Androgen signaling in LMAN regulates song stereotypy in male canaries.

During breeding when testosterone concentrations are high, male songbirds that are open-ended vocal learners like canaries (Serinus canaria) tend to produce a stable, stereotyped song that facilitates mate attraction or territory defense. Outside breeding contexts, song becomes more variable. The neuroendocrine mechanisms controlling this vocal variability across seasons are not entirely clear. We tested whether androgen signaling within the lateral magnocellular nucleus of the anterior nidopallium (LMAN), a cortical-like brain region of the vocal control system known as a vocal variability generator, plays a role in seasonal vocal variability. We first characterized song in birds housed alone on a short day (SD) photoperiod, which simulates non-breeding conditions. Then, cannulae filled with the androgen receptor (AR) blocker flutamide or left empty as control were implanted bilaterally in LMAN. Birds were then transferred to long days (LD) to simulate the breeding season and song was analyzed again. Blocking AR in LMAN increased acoustic variability of song and the acoustic variability of syllables. However, blocking AR in LMAN did not impact the variability of syllable usage nor their sequencing in LD birds, song features that are controlled by androgen signaling in a somatosensory brain region of the vocal control system called HVC. These findings highlight the multifactorial, non-redundant actions of steroid hormones in controlling complex social behaviors such as birdsong. They also support the hypothesis that LMAN is a key brain area for the effects of testosterone on song plasticity both seasonally in adults and during the song crystallization process at sexual maturity.

Female cichlids mate with novel androgen receptor mutant males that lack coloration.

A key challenge in animal behavior is disentangling the social stimuli that drive conspecific behaviors. For some species, like teleost fish, putative sexual signaling cues are inextricably linked to others, making it difficult to parse the precise roles distinct signals play in driving conspecific behaviors. In the African cichlid Astatotilapia burtoni, males are either dominant or subordinate, wherein bright coloration, territoriality, and courtship behavior inextricably correlate positively with rank. Here, we leveraged androgen receptor (AR) mutant male A. burtoni that lack dominance-typical coloration but not behavior to isolate the role of male coloration in driving female mating behaviors in this species. We found in independent behavioral assays that females behave aggressively towards AR mutant but not WT males, yet still mated with both types of males. Females showed enhanced activation of esr2b + cells in the hypothalamus when housed with either mutant or WT males and this activation scaled with spawning activities. Therefore, there is not a simple relationship between male coloration and female mating behaviors in A. burtoni, suggesting independent sensory mechanisms converge on hypothalamic esr2b cells to coordinate behavioral output.

A phylogenetics-based nomenclature system for steroid receptors in teleost fishes.

Teleost fishes have emerged as tractable models for studying the neuroendocrine regulation of social behavior via molecular genetic techniques, such as CRISPR/Cas9 gene editing. Moreover, teleosts provide an opportunity to investigate the evolution of steroid receptors and their functions, as species within this lineage possess novel steroid receptor paralogs that resulted from a teleost-specific whole genome duplication. Although teleost fishes have grown in popularity as models for behavioral neuroendocrinology, there is not a consistent nomenclature system for steroid receptors and their genes, which may impede a clear understanding of steroid receptor paralogs and their functions. Here, we used a phylogenetic approach to assess the relatedness of protein sequences encoding steroid receptor paralogs in 18 species from 12 different orders of the Infraclass Teleostei. While most similarly named sequences grouped based on the established phylogeny of the teleost lineage, our analysis revealed several inconsistencies in the nomenclature of steroid receptor paralogs, particularly for sequences encoding estrogen receptor beta (ERß). Based on our results, we propose a nomenclature system for teleosts in which Greek symbols refer to proteins and numbers refer to genes encoding different subtypes of steroid receptors within the five major groups of this nuclear receptor subfamily. Collectively, our results bridge a critical gap by providing a cohesive naming system for steroid receptors in teleost fishes, which will serve to improve communication, promote collaboration, and enhance our understanding of the evolution and function of steroid receptors across vertebrates.

