Statistical analysis

Statistical analysis

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Hormones and Behavior

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Sex-specific hypothalamic-pituitary-gonadal axis sensitivity in migrating songbirds

Kristen M. Covinoa,b,c,⁎, Jodie M. Jawora,1, Sara R. Morrisb,c, Frank R. Moorea

a Biology Department, Canisius College, 2001 Main St., Buffalo, NY 14208, USA bDepartment of Biological Sciences, University of Southern Mississippi, Hattiesburg, MS 39406, USA c Shoals Marine Laboratory, 102 Chase Ocean Engineering Lab, 24 Colovos Rd, Durham, NH 03824, USA

A R T I C L E I N F O

Keywords: Testosterone GnRH bioassay Migration HPG axis Breeding development Rpotential

A B S T R A C T

In seasonally migratory species, the overlap between the migratory and breeding life history stages is a balance between the physiological and behavioral requirements of each stage. Previous studies investigating the degree to which songbirds prepare for breeding during spring migration have focused on either circulating hormone levels or direct measures of gonadal recrudescence. In this study, we evaluated the phenology of breeding preparation in a long-distance migratory songbird, the Swainson’s Thrush (Catharus ustulatus), by assessing hypothalamic-pituitary-gonadal (HPG) axis sensitivity with gonadotropin-releasing hormone (GnRH) bioassays throughout the migratory period. During spring migration both males and females had a significant response to GnRH injections as reflected in elevated testosterone levels. The magnitude of response to GnRH injections, Rpotential, in females stayed consistent throughout spring migration; however, Rpotential in males increased as the migratory season progressed. It is clear that at least some degree of endocrinological breeding development occurs either before or during spring migration in both sexes, however the phenology appears to be sex specific. In males this breeding development continues at a relatively steady pace throughout the migratory period while in females, relatively little endocrine breeding development occurs during migration. These sex-specific differ- ences in the phenology of the endocrine breeding development warrant future investigations for both male and female songbirds. Moreover, research focused on how physiological breeding development is balanced with the expression of migratory traits in long-distance songbird migrants is needed.

1. Introduction

Testosterone (T) is a steroid hormone that mediates a wide-range of behavioral and physiological effects in songbirds and other vertebrates (Wingfield and Silverin, 2009; reviewed in Adkins-Regan, 2005). While T is linked to many actions throughout the annual cycle, it has been heavily studied in the context of the breeding season (e.g. Ramenofsky and Wingfield, 2006; but see DeVries et al., 2011; Ramenofsky et al., 1992, 1999; Schwabl et al., 1988; Soma, 2006). Many of the effects mediated by T, or its metabolites, are central to the expression of male breeding behaviors, including singing and aggressive territory defense (Adkins-Regan, 2005; Goymann and Wingfield, 2014; Wingfield et al., 2001). Testosterone, either as a prohormone for estradiol or in its own right, is also important for female reproduction and reproductive be- haviors (Ketterson et al., 2005; Norris, 1997; Rosvall, 2013; Staub and De Beer, 1997). Typically peak levels of circulating T occur during the early breeding season in male songbirds, and several studies have

focused on circulating levels of T throughout the annual cycle to in- vestigate breeding preparation and hypothalamic-pituitary-gonadal (HPG) axis activity (e.g. Jawor et al., 2006; Wingfield, 1984; see Ketterson et al., 2005). While only a few studies have investigated T variation in female songbirds, detectable levels are generally found at some point during the annual cycle and show within season variability in at least some species (e.g. Jawor et al., 2007; see Ketterson et al., 2005).

The coordination of physiological and behavioral traits associated with each stage of the annual cycle allows adequate time and energy for development and expression of those traits (Jacobs and Wingfield, 2000; Ramenofsky, 2011). In addition to the stages expressed by all birds (e.g., breeding, molt, non-breeding), migratory birds face the additional challenge of expressing spring and fall migration stages and, in some species, an additional molt. This increase in the number of stages in the annual cycle results in less flexibility in the timing of each stage and more overlap between successive stages (Ramenofsky, 2011).

https://doi.org/10.1016/j.yhbeh.2017.11.003 Received 30 January 2017; Received in revised form 13 October 2017; Accepted 3 November 2017

⁎ Corresponding author at: Biology Department, Canisius College, 2001 Main St., Buffalo, NY 14208, USA.

1 Present Address: Department of Biology, New Mexico State University, Las Cruces, NM 88003, USA. E-mail address: covinok@gmail.com (K.M. Covino).

Hormones and Behavior 97 (2018) 112–120

Available online 14 November 2017 0018-506X/ © 2017 Elsevier Inc. All rights reserved.

