Whenever established between animals, dominance hierarchies usually produce relevant behavioral adaptations in both dominant and subordinate individuals. These adaptations are examples of social plasticity, which is the capacity to adaptively change behavior according to previous experience and to social context (Maruska et al., 2019). Across fish species, dominance hierarchies have been shown to increase the fitness of dominant individuals by allowing greater access to resources and mating opportunities (Oliveira, 2012; Maruska et al., 2019). However, little is known about the effects of dominant-subordinate interactions on the behavior of subordinate individuals.

In rodents, the experience of subordination is sometimes used as a model of psychosocial stress, as many stressors that are relevant to human mental health involve experiencing social subordination (Blanchard et al., 1993; Read and Harper, 2020). Another relevant model organism for behavioral neuroscience and biological psychiatry is the zebrafish (Stewart et al., 2014), which has been widely used in behavioral tests and as a model of anxiety-like behavior (Kysil et al., 2017). In this regard, zebrafish seem to be an ideal fish species to understand the effects of social subordination on anxiety-like behavior.

Moreover, neurobehavioral and physiological changes have been observed to occur in subordinate zebrafish, regarding pathways associated with stress responses. For instance, male subordinate zebrafish showed higher plasma cortisol levels, as well as higher expression of corticotropin-releasing hormone, neuropeptide Y, and glucocorticoid receptor in the telencephalon (Filby et al., 2010a). On the other hand, subordinate male zebrafish exhibited elevated plasma cortisol concentrations and impaired neurogenesis in the dorsal but not ventral regions of the telencephalon, an effect that was attenuated by treatment with metyrapone, a cortisol synthesis inhibitor (Tea et al., 2019). Both male and female subordinate zebrafish showed higher serotonergic activity in the hindbrain (Dahlbom et al., 2012). Finally, subordinate males showed an up-regulation in the expression of glucocorticoid and mineralocorticoid receptors, brain-derived neurotrophic factor (BDNF), the 5-HT2B receptor, and isoform B of the serotonin transporter (Theodoridi et al., 2017). Overall, these findings suggest that the experience of subordination activates pathways related to arousal and defensive (anxiety-like) behavior in zebrafish, which is likely to lead to increased anxiety-like behavior.

But are sex differences significant to both the establishment of dominance hierarchies and to the effects of this particular psychosocial stressor? From the point of view of psychopathology, human responses to threat more closely resemble the effects of social subordination (e.g., anxiety and depression) and are more prevalent in women (Riecher-Rössler, 2017), with women also more likely to suffer social violence and imposed subordination than men in Western societies (Oram et al., 2017). From the point of view of animal behavior, this question remains relevant because, in spite of anthropocentric biases, female individuals have been shown to enter dominance hierarchies in many species, including zebrafish (Filby et al., 2010a; Paull et al., 2010; Dahlbom et al., 2012; Tea et al., 2019). In zebrafish, anxiety-like behavior has been described in a now growing amount of tests, the most used of which are the light/dark test (Maximino et al., 2010) and the novel tank test (Levin et al., 2007; Bencan et al., 2009; Egan et al., 2009). In the latter, a neophobic response is elicited by introducing the fish to a novel tank and usually is observed as bottom-dwelling (i.e., spending most of the trial in the bottom of the tank) associated with freezing and erratic swimming (Egan et al., 2009). The main controlling stimulus in the cast of the novel tank test is escape from the top of the tank (Maximino et al., 2012), which could be related to antipredator strategies employed by zebrafish in the wild (Engeszer et al., 2007). Moreover, the novel tank test is relatively sensitive to anxiolytic and anxiogenic manipulations (Kysil et al., 2017), showing potential as a way of detecting the effects of social stress on anxiety-like behavior, as well as sex differences in these effects.

While female zebrafish have been observed to form dominant-subordinate hierarchies with other females, neurochemical effects appear to be different in female subordinates compared to males; for example, Tea et al. (2019) observed the formation of hierarchies among female zebrafish without elevations in plasma cortisol and changes in neurogenesis, differently from males. Likewise, Filby et al. (2010a) did not observe, in female subordinate zebrafish, any changes in the expression arousal of pathway genes that were also observed in males. Thus, sex differences in the physiological consequences of psychosocial stress suggest that female zebrafish would be less sensitive than males to possible anxiogenic-like effects of subordination stress.

After 5 days of co-housing, a clear dominance hierarchy was established across all dyads, irrespective of sex (c_quad = 0.599, p = 0.74, df = 2). Sex differences were found in the number of attacks, with females, in general, attacking more than males, but no differences were seen between males and females in other variables used to classify subjects as dominant or subordinate (i.e., chasing, bottom-dwelling, immobility, and retreats). A POSET-test for all variables did not find significant differences between males and females (Z = 0.6782, p = 0.619), reinforcing the idea that both male and female zebrafish can enter into dominant-subordinate hierarchies. Table 1 shows the effect sizes for each endpoint.

