Beyond a biased binary: A perspective on the misconceptions, challenges, and implications of studying females in avian behavioral endocrinology.

For decades, avian endocrinology has been informed by male perspectives and male-focused research, leaving significant gaps in our understanding of female birds. Male birds have been favored as research subjects because their reproductive behaviors are considered more conspicuous and their reproductive physiology is presumably less complex than female birds. However, female birds should not be ignored, as female reproductive behavior and physiology are essential for the propagation of all avian species. Endocrine research in female birds has made much progress in the last 20 years, but a substantial disparity in knowledge between male and female endocrinology persists. In this perspective piece, we provide examples of why ornithology has neglected female endocrinology, and we propose considerations for field and laboratory techniques to facilitate future studies. We highlight recent advances that showcase the importance of female avian endocrinology, and we challenge historic applications of an oversimplified, male-biased lens. We further provide examples of species for which avian behavior differs from the stereotypically described behaviors of male and female birds, warning investigators of the pitfalls in approaching endocrinology with a binary bias. We hope this piece will inspire investigators to engage in more comprehensive studies with female birds, to close the knowledge gap between the sexes, and to look beyond the binary when drawing conclusions about what is 'male' versus 'female' biology.

Bidirectional relationships between testosterone and aggression: a critical analysis of four predictions.

Experimentally elevated testosterone (T) often leads to enhanced aggression, with examples across many different species, including both males and females. Indeed, the relationship between T and aggression is among the most well-studied and fruitful areas of research at the intersection of behavioral ecology and endocrinology. This relationship is also hypothesized to be bidirectional (i.e., T influences aggression, and aggression influences T), leading to four key predictions: (1) Individuals with higher T levels are more aggressive than individuals with lower T. (2) Seasonal changes in aggression mirror seasonal changes in T secretion. (3) Aggressive territorial interactions stimulate increased T secretion. (4) Temporary elevations in T temporarily increase aggressiveness. These predictions cover a range of timescales, from a single snapshot in time, to rapid fluctuations, and to changes over seasonal timescales. Adding further complexity, most predictions can also be addressed by comparing among individuals or with repeated sampling within-individuals. In our review, we explore how the spectrum of results across predictions shapes our understanding of the relationship between T and aggression. In all cases, we can find examples of results that do not support the initial predictions. In particular, we find that predictions 1-3 have been tested frequently, especially using an among-individual approach. We find qualitative support for all three predictions, though there are also many studies that do not support predictions 1 and 3 in particular. Prediction 4, on the other hand, is something that we identify as a core underlying assumption of past work on the topic, but one that has rarely been directly tested. We propose that when relationships between T and aggression are individual-specific or condition-dependent, then positive correlations between the two variables may be obscured or reversed. In essence, even though T can influence aggression, many assumed or predicted relationships between the two variables may not manifest. Moving forward, we urge greater attention to understanding how and why it is that these bidirectional relationships between T and aggression may vary among timescales and among individuals. In doing so, we will move towards a deeper understanding on the role of hormones in behavioral adaptation.

How experimental competition changes ovarian gene activity in free-living birds: Implications for steroidogenesis, maternal effects, and beyond.

The ovary plays an important role in mediating both a female's response to her social environment and communicating it to her developing offspring via maternal effects. Past work has focused on how ovarian hormones respond to competition, but we know little about how the broader ovarian transcriptomic landscape changes, either during or after competition, giving us a narrow perspective on how socially induced phenotypes arise. Here, we experimentally generated social competition among wild, cavity-nesting female birds (tree swallows, Tachycineta bicolor), a species in which females lack a socially induced rise in circulating testosterone but they nevertheless increase allocation to eggs. After territory settlement, we reduced availability of nesting cavities, generating heightened competition; within 24 h we reversed the manipulation, causing aggressive interactions to subside. We measured ovarian transcriptomic responses at the peak of competition and 48 h later, along with date-matched controls. Network analyses indicated that competing females experienced an immediate and temporary decrease in the expression of genes involved in the early stages of steroidogenesis, and this was moderately correlated with plasma testosterone; however, two days after competition had ended, there was a marked increase in the expression of genes involved in the final stages of steroidogenesis, including HSD17B1. Gene networks related to the cell cycle, muscle performance, and extracellular matrix organization also displayed altered activity. Although the functional consequences of these findings are unclear, they shed light on socially responsive ovarian genomic mechanisms that could potentially exert lasting effects on behavior, reproduction, and maternal effects.

