Animals communicate by exchanging signals frequently in the proximity of other conspecifics that may detect and intercept signals not directed to them. There is evidence that the presence of these bystanders modulates the signaling behavior of interacting individuals, a phenomenon that has been named audience effect. Research on the audience effect has predominantly focused on its function rather than on its proximate mechanisms. Here, we have investigated the physiological and neuromolecular correlates of the audience effect in a cichlid fish (Mozambique tilapia, Oreochromis mossambicus). A male was exposed to a territorial intrusion in the presence or absence of a female audience. Results showed that the presence of the female audience increased territorial defense, but elicited a lower androgen and cortisol response to the territorial intrusion. Furthermore, analysis of the expression of immediate early genes, used as markers of neuronal activity, in brain areas belonging to the social decision-making network (SDMN) revealed different patterns of network activity and connectivity across the different social contexts (i.e., audience × intrusion). Overall, these results suggest that socially driven plasticity in the expression of territorial behavior is accommodated in the central nervous system by rapid changes in functional connectivity between nodes of relevant networks (SDMN) rather than by localized changes of activity in specific brain nuclei.

The Challenge Hypothesis (Wingfield et al. Am. Nat. 136,(829)(830)(831)(832)(833)(834)(835)(836)(837)(838)(839)(840)(841)(842)(843)(844)(845)(846) aims to explain the complex relationship between androgens and social interactions. Despite its well acceptance in the behavioral endocrinology literature, several studies have failed to found an androgen response to staged social interactions. Possible reasons for these inconsistencies are the use of single sampling points that may miss the response peak, and the occurrence of inter-individual variability in the androgen response to social interactions. In this study we addressed these two possible confounding factors by characterizing the temporal pattern of the androgen response to social interactions in the African cichlid, Oreochromis mossambicus, and relating it to inter-individual variation in terms of the individual scope for androgen response (i.e. the difference between baseline and maximum physiological levels for each fish) and behavioral types. We found that the androgen response to territorial intrusions varies between individuals and is related to their scope for response. Individuals that have a lower scope for androgen response did not increase androgens after a territorial intrusion but were more aggressive and exploratory. In contrast males with a higher scope for response had fewer aggressive and exploratory behaviors and exhibited two peaks of KT, an early response 2-15 min after the interaction and a late response at 60-90 min post-interaction. Given that the pharmacological challenge of the Hypothalamic-Pituitary-Gonad axis only elicits the late response, we suggest that these two peaks may be regulated by different physiological mechanisms, with the early response being mediated by direct brain-gonad neural pathways. In summary, we suggest that determining the temporal pattern of the androgen response to social interactions and considering inter-individual variation may be the key to understanding the contradictory results of the Challenge Hypothesis.

21It has been hypothesized that androgens respond to the social interactions as a way to adjust 22 the behaviour of individuals to the challenges of the social environment in an adaptive 23 manner. 24 Therefore, it is expected that transient changes in circulating androgen levels within 25 physiological scope should impact the state of the brain network that regulates social 26 behavior, which should translate into adaptive behavioural changes. Here, we examined the 27 effect that a transient peak in androgen circulating levels, which mimics socially driven 28 changes in androgen levels, has on the forebrain state, which harbors most nuclei of the social 29 decision-making network. For this purpose, we successfully induced transient changes in 30 circulating androgen levels in an African cichlid fish (Mozambique tilapia, Oreochromis 31 mossambicus) commonly used as a model in behavioral neuroendocrinology by injecting 11-32 ketotestosterone or testosterone, and compared the forebrain transcriptome of these 33 individuals to control fish injected with vehicle. Forebrain samples were collected 30 min and 34 60 min after injection and analyzed using RNAseq. Our results showed that a transient peak in 35 11-ketotestosterone drives more accentuated changes in forebrain transcriptome than 36 testosterone, and that transcriptomic impact was greater at the 30 min than at the 60 min post-37 androgen administration. Several genes involved in the regulation of translation, steroid 38 metabolism, ion channel membrane receptors, and genes involved in epigenetic mechanisms 39 were differentially expressed after 11-ketotestosterone or testosterone injection. In summary, 40 this study identified specific candidate genes that may regulate socially driven changes in 41 behavioural flexibility mediated by androgens. 42 130 Animals and housing 131O. mossambicus adult males from a stock held at ISPA were used in this experiment. Fish 132 were maintained in glass tanks (120 x 40 x 50 cm, 240 l) with a fine gravel substrate. Tanks 133 were supplied with a double filtering system (gravel and external biofilter) and constant 134 aeration. Water quality was analyzed twice per month for nitrites (0.2-0.5 ppm), ammonia 135 (<0.5 ppm, Pallintest kit) and pH (6.0 -6.2). Fish were kept at a temperature of 26 ± 2 ºC, a 136 12L:12D photoperiod, and fed with commercial cichlid floating sticks. Ninety-nine focal 137