Associated and disassociated patterns in hormones, song, behavior and brain receptor expression between life-cycle stages in male black redstarts, Phoenicurus ochruros
Highlights
► Male black redstarts defend territories during breeding and nonbreeding. ► Testosterone is elevated during breeding, but low during nonbreeding. ► Song structure differs between seasons, but independent of testosterone. ► Song output in territorial context differs between seasons and depends on testosterone. ► Aromatase in preoptic area is up-regulated during breeding.
Introduction
Most animals live in highly seasonal environments that vary, for example, in temperature and precipitation and consequently in the availability of resources such as food. To survive and maximize their reproductive success animals need to cope with these largely predictable changes and time their life-cycle accordingly. Hormones play a major role in the development and transition between life-cycle stages (e.g. Jacobs and Wingfield, 2000, Wingfield et al., 2001) and testosterone has been identified as an important player in regulating the breeding life-cycle stage of male vertebrates: it is required for spermatogenesis, the development of secondary sex characteristics and it facilitates sexual and territorial behaviors (Adkins-Regan, 2005, Nelson, 2005). Accordingly, testosterone levels are often highest at the beginning of the breeding season when interactions among males, song and sexual activities are most intense (Ball and Wingfield, 1987, Dawson, 1983, Morton et al., 1990, Silverin, 1993, Silverin et al., 1986, Van Duyse et al., 2003, Wingfield et al., 1990). However, the correlation between behaviors expressed in a territorial context and testosterone levels is not always that straight-forward: many temperate-zone songbird species, for example, defend territories and sing outside the breeding season when testosterone levels are low (Apfelbeck and Goymann, 2011, Canoine and Gwinner, 2002, Landys et al., 2010, Schwabl, 1992, Wingfield, 1994). Testosterone could still facilitate territoriality in these species: testosterone precursors may be derived from non-gonadal sources and metabolized to testosterone directly in the brain (e.g. Soma et al., 2000, Soma and Wingfield, 2001) or the brain may have a higher sensitivity for low levels of the hormone (Canoine et al., 2007). In some species, however, testosterone facilitates territorial behavior during the breeding period, but does not appear to facilitate territorial behavior during the nonbreeding season (Canoine and Gwinner, 2002, Hau and Beebe, 2011, Hau et al., 2000, Landys et al., 2010, Marasco et al., 2011, Schwabl and Kriner, 1991). In rufous-collared sparrows (Zonotrichia capensis) territorial behavior seems to be independent of testosterone even during breeding (Moore et al., 2004, Moore et al., 2004). In other species, that defend territories and sing both in- and outside the breeding season, these behaviors seem to differ in these two contexts (Moore, 1988). For example, during breeding song may contain more repetitive elements (DeWolfe et al., 1974, Leitner et al., 2001, Smith et al., 1997, Voigt and Leitner, 2008), longer songs (Riters et al., 2000) or song may be more stereotyped (Smith et al., 1997) than during nonbreeding. In the latter cases testosterone during breeding may activate these changes in territorial behaviors and song. In song sparrows (Melospiza melodia), for example, seasonal changes in song have been correlated with the size of the HVC, a brain nucleus of the song control system in songbirds (Nottebohm et al., 1976) that is considered to control motor output during singing (Brenowitz et al., 1997, Yu and Margoliash, 1996). The HVC is sensitive to androgens (Gahr and Metzdorf, 1997) and its size is thought to depend at least partly on circulating testosterone levels (Nottebohm, 1980, Sartor et al., 2005). Furthermore, the sensitivity to testosterone may change within the HVC depending on season (Gahr and Metzdorf, 1997, Soma et al., 1999). It has been argued that a larger HVC during breeding is related to a larger song repertoire, a higher song rate and facilitation of a more complex song in song sparrows (Brenowitz, 1997, Smith et al., 1997, but see (Gahr, 1997). In other species such as canaries (Serinus canaries) and black-capped chickadees (Poecile atricapillus), however, seasonal changes in song are not related to HVC size (Fusani et al., 2000, Smulders et al., 2006). Furthermore, testosterone may influence the motivation to sing and the song rate in a reproductive context by activating song areas outside the song control system, e.g. by aromatization of testosterone to estrogens in the preoptic area (Foidart et al., 1998, Riters et al., 2000, Soma et al., 2003). The preoptic area has been shown to play an important role in the regulation of estrogen-dependent aggressive behavior (Schlinger and Callard, 1990, Silverin et al., 2004).