Genetic dissection of steroid-hormone modulated social behavior: Novel paralogous genes are a boon for discovery.

Research across species has led to important discoveries on the functions of steroid hormones in the regulation of behavior. However, like in many fields, advancements in transgenic and mutagenic technology allowed mice to become the premier genetic model for conducting many experiments to understand how steroids control social behavior. Since there has been a general lack of parallel methodological developments in other species, many of the findings cannot be generalized. This is especially the case for teleost fish, in which a whole-genome duplication produced novel paralogs for key steroid hormone signaling genes. In this review, we summarize technical advancements over the history of the field of neuroendocrinology that have led to important insights in our understanding of the control of social behavior by steroids. We demonstrate that early mouse genetic models to understand these mechanisms suffered from several issues that were remedied by more precise transgenic technological advancements. We then highlight the importance of CRISPR/Cas9 gene editing tools that will in time bridge the gap between mice and non-traditional model species for understanding principles of steroid hormone action in the modulation of social behavior. We specifically highlight the role of teleost fish in bridging this gap because they are 1) highly genetically tractable and 2) provide a novel advantage in achieving precise genetic control. The field of neuroendocrinology is entering a new "gene editing revolution" that will lead to novel discoveries about the roles of steroid hormones in the regulation and evolutionary trajectories of social behavior.

Modular genetic control of social status in a cichlid fish.

Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fish Astatotilapia burtoni is a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. Dominant A. burtoni males possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status in A. burtoni is in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication, A. burtoni possess two androgen receptor (AR) paralogs, ARα and ARß, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutant A. burtoni and performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find that ARß, but not ARα, is required for testes growth and bright coloration, while ARα, but not ARß, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status in A. burtoni is inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity in A. burtoni and other species whose social status relies on social experience.

Hormonal regulation of social ascent and temporal patterns of behavior in an African cichlid.

For many species, social rank determines which individuals perform certain social behaviors and when. Higher ranking or dominant (DOM) individuals maintain status through aggressive interactions and perform courtship behaviors while non-dominant (ND) individuals do not. In some species ND individuals ascend (ASC) in social rank when the opportunity arises. Many important questions related to the mechanistic basis of social ascent remain to be answered. We probed whether androgen signaling regulates social ascent in male Astatotilapia burtoni, an African cichlid whose social hierarchy can be readily controlled in the laboratory. As expected, androgen receptor (AR) antagonism abolished reproductive behavior during social ascent. However, we discovered multiple AR- and status-dependent temporal behavioral patterns that typify social ascent and dominance. AR antagonism in ASC males increased the time between successive behaviors compared to DOM males. Socially ascending males, independent of AR activation, were more likely than DOM males to follow aggressive displays with another aggressive display. Further analyses revealed differences in the sequencing of aggressive and courtship behaviors, wherein DOM males were more likely than ASC males to follow male-directed aggression with courtship displays. Strikingly, this difference was driven mostly by ASC males taking longer to transition from aggression to courtship, suggesting ASC males can perform certain DOM-typical temporal behavioral patterns. Our results indicate androgen signaling is necessary for social ascent and hormonal signaling and social experience may shape the full suite of DOM-typical behavioral patterns.

Dissociable Effects on Birdsong of Androgen Signaling in Cortex-Like Brain Regions of Canaries.