Testing hormonal responses to real and simulated social challenges in a competitive female bird.

Competitive interactions often occur in series; therefore animals may respond to social challenges in ways that prepare them for success in future conflict. Changes in the production of the steroid hormone testosterone (T) are thought to mediate phenotypic responses to competition, but research over the past few decades has yielded mixed results, leading to several potential explanations as to why T does not always elevate following a social challenge. Here, we measured T levels in tree swallows (Tachycineta bicolor), a system in which females compete for limited nesting cavities and female aggression is at least partially mediated by T. We experimentally induced social challenges in two ways: (1) using decoys to simulate territorial intrusions and (2) removing subsets of nesting cavities to increase competition among displaced and territory-holding females. Critically, these experiments occurred pre-laying, when females are physiologically capable of rapidly increasing circulating T levels. However, despite marked aggression in both experiments, T did not elevate following real or simulated social challenges, and in some cases, socially challenged females had lower T levels than controls. Likewise, the degree of aggression was negatively correlated with T levels following a simulated territorial intrusion. Though not in line with the idea that social challenges prompt T elevation in preparation for future challenges, these patterns nevertheless connect T to territorial aggression in females. Coupled with past work showing that T promotes aggression, these results suggest that T may act rapidly to allow animals to adaptively respond to the urgent demands of a competitive event.

Experimental competition induces immediate and lasting effects on the neurogenome in free-living female birds.

Periods of social instability can elicit adaptive phenotypic plasticity to promote success in future competition. However, the underlying molecular mechanisms have primarily been studied in captive and laboratory-reared animals, leaving uncertainty as to how natural competition among free-living animals affects gene activity. Here, we experimentally generated social competition among wild, cavity-nesting female birds (tree swallows, Tachycineta bicolor). After territorial settlement, we reduced the availability of key breeding resources (i.e., nest boxes), generating heightened competition; within 24 h we reversed the manipulation, causing aggressive interactions to subside. We sampled females during the peak of competition and 48 h after it ended, along with date-matched controls. We measured transcriptomic and epigenomic responses to competition in two socially relevant brain regions (hypothalamus and ventromedial telencephalon). Gene network analyses suggest that processes related to energy mobilization and aggression (e.g., dopamine synthesis) were up-regulated during competition, the latter of which persisted 2 d after competition had ended. Cellular maintenance processes were also down-regulated after competition. Competition additionally altered methylation patterns, particularly in pathways related to hormonal signaling, suggesting those genes were transcriptionally poised to respond to future competition. Thus, experimental competition among free-living animals shifts gene expression in ways that may facilitate the demands of competition at the expense of self-maintenance. Further, some of these effects persisted after competition ended, demonstrating the potential for epigenetic biological embedding of the social environment in ways that may prime individuals for success in future social instability.

A single GnRH challenge promotes paternal care, changing nestling growth for one day.

Decades of comparative and experimental work suggest that testosterone (T) promotes mating effort at the expense of parental effort in many vertebrates. There is abundant evidence that T-mediated trade-offs span both evolutionary and seasonal timescales, as T is often higher in species or breeding stages with greater mating competition and lower in association with parental effort. However, it is less clear whether transient elevations in T within a male's own reactive scope can affect parental effort in the same way, with effects that are visible to natural selection. Here, we injected free-living male tree swallows (Tachycineta bicolor) with gonadotropin-releasing hormone (GnRH), thus temporarily maximizing T production within an individual's own limit. Passive loggers at each nest showed that GnRH-injected males provisioned more frequently than saline males for the subsequent day, and their offspring gained more mass during that time. The degree of offspring growth was positively correlated with the father's degree of T elevation, but provisioning was not proportional to changes in T, and GnRH- and saline-injected males did not differ in corticosterone secretion. These results suggest that prior knowledge of T-mediated trade-offs garnered from seasonal, evolutionary, and experimental research cannot necessarily be generalized to the timescale of transient fluctuations in T secretion within an individual. Instead, we propose that GnRH-induced T fluctuations may not result in visible trade-offs if selection has already sculpted an individual male's reactive scope based on his ability to handle the competing demands of mating and parental care.

How research on female vertebrates contributes to an expanded challenge hypothesis.