T

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According to (Jacobs and Wingfield, 2000; Ramenofsky, 2011), each stage of the annual cycle can be represented by three distinct phases; development, mature expression, and termination. Due to energetic limitations, the mature expression of any two stages cannot overlap, although the development of one stage may overlap with the mature expression of or termination of preceding stages (Jacobs and Wingfield, 2000; Ramenofsky, 2011). In long-distance migrants, especially those breeding at northern latitudes, the opportunity to breed is limited to a relatively narrow time frame; thus proper timing of the mature ex- pression of breeding upon arrival is crucial (Jacobs and Wingfield, 2000; Ramenofsky and Wingfield, 2006). Estimates suggest that phy- siological development of the breeding stage, including full activation of the HPG axis and complete gonadal recrudescence, takes approxi- mately one month for songbirds before mature expression of breeding activity (e.g. Ramenofsky, 2011). Given the time constraints restricting long-distance migrants upon arrival on the breeding grounds, HPG-axis activity, leading to the production of gonadal steroids and gametes, should increase before or during spring migration. Males should be prepared to transition quickly to expression of breeding behavior once on the breeding grounds, including establishment of territory and mate attraction, if environmental conditions are conducive. Similarly, if en- vironmental conditions are appropriate for breeding, females should arrive on the breeding grounds receptive to courting males since early initiation of breeding increases reproductive success (Smith and Moore, 2005; Wingfield and Ramenofsky, 2011). Thus, arriving individuals must have sufficiently developed HPG axis activity to promote the ex- pression of these behaviors and to facilitate a quick transition to the breeding life-history stage. This development of the HPG axis and re- sulting gonadal steroid levels are an essential component of seasonal breeding preparation that may have other downstream effects on re- production including recrudescence of gonads and expression of re- productive behaviors. While some male songbirds have detectable in- creases in T during their vernal migration towards the breeding grounds (Covino et al., 2015, 2016; Wingfield and Farner, 1978a, 1978b) this pattern is not universal (Bauchinger et al., 2007; Covino et al., 2015). Studies of breeding hormone fluctuations in migrating female songbirds are relatively rare but indicate that hormone levels, including T and estradiol, may not increase until after females reach the breeding grounds (Covino et al., 2015, 2016, Wingfield and Farner, 1978a, 1978b). Since circulating T levels do not reliably predict breeding readiness or timing in all species (Caro et al., 2006; Davies et al., 2015; Partecke et al., 2005; Schaper et al., 2012) studies incorporating more direct measures of HPG axis activity are necessary.

Although several studies have noted links between endocrine breeding development (e.g. increasing T levels) and expression of mi- gratory characteristics (Owen et al., 2014; Ramenofsky and Németh, 2014; Tonra et al., 2011), some of the behavioral and physiological effects linked to high T levels may be antagonistic to the expression of the migratory syndrome (Ketterson et al., 2015) and a successful mi- gratory journey. For example, T-mediated territoriality while en route may be costly when a migrant is time constrained, which is especially likely during spring passage. High concentrations of T may negatively impact lipogenesis and fuel deposition rates potentially interrupting migratory behavior (Deviche, 1995; Ketterson et al., 1991). In contrast, several studies have indicated that the expression of various migratory traits is linked to increasing T levels (Deviche, 1995; Rowan, 1925, 1929; Weise, 1967). For example, the expression of migratory char- acteristics including fattening, mass gains, and migratory restlessness, may be dampened or delayed when T is absent (King and Farner, 1962; Lofts and Marshall, 1961; Morton and Mewaldt, 1962; Schwabl et al., 1988; Schwabl and Farner, 1989; Stetson and Erickson, 1972). Further, the administration of exogenous T induces the expression of migratory behaviors in captive birds (Owen et al., 2014; Tonra et al., 2011, 2013).

Increases in HPG-axis sensitivity that outpace increases in circu- lating T levels may reflect an overlap between migration and breeding life history stages that avoids the antagonistic effects of prolonged T

elevation during migration (see Covino et al., 2016). While levels of circulating T or other breeding hormones (luteinizing hormone, estra- diol, dihydrotestosterone) may reflect reproductive condition, in- dividuals may express short-term fluctuations in hormone levels for many reasons (Deviche et al., 2006; Wingfield et al., 1990). These short- term fluctuations may mask seasonal changes, especially in studies of free-living birds where confounding variables may be difficult to con- trol. Gonadotropin-releasing hormone (GnRH) bioassays in which exogenous GnRH is administered, sometimes called GnRH challenges, test the sensitivity of the HPG axis vis-á-vis determining the physiolo- gical capacity of an individual to elevate T (Jawor et al., 2006; Schoech et al., 1996; Wingfield et al., 1979, 1991). Through the administration of a large dose of GnRH, researchers, in essence, flood the GnRH re- ceptors of the anterior pituitary (Bentley et al., 2006; Goymann and Wingfield, 2004; Wingfield et al., 1991). This effectively tests the sen- sitivity of the anterior pituitary to GnRH and, subsequently, that of the gonads to LH. The resulting elevation in T, termed the Rpotential of an individual (Goymann et al., 2007), reveals the overall sensitivity of the HPG axis and, since this approach requires only a single capture, it is easy to use with free-living individuals (e.g. Covino et al., 2016; DeVries et al., 2011, 2012). Variation in Rpotential may be due to changing sensitivity to GnRH, LH, or a combination of the two. That said, this technique provides reasonable quantification of en- docrinological breeding development and previous studies have found Rpotential to relate to ornament size (McGlothlin et al., 2008), breeding success (Cain and Pryke, 2017; McGlothlin et al., 2010), and body condition (Davies et al., 2015).

In this study we sought to investigate one component of en- docrinological breeding development represented by HPG axis sensi- tivity, in male and female birds during their spring migration. To do so we determined the Rpotential in Swainson’s Thrushes (Catharus ustulatus) at different points along their migratory route as they traveled towards their breeding grounds. Although an earlier study did not find changes in the seasonal levels of baseline T (non-GnRH stimulated T) in this species during migration (Covino et al., 2015), we predicted that their HPG axis sensitivity (tested via GnRH challenge; Rpotential) would in- crease during migration (Goymann et al., 2007). Specifically, we ex- pected that Rpotential would increase as birds migrate towards their breeding destination, where higher levels of T are required for the ex- pression of breeding traits. Given that both sexes must be prepared for breeding activities and behaviors upon arrival on the breeding grounds, we also predicted that both males and females would have a higher Rpotential when sampled closer to their breeding grounds.

