Effects of testosterone on urogenital tract morphology and androgen receptor expression in immature Eastern Fence lizards (Sceloporus undulatus).

In non-avian reptiles, the onset of sexual dimorphism of the major structures of the urogenital tract varies temporally relative to gonadal differentiation, more so than in other amniote lineages. In the current study, we used tonic-release implants to investigate the effects of exogenous testosterone (T) on postnatal development of the urogenital tract in juvenile Eastern Fence Lizards (Sceloporus undulatus) to better understand the mechanisms underlying the ontogeny of sexual differentiation in reptiles. We examined gonads, mesonephric kidneys and ducts (male reproductive tract primordia), paramesonephric ducts (oviduct primordia), sexual segments of the kidneys (SSKs), and hemiphalluses to determine which structures were sexually dimorphic independent of T treatment and which structures exhibited sexually dimorphic responses to T. To better understand tissue-level responsiveness to T treatment, we also characterized androgen receptor (AR) expression by immunohistochemistry. At approximately 4 months after hatching in control animals, gonads were well differentiated but quiescent; paramesonephric ducts had fully degenerated in males; mesonephric kidneys, mesonephric ducts, and SSKs remained sexually undifferentiated; and hemiphalluses could not be everted in either sex. Exogenous T caused enlargement, regionalization, and secretory activity of the mesonephric ducts and SSKs in both sexes; enlargement and regionalization of the oviducts in females; and enlargement of male hemipenes. The most responsive tissues exhibited moderate but diffuse staining for AR in control lizards and intense nuclear staining in T-treated lizards, suggestive of autoregulation of AR. The similarity between sexes in the responsiveness of the mesonephric ducts and SSK to T indicates an absence of sexually dimorphic organizational effects in these structures prior to treatment, which was initiated approximately 2 months after hatching. In contrast, the sex-specific responses in oviducts and hemipenes indicate that significant organization and/or differentiation had taken place prior to treatment.

Species differences in hormonally mediated gene expression underlie the evolutionary loss of sexually dimorphic coloration in Sceloporus lizards.

Phenotypic sexual dimorphism often involves the hormonal regulation of sex-biased expression for underlying genes. However, it is generally unknown whether the evolution of hormonally mediated sexual dimorphism occurs through upstream changes in tissue sensitivity to hormone signals, downstream changes in responsiveness of target genes, or both. Here, we use comparative transcriptomics to explore these possibilities in 2 species of Sceloporus lizards exhibiting different patterns of sexual dichromatism. Sexually dimorphic S. undulatus develops blue and black ventral coloration in response to testosterone, while sexually monomorphic S. virgatus does not, despite exhibiting similar sex differences in circulating testosterone levels. We administered testosterone implants to juveniles of each species and used RNAseq to quantify gene expression in ventral skin. Transcriptome-wide responses to testosterone were stronger in S. undulatus than in S. virgatus, suggesting species differences in tissue sensitivity to this hormone signal. Species differences in the expression of genes for androgen metabolism and sex hormone-binding globulin were consistent with this idea, but expression of the androgen receptor gene was higher in S. virgatus, complicating this interpretation. Downstream of androgen signaling, we found clear species differences in hormonal responsiveness of genes related to melanin synthesis, which were upregulated by testosterone in S. undulatus, but not in S. virgatus. Collectively, our results indicate that hormonal regulation of melanin synthesis pathways contributes to the development of sexual dimorphism in S. undulatus, and that changes in the hormonal responsiveness of these genes in S. virgatus contribute to the evolutionary loss of ventral coloration.

Evolution of hormone-phenotype couplings and hormone-genome interactions.

When selection favors a new relationship between a cue and a hormonally mediated response, adaptation can proceed by altering the hormonal signal that is produced or by altering the phenotypic response to the hormonal signal. The field of evolutionary endocrinology has made considerable progress toward understanding the evolution of hormonal signals, but we know much less about the evolution of hormone-phenotype couplings, particularly at the hormone-genome interface. We briefly review and classify the mechanisms through which these hormone-phenotype couplings likely evolve, using androgens and their receptors and genomic response elements to illustrate our view. We then present two empirical studies of hormone-phenotype couplings, one rooted in evolutionary quantitative genetics and another in comparative transcriptomics, each focused on the regulation of sexually dimorphic phenotypes by testosterone (T) in the brown anole lizard (Anolis sagrei). First, we illustrate the potential for hormone-phenotype couplings to evolve by showing that coloration of the dewlap (an ornament used in behavioral displays) exhibits significant heritability in its responsiveness to T, implying that anoles harbor genetic variance in the architecture of hormonal pleiotropy. Second, we combine T manipulations with analyses of the liver transcriptome to ask whether and how statistical methods for characterizing modules of co-expressed genes and in silico techniques for identifying androgen response elements (AREs) can improve our understanding of hormone-genome interactions. We conclude by emphasizing important avenues for future work at the hormone-genome interface, particularly those conducted in a comparative evolutionary framework.