The neural basis of how learned vocalizations change during development and in adulthood represents a major challenge facing cognitive neuroscience. This plasticity in the degree to which learned vocalizations can change in both humans and songbirds is linked to the actions of sex steroid hormones during ontogeny but also in adulthood in the context of seasonal changes in birdsong. We investigated the role of steroid hormone signaling in the brain on distinct features of birdsong using adult male canaries (Serinus canaria), which show extensive seasonal vocal plasticity as adults. Specifically, we bilaterally implanted the potent androgen receptor antagonist flutamide in two key brain regions that control birdsong. We show that androgen signaling in the motor cortical-like brain region, the robust nucleus of the arcopallium (RA), controls syllable and trill bandwidth stereotypy, while not significantly affecting higher order features of song such syllable-type usage (i.e., how many times each syllable type is used) or syllable sequences. In contrast, androgen signaling in the premotor cortical-like brain region, HVC (proper name), controls song variability by increasing the variability of syllable-type usage and syllable sequences, while having no effect on syllable or trill bandwidth stereotypy. Other aspects of song, such as the duration of trills and the number of syllables per song, were also differentially affected by androgen signaling in HVC versus RA. These results implicate androgens in regulating distinct features of complex motor output in a precise and nonredundant manner.SIGNIFICANCE STATEMENT Vocal plasticity is linked to the actions of sex steroid hormones, but the precise mechanisms are unclear. We investigated this question in adult male canaries (Serinus canaria), which show extensive vocal plasticity throughout their life. We show that androgens in two cortex-like vocal control brain regions regulate distinct aspects of vocal plasticity. For example, in HVC (proper name), androgens regulate variability in syntax but not phonology, whereas androgens in the robust nucleus of the arcopallium (RA) regulate variability in phonology but not syntax. Temporal aspects of song were also differentially affected by androgen signaling in HVC versus RA. Thus, androgen signaling may reduce vocal plasticity by acting in a nonredundant and precise manner in the brain.

Sex differences in hippocampal mineralocorticoid and glucocorticoid receptor mRNA expression in response to acute mate pair separation in zebra finches (Taeniopygia guttata).

Mate separation has been shown to mediate changes in physiological and behavioral processes via activation of the hypothalamo-pituitary-adrenal (HPA) axis in both mammalian and avian species. To elucidate the neural mechanisms associated with changes in the HPA axis in response to social stress, we investigated the effects of mate pair separation on circulating corticosterone concentrations as well as gene expression levels of mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and corticotropin releasing hormone (CRH) in the hypothalamus and hippocampus of both male and female zebra finches, a species that forms strong pair bonds. Zebra finches (Taeniopygia guttata) were housed three to a cage (a mated pair plus a stimulus female), and were assigned to one of three new housing treatment groups: (1) male or female removed from their respective mate and placed in a cage with a new opposite sex conspecific and stimulus female (2) male or female that remained with their mate, but a new stimulus female was introduced, or (3) the subjects were handled but not separated from their mate or the stimulus female. After 48 hr in the new housing condition, we observed significant increases in plasma corticosterone concentrations in response to both mate pair and stimulus female separation. No significant differences in MR, GR, or CRH mRNA expression in the hypothalamus were observed in response to any treatment for both males and females. Females exhibited a significant up regulation in hippocampal MR, but not GR mRNA, whereas males exhibited a significant down regulation of both hippocampal MR and GR mRNA in response to mate pair separation. Thus, the hippocampus appears to play a key role in regulating sex specific responses to social stressors.

The regulation of birdsong by testosterone: Multiple time-scales and multiple sites of action.

Contribution to Special Issue on Fast effects of steroids. Sex steroid hormones act during early development to shape the circuitry upon which these same hormones act in adulthood to control behavioral responses to various stimuli. The "organizational" vs. "activational" distinction was proposed to explain this temporal difference in hormone action. In both of these cases steroids were thought to act genomically over a time-scale of days to weeks. However, sex steroids can affect behavior over short (e.g., seconds or minutes) time-scales. Here, we discuss how testosterone controls birdsong via actions at different sites and over different time-scales, with an emphasis on this process in canaries (Serinus canaria). Our work shows that testosterone in the medial preoptic nucleus regulates the motivation to sing, but not aspects of song performance. Instead, different aspects of song performance are regulated by long-term actions of testosterone in steroid-sensitive cortical-like brain regions and the syrinx, the avian vocal production organ. On the other hand, acute aromatase inhibition rapidly reduces the availability of estrogens and this reduction is correlated with reductions in the motivation to sing and song performance. Thus, testosterone and its estrogenic metabolites regulate distinct features of birdsong depending on the site and temporal window of action. The number of brain areas expressing androgen receptors is higher in species producing learned vocalization as compared to species that produce unlearned calls. An appealing scenario is that rapid effects of steroids in specific brain regions is a derived trait secondary to the widespread genomic effects of steroids in systems where steroids coordinate morphological, physiological, and behavioral traits.