2. Materials and methods

2.1. Study species and study sites

Our focal species was the Swainson’s Thrush (hereafter thrush), a long-distance Nearctic-Neotropical migrant that breeds in the Rocky Mountains, throughout North American boreal forests, and along the Pacific coast and winters in southern Central America and eastern South America (Fig. 1). As in Covino et al. (2015), we captured migrating thrushes throughout vernal migration at one southern site (from 6 April through 12 May) and one northern site (from 10 May through 2 June) during 2012–2014. Our study encompassed the entirety of thrush mi- gration at both study locations. Our southern site is relatively far from the Swainson’s Thrush breeding grounds (at least 1400 km to closest edge of the breeding range) and is located along the northern coast of the Gulf of Mexico in Cameron Parish, Louisiana (29° 45′ N 93° 30′ W). Our northern site is relatively close to the breeding grounds for this species (< 200 km to closest edge of the breeding range) and is located on an island off the New England coast in York County, Maine (42°58′ N, 70°36′ W; Fig. 1). We also captured birds at a site along the Gulf of Mexico coast in Mobile County, Alabama (30° 15′ N 88° 05′ W), during fall migration from 20 September through 8 October 2014, to enable

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sample comparisons outside of either spring migration or the breeding season.

2.2. Capture and sampling

We captured birds using mist-nets which were operated daily, weather permitting, throughout each migratory season and checked at least every 30 min. Within 10 min (Mean ± SD; 4.6 ± 1.9 min) of removing a thrush from a net, we obtained a blood sample (approxi- mately 70–100 μl) via the brachial vein with a 26-gauge needle and collected blood into heparinized capillary tubes. After initial sampling, we administered a 100 μl injection into the pectoralis major of either 1 M phosphate-buffered saline (PBS; control birds with no predicted increase in T; Male N = 24, Female N = 14) or a GnRH solution made from 2.5 μg of chicken GnRH-I (American Peptide Co., #54-8-23; Male N = 89, Female N = 93) dissolved in 100 μl of PBS (following Jawor et al., 2006, 2007). This dosage of GnRH has been used previously to stimulate T production in a similarly sized species, the Northern Car- dinal (Cardinalis cardinalis; DeVries et al., 2011, 2012). Injected thru- shes were held in either a wooden holding box or a cloth bag until a post-injection blood sample was obtained. We studied the timing of maximum Rpotential on a subset of thrushes (Male N = 12, Female N = 10) during spring migration at our southern study site by ob- taining post-injection T samples at 15, 30, 45, or 60 min. Based on our results (reported in Results below), all other birds were held for 30 min between administering the injection and obtaining the post-injection sample. After completion of the GnRH bioassay, each bird was banded with a USGS aluminum leg band and measurements of wing length (unflattened wing chord; nearest 0.5 mm), and body mass (nearest 0.01 g) were taken. All blood samples (pre- and post-injection) were kept in a cooler on ice until they were centrifuged at 14,000 rpm for 10 min. The plasma portion of each sample was drawn off and stored at −20 °C and the cellular portion was placed in approximately 500 μl of lysis buffer (50 mM TRIS, 10 mM EDTA, 1% SDS, 0.1 M NaCl) and

stored at either 4 °C or−20 °C. As described in Covino et al. (2015) and the cellular portion was used to determine sex genetically based on methods in Griffiths et al. (1996, 1998).

2.3. Testosterone assays

We used an enzyme immunoassay to determine T levels (Enzo Life Sciences Inc., #901–065; assay sensitivity = 5.67 pg/ml; (Covino et al., 2015; DeVries et al., 2011; Jawor, 2007). To facilitate determination of extraction recovery, tritium-labeled T (approximately 2000 cpm; Per- kinElmer) was added to 25–30 μl of each plasma sample (depending on available sample volume). After three extractions with diethyl ether, samples were re-suspended with 50 μl of ethanol and 300 μl of the assay buffer provided in the assay kit. Samples were run in duplicate on the assay plate using 100 μl of the reconstituted in sample each well pair. Pre- and post-injection samples from the same individual were run se- quentially on the same assay plate. Recoveries were determined using 100 μl from each reconstituted sample measured on a calibrated scin- tillation counter and compared to a retained reference sample (mean recoveries = 85%). After correcting for incomplete recoveries and in- itial plasma volume, a logistic curve was used to determine T con- centrations (Microplate Manager; Bio-Rad Laboratories, Inc., Hercules, California). Intra- and inter-assay variations were determined based on three standard samples of known T concentration placed in each assay plate (N = 19). Three samples from our fall sampling period contained plasma T at a concentration lower than our standard curve: the pre- and post-injection samples from a male thrush and the pre-injection sample from a female thrush. All three samples were slightly below the assay standard curve but were well within the standard deviation produced from the curve replicates. Thus, we opted to include the extrapolated values for these three samples rather than drop them from our analyses. Intra-assay variation ranged from 0.7% to 11.5% and inter-assay var- iation was 12.0%. Inter-assay variation may have been inflated due to the use of EIA kits from multiple kit lots.

Fig. 1. The breeding range of the Swainson’s Thrush (Catharus ustulatus) is limited to the boreal forests of North America. Its wintering range extends from Central American through western South America. The sampling locations in Louisiana and Maine are indicated. This map was generated using data from BirdLife International (BirdLife-International and NatureServe 2014).

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2.4. Statistical analyses

Based on the standards placed within each assay plate, T data were corrected for inter-assay variation before analyses and were cube-root transformed to compensate for their deviation from a normal distribu- tion. For analyses of seasonal variation in the GnRH response, data were organized into four sampling periods: fall migration, south-early spring migration, south-late spring migration, and north spring migration. South represents birds sampled in Louisiana and north represents birds captured in Maine. Louisiana birds were categorized as ‘early’ or ‘late’ based on when they were captured relative to the median date of capture for each sex because the migratory period is relatively pro- tracted there compared to Maine. This allowed us to further investigate temporal changes to HPG axis sensitivity since the end of the migratory season at our southern site overlaps slightly with that of our northern site.

