker patterns. Our study suggests that plasma markers of oxidative stress are poor predictors of individual oxidative stress levels in specific brain divisions.

The multistress concept is a recent approach developed to better understand the effects of environmental stress in animals under real-world conditions. In nature, animals are exposed to simultaneous environmental fluctuations on daily and seasonal time scales, and interactions among stressors can produce antagonistic, additive, or synergistic physiological responses. Consequently, studies examining only one stressor under controlled laboratory conditions may fail to reflect responses in natural habitats. This review discusses the effects of multiple stressors on redox metabolism and heat shock protein (HSP) responses, focusing on aquatic environments. We discuss the multistress approach in laboratory and field studies, highlighting major abiotic stressors such as salinity changes, solar radiation, temperature, and low oxygen availability, including hypoxia and aerial exposure. The effects of multiple stressors on HSPs and antioxidants are summarized using examples from the literature. Finally, the role of social stress and life history stage in shaping responses to multiple abiotic stressors is considered. Overall, the review highlights the application of the multistress approach in laboratory and field experiments, emphasizing key stress responses involving endogenous antioxidants and HSPs, both initiated by reactive oxygen species (ROS).

Neuroendocrine stressors can disrupt the brain’s redox equilibrium by generating high levels of reactive oxygen species (ROS) that lead to oxidative stress. The magnitude of the effect of neuroendocrine stressors on brain redox equilibrium can be influenced by many internal and external factors. To what extent the relationship between neuroendocrine and oxidative stress is modulated by an individual’s stress coping style is only beginning to be understood. To explore this, we subjected proactive and reactive zebrafish to an acute novelty stressor and subsequently quantified changes in behavior and whole brain biomarkers of oxidative stress and antioxidants (DNA damage, total glutathione (GSH), glutathione ratio, oxygen radical absorbance capacity (ORAC), and superoxide dismutase (SOD). Stressed fish had significantly higher total glutathione, trends higher ORAC, DNA damage, and glutathione ratio, and trend for lower SOD levels compared to controls. In addition, individuals with a reactive stress coping style exhibited significantly higher levels of SOD and glutathione ratio, and a trend for ORAC compared to proactive individuals. From a principal component analysis, we also found that the reactive individuals had significantly higher PC1 scores (antioxidant axis) compared to the proactive, and a trend for stressed fish having higher PC1 scores than control. The oxidative stress axis (PC2) showed that the stressed fish had a significantly higher PC2 score relative to control fish. Our results show that neuroendocrine stress-induced disruption of redox equilibrium in the brain differs by stress coping style. Those with a reactive stress coping style have elevated antioxidant capabilities and capacities. Overall, our findings suggest that elevated reactivity to neuroendocrine stressors commonly seen in reactive stress coping styles may be mitigated through the glutathione buffering system and other antioxidants.

Aggression is commonly observed in animals in captive environments. When left unchecked, aggression can lead to injury and death among captive animals. The negative effects of aggression may be mitigated by providing environmental enrichment appropriate for the species. However, environmental enrichment may also promote aggression due to the added structure enhancing territory value. The African cichlid Astatotilapia burtoni, is a popular model organism that exhibits a social structure resembling a lek, in which males compete for territories used to attract mates, resulting in high levels of aggression. To determine methods of managing aggression in captive settings, we studied the effect of artificial aquarium plants on welfare, aggression, and reproduction in groups containing mixed-sex A. burtoni. We found that females in planted tanks did not experience lowered mortality or injury rates than those in unplanted tanks. However, plant treatment impacted breeding behavior, with females in unplanted tanks brooding higher numbers of eggs at the end of the experiment. We also found that males in planted tanks were more aggressive and that aggressive behavior was negatively linked to reproductive output amongst female fish. These findings indicate that increased habitat complexity may not result in improved welfare but it may alter breeding behavior and male territoriality among captive A. burtoni.

Oldfield & Bonano’s (2024) [O&B] target article discusses the wellbeing and sociality of bony fishes. Many fish species, such as cichlids, are not only social but territorial. Aggression is normal, but territorial aggression in a captive environment can have a negative impact on welfare. Aggression-related injuries and deaths are prevalent in pet stores as well as research settings. We discuss the need to develop effective methods to minimize the negative consequences of aggression.