Differential effects of global versus local testosterone on singing behavior and its underlying neural substrate.

Steroid hormones regulate multiple but distinct aspects of social behaviors. Testosterone (T) has multiple effects on learned courtship song in that it regulates both the motivation to sing in a particular social context as well as the quality of song produced. The neural substrate(s) where T acts to regulate the motivation to sing as opposed to other aspects of song has not been definitively characterized. We show here that T implants in the medial preoptic nucleus (POM) of castrated male canaries (Serinus canaria) increase song rate but do not enhance acoustic features such as song stereotypy compared with birds receiving peripheral T that can act globally throughout the brain. Strikingly, T action in the POM increased song control nuclei volume, consistent with the hypothesis that singing activity induces neuroplasticity in the song control system independent of T acting in these nuclei. When presented with a female canary, POM-T birds copulated at a rate comparable to birds receiving systemic T but produced fewer calls and songs in her presence. Thus, POM is a key site where T acts to activate copulation and increase song rate, an appetitive sexual behavior in songbirds, but T action in other areas of the brain or periphery (e.g., HVC, dopaminergic cell groups, or the syrinx) is required to enhance the quality of song (i.e., stereotypy) as well as regulate context-specific vocalizations. These results have broad implications for research concerning how steroids act at multiple brain loci to regulate distinct sociosexual behaviors and the associated neuroplasticity.

Dissociable effects of social context on song and doublecortin immunoreactivity in male canaries.

Variation in environmental factors such as day length and social context greatly affects reproductive behavior and the brain areas that regulate these behaviors. One such behavior is song in songbirds, which males use to attract a mate during the breeding season. In these species the absence of a potential mate leads to an increase in the number of songs produced, while the presence of a mate greatly diminishes singing. Interestingly, although long days promote song behavior, producing song itself can promote the incorporation of new neurons in brain regions controlling song output. Social context can also affect such neuroplasticity in these song control nuclei. The goal of the present study was to investigate in canaries (Serinus canaria), a songbird species, how photoperiod and social context affect song and the incorporation of new neurons, as measured by the microtubule-associated protein doublecortin (DCX) in HVC, a key vocal production brain region of the song control system. We show that long days increased HVC size and singing activity. In addition, male canaries paired with a female for 2 weeks showed enhanced DCX-immunoreactivity in HVC relative to birds housed alone. Strikingly, however, paired males sang fewer songs that exhibited a reduction in acoustic features such as song complexity and energy, compared with birds housed alone, which sang prolifically. These results show that social presence plays a significant role in the regulation of neural and behavioral plasticity in songbirds and can exert these effects in opposition to what might be expected based on activity-induced neurogenesis.

Androgen receptor deficiency is associated with reduced aromatase expression in the ventromedial hypothalamus of male cichlids.

Social behaviors are regulated by sex steroid hormones, such as androgens and estrogens. However, the specific molecular and neural processes modulated by steroid hormones to generate social behaviors remain to be elucidated. We investigated whether some actions of androgen signaling in the control of social behavior may occur through the regulation of estradiol synthesis in the highly social cichlid fish, Astatotilapia burtoni. Specifically, we examined the expression of cyp19a1, a brain-specific aromatase, in the brains of male A. burtoni lacking a functional ARα gene (ar1), which was recently found to be necessary for aggression in this species. We found that cyp19a1 expression is higher in wild-type males compared to ar1 mutant males in the anterior tuberal nucleus (ATn), the putative fish homolog of the mammalian ventromedial hypothalamus, a brain region that is critical for aggression across taxa. Using in situ hybridization chain reaction, we determined that cyp19a1+ cells coexpress ar1 throughout the brain, including in the ATn. We speculate that ARα may modulate cyp19a1 expression in the ATn to govern aggression in A. burtoni. These studies provide novel insights into the hormonal mechanisms of social behavior in teleosts and lay a foundation for future functional studies.