To estimate both interactive and main effects, we used linear mixed effects models which allow for modeling of random effects (Bates, 2010; Bates et al., 2014). To determine the significance of model in- teraction terms, we compared models with interactions to those without them using likelihood ratio tests. We also used likelihood ratio tests to determine the significance of fixed effects by comparing full models to similar models without the variable in question. We used repeated measures models structured with individual as the random effect in three sets of analyses. (1) A time-series analysis for post-bleed time with T as the response variable and with sex and sample (0, 15, 30, 45, 60 min) as fixed effects. (2) An analysis of the effect of the injection on both control (PBS-injected) individuals and GnRH-injected individuals. The fall sampling period was not included in this analysis because sampling constraints prevented inclusion of control birds. Testosterone was the response variable, sampling period and sample (pre- or post- injection) were evaluated as fixed effects. Data from males and females were analyzed separately. (3) An analysis of the effect of the GnRH injection for all sampling periods (fall, spring south-early, spring south- late, and north) with T as the response variable and with sampling period, sample (pre- or post-injection), and sex as fixed effects. A fourth analysis investigated temporal variation across spring migration. In that analysis, we calculated “Response” to the GnRH bioassay as the post- injection T level less the pre-injection T level for each individual. We analyzed GnRH Response for males and females separately using gen- eralized linear models with date (day of year) as the predictor variable with data pooled across both sites. For all analyses, we analyzed pair- wise effects using least squares means tests with a Tukey correction for multiple comparisons when necessary (R package lsmeans). R-squared values were calculated for general linear models using R package sjstata and Cohen’s d for paired comparisons were calculated using R package effsize.

3. Results

3.1. Controls and time series trials

Post-GnRH-injection T levels depended upon the time when the post-injection sample was taken (χ2 = 19.48, df = 3, P < 0.001). In both males and females, samples taken at 15, 45, or 60 min post-in- jection were no different from pre-injection samples (P > 0.80 for each pairwise comparison; Fig. 2). Samples taken at 30 min post-in- jection were significantly higher than initial T levels in both sexes (P < 0.05 for each).

In males, there was a significant interaction between sample (pre- or post-injection) and injection type (PBS or GnRH) on T (χ2 = 6.03, df = 1, P = 0.014). There was no difference in the T levels in PBS in- jected males before or after the injection (t = 1.29, df = 109, P = 0.57; d = 0.37) or between post-injection samples from PBS in- jected males and pre-injection levels from GnRH injected males (t = 0.13, df = 187, P = 0.99; d = 0.03). Testosterone levels taken

from GnRH injected males 30 min after the injection were higher than levels in the same males prior to the injection (t = 7.61, df = 109, P < 0.001; d = 0.78) and higher than PBS injected males both before (t = 4.89, df = 187, P < 0.001; d = 1.10) and after (t = 3.69, df = 187, P < 0.01; d = 0.83) their injection.

In females, there was also a significant interaction between sample and injection type on T (χ2 = 9.04, df = 1, P < 0.01). Testosterone levels were similar in females injected with PBS before and after the injection (t = 0.37, df = 107, P = 0.98; d = 0.10). Additionally, post- injection T levels from PBS injected females and pre-injection T levels from GnRH injected females were also similar (t = 0.19, df = 182, P = 0.99; d = 0.05). However, T levels taken from GnRH injected fe- males after the injection were higher than PBS injected females before (t = 3.72, df = 182, P < 0.01; d = 0.98) and after (t = 3.35, df = 182, P < 0.01; d = 0.96) the injected. Testosterone levels from GnRH injected females were also higher after the injection compared to prior to the injection (t = 9.29, df = 107, P < 0.001; d = 0.98).

3.2. Seasonal variation in GnRH response

Data for males and females were analyzed separately for the ana- lyses of T and sampling period because third-order interactions were evident when data from males and females were analyzed together with sex as a fixed effect (χ2 = 41.00, df = 10, P < 0.0001). In males, there was a significant interaction between sample (pre- or post-injec- tion) and sampling period on T (χ2 = 22.23, df = 3, P < 0.0001). Pairwise analyses revealed a number of differences (Fig. 3). There was no pattern of increasing pre-injection (circulating) T levels across mi- gratory stage (Fig. 3). Post-injection T was lower in fall relative to all spring samples and was highest at the northern sampling location overall (P < 0.01 for each comparison). Additionally, post-injection T levels at the northern sampling location were higher than pre-injection (circulating) levels at all migratory stages (P < 0.001 for each com- parison).

The effect of the injection on T did not differ across sampling period in females (χ2 = 3.03, df = 3, P = 0.39; Fig. 4). There was a sig- nificant effect of sample (pre- or post-injection) where post-injections samples were overall higher than pre-injection samples (χ2 = 66.30, df = 1, P < 0.0001). Migratory stage also had a significant effect on T (χ2 = 15.63, df = 3, P < 0.01). As determined by pairwise evaluation of effects, pre-injection T levels were consistent across the three spring sampling periods (Fig. 4; P > 0.50 for each comparison), but all values were higher than pre-injection levels during the fall sampling period (P < 0.05 for each comparison). Post-injection levels were higher than pre-injection levels during the spring sampling periods but not during the fall. While higher than the post-injection levels during fall (P < 0.05 for each comparison), post-injection levels across all three spring sampling periods were similar (P > 0.90 for each comparison).

3.3. Temporal variation

In males, Rpotential (response to GnRH injection) increased with date such that many individuals had low or no response early in the spring season while individuals sampled later had a strong and significant response (t = 3.81, df = 81, P < 0.001; R2 = 0.18; Fig. 5). Rpotential did not change with date in females (t = 0.78, df = 89, P = 0.44; R2 = 0.008; Fig. 5).

4. Discussion

This study examined HPG-axis activity in migrating male and fe- male thrushes during spring passage to determine the degree of overlap between the endocrine breeding development and spring migration in songbirds. Male thrushes responded to a GnRH injection with increases in T levels and this observed Rpotential increased both over time and with geographic proximity to the breeding grounds. These results indicate

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that endocrine breeding development occurs during spring migration in male thrushes. The GnRH bioassays elicited significant elevations in T levels in female thrushes but their Rpotential remained constant throughout spring migration. The results reveal overlap between the endocrinological development of breeding and spring migration in male songbirds, while the pattern in females suggests less overlap between endocrine breeding development and spring migration and raises im- portant questions about the transition from migration to breeding in female migratory songbirds (Fig. 6). The possibility of sex-dependent differences in the overlap between migration and physiological breeding development (breeding hormones, HPG axis activity, gonadal recrudescence) has been supported by previous studies. One such study examining HPG axis activity in Black-and-white Warblers (Mniotilta varia) at a single migratory site revealed a sex-dependent pattern si- milar to the results reported here: Males closer to their breeding

destination showed greater Rpotential compared to those that were fur- ther away but Rpotential was unrelated to breeding ground proximity in females (Covino et al., 2016).

Several previous studies have also detected changes to hormones of the HPG axis subsequent to photostimulation in migratory birds (Dawson et al., 2001; Deviche and Small, 2001; Leska and Dusza, 2007). For example, Dawson et al. (2002) demonstrated that hy- pothalamic GnRH levels increased after photostimulation in male starlings. While Wingfield and Farner (1978b) found similar increases in LH in migrating male White-crowned Sparrows (Zonotrichia leu- cophrys gambelii), Bauchinger et al. (2007) did not detect any changes in the LH levels of migrating Garden Warblers. Moreover, testicular re- crudescence has also been shown to occur throughout spring migration in both free-living (Bauchinger et al., 2005, 2007; Wingfield and Farner, 1978a, 1978b) and captive migrants when photostimulated

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Fig. 2. Testosterone levels in Swainson’s Thrushes before (Time = 0) and at various time points after an injection of GnRH administered during spring migration in Louisiana. Data for males and females at each time point are slightly offset to facilitate comparison. Error bars represent standard error of the mean. Sample sizes within each sex and time point are provided below the error bars.

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Fig. 3. Testosterone levels in male migrating Swainson’s Thrushes before (open circles) and 30 min after (filled circles) an injection of GnRH. Back transformed means and standard errors are presented. Sampling periods corresponds to migration season (fall or spring), the location of sampling (see Fig. 1), and/or the time period of passage. Sample sizes within each sampling period are provided. Groups that are significantly different are indicated with different capital letters as determined by least squares means pairwise comparisons.

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Fig. 4. Testosterone levels in female migrating Swainson’s Thrushes before (open circles) and 30 min after (filled circles) an injection of GnRH. Back transformed means and standard errors are presented. Sampling periods corresponds to migration season (fall or spring), the location of sampling (see Fig. 1), and/or the time period of passage. Sample sizes within each sampling period are provided. Groups that are significantly different are indicated with different capital letters as determined by least squares means pairwise comparisons.

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(Bluhm et al., 1991; Wingfield et al., 1996, 1997). Collectively studies focused on non-stimulated circulating T during migration have yielded conflicting results regarding whether T levels increase in male song- birds during spring migration (Bauchinger et al., 2007; Bluhm et al., 1991; Covino et al., 2015; Wingfield and Farner, 1978a, 1978b). However, investigations of HPG axis activity via measures of hy- pothalamic GnRH, gonadotropins, or using GnRH bioassays, and gonad recrudescence provide strong evidence for the overlap between spring migration and physiological breeding development (increasing HPG axis activity). Additionally, the increases seen in HPG axis sensitivity outpace increases in unstimulated circulating T levels (this study; Covino et al., 2015). If the sensitivity of the HPG axis increases without substantial increases in general circulating T levels, a balance between the necessity for endocrinological breeding development to occur during spring migration and the potential antagonistic effects of high levels of T on migration itself is achieved. Although not addressed in the present study, it is possible that gonadotropin-inhibitory hormone regulates gonadal steroid production during the migratory period (Ernst et al., 2016). Results from the present study, those described above, and that of Tonra et al. (2013), which detected increasing T levels late in the wintering period in male American Redstarts (Setophaga ruticilla), also a long-distance inter-continental migrant, indicate that at least some physiological breeding development occurs prior to the beginning of spring migration. Based on these studies, we suggest the relationship

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Fig. 5. Testosterone “Response” to gonadotropin-releasing hormone bioassays in migrating (A) male and (B) female Swainson’s Thrushes sampled during spring migration (both sites combined). Response was calculated as the levels of testosterone 30 min after an injection with gonadotropin-releasing hormone minus initial testosterone levels. Day of year is used as a temporal variable and refers to the number of days since 1 January. Solid lines indicate the best fit relationship and the shaded areas are the 95% confidence intervals. Response increased temporally in male migrants (t = 3.81, df = 81, P < 0.001; R2 = 0.18) but there was no temporal change in Response in female migrants (t = 0.78, df = 89, P = 0.44; R2 = 0.008).

Fig. 6. Theoretical schedule for physiological breeding development in male (blue line) and female (pink line) Nearctic-Neotropical migrants. Based on data in this study and others (see text) we propose that both males and females begin physiological develop- ment of breeding prior to or very soon after departure on spring migration. Males, however, continue this process throughout the migratory period while females may not continue physiological development of breeding during the migratory period and may delay the final stages until arrival on the breeding grounds. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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depicted in Fig. 6 as a model for physiological breeding development in male Nearctic-Neotropical migrants.

Evidence suggests that some degree of endocrinological breeding development occurs either before or during spring migration in female songbirds. That said, research has failed to produce consistent results across all hormones investigated, including T, LH, FSH, and estradiol, or across species studied (Covino et al., 2015, 2016; Wingfield et al., 1996, 1997; Wingfield and Farner, 1978a, 1978b). For example, in a study of female White-crowned Sparrows (Z. l. pugetensis), Wingfield and Farner (1978a) detected increases in both T and estradiol but not LH. Additionally, while Covino et al. (2015) showed that T levels in- creased during migration in female Northern Waterthrushes (Parkesia noveboracensis), this pattern was not found in the two other songbird species including the Swainson’s Thrush. In the current study, even though the degree to which female thrushes could elevate T did not vary throughout spring migration, their ability to significantly elevate T during spring migration contrasted with responses during other non- breeding time periods (i.e. fall migration). Some studies have demon- strated that the initial stages of follicular development occur during the vernal migratory period with the caveat that estradiol stimulation for final maturation of the ovary, yolk production, and yolk deposition in the follicles does not occur until females reach the breeding grounds (Wingfield and Farner, 1978a, 1978b). The Wingfield and Farner work on ovarian development and the results presented in the present study indicate that while gonadal development and increasing HPG axis sensitivity in females occurs prior to the breeding season, the full pro- gression of physiological breeding development may be delayed, in part, until females arrive on the breeding grounds.

Photoperiod serves as the initial cue for HPG axis activation and the development of migratory traits in both males and females of many temperate breeding songbird species (Ball and Ketterson, 2008; Helm et al., 2009; Ramenofsky, 2011; Rowan, 1925). In addition, supple- mentary factors, temperature and behavioral interactions, provide fine- tuning of the HPG axis for complete maturation of the female re- productive cycle (Ball and Ketterson, 2008). In this study, Rpotential in female thrushes did not vary during migration, whereas Rpotential in males increased as the migratory season progressed. Thus males and females have different patterns regarding the degree of overlap between spring migration and their physiological development of breeding. In males, endocrinological breeding development vis-à-vis increasing HPG axis sensitivity occurs at a steady pace throughout the migratory period, which would ensure that they arrive on the breeding grounds prepared to fully express breeding traits. The female pattern, however, may in- volve more fine-tuning by requiring supplementary and local cues from the breeding grounds and delaying the final stages of physiological breeding development until arrival. The supplementary cues that reg- ulate HPG axis action in females may include behavioral interactions with courting males as well as environmental cues such as temperature and food availability (Ball and Ketterson, 2008). Thus, the final stages of HPG axis activation and ovarian development may be delayed until females reach the breeding grounds where these supplementary cues would be available to them. Based on results from our current study and that of previous studies, we propose that female Nearctic-Neotropical migrants begin their physiological breeding development prior to or early during spring migration but that development does not continue throughout the migratory period and the final stages for of physiolo- gical breeding development are delayed until arrival on the breeding grounds (Fig. 6).

Both male and female thrushes had higher Rpotential during spring migration than fall migration. While sample sizes for fall birds were low, these results support the idea that T and HPG axis sensitivity are increasing during spring migration as a function of photoinduction and endocrine breeding development rather than due to their necessity for migration itself. Similarly, in some migratory species, migratory beha- viors are expressed prior to gonadal recrudescence indicating an asynchrony between the development of migratory and breeding

characteristics (Hahn et al., 1995). In fact, the photoperiodic cues for development of spring migration and breeding may be separate (Wang et al., 2013), and so development of one stage (e.g. migration) would not be required for the development of the subsequent stage (e.g. breeding). In this study, T levels did not elevate in response to the GnRH bioassay in either male or female thrushes tested during fall migration indicating that this ability is not a requirement for migration.

5. Conclusions

In our study, endocrinological breeding development was de- termined vis-à-vis measures of HPG axis sensitivity (Rpotential) in re- sponse to injections of GnRH. Our findings indicate that endocrine breeding development continues during spring migration in male mi- grants. This overlap between migration and endocrine breeding de- velopment was observed throughout the migratory season as males traveled towards their breeding destination. In female migrants, our results indicate that endocrinological breeding development did not occur throughout their migratory journey, however, data indicate that some endocrinological breeding development did occur prior to (or early during) spring migration. Additionally, our results indicate that Rpotential is higher during spring migration compared to fall migration in both male and female migrants. The difference in the phenology of changes to HPG axis sensitivity between males and females indicates sex-specific differences in physiological breeding development patterns (Fig. 6).

Acknowledgements

Funding for this research was provided by the Eastern Bird Banding Association, the Inland Bird Banding Association, the Louisiana Ornithological Society, the Alabama Ornithological Society, and the Wilson Ornithological Society. KMC was supported by an NSF GK-12 Fellowship (#0947944) during the 2013–2014 academic year. Support was also provided by the Shoals Marine Laboratory and by an anon- ymous donor to the Appledore Island Migration Station. We wish to thank the Migratory Bird Research Group, the field technicians and volunteers at Johnson’s Bayou, Appledore Island, and Dauphin Island and Matthew Capps at the Dauphin Island Parks and Beach Board without whom the field work would not have been possible. We are also grateful to B.R. Kreiser at USM for helpful advice that the use of la- boratory space. This research was conducted under Federal USGS Bird Banding Permits to FRM (21221) and SRM (22243), Federal Fish and Wildlife Scientific Collecting Permit MB425364-3, Alabama Scientific Collecting Permit 7217, Louisiana State Scientific Collecting Permit LNHP-11-058, Maine State Scientific Collecting Permit (2011-56), and University of Southern Mississippi IACUC permit (11092210). This is contribution 24 of the Appledore Island Migration Station and 186 of the Shoals Marine Laboratory.

References

Adkins-Regan, E., 2005. Hormones and Animal Social Behavior. Princeton University Press, Princeton, NJ.

Ball, G.F., Ketterson, E.D., 2008. Sex differences in the response to environmental cues regulating seasonal reproduction in birds. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 363, 231–246. http://dx.doi.org/10.1098/rstb.2007.2137.

Bates, D., 2010. lmr4: Mixed Effects Modeling With R. Springer, New York. Bates, D., Mächler, M., Bolker, B., Walker, S., 2014. Fitting linear mixed-effects models

using lme4. J. Stat. Softw. 67, 1–48. http://dx.doi.org/10.18637/jss.v067.i01. Bauchinger, U., Wohlmann, A., Biebach, H., 2005. Flexible remodeling of organ size

during spring migration of the garden warbler (Sylvia borin). Zoology 108, 97–106. http://dx.doi.org/10.1016/j.zool.2005.03.003.

Bauchinger, U., Hof, T., Biebach, H., 2007. Testicular development during long-distance spring migration. Horm. Behav. 51, 295–305. http://dx.doi.org/10.1016/j.yhbeh. 2006.10.010.

Bentley, G., Kriegsfeld, L., Osugi, T., Ukena, K., O’Brien, S., Perfito, N., Moore, I.T., Tsutsui, K., Wingfield, J.C., 2006. Interactions of gonadotropin-releasing hormone (GnRH) and gonadotropin-inhibitory hormone (GnIH) in birds and mammals. J. Exp. Zool. 305A, 807–814. http://dx.doi.org/10.1002/jez.a.

K.M. Covino et al. Hormones and Behavior 97 (2018) 112–120

118

http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0005
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0005
http://dx.doi.org/10.1098/rstb.2007.2137
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0015
http://dx.doi.org/10.18637/jss.v067.i01
http://dx.doi.org/10.1016/j.zool.2005.03.003
http://dx.doi.org/10.1016/j.yhbeh.2006.10.010
http://dx.doi.org/10.1016/j.yhbeh.2006.10.010
http://dx.doi.org/10.1002/jez.a
Bluhm, C., Schwabl, H., Schwabl, I., Perera, A., Follett, B., Goldsmith, A., Gwinner, E., 1991. Variation in hypothalamic gonadotrophin-releasing hormone content, plasma and pituitary LH, and in-vitro testosterone release in a long-distance migratory bird, the garden warbler (Sylvia borin), under constant photoperiods. J. Endocrinol. 128, 339–345.

Cain, K.E., Pryke, S.R., 2017. Testosterone production ability predicts breeding success and tracks breeding stage in male finches. J. Evol. Biol. 30, 430–436. http://dx.doi. org/10.1111/jeb.13005.

Caro, S.P., Lambrechts, M.M., Chastel, O., Sharp, P.J., Thomas, D.W., Balthazart, J., 2006. Simultaneous pituitary-gonadal recrudescence in two Corsican populations of male blue tits with asynchronous breeding dates. Horm. Behav. 50, 347–360. http://dx. doi.org/10.1016/j.yhbeh.2006.03.001.

Covino, K.M., Morris, S.R., Moore, F.R., 2015. Patterns of testosterone in three Nearctic–Neotropical migratory songbirds during spring passage. Gen. Comp. Endocrinol. http://dx.doi.org/10.1016/j.ygcen.2015.08.012.

Covino, K.M., Jawor, J.M., Kelly, J.F., Moore, F.R., 2016. Overlapping life-history stages in migrating songbirds: variation in circulating testosterone and testosterone pro- duction capacity. J. Ornithol. http://dx.doi.org/10.1007/s10336-016-1385-8.

Davies, S., Behbahaninia, H., Giraudeau, M., Meddle, S.L., Waites, K., Deviche, P., 2015. Advanced seasonal reproductive development in a male urban bird is reflected in earlier plasma luteinizing hormone rise but not energetic status. Gen. Comp. Endocrinol. 224, 1–10. http://dx.doi.org/10.1016/j.ygcen.2015.05.005.

Dawson, A., King, V., Bentley, G.E., Ball, G., 2001. Photoperiodic control of seasonality in birds. J. Biol. 16, 365–380. http://dx.doi.org/10.1177/074873001129002079.

Dawson, A., Talbot, R.T., Dunn, I.C., Sharp, P.J., 2002. Changes in Basal Hypothalamic Chicken Gonadotropin-releasing Hormone-I and Vasoactive Intestinal Polypeptide Associated with a Photo-induced Cycle in Gonadal Maturation and Prolactin Secretion in Intact and Thyroidectomized Starlings (Sturnus vulgaris). 14. pp. 533–539.

Deviche, P., 1995. Androgen regulation of avian premigratory hyperphagia and fattening: from eco-physiology to neuroendocrinology. Am. Zool. 35, 234–245.

Deviche, P., Small, T., 2001. Photoperiodic control of seasonal reproduction: neu- roendocrine mechanisms and adaptations. In: Dawson, A., Chaturvedi, C.M. (Eds.), Avian Endocrinology. Narosa Publishing House, New Delhi, pp. 113–128.

Deviche, P., Small, T., Sharp, P., Tsutsui, K., 2006. Control of luteinizing hormone and testosterone secretion in a flexibly breeding male passerine, the Rufous-winged Sparrow, Aimophila carpalis. Gen. Comp. Endocrinol. 149, 226–235. http://dx.doi. org/10.1016/j.ygcen.2006.06.004.

DeVries, M.S., Holbrook, A.L., Winters, C.P., Jawor, J.M., 2011. Non-breeding gonadal testosterone production of male and female Northern Cardinals (Cardinalis cardinalis) following GnRH challenge. Gen. Comp. Endocrinol. 174, 370–378. http://dx.doi.org/ 10.1016/j.ygcen.2011.09.016.

DeVries, M.S., Winters, C.P., Jawor, J.M., 2012. Testosterone elevation and response to gonadotropin-releasing hormone challenge by male Northern Cardinals (Cardinalis cardinalis) following aggressive behavior. Horm. Behav. 62, 99–105. http://dx.doi. org/10.1016/j.yhbeh.2012.05.008.

Ernst, D.K., Lynn, S.E., Bentley, G.E., 2016. Differential response of GnIH in the brain and gonads following acute stress in a songbird. Gen. Comp. Endocrinol. 227, 51–57. http://dx.doi.org/10.1016/j.ygcen.2015.05.016.

Goymann, W., Wingfield, J., 2004. Competing females and caring males. Sex steroids in African black coucals. Anim. Behav. 68, 733–740. http://dx.doi.org/10.1016/j. anbehav.2003.12.012.

Goymann, W., Wingfield, J.C., 2014. Male-to-female testosterone ratios, dimorphism, and life history—what does it really tell us? Behav. Ecol. 25, 685–699. http://dx.doi.org/ 10.1093/beheco/aru019.

Goymann, W., Landys, M.M., Wingfield, J.C., 2007. Distinguishing seasonal androgen responses from male—male androgen responsiveness—revisiting the challenge hy- pothesis. Horm. Behav. 51, 463–476. http://dx.doi.org/10.1016/j.yhbeh.2007.01. 007.

Griffiths, R., Daan, S., Dijkstra, C., 1996. Sex identification in birds using two CHD genes. Proc. Biol. Sci. 263, 1251–1256.

Griffiths, R., Double, M.C., Orr, K., Dawson, R.J.G., 1998. A DNA test to sex most birds. Mol. Ecol. 7, 1071–1075.

Hahn, T.P., Wingfteld, J.C., Mullen, R., Deviche, P.J., 1995. Endocrine bases of spatial and temporal opportunism in arctic-breeding birds. Integr. Comp. Biol. 35, 259–273. http://dx.doi.org/10.1093/icb/35.3.259.

Helm, B., Schwabl, I., Gwinner, E., 2009. Circannual basis of geographically distinct bird schedules. J. Exp. Biol. 212, 1259–1269. http://dx.doi.org/10.1242/jeb.025411.

Jacobs, J.D., Wingfield, J.C., 2000. Endocrine control of life-cycle stages: a constraint on response to the environment? Condor 102, 35–51. http://dx.doi.org/10.1650/0010- 5422(2000)102[0035:ECOLCS]2.0.CO;2.

Jawor, J.M., 2007. Testosterone in northern cardinals (Cardinalis cardinalis): possible influence of prolonged territorial behavior. Auk 124, 331–338.

Jawor, J.M., McGlothlin, J.W., Casto, J.M., Greives, T.J., Snajdr, E. a, Bentley, G.E., Ketterson, E.D., 2006. Seasonal and individual variation in response to GnRH chal- lenge in male dark-eyed juncos (Junco hyemalis). Gen. Comp. Endocrinol. 149, 182–189. http://dx.doi.org/10.1016/j.ygcen.2006.05.013.

Jawor, J.M., Mcglothlin, J.W., Casto, J.M., Greives, T.J., Snajdr, E.a., Bentley, G.E., Ketterson, E.D., 2007. Testosterone response to GnRH in a female songbird varies with stage of reproduction: implications for adult behaviour and maternal effects. Funct. Ecol. 21, 767–775. http://dx.doi.org/10.1111/j.1365-2435.2007.01280.x.

Ketterson, E.D., Nolan, V., Wolf, L., Ziegenfus, C., Dufty, A.M., Ball, G.F., Johnsen, T.S., 1991. Testosterone and avian life histories: the effect of experimentally elevated testosterone on corticosterone and body mass in dark-eyed juncos. Horm. Behav. 25, 489–503. http://dx.doi.org/10.1016/0018-506X(91)90016-B.

Ketterson, E.D., Nolan, V., Sandell, M., 2005. Testosterone in females: mediator of

adaptive traits, constraint on sexual dimorphism, or both? Am. Nat. 166 (Suppl), S85–S98. http://dx.doi.org/10.1086/444602.

Ketterson, E.D., Fudickar, A.M., Atwell, J.W., Greives, T.J., 2015. Seasonal timing and population divergence: when to breed, when to migrate. Curr. Opin. Behav. Sci. 6, 50–58. http://dx.doi.org/10.1016/j.cobeha.2015.09.001.

King, J.R., Farner, D.S., 1962. The relationship of fat deposition to Zugunruhe and mi- gration. Condor 65, 200–223.

Leska, A., Dusza, L., 2007. Seasonal Changes in the Hypothalamo-pituitary-gonadal Axis in Birds. 7. pp. 99–126.

Lofts, B., Marshall, A.J., 1961. Zugunruhe activity in castrated bramblings Fringilla montifringill. Ibis 103, 189–194 (Lond. 1859).

McGlothlin, J.W., Jawor, J.M., Greives, T.J., Casto, J.M., Phillips, J.L., Ketterson, E.D., 2008. Hormones and honest signals: males with larger ornaments elevate testos- terone more when challenged. J. Evol. Biol. 21, 39–48. http://dx.doi.org/10.1111/j. 1420-9101.2007.01471.x.

McGlothlin, J.W., Whittaker, D.J., Schrock, S.E., Gerlach, N.M., Jawor, J.M., Snajdr, E.A., Ketterson, E.D., 2010. Natural selection on testosterone production in a wild songbird population. Am. Nat. 175, 687–701. http://dx.doi.org/10.1086/652469.

Morton, M.L., Mