That hormones both modulate and respond to behavior is well-established (Alward et al., 2018; Wingfield et al., 2019). While investigating the latter directionality is challenging due to the need to reliably manipulate behavior, there have been many studies identifying secretory changes related to reproduction and social competition. Cheng, 1986, Cheng, 1992, for example, discovered that the combined sensory inputs of female ring doves hearing their own coos and the head-down posture of vocal production activate the hypothalamic-pituitary-gonadal (HPG) axis, which then triggers egg laying. Studies on the Challenge Hypothesis (Wingfield et al., 1990) have shown aggressive encounters can increase androgens (Wingfield et al., 2019), which can in turn prime animals to win future fights (Fuxjager et al., 2011). Along with the HPG axis, activity in the hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis is also upregulated during the breeding season in amphibians, reptiles, and birds to support energy mobilization during mating and reproduction (Moore and Jessop, 2003; Romero, 2002).

Frog reproduction presents an excellent opportunity to examine hormonal responses to behavior. Before mating, frogs typically first attract a mate by producing advertisement calls. The Energetics-Hormone Vocalization hypothesis proposes androgens maintain the high vocal efforts male frogs use to attract females, but due to energetic demands of calling, glucocorticoids should increase until they inhibit androgens, which should reduce calling (Emerson, 2001). Relationships between androgens, glucocorticoids, and calling effort in male frogs, however, do not always support this hypothesis (Leary et al., 2015). Unlike calling, which is highly variable, amplexus permits investigation of a discrete behavior and its secretory consequences. In many frog species, mating occurs only after the male performs the simple, but energetically demanding task of clasping the female in amplexus (Fig. 1), which often lasts for several hours before fertilization (McLister, 2003). In many amphibians, steroid hormone levels are higher in amplexed males compared to unpaired males (Harvey et al., 1997; Orchinik et al., 1988; Reedy et al., 2014). What causes elevated hormones in amplexus, however, is unknown.

Similar to other amphibians, males of Cope's gray treefrog (Hyla chrysoscelis) collected in amplexus have higher levels of estradiol (E2), testosterone (T), and corticosterone (CORT) compared to unpaired calling males (Baugh, 2024). In this study, we investigated the causal relationship between amplexus and circulating steroid hormones in male H. chrysoscelis. To test the hypothesis that the behavioral act of amplexus elevates male hormone levels, we compared E2, T, and CORT levels after 90 min across three groups of males that were either induced to enter amplexus, removed from amplexus, or prevented from entering amplexus.