Expression of novel androgen receptors in three GnRH neuron subtypes in the cichlid brain.

Within a social hierarchy, an individuals' social status determines its physiology and behavior. In A. burtoni, subordinate males can rise in rank to become dominant, which is accompanied by the upregulation of the entire HPG axis, including activation of GnRH1 neurons, a rise in circulating androgen levels and the display of specific aggressive and reproductive behaviors. Cichlids possess two other GnRH subtypes, GnRH2 and GnRH3, the latter being implicated in the display of male specific behaviors. Interestingly, some studies showed that these GnRH neurons are responsive to fluctuations in circulating androgen levels, suggesting a link between GnRH neurons and androgen receptors (ARs). Due to a teleost-specific whole genome duplication, A. burtoni possess two AR paralogs (ARα and ARß) that are encoded by two different genes, ar1 and ar2, respectively. Even though social status has been strongly linked to androgens, whether ARα and/or ARß are present in GnRH neurons remains unclear. Here, we used immunohistochemistry and in situ hybridization chain reaction (HCR) to investigate ar1 and ar2 expression specifically in GnRH neurons. We find that all GnRH1 neurons intensely express ar1 but only a few of them express ar2, suggesting the presence of genetically-distinct GnRH1 subtypes. Very few ar1 and ar2 transcripts were found in GnRH2 neurons. GnRH3 neurons were found to express both ar genes. The presence of distinct ar genes within GnRH neuron subtypes, most clearly observed for GnRH1 neurons, suggests differential control of these neurons by androgenic signaling. These findings provide valuable insight for future studies aimed at disentangling the androgenic control of GnRH neuron plasticity and reproductive plasticity across teleosts.

Expression of novel androgen receptors in three GnRH neuron subtypes in the cichlid brain.

In teleosts, GnRH1 neurons stand at the apex of the Hypothalamo-Pituitary-Gonadal (HPG) axis, which is responsible for the production of sex steroids by the gonads (notably, androgens). To exert their actions, androgens need to bind to their specific receptors, called androgen receptors (ARs). Due to a teleost-specific whole genome duplication, A. burtoni possess two AR paralogs (ARα and ARß) that are encoded by two different genes, ar1 and ar2, respectively. In A. burtoni, males stratify along dominance hierarchies, in which an individuals' social status determines its physiology and behavior. GnRH1 neurons have been strongly linked with dominance and circulating androgen levels. Similarly, GnRH3 neurons are implicated in the display of male specific behaviors. Some studies have shown that these GnRH neurons are responsive to fluctuations in circulating androgens levels, suggesting a link between GnRH neurons and ARs. While female A. burtoni do not naturally form a social hierarchy, their reproductive state is positively correlated to androgen levels and GnRH1 neuron size. Although there are reports related to the expression of ar genes in GnRH neurons in cichlid species, the expression of each ar gene remains inconclusive due to technical limitations. Here, we used immunohistochemistry, in situ hybridization chain reaction (HCR), and spatial transcriptomics to investigate ar1 and ar2 expression specifically in GnRH neurons. We find that all GnRH1 neurons intensely express ar1 but only a few of them express ar2, suggesting the presence of genetically-distinct GnRH1 subtypes. Very few ar1 and ar2 transcripts were found in GnRH2 neurons. GnRH3 neurons were found to express both ar genes. The presence of distinct ar genes within GnRH neuron subtypes, most clearly observed for GnRH1 neurons, suggests differential control of these neurons by androgenic signaling. These findings provide valuable insight for future studies aimed at disentangling the androgenic control of GnRH neuron plasticity and reproductive plasticity across teleosts.

Androgen receptor alpha deficiency impacts aromatase expression in the female cichlid brain.