Neuroendocrine stressors can disrupt the brain’s redox equilibrium by generating high levels of reactive oxygen species (ROS) that lead to oxidative stress. To what extent the relationship between neuroendocrine and oxidative stress is modulated by stress coping styles remain unclear. We subjected proactive and reactive zebrafish to an acute novelty stressor (i.e. neuroendocrine stressor), and quantified changes in behavior and whole brain biomarkers of oxidative stress and antioxidants (DNA damage, total glutathione (GSH), glutathione ratio, oxygen radical absorbance capacity (ORAC), and superoxide dismutase (SOD). Fish exposed to the novelty stressor had significantly higher total glutathione, trend for higher ORAC, DNA damage, and glutathione ratio, and trend for lower SOD levels than controls. Those with a reactive stress coping style exhibited significantly higher levels of SOD and glutathione ratio, and a trend for higher ORAC compared to proactive. Principal component analysis showed that reactive individuals that experienced the novelty stressor had significantly higher composite antioxidant scores than proactive. Our data show that exposure to a novelty stressor results in changes to antioxidant and oxidative stress levels in the brain that differs by stress coping style. Reactive individuals showed elevated antioxidant capacity. Overall, our findings suggest that elevated reactivity to neuroendocrine stressors commonly seen in reactive stress coping styles may be mitigated through the glutathione buffering system and other antioxidants.

Phenotypic plasticity allows organisms to adapt to changing environments within their lifetimes. However, environmentally induced changes in the plastic trait of interest may influence a range of fitness-related traits due to trade-offs, pleiotropy, linkage, or epistasis of genes regulating the plastic trait. These correlated responses may constrain or facilitate the evolution of plasticity, but their evolutionary implications are often poorly understood due to a lack of data on their direction and magnitude. Males in the African cichlid Astatotilapia burtoni are blue or yellow, and males are able to adjust their body coloration to the color of the background, presumably to increase crypsis. To test whether background color influences fitness-related traits, we raised mix-sex groups of juvenile A. burtoni to adulthood in yellow or blue tanks. We found that more males adopted the blue phenotype in blue tanks while more males adopted the yellow phenotype in the yellow tank, though the degree of background color matching decreased with age. Males, but not females, from blue tanks showed earlier sexual maturation than those held in yellow tanks. However, across the duration of the experiment, there was a higher occurrence of breeding in females housed in yellow tanks than those that were housed in blue tanks. In addition, fish in blue tanks exhibited reduced growth rate but higher survivorship relative to their yellow-reared counterparts. Our data suggest that background color affects important fitness-related traits in a color polymorphic cichlid, which may influence the evolution of phenotypic plasticity. Adjusting body coloration to specific backgrounds may increase crypsis but could also affect other fitness-related traits. We housed groups of the cichlid fish Astatotilapia burtoni in yellow or blue aquaria and find that background color not only influenced the expression of blue or yellow body coloration, but also several fitness-related traits, including growth, survival, and breeding behavior. These environment-specific effects may influence the evolution of plasticity in body coloration.

Male-male competition and female-female competition can play important roles in the origin and maintenance of phenotypic polymorphism and speciation. If territory owners bias aggression towards others of their own phenotype, rare male phenotypes will be involved in fewer costly fights, facilitating the evolution of diversity, and stabilizing the coexistence of distinct phenotypes or species. However, the mechanisms that regulate aggression biases have received little attention. We discuss how learning and plasticity in behavioral biases may dramatically influence how aggression biases evolve, which in turn may have important consequences for clarifying the role of intrasexual competition in the process of speciation. We then present data from a field study of two cichlid species in Lake Victoria and illustrate how the social environment could modulate aggression biases of territorial males towards specific intruder phenotypes. Specifically, in Pundamilia nyererei (males are red) and P. ‘pink anal’ (males are blue), blue territory holders showed a tendency to shift their aggression bias more towards red intruder (stimulus) males relative to blue intruder males when these territory holders had more red territorial neighbors. By contrast, red territory holders tended to reduce aggression towards red intruder males relative to blue intruder males when they were surrounded by more red territorial neighbors. Although sample sizes are small, our data suggest that social context may shape aggression biases in the Pundamilia species complex and that these effects may vary between species. We conclude that considering the social environment and experience in shaping aggression biases may advance our understanding of how mate competition shapes evolutionary patterns of phenotypic diversification.

Environmental enrichment can have complex, contradictory effects on aggression and animal welfare. Although increasing enrichment may reduce aggression by limiting the visibility of competitors, it may also intensify territorial defense and harm the welfare of subordinates who are the target of territorial aggression. However, it remains unclear how variation in the complexity of an enrichment structure defended by a single dominant individual influences welfare outcomes for subordinates. In the cichlid fish Astatotilapia burtoni, dominant males defend structures as mating territories, whereas subordinate males do not. To establish social hierarchies, we housed two differently sized males (larger become dominant) and six females in one compartment containing one defendable structure. We manipulated structure complexity by placing 1, 2, or 3 halves of terracotta pots clumped together. Increasing cave number did not enhance territoriality: dominant males showed similar aggression rates, relative gonad sizes, and testosterone levels across treatments. Cave number did not significantly affect mortality, body condition, growth rate, or fin damage in subordinate males. Although relative gonad size and testosterone levels were generally higher in dominants, these differences were not always significant in the 2 and/or 3-cave treatments, suggesting weaker physiological differentiation between social states. The behavioral data supported this pattern, with status-specific differences in chase rates declining with increasing cave number. Overall, increasing environmental enrichment had little effect on welfare, but resulted in reduced physiological and behavioral distinctions between dominant and subordinate males. Therefore, careful consideration of enrichment strategies is essential for accurately interpreting status-specific outcomes in laboratory settings. ### Competing Interest Statement The authors have declared no competing interest. American Association for Laboratory Animal Science National Institute of General Medical Sciences, R15GM150286 National Science Foundation, 2444902

High social rank has many benefits, including priority access to mates and resources. However, maintaining high rank or social dominance can be physiologically costly, particularly in species that engage in agonistic competition to maintain their dominance status. Previous studies have rarely manipulated the intensity of agonistic competition in controlled settings to isolate the cost of social dominance. We utilized males of the cichlid fish Astatotilapia burtoni to investigate how the degree of territoriality influences patterns of oxidative stress. In this species, high-rank males compete for territories to attract mates. We housed males individually with a defendable structure and used clear tank dividers to allow visual access to a size-matched male neighbor housed in an identical setup to manipulate territoriality. Defendable structures were placed either close to the divider (proximal treatment) or further away (distal treatment). We found that males in the distal treatment expressed higher levels of territorial aggression than those in the proximal treatment. While males in the distal treatment did not display higher levels of oxidative stress, territory proximity influenced the relationships between physiological markers of dominance (i.e., testosterone, gonad size) and oxidative balance. Although our work is not consistent with higher competition increasing the physiological cost of high rank, it suggests that the level of competition influences the regulation of oxidative balance relative to social dominance characteristics. We suggest that manipulating the degree of territoriality in a controlled setting provides a unique opportunity to further our understanding of how individuals manage the trade-offs associated with high rank. Summary Statement Manipulating the amount of space between territories results in variability in the degree of territorial aggression and the interaction of physiological markers of social dominance and oxidative stress.

Attaining large body size has several selective benefits, however, increased growth rate has potential costs that can constrain investment in other life history traits, such as reproductive output and territorial defense. Oxidative stress can both constrain and result from growth, potentially mediating life history trade-offs between growth rate and other life history traits. Studies on the oxidative cost of growth have provided mixed evidence, in part because components of oxidative balance, including oxidative damage and antioxidant function, is influenced by investment in other activities in a tissue-specific manner. Here, I examined how among-individual variation in growth rate is linked to oxidative stress, and how this relationship is influenced by markers of social dominance (aggressiveness and relative gonad size) in males of the cichlid fish Astatotilapa burtoni. To this end, 7 markers of oxidative damage and antioxidant function in various tissue types (total of 14 measurements) were assessed in dominant and subordinate males. I found that dominant males grew faster than subordinate males. However, increased growth was linked to reduced oxidative stress. This effect was independent of social status but modulated by the degree of social dominance. Overall, the results are consistent with oxidative stress mediating the link between growth and other life history traits. However, my findings challenge the idea that increased growth rate results in elevated oxidative stress, perhaps due to effective protective mechanisms that can neutralize the oxidative challenge of growth.

