N the modulation of song characteristics in this species. We did

N the modulation of song characteristics in this species. We did so by implanting birds with an androgen receptor blocker (flutamide) and an aromatase inhibitor (letrozole) which inhibits the conversion of testosterone to estradiol, as testosterone can modulate behavior either directly by binding to androgen receptors or indirectly by conversion to estradiol and binding to estrogenFigure 1. A song of a black redstart illustrating the acoustic measures analyzed (Spectrogram: Avisoft-SASLab Pro, sample rate 22, 050 Hz, FFT = 256 points, Hamming-Window, Overlap: 50 ). doi:10.1371/journal.pone.0052009.gTestosterone Affects Song ModulationFigure 2. Song rate before, during and after the STI. Depicted separately for males treated with flutamide and letrozole (`Flut/Let’) and placebo treated males (`placebo’) in A) spring (n = 10 per group) and B) in fall (n = 6 per group). Each circle represents one individual male and measurements of the same male are connected by a line. Asterisks order Nafarelin indicate significance (*** p,0.001) and are according to a priori set contrasts (before vs. STI and before vs. after the STI). Mind the different scales in A and B. doi:10.1371/journal.pone.0052009.greceptors [52]. As controls we used birds treated with placebo implants. After implantation, we first recorded the spontaneous song of territorial males in an undisturbed context and then conducted a playback experiment simulating a territorial intrusion (STI) by a foreign male. This procedure was conducted in spring during the early breeding season, and again in fall during nonbreeding, using a different set of birds. The aim of our study was threefold. First, we wanted to investigate whether black redstarts change structural song parameters in an aggressive context, i.e. whether song parameters differ between a non-challenged context before the STI and during/ after the STI. Based on prior studies on black redstart song and in particular on a playback-study on song and age ( [47], see above) we expected to find changes in song output measures and structural song characteristics. Index signals that honestly communicate a physical trait related to male quality [19] are good candidates here. Thus, we expected those structural songparameters to change in the agonistic context that have been ` shown to be characteristic for adult males song, that is the number of song elements and the frequency-range of song parts [47]. Specifically we would expect focal males to sing longer song parts with trills, higher frequencies and/or with broader frequency bandwidth during a territorial encounter than in an undisturbed situation. Essed between all patients (groups HAT-1 and HAT-2) and the control Second, by blocking the actions of testosterone, we attempted to determine the role of this hormone in context-dependent vocal plasticity. If testosterone is playing a key role in the resource allocation for competitive behavior (e.g. [53]) during the breeding season in spring, we would expect flutamide/letrozole-treated males (thereafter termed Flut/Let males) to invest less in those behaviors and song patterns that are relevant in such situations than placebo-males. Thus, changes in song during territorial encounters (see above) should be less pronounced or absent in Flut/Let males in contrast to placebo treated males.Testosterone Affects Song ModulationTable 1. Linear mixed model results for the effects of context and Flut/Let-treatment on song output and structure in spring.elementtreatmentcontextinteractionCohens d [95 CI] placebo Flut/Let 1.4 [0.4,.N the modulation of song characteristics in this species. We did so by implanting birds with an androgen receptor blocker (flutamide) and an aromatase inhibitor (letrozole) which inhibits the conversion of testosterone to estradiol, as testosterone can modulate behavior either directly by binding to androgen receptors or indirectly by conversion to estradiol and binding to estrogenFigure 1. A song of a black redstart illustrating the acoustic measures analyzed (Spectrogram: Avisoft-SASLab Pro, sample rate 22, 050 Hz, FFT = 256 points, Hamming-Window, Overlap: 50 ). doi:10.1371/journal.pone.0052009.gTestosterone Affects Song ModulationFigure 2. Song rate before, during and after the STI. Depicted separately for males treated with flutamide and letrozole (`Flut/Let’) and placebo treated males (`placebo’) in A) spring (n = 10 per group) and B) in fall (n = 6 per group). Each circle represents one individual male and measurements of the same male are connected by a line. Asterisks indicate significance (*** p,0.001) and are according to a priori set contrasts (before vs. STI and before vs. after the STI). Mind the different scales in A and B. doi:10.1371/journal.pone.0052009.greceptors [52]. As controls we used birds treated with placebo implants. After implantation, we first recorded the spontaneous song of territorial males in an undisturbed context and then conducted a playback experiment simulating a territorial intrusion (STI) by a foreign male. This procedure was conducted in spring during the early breeding season, and again in fall during nonbreeding, using a different set of birds. The aim of our study was threefold. First, we wanted to investigate whether black redstarts change structural song parameters in an aggressive context, i.e. whether song parameters differ between a non-challenged context before the STI and during/ after the STI. Based on prior studies on black redstart song and in particular on a playback-study on song and age ( [47], see above) we expected to find changes in song output measures and structural song characteristics. Index signals that honestly communicate a physical trait related to male quality [19] are good candidates here. Thus, we expected those structural songparameters to change in the agonistic context that have been ` shown to be characteristic for adult males song, that is the number of song elements and the frequency-range of song parts [47]. Specifically we would expect focal males to sing longer song parts with trills, higher frequencies and/or with broader frequency bandwidth during a territorial encounter than in an undisturbed situation. Second, by blocking the actions of testosterone, we attempted to determine the role of this hormone in context-dependent vocal plasticity. If testosterone is playing a key role in the resource allocation for competitive behavior (e.g. [53]) during the breeding season in spring, we would expect flutamide/letrozole-treated males (thereafter termed Flut/Let males) to invest less in those behaviors and song patterns that are relevant in such situations than placebo-males. Thus, changes in song during territorial encounters (see above) should be less pronounced or absent in Flut/Let males in contrast to placebo treated males.Testosterone Affects Song ModulationTable 1. Linear mixed model results for the effects of context and Flut/Let-treatment on song output and structure in spring.elementtreatmentcontextinteractionCohens d [95 CI] placebo Flut/Let 1.4 [0.4,.