A key goal in animal behavior and sensory biology research has been to disentangle the specific sensory stimuli that control behaviors across species (Searcy and Nowicki, 2010). Concerning reproductive behaviors, much focus has historically been given to the role of male sexual signaling systems in driving female mating behavior. Decades of research have revealed that auditory cues, chemosensory signals, behavioral displays, coloration, and hormonal factors are all known to impact female mate choice in fish, reptiles, insects, birds, mammals, and humans (Byers et al., 2010; Maney et al., 2006; McComb, 1991; Waitt et al., 2003).

Identifying the role of specific male signals in guiding female mating behaviors, however, can be challenging. For instance, if variation in signaling traits along multiple modalities correlate with female mating responses, studying the role of individual signaling traits may prove difficult. Thus, precisely identifying the roles of distinct sensory modalities in modifying behavior is challenging. Therefore, a major goal of research into the influence of different signaling modalities affecting conspecific behavior has been to experimentally isolate variation only in a single trait of interest.

Astatotilapia burtoni, an African cichlid fish, develop plastic social hierarchies in which an individual male's social status is reflected by his body coloration and behavior (Fernald, 2012; Fernald and Maruska, 2012; Maruska, 2015). Dominant males are brightly colored with a dark eye bar, defend a territory, and mate frequently, while non-dominant males are drably colored, do not defend a territory, and do not mate. The unique ability of male A. burtoni to quickly alter the intensity of their coloration based on their social status or environment makes these fish a fascinating model for exploring the multifaceted systems controlling social behavior and mate preference (Alward et al., 2023; Jackson et al., 2023; Maruska and Fernald, 2018; Stevenson et al., 2018). A. burtoni are a mouthbrooding species whose courtship ritual involves a male performing a suite of behaviors that culminate in spawning and the fertilization of eggs (Juntti and Fernald, 2016). First, the male will chase the female; then he will perform a quiver towards the female, in which he arches his body, displays egg spots on his fin, and rapidly vibrates his whole body. Following quivers, the male will typically lead the female back to his territory. If she follows, this may result in circling behaviors where the male and female peck at each other's anogenital regions to stimulate the release of gametes. The female will then scoop up her released eggs into her mouth, which are fertilized by sperm the male releases into the water. This process in females is thought to be initiated and mediated by multiple sensory cues from the male, including chemosensory signals, auditory cues, visual cues, social status, coloration, and behavior (King et al., 2022; Maruska and Butler, 2021).

One of the most striking male features that has been assumed to guide female behavior is the bright coloration displayed by dominant males. However, no study has provided evidence to support the hypothesis that male coloration mediates female mate preference in A. burtoni. This is because coloration correlates tightly with all aspects of social status, which can change rapidly. Indeed, within minutes after being isolated, an initially subordinate male will begin to express vibrant colors and behaviors typical of a dominant male The only way to mute body coloration in A. burtoni is to socially suppress the male, which suppresses other aspects of social status, including behavior. Therefore, current experimental manipulations in male A. burtoni do not allow for an accurate interpretation of the role of coloration in guiding female mating behaviors given the inextricable confounds of behavioral changes associated with social status suppression.

Using androgen receptor (AR) mutant A. burtoni engineered using CRISPR/Cas9 gene editing, we have previously shown that two distinct AR genes are required for non-overlapping traits of social dominance in males such as coloration, behavior, and reproductive physiology (Alward et al., 2020). ARβ mutant males perform normal levels of mating and territorial behaviors, but lack the bright yellow or blue body coloration typical of wild-type (WT) dominant males. Specifically, in a dyadic assay in which focal ARβ males were housed with a smaller stimulus male and three females, ARβ mutant males showed drab coloration, possessed small testes, but maintained normal levels of mating and territorial behavior like quivers and lateral displays. Moreover, ARβ mutant males possess testosterone levels indistinguishable from WT males and slightly higher levels of 11-ketotestosterone than WT males. Using ARβ mutants, this study aimed to determine if male body coloration impacts female behavior in A. burtoni. While we do not know whether ARβ mutant males differ in the release of pheromonal or other chemical cues, the presence of normal levels of male-typical mating behaviors in these drably colored ARβ mutants presents a unique opportunity to study the potential role of coloration in driving mating behaviors in female A. burtoni.

In our current study, we also examined possible molecular mechanisms in the brain underlying female responses to color by quantifying the activation of esr2b+ cells in the hypothalamus. Esr2b, the gene that encodes estrogen receptor (ER) βb (ERβb) was recently shown to be necessary for female mating behaviors in Medaka (Oryzias latipes) (Nishiike et al., 2021) and is expressed higher in the ventral telencephalon and preoptic area (POA), regions of the hypothalamus known to be functionally related to mating behavior. Recent work has shown that visual signals enhance neural activation as measured by expression of the immediate early gene c-fos (Fos proto-oncogene, AP-1 transcription factor subunit) in the A. burtoni hypothalamus. For example, visual signals alone from females enhanced neural activation in the ventral nucleus of the ventral telencephalon (Vv) and POA of males (Field and Maruska, 2017), while visual signals alone from males enhanced neural activation in the Vv of females (King et al., 2022). While it is not known whether esr2b specifically is involved in the processing of social information, previous work has shown estradiol sensitizes female songbirds to courtship song from males (Dabelsteen, 1988; Maney and Pinaud, 2011; Searcy and Capp, 1997; Straughan et al., 1981) and retinal sensitivity in female frogs that assess socio-sexual visual cues from males (Leslie et al., 2021), suggesting estrogen signaling plays a role in processing social signals related to mating. Here, activation of esr2b+ cells was quantified by the expression of the immediate early gene egr1 (early growth response factor 1), a marker of cellular activation in the brain (Clayton, 2000). Because coloration has proved to be an important factor in mating for many species (Byers et al., 2010; Waitt et al., 2003), we expected to see behavioral measures indicating a female preference towards WT males opposed to ARβ mutant males that would correspond to higher activation of esr2b + cells in the hypothalamus.