In some animal species, body coloration can change dramatically during the lifetime of an individual in response to environmental conditions. Body coloration is often a target in sexual selection, but how competition for mates influences phenotypic plasticity in body coloration is rarely considered. Here, we provide experimental evidence in the polymorphic cichlid fish Astatotilapia burtoni that male-male competition for mating territories influences the expression of body coloration. In this species, males can express yellow or blue body coloration and may change color. We housed males individually with visual access to a neighboring male and placed halved terracotta pots as defendable structures either near the neighbor (proximal treatment) or further away from the neighbor (distal treatment). We found that males were more aggressive in the distal treatment, and that a higher proportion of males expressed the yellow phenotype in this setting compared to those housed in the proximal treatment towards the end of the six-week experiment. Unexpectedly, we found that males in the proximal treatment who were smaller than their neighbor were more likely to express the blue phenotype while larger males tended to express the yellow phenotype at the beginning of the experiment. We also found that oxidative stress levels were higher in blue males compared to yellow males. As previous studies show that yellow males are better fighters and blue males more attractive to females, our findings suggest that males can adjust their color phenotype depending on the level of competition or resource holding potential. Our results support the notion that male-male competition may be an important factor influencing the evolution of phenotypic plasticity in coloration.

Phenotypic plasticity allows organisms to adapt to changing environments within their lifetimes. However, environmentally induced changes in the plastic trait of interest may influence a range of fitness-related traits due to trade-offs, pleiotropy, linkage, or epistasis of genes regulating the plastic trait. These correlated responses may constrain or facilitate the evolution of plasticity, but their evolutionary implications are often poorly understood due to a lack of data on their direction and magnitude. Males in the African cichlid Astatotilapia burtoni are blue or yellow, and males are able to adjust their body coloration to the color of the background, presumably to increase crypsis. To test whether background color influences fitness-related traits, we raised mix-sex groups of juvenile A. burtoni to adulthood in yellow or blue tanks. We found that more males adopted the blue phenotype in blue tanks while more males adopted the yellow phenotype in the yellow tank, though the degree of background color matching decreased with age. Males, but not females, from blue tanks showed earlier sexual maturation than those held in yellow tanks. However, across the duration of the experiment, there was a higher occurrence of breeding in females housed in yellow tanks than those that were housed in blue tanks. In addition, fish in blue tanks exhibited reduced growth rate but higher survivorship relative to their yellow-reared counterparts. Our data suggest that background color affects important fitness-related traits in a color polymorphic cichlid, which may influence the evolution of phenotypic plasticity.

The expression of sexually selected traits, such as ornaments or body coloration, is often influenced by environmental conditions. While such phenotypic plasticity is often thought to precede evolutionary change, plasticity itself can also be a target of selection. However, the selective forces supporting the evolution and persistence of plasticity in sexual traits are often unclear. Using the cichlid fish Astatotilapia burtoni , we show that variation in the level of mate competition may promote plasticity in body coloration. In this species, males can change between yellow and blue colour. We found that experimentally increased competition over mating territories led to a higher proportion of males expressing the yellow phenotype. The expression of yellow coloration was found to be beneficial because yellow males won more staged dyadic contests and exhibited a lower level of oxidative stress than blue males. However, females were more likely to spawn with blue males in mate choice experiments, suggesting that expression of blue coloration is sexually more attractive. The ability to adjust colour phenotype according to the local competitive environment could therefore promote the persistence of plasticity in coloration.

In females of the mouthbrooding cichlid fish Astatotilapia burtoni , we recently found a positive relationship between liver antioxidant function and filial cannibalism. Here, we manipulated the level of fry consumption in A. burtoni females to assess how the level of fry consumption affects liver antioxidant function. Feeding treatment did not affect liver antioxidant function, but feeding treatment significantly influenced the relationship between gonadal development and antioxidant function. Future studies should isolate the effects of gonadal development and fry consumption on antioxidant function.

