723 INTRODUCTION Secondary sexual signals (coloration, morphology, scents) mostly serve as honest indicators of an individual’s genetic sex and potential mate quality. Many vertebrate secondary sexual signals are directly controlled by sex steroid hormones, which can reinforce their value as honest signals because primary sex steroids, such as testosterone and 17-estradiol, exhibit sexually dimorphic patterns that correlate with an individual’s genetic sex and quality/health. Such signals can be expressed seasonally (e.g. breeding plumage, skin coloration) or for the duration of the annual cycle (e.g. electric waveforms) and are often subject to modulation and/or regulation by sex steroids. Sexual signals and the sensory systems required for their perception (visual, auditory, olfactory) are directly influenced by sex steroid hormones during development. Key experiments done by Phoenix et al. demonstrated that sex steroids are crucial for guiding the organization of neural centers controlling sex-specific behavior and that the presence of those same sex steroids or others are necessary later in life to activate sexual behavior controlled by these centers (Phoenix et al., 1959). Their results established that sex steroids have two major effects in vertebrate reproduction: permanent (organizational) and reversible (activational). Although this generalized verbal model has been adjusted to accommodate other, more complicated pathways through which sex hormones act (e.g. Moore et al., 1998), the concept of activational/organizational effects of steroids generally fits most systems and has repeatedly passed experimental tests [reviewed extensively in a special issue of Hormones and Behavior (Wallen, 2009)]. Organizational and activational effects of steroids on sexual signals (e.g. behavior, coloration) have been demonstrated in numerous vertebrate groups. For testing organizational effects, individuals or whole clusters of offspring are exposed either in utero or at birth to exogenous sex steroids and then followed into adulthood to examine the effects of treatment on specific secondary sexual signals. Largely, these experiments study changes in behaviors and their neurological centers following steroid treatment [e.g. singing behavior in zebra finches, Taeniopygia guttata (Gurney and Konishi, 1980) and courtship behavior in Japanese quail, Coturnix japonica (Adkins, 1979)]. Activational effects of steroid hormones on secondary sexual signals, however, focus on the signals themselves and how they respond to steroid treatment over smaller windows of time, typically in adults. The activation of conspicuous and/or powerful sexual signals by primary sex steroids is present in all vertebrate groups [e.g. fish (Liley and Stacey, 1983), amphibians (Kelley and Pfaff, 1976), reptiles (Cooper et al., 1987), birds (van Oordt and Junge, 1934) and mammals (Carlisle et al., 1981)]. The Journal of Experimental Biology 215, 723-730 © 2012. Published by The Company of Biologists Ltd doi:10.1242/jeb.064923 RESEARCH ARTICLE How to make a sexy snake: estrogen activation of female sex pheromone in male red-sided garter snakes M. Rockwell Parker* and Robert T. Mason Department of Zoology, Oregon State University, 3029 Cordley Hall, Corvallis, OR 97331, USA *Author for correspondence at present address: Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104, USA (rparker@monell.org) Accepted 15 November 2011 SUMMARY Vertebrates indicate their genetic sex to conspecifics using secondary sexual signals, and signal expression is often activated by sex hormones. Among vertebrate signaling modalities, the least is known about how hormones influence chemical signaling. Our study species, the red-sided garter snake (Thamnophis sirtalis parietalis), is a model vertebrate for studying hormonal control of chemical signals because males completely rely on the female sex pheromone to identify potential mates among thousands of individuals. How sex hormones can influence the expression of this crucial sexual signal is largely unknown. We created two groups of experimental males for the first experiment: Sham (blank implants) and E2 (17-estradiol implants). E2 males were vigorously courted by wild males in outdoor bioassays, and in a Y-maze E2 pheromone trails were chosen by wild males over those of small females and were indistinguishable from large female trails. Biochemically, the E2 pheromone blend was similar to that of large females, and it differed significantly from Shams. For the second experiment, we implanted males with 17-estradiol in 2007 but removed the implants the following year (2008; Removal). That same year, we implanted a new group of males with estrogen implants (Implant). Removal males were courted by wild males in 2008 (implant intact) but not in 2009 (removed). Total pheromone quantity and quality increased following estrogen treatment, and estrogen removal re-established male-typical pheromone blends. Thus, we have shown that estrogen activates the production of female pheromone in adult red-sided garter snakes. This is the first known study to quantify both behavioral and biochemical responses in chemical signaling following sex steroid treatment of reptiles in the activation/organization context. We propose that the homogametic sex (ZZ, male) may possess the same targets for activation of sexual signal production, and the absence of the activator (17-estradiol in this case) underlies expression of the male phenotype. Key words: activation, pheromone, chemical ecology, endocrinology, reproduction, snake. THE฀JOURNAL฀OF฀EXPERIMENTAL฀BIOLOGY