Stratification of status (dominant/subordinate) by sex revealed a significant effect on behavior in the NTT (POSET test, maxT = 6.175, p < 0.0001), with no effects of sex (step-down adjusted p = 0.649), but a significant effect of status (step-down adjusted p < 0.0001). Overall, subordinates spent more time at the bottom of the tank (Figure 2A), less time at the top of the tank (Figure 2B), increased absolute turn angles (Figure 2C), and increased freezing (Figure 2D) than both controls and dominant individuals, but no differences in swimming speed were observed (Figure 2E).

The present work tested the hypothesis that status in a dominance-subordinate hierarchy increases anxiety-like behavior in the zebrafish. Overall, both male and female zebrafish were capable of establishing dominance, with subordinate males and females displaying increased anxiety-like behavior in the novel tank test after the establishment of the hierarchy.

While adaptive changes in the physiology of subordinate zebrafish has been observed before, which are consistent with increased defensive/anxiety-like behavior, especially in the up-regulation of expression and activity of the hypothalamus-pituitary-internal (stress) and serotonergic pathways, behavioral adjustments have not been reported before. Dahlbom et al. (2011) reported that zebrafish with a “bolder” profile, spending more time in the center of an open tank and being more active in the novel object test, were more likely to become dominant in following tournaments, suggesting that pre-existing differences in anxiety-like behavior in regards to novelty are predictive of the animal’s ability to attain dominance. Indeed, a boldness-aggression syndrome has been repeatedly observed in zebrafish across stocks, strains, and contexts (Moretz et al., 2007; Norton et al., 2011; Ariyomo et al., 2013; Martins and Bhat, 2014; Mustafa et al., 2019), suggesting that lower anxiety-like behavior is associated with higher aggression, which in its turn is predictive of the capacity to become dominant. However, in the present study subordination elicited increased anxiety-like behavior in the NTT in comparison to controls, suggesting that heightened anxiety was not a pre-existing difference across conditions.

Interestingly, both males and females were capable of establishing hierarchies. While the number of mixed-sex pairs was relatively small, and therefore it’s not possible to ascertain whether male-female dyads consistently produce hierarchies instead of simply leading to attempts to courtship and/or male coercion, in two of the pairs, the female was indeed dominant, patrolling the top of the tank and chasing the subordinate male. Further experiments are needed to confirm whether females commonly become dominant over males.

Differences between sexes were also apparently absent in the effects of subordination on anxiety-like behavior. Sex differences have been observed before in zebrafish in both directions. For example, while male subordinate zebrafish were observed to display higher cortisol levels and impaired neurogenesis, female subordinate zebrafish did not display such an effect (Tea et al., 2019), suggesting that females might not be susceptible to the anxiogenic-like effect of subordination stress. However, baseline anxiety-like behavior was observed to be higher in females than in male zebrafish (Genario et al., 2020). Both results, which point to different directions, are inconsistent with the apparent lack of effect of sex on anxiety-like behavior. Regarding effect sizes, it appears that female subordinates showed a larger difference in absolute turn angle (erratic swimming) than subordinate males, suggesting sex differences in specific behaviors. Direct and conceptual replication attempts at other laboratories and contexts could further clarify the robustness of the lack of sex differences in the anxiogenic-like effects of subordination in zebrafish.

Overall, the present study shows that, in zebrafish, subordination is a psychosocial stressor that can lead to increased anxiety-like behavior. This has been observed previously in mammals (Blanchard et al., 1993), an effect that has been further exploited as a model to understand the neurobehavioral effects of social stress and its relations to anxiety and depression. While previously subordinate zebrafish have been shown to display behaviors which are consistent with an anxiety-like state during dyadic encounters (Filby et al., 2010a), systematic changes in behavior in a different context (to rule out behavioral displays of submission) and in tests explicitly designed to assess anxiety-like behavior were not previously used. The convergent evidence from rodents and zebrafish referring to dominant/subordinate stress effects to anxiety-like subordinate behavior points towards the existence of a conserved trend across social species, which should be further exploited to understand the extent of similarity/dissimilarity of the underlying mechanisms (Gerlai, 2020).

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://github.com/lanec-unifesspa/zebrafish_subordination.

The animal study was reviewed and approved by CEUA/UEPA.

BB: conceptualization, methodology, investigation, data curation, formal analysis, visualization, and writing—original draft. JR: conceptualization, investigation, and formal analysis. ML-M, DS-S, and MS: conceptualization, formal analysis, and writing—original draft. CM: conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, software, supervision, validation, visualization, writing—original draft, writing—review and editing. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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