Social stress can increase reactive oxygen species and derail antioxidant function in the brain, which may contribute to the onset and progression of mental health disorders. In hierarchical species, repeated social defeat can raise oxidative stress in the brain. However, how oxidative balance in the brain is regulated across different levels in a social hierarchy is unknown. Here, we study the effect of social status on patterns of oxidative stress across several brain divisions in a highly social cichlid fish, Astatotilapia burtoni. In this species, dominant males are territorial, brightly colored, and reproductively active while subordinate males are not. We measured several markers of oxidative stress in macrodissected brain divisions in dominant and subordinate males. We found that dominant individuals had lower oxidative DNA damage (8-OhdG) in the midbrain while also having increased total antioxidant capacity in the midbrain and hypothalamus. However, in dominant males, oxidative DNA damage tended to be higher in the hypothalamus while total glutathione levels were lower in the telencephalon compared to subordinate males. Finally, we found that indicators of reproductive activity (gonadosomatic index and social behavior) were co-regulated with antioxidant function or oxidative damage in the telencephalon. Combined, our results suggest that social status and activation of the reproductive system regulate oxidative balance in the brain in a highly brain division specific manner.

An individual's social environment can have widespread effects on their physiology, including effects on oxidative stress and hormone levels. Many studies have suggested that variation in oxidative stress experienced by individuals of different social statuses might be due to endocrine differences, however, few studies have evaluated this hypothesis. Here, we assessed whether a suite of markers associated with oxidative stress in different tissues (blood/plasma, liver, and gonads) had social status-specific relationships with circulating testosterone or cortisol levels in males of a cichlid fish, Astatotilapia burtoni. Across all fish, blood DNA damage (a global marker of oxidative stress) and gonadal synthesis of reactive oxygen species [as indicated by NADPH-oxidase (NOX) activity] were lower when testosterone was high. However, high DNA damage in both the blood and gonads was associated with high cortisol in subordinates, but low cortisol in dominants. Additionally, high cortisol was associated with greater production of reactive oxygen species (greater NOX activity) in both the gonads (dominants only) and liver (dominants and subordinates). In general, high testosterone was associated with lower oxidative stress across both social statuses, whereas high cortisol was associated with lower oxidative stress in dominants and higher oxidative stress in subordinates. Taken together, our results show that differences in the social environment can lead to contrasting relationships between hormones and oxidative stress.

Within social dominance hierarchies, rank has a major impact on health and fitness. Dominance hierarchies are rarely stable as individuals may change rank due to changes in the social environment. Here we describe general group-level social network properties and changes in social ties and behavior during rank transitions in 16 communities of the cichlid Astatotilapia burtoni. Social networks based on chases were dense with dominant males frequently chasing subordinate males and females. This intense territoriality of dominant males was also reflected by a high degree of 'power' inequality. Compared to chase-based networks, display-based networks were characterized by a high degree of reciprocity due to display behaviors mostly occurring bidirectionally among a few highly ranked males. Territory ownership is tightly linked to social dominance and territory loss (i.e., social descent) was, as expected, associated with a sharp reduction in aggression level and an increase in chases received. However, although territory acquisition (i.e., social ascent) was an abrupt process, ascending males displayed elevated aggression prior to ascent, in sharp contrast to previous studies carried out in less complex settings. Together, our results provide novel insights into dynamic changes in behavior in cichlid dominance hierarchies.

Investment in current reproduction may negatively influence subsequent fitness. Oxidative stress has been proposed as a potential mediator of this trade-off between current and future reproductive success. However, evidence of reproduction causing oxidative stress is limited, possibly owing to compensatory mechanisms that counteract oxidative insults. Here we test the idea that organisms protect against oxidative challenges through a dynamic interaction between behavioural and physiological adjustments at different stages of reproduction. To test this idea, we manipulated maternal care in the mouthbrooding cichlid fish Astatotilapia burtoni by allowing females to continue care (brooders) or by preventing care (non-brooders). We found that brooders depleted the pool of antioxidants as brood care progressed; however, we only observed increased oxidative DNA damage at the early stage of care relative to non-brooders, possibly owing to upregulated antioxidant protection during later stages of care. Most brooders adjusted parental investment by consuming some of their offspring during mouthbrooding. Intriguingly, the level of filial cannibalism was positively related to liver antioxidant function. These changes in antioxidant function and filial cannibalism allow parents to manage the cost of reproduction and are important for our understanding of how oxidative stress mediates life-history trade-offs.