Hence, although it is well accepted that testosterone plays a role in the organization and activation of song (Bolhuis and Gahr, 2006) and territorial behavior (Wingfield et al., 2006) in the breeding season, it is still unclear to what extent testosterone facilitates these behaviors in species that sing and defend territories outside the breeding season.
The black redstart (Phoenicurus ochruros) is a temperate-zone song bird species that defends a territory and sings during the breeding season in spring and also during nonbreeding in fall. Black redstarts are socially monogamous and both females and males provide parental care (Draganoiu et al., 2005, Landmann, 1996). Males defend a territory and sing during the breeding season in spring and early summer. During late summer (mid-August–mid-September) they molt and show a decrease in singing activity. Afterwards they express a pronounced period of fall territoriality and song activity until the end of October just before they start migration (Nicolai, 2005, Weggler, 2000).
To investigate whether testosterone may facilitate song and territorial behavior in different life-cycle stages, we compared testosterone (obtained via blood samples), territorial behavior, spontaneous song, and the distribution of hormone receptors in the brain of male black redstarts during breeding and nonbreeding territoriality. We focused on brain areas relevant for singing (forebrain song control nuclei) and aggressive behavior (diencephalon). We describe the expression pattern of androgen receptor-, estrogen receptor- and aromatase mRNA of male black redstarts during breeding and nonbreeding territoriality and determine whether HVC size and aromatase expression in the preoptic area differ between life-cycle stages and correlate with testosterone levels, territorial behavior and spontaneous song. If song output and structure differ with life-cycle stage, we expected males to have a larger HVC during breeding than outside the breeding season (e.g. Smith et al., 1997). As aromatase expression in the preoptic area has been shown to play an important role in the expression of reproductive behaviors (Balthazart et al., 2010), we expected a higher expression of aromatase mRNA in that area during breeding compared to the nonbreeding fall territorial phase.
We compared song output and song structure between breeding and nonbreeding territoriality to test if they differ between life-cycle stages. If testosterone activates song during the breeding season we expected that males produce more spontaneous song during breeding in spring than during nonbreeding in fall. Similar to other species, parts of the song of black redstarts contain repetitive elements and males increase the number of these elements in an agonistic context (Apfelbeck et al., 2012). If testosterone changes the structure of song during breeding, we expected to find significant differences in the structure of spontaneous song between the breeding and the nonbreeding season.
Section snippets
Study period and study site
Free-living male black redstarts were challenged with simulated territorial intrusions and caught in 2008 (April 1–June 12; September 19–October 6), 2009 (July 3–August 13) and, 2010 (June 28–July 31) in Upper Bavaria, Germany (N 47°, E 11°, 500–600 m above sea level). These males contributed to different experiments (see below); however most of them were bled and contributed to the seasonal testosterone profile presented. Furthermore, some of the hormone data from the early breeding season and
Seasonal testosterone profile
Testosterone levels of males caught at various times of the year (see next sentence) differed significantly (F5,193 = 34, p < 0.0001, Fig. 1). A priori set contrasts revealed that testosterone levels of males during all other phases of the life-cycle were significantly lower than during territory establishment (incubation: t = −2.3, p = 0.02, nestlings (first and second brood combined): t = −6.4, p < 0.0001; fledglings (first and second brood combined): t = −8.5, p < 0.0001; molt: t = −3.1, p = 0.002; nonbreeding
Non-vocal territorial behavior and testosterone
In black redstarts, the expression of non-vocal territorial behaviors did not correlate with differences in testosterone levels and aromatase expression in the preoptic area. Because males vigorously defended territories both during breeding and during fall nonbreeding while testosterone levels and aromatase expression in the preoptic area were higher during breeding than during fall nonbreeding territoriality. Thus, territorial behavior does not seem to be maintained by an increased
Conclusions
Our studies show that the relationship between testosterone and territorial behavior in male black redstarts is complex: testosterone does not seem to modulate non-vocal territorial behaviors, but the hormone may be involved in the regulation of context-dependent song through aromatization in the preoptic area. Our studies on black redstarts also suggest that there is no one unique mechanism by which sex steroids regulate territorial and song behavior in songbirds. Rather there seems to be a
Acknowledgments
We are grateful to Johanna Stegherr, Alexander Weber, Lisa Trost, Benjamin Wasmer and Stefan Leitner for their help in the field, to Ingrid Schwabl and Monika Trappschuh for support in the hormone analysis, to Christina Wolf for conducting the in situ-hybridizations and to Cornelia Voigt and Harriet Windley for their help in the analysis of the in situ-hybridization results. We also want to thank two anonymous reviewers for valuable comments on a previous version of this manuscript.
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