Steroid hormones bind to specific receptors that act as transcription factors to modify gene expression in the brain to regulate physiological and behavioural processes. The specific genes controlled by steroid hormones in the brain are not fully known. Identifying these genes is integral to establishing a comprehensive understanding of how hormones impact physiology and behaviour. A popular organism for answering this question is the cichlid fish Astatotilapia burtoni. Recently, CRISPR/Cas9 was used to engineer A. burtoni that lack functional androgen receptor (AR) genes encoding ARα. ARα mutant male A. burtoni produced fewer aggressive displays and possessed reduced expression of the gene encoding brain-specific aromatase, cyp19a1, in the ventromedial hypothalamus (VMH), an aggression locus. As a follow-up, we investigated whether ARα deficiency affected cyp19a1 expression in female A. burtoni using the same genetic line. We find that female A. burtoni possessing one or two non-functional ARα alleles had much higher expression of cyp19a1 in the preoptic area (POA), while females with one non-functional ARα allele possessed lower expression of cyp19a1 in the putative fish homologue of the bed nucleus of the stria terminalis (BNST). Thus, ARα may have a sex-specific role in modifying cyp19a1 expression in the teleost POA and BNST, regions that underlie sex differences across vertebrates.

Sexually dimorphic control of aggression by androgen signaling in a cichlid.

Innate social behaviors like aggression are modulated by sex steroid hormones such as androgens and estrogens. However, we know little about how the same hormone regulates similar behaviors in both sexes. We investigated the role of androgenic signaling in the regulation of aggression in Astatotilapia burtoni, a social fish in which males and females perform similar aggressive behaviors. We used androgen receptor (AR) α knockout (KO) animals for this study since this gene was recently shown to be required for male-typical aggression and mating. Surprisingly, ARα KO females did not show deficits in aggression. We also determined that females lacking the other AR, ARß, showed normal levels of aggression. Blocking both ARs pharmacologically confirmed that neither AR is necessary for aggression in females. However, ARα KO males showed clear deficits in attacks. Thus, in A. burtoni there appears to be a sexual dimorphism in the role of ARα in the control of aggression.

Sexually dimorphic control of aggression by androgen signaling in a cichlid.

Innate social behaviors like aggression are modulated by sex steroid hormones such as androgens and estrogens. However, we know little about how the same hormone regulates similar behaviors in both sexes. We investigated the role of androgenic signaling in the regulation of aggression in Astatotilapia burtoni, a social fish in which males and females perform similar aggressive behaviors. We used ARa knockout (KO) animals for this study, which was recently shown to be required for male-typical aggression and mating. Surprisingly, ARα KO females did not show deficits in aggression. We also determined that females lacking the other AR, ARß, showed normal levels of aggression. Blocking both ARs pharmacologically confirmed that neither AR is necessary for aggression in females. However, ARα KO males showed clear deficits in attacks. Thus, in A. burtoni there appears to be a sexual dimorphism in the role of ARα in the control of aggression.

Sex differences in aggression and its neural substrate in a cichlid fish.

Aggression is ubiquitous among social species and functions to maintains social dominance hierarchies. The African cichlid fish Astatotilapia burtoni is an ideal study species for studying aggression due to their unique and flexible dominance hierarchy. However, female aggression in this species and the neural mechanisms of aggression in both sexes is not well understood. To further understand the potential sex differences in aggression in this species, we characterized aggression in male and female A. burtoni in a mirror assay. We then quantified neural activation patterns in brain regions of the social behavior network (SBN) to investigate if differences in behavior are reflected in the brain with immunohistochemistry by detecting the phosphorylated ribosome marker phospho-S6 ribosomal protein (pS6), a marker for neural activation. We found that A. burtoni perform both identical and sex-specific aggressive behaviors in response to a mirror assay. We observed sex differences in pS6 immunoreactivity in the Vv, a homolog of the lateral septum in mammals. Males but not females had higher ps6 immunoreactivity in the ATn after the aggression assay. The ATn is a homolog of the ventromedial hypothalamus in mammals, which is strongly implicated in the regulation of aggression in males. Several regions also have higher pS6 immunoreactivity in negative controls than fish exposed to a mirror, implicating a role for inhibitory neurons in suppressing aggression until a relevant stimulus is present. Male and female A. burtoni display both similar and sexually dimorphic behavioral patterns in aggression in response to a mirror assay. There are also sex differences in the corresponding neural activation patterns in the SBN. In mirror males but not females, the ATn clusters with the POA, revealing a functional connectivity of these regions that is triggered in an aggressive context in males. These findings suggest that distinct neural circuitry underlie aggressive behavior in male and female A. burtoni, serving as a foundation for future work investigating the molecular and neural underpinnings of sexually dimorphic behaviors in this species to reveal fundamental insights into understanding aggression.