Global review reveals how disparate study motivations, analytical designs, and focal ions limit understanding of salinization effects on freshwater animals.

Global salinization of freshwaters is adversely affecting biotic communities and ecosystem processes. We reviewed six decades (1960-2020) of literature published on animal responses to increased salinities across different taxonomic and ecological contexts and identified knowledge gaps. From 585 journal articles, we characterized 5924 responses of mollusks, crustaceans, zooplankton, non-arthropod invertebrates (NAI), insects, fishes, and amphibians to salinization. Insects and fishes were the most studied taxa; Na+ and Cl- were the most studied ions-. Collectively, concentrations of the ions examined typically spanned five orders of magnitude. Species' invasiveness was a key motivation for studying mollusks, crustaceans, and fishes; threats of urbanization and road salts were key motivations for studying NAI, zooplankton, and amphibians. Laboratory studies were more common than field studies for most taxa. Focal life stages in laboratory studies varied widely but juveniles and adults were represented similarly in field studies. Studies of mollusks, NAI, and crustacean focused on adults; studies of zooplankton, insects, fishes, and amphibians focused on juveniles. Organismal- and population-level responses measuring solute uptake, internal chemistry, body condition, or ion concentrations predominated laboratory studies; population- and assemblage-level responses measuring abundance, spatial distribution, or assemblage composition predominated field studies. Negative responses to salinization predominated but positive and unimodal responses were apparent across all taxa and organizational levels. Key topics for further research include a) salinity responses by more taxa, b) responses to especially toxic ions (i.e., potassium, bicarbonate, sulfate, magnesium), c) mechanisms causing positive and unimodal responses, d) traits underpinning responses, e) effects transcending organizational levels, f) ion-specific response thresholds, and g) interactions between salinity and other stressors. Our review suggests inter-taxa variation in sensitivity to salinization reflects occurrence of certain biological traits, including gill-breathing, semi-permeable skin, multiple life stages, and limited mobility. We propose a traits-based framework to predict salinization sensitivity from shared traits. This evolutionary approach could inform management aimed at preventing or reducing adverse impacts of freshwater salinization.

Ontogenetic Change in Male Expression of Testosterone-Responsive Genes Contributes to the Emergence of Sex-Biased Gene Expression in Anolis sagrei.

Sex differences in gene expression tend to increase with age across a variety of species, often coincident with the development of sexual dimorphism and maturational changes in hormone levels. However, because most transcriptome-wide characterizations of sexual divergence are framed as comparisons of sex-biased gene expression across ages, it can be difficult to determine the extent to which age-biased gene expression within each sex contributes to the emergence of sex-biased gene expression. Using RNAseq in the liver of the sexually dimorphic brown anole lizard (Anolis sagrei), we found that a pronounced increase in sex-biased gene expression with age was associated with a much greater degree of age-biased gene expression in males than in females. This pattern suggests that developmental changes in males, such as maturational increases in circulating testosterone, contribute disproportionately to the ontogenetic emergence of sex-biased gene expression. To test this hypothesis, we used four different experimental contrasts to independently characterize sets of genes whose expression differed as a function of castration and/or treatment with exogenous testosterone. We found that genes that were significantly male-biased in expression or upregulated as males matured tended to be upregulated by testosterone, whereas genes that were female-biased or downregulated as males matured tended to be downregulated by testosterone. Moreover, the first two principal components describing multivariate gene expression indicated that exogenous testosterone reversed many of the feminizing effects of castration on the liver transcriptome of maturing males. Collectively, our results suggest that developmental changes that occur in males contribute disproportionately to the emergence of sex-biased gene expression in the Anolis liver, and that many of these changes are orchestrated by androgens such as testosterone.

Experimental removal of nematode parasites increases growth, sprint speed, and mating success in brown anole lizards.