The bi-directional links between hormones and behavior have been a rich area of research for decades. Theory on the evolution of testosterone (T) was greatly advanced by the challenge hypothesis, which presented a framework for understanding interspecific, seasonal, and social variation in T levels in males, and how they are shaped by the competing demands of parental care and male-male competition. Female competition is also widespread in nature, although it is less clear whether or how the challenge hypothesis applies to females. Here, we evaluate this issue in four parts: (1) We summarize and update prior analyses of seasonal plasticity and interspecific variation in T in females. (2) We evaluate experimental links between T and female aggression on shorter timescales, asking how T manipulations affect aggression and conversely, how social manipulations affect T levels in female mammals, birds, lizards, and fishes. (3) We examine alternative mechanisms that may link aggression to the social environment independently of T levels in circulation. (4) We present a case study, including new data analyses, in an aggressive female bird (the tree swallow, Tachycineta bicolor) to explore how variation in tissue-level processing of T may bridge the gap between circulating T and variation in behavior that is visible to natural selection. We close by connecting these multivariate levels of sex steroid signaling systems alongside different temporal scales (social, seasonal, and evolutionary) to generate broadly applicable insights into how animals respond to their social environment, regardless of whether they are male or female.

SGLT2 Inhibitors Increase the Risk of Diabetic Ketoacidosis Developing in the Community and During Hospital Admission.

CONTEXT: Diabetic ketoacidosis (DKA) has been associated with the use of sodium glucose cotransporter 2 inhibitors (SGLT2is). OBJECTIVE: To determine the incidence, characteristics, and outcomes of DKA in SGLT2i users vs nonusers with type 2 diabetes. DESIGN: Retrospective, multicenter, controlled cohort study. SETTING: All public hospitals in Melbourne and Geelong (combined population of 5 million), Australia, from 1 September 2015 to 31 October 2017. PATIENTS: Consecutive cases of DKA that developed in the community, or during the course of hospital admission, in patients with type 2 diabetes. MAIN OUTCOME MEASURES: In SGLT2i users vs nonusers: (i) OR of DKA developing during hospital admission, and (ii) incidence of DKA. RESULTS: There were 162 cases of DKA (37 SGLT2i users and 125 non-SGLT2i users) with a physician-adjudicated diagnosis of type 2 diabetes. Of these, DKA developed during the course of inpatient admission in 14 (38%) SGLT2i users vs 2 (2%) non-SGLT2i users (OR, 37.4; 95% CI, 8.0 to 175.9; P < 0.0001). The incidence of DKA was 1.02 per 1000 (95% CI, 0.74 to 1.41 per 1000) in SGLT2i users vs 0.69 per 1000 (95% CI, 0.58 to 0.82 per 1000) in non-SGLT2i users (OR, 1.48; 95% CI, 1.02 to 2.15; P = 0.037). Fifteen SGLT2i users (41%) had peak blood glucose <250 mg/dL (14 mmol/L) compared with one (0.8%) non-SGLT2i user (P < 0.001). CONCLUSIONS: SGLT2i users were more likely to develop DKA as an inpatient compared with non-SGLT2i users. SGLT2i use was associated with a small but significant increased risk of DKA.

Testosterone production and social environment vary with breeding stage in a competitive female songbird.

In many vertebrates, males increase circulating testosterone (T) levels in response to seasonal and social changes in competition. Females are also capable of producing and responding to T, but the full extent to which they can elevate T across life history stages remains unclear. Here we investigated T production during various breeding stages in female tree swallows (Tachycineta bicolor), which face intense competition for nesting sites. We performed GnRH and saline injections and compared changes in T levels 30 min before and after injection. We found that GnRH-injected females showed the greatest increases in T during territory establishment and pre-laying stages, whereas saline controls dramatically decreased T production during this time. We also observed elevated rates of conspecific aggression during these early stages of breeding. During incubation and provisioning, however, T levels and T production capabilities declined. Given that high T can disrupt maternal care, an inability to elevate T levels in later breeding stages may be adaptive. Our results highlight the importance of saline controls for contextualizing T production capabilities, and they also suggest that social modulation of T is a potential mechanism by which females may respond to competition, but only during the period of time when competition is most intense. These findings have broad implications for understanding how females can respond to their social environment and how selection may have shaped these hormone-behavior interactions.