Breaking Through the Bottleneck: Krogh's Principle in Behavioral Neuroendocrinology and the Potential of Gene Editing.

In 1929, August Krogh wrote that for every question in biology, there is a species or collection of species in which pursuing such questions is the most appropriate for achieving the deepest insights. Referred to as "Krogh's Principle," these words are a guiding force for many biologists. In practice, Krogh's principle might guide a biologist interested in studying bi-parental care to choose not to use lab mice, in which the female does most of the parenting, but instead study species in which bi-parental care is present and clearly observable, such as in certain poison dart frogs. This approach to pursuing biological questions has been fruitful, with more in-depth insights achievable with new technologies. However, up until recently, an important limitation of Krogh's principle for biologists interested in the functions of certain genes, was certain techniques were only available for a few traditional model organisms such as lab mice, fruit flies (Drosophila melanogaster), zebrafish (Danio rerio) and C. elegans (Caenorhabditis elegans), in which testing the functions of molecular systems on biological processes can be achieved using genetic knockout (KO) and transgenic technology. These methods are typically more precise than other approaches (e.g., pharmacology) commonly used in nontraditional model organisms to address similar questions. Therefore, some of the most in-depth insights into our understanding of the molecular control of these mechanisms have come from a small number of genetically tractable species. Recent advances in gene editing technology such as CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)/Cas9 gene editing as a laboratory tool has changed the insights achievable for biologists applying Krogh's principle. In this review, we will provide a brief summary on how some researchers of nontraditional model organisms have been able to achieve different levels of experimental precision with limited genetic tractability in their non-traditional model organism in the field of behavioral neuroendocrinology, a field in which understanding tissue and brain-region specific actions of molecules of interest has been a major goal. Then, we will highlight the exciting potential of Krogh's principle using discoveries made in a popular model species of social behavior, the African cichlid fish Astatotilapia burtoni. Specifically, we will focus on insights gained from studies of the control of social status by sex steroid hormones (androgens and estrogens) in A. burtoni that originated during field observations during the 1970s, and have recently culminated in novel insights from CRISPR/Cas9 gene editing in laboratory studies. Our review highlighting discoveries in A. burtoni may function as a roadmap for others using Krogh's principle aiming to incorporate gene editing into their research program. Gene editing is thus a powerful complimentary laboratory tool researchers can use to yield novel insights into understanding the molecular mechanisms of physiology and behavior in non-traditional model organisms.

Female cichlids attack and avoid-but will still mate with-androgen receptor mutant males that lack male-typical body coloration.

A key challenge in animal behavior is disentangling the social stimuli that drive conspecific behaviors. For behaviors like birdsong, insights can be made through the experimental isolation of relevant cues that affect behavior. However, for some species like teleost fish, putative sexual signaling cues are inextricably linked to others, making it difficult to parse the precise roles distinct signals play in driving conspecific behaviors. In the African cichlid Astatotilapia burtoni, males are dominant or subordinate, wherein bright coloration and territorial and courtship behavior inextricably correlate positively with rank. Here, we leveraged androgen receptor (AR) mutant male A. burtoni that lack dominance-typical coloration but not behavior to isolate the role of male coloration in driving female mating behaviors in this species. We found in independent behavioral assays that females behave aggressively towards AR mutant but not WT males but still mated with both types of males. Females showed enhanced activation of esr2b+ cells in the hypothalamus when housed with either mutant or WT males and this activation scaled with spawning activities. Therefore, there is not a simple relationship between male coloration and female mating behaviors in A. burtoni, suggesting independent sensory mechanisms converge on hypothalamic esr2b+ cells to coordinate behavioral output.