Parasites interact with nearly all free-living organisms and can impose substantial fitness costs by reducing host survival, mating success, and fecundity. Parasites may also indirectly affect host fitness by reducing growth and performance. However, experimentally characterizing these costs of parasitism is challenging in the wild because common antiparasite drug formulations require repeated dosing that is difficult to implement in free-living populations, and because the extended-release formulations that are commercially available for livestock and pets are not suitable for smaller animals. To address these challenges, we developed a method for the long-term removal of nematode parasites from brown anole lizards (Anolis sagrei) using an extended-release formulation of the antiparasite drug ivermectin. This treatment eliminated two common nematode parasites in captive adult males and dramatically reduced the prevalence and intensity of infection by these parasites in wild adult males and females. Experimental parasite removal significantly increased the sprint speed of captive adult males, the mating success of wild adult males, and the growth of wild juveniles of both sexes. Although parasite removal did not have any effect on survival in wild anoles, parasites may influence fitness directly through reduced mating success and indirectly through reduced growth and performance. Our method of long-term parasite manipulation via an extended-release formulation of ivermectin should be readily adaptable to many other small vertebrates, facilitating experimental tests of the extent to which parasites affect host phenotypes, fitness, and eco-evolutionary dynamics in the wild.

Hormonal pleiotropy structures genetic covariance.

Quantitative genetic theory proposes that phenotypic evolution is shaped by G, the matrix of genetic variances and covariances among traits. In species with separate sexes, the evolution of sexual dimorphism is also shaped by B, the matrix of between-sex genetic variances and covariances. Despite considerable focus on estimating these matrices, their underlying biological mechanisms are largely speculative. We experimentally tested the hypothesis that G and B are structured by hormonal pleiotropy, which occurs when one hormone influences multiple phenotypes. Using juvenile brown anole lizards (Anolis sagrei) bred in a paternal half-sibling design, we elevated the steroid hormone testosterone with slow-release implants while administering empty implants to siblings as a control. We quantified the effects of this manipulation on the genetic architecture of a suite of sexually dimorphic traits, including body size (males are larger than females) and the area, hue, saturation, and brightness of the dewlap (a colorful ornament that is larger in males than in females). Testosterone masculinized females by increasing body size and dewlap area, hue, and saturation, while reducing dewlap brightness. Control females and males differed significantly in G, but treatment of females with testosterone rendered G statistically indistinguishable from males. Whereas B was characterized by low between-sex genetic correlations when estimated between control females and males, these same correlations increased significantly when estimated between testosterone females and either control or testosterone males. The full G matrix (including B) for testosterone females and either control or testosterone males was significantly less permissive of sexually dimorphic evolution than was G estimated between control females and males, suggesting that natural sex differences in testosterone help decouple genetic variance between the sexes. Our results confirm that hormonal pleiotropy structures genetic covariance, implying that hormones play an important yet overlooked role in mediating evolutionary responses to selection.

Covariation between Thermally Mediated Color and Performance Traits in a Lizard.

Physiological changes in response to environmental cues are not uncommon. Temperature has strong, predictable effects on many traits, such that many traits in ectotherms follow stereotyped thermal performance curves in response to increasing temperature. The prairie lizard-an abundant lizard throughout the central United States-has thermally sensitive, blue abdominal and throat patches. Currently, the role of these patches is not well understood. In this study, we set out to investigate whether individual plasticity in patch color paralleled individual plasticity in sprint speed (do they covary), and if the plasticity in these two patches signal redundant or independent information, testing competing hypotheses suggested for the evolution of multiple signals. We found that both abdominal and throat patch hue follow classical thermal performance curves, suggesting that at the species level hue is a good predictor of sprinting ability. At the individual level, we found that color and performance were statistically repeatable, so individuals with relatively high phenotypic values maintain relatively high phenotypic values across all temperatures. Additionally, we found that abdominal and patch hue covary with sprinting speed at the individual level. Together, these results suggest that the bluest individuals are the fastest individuals across temperatures. However, we found that abdominal and throat patch hue do not covary with each other at the individual level, suggesting that these signals may have independent functions. The importance of examining the function of individual variation cannot be overstated, and overall, more work is needed to better understand both the proximate and ultimate mechanisms underlying signal plasticity in this species and others.

Vision testing is additive to the sideline assessment of sports-related concussion.

We examined the King-Devick (K-D) test, a vision-based test of rapid number naming, as a complement to components of the Sport Concussion Assessment Tool, 3rd edition (SCAT3) for diagnosis of concussion. Baseline and postconcussion data for the University of Florida men's football, women's soccer, and women's lacrosse teams were collected, including the K-D test, Standardized Assessment of Concussion (SAC), and Balance Error Scoring System (BESS). Among 30 athletes with first concussion during their athletic season (n = 217 total), differences from baseline to postinjury showed worsening of K-D time scores in 79%, while SAC showed a ≥2-point worsening in 52%. Combining K-D and SAC captured abnormalities in 89%; adding the BESS identified 100% of concussions. Adding a vision-based test may enhance the detection of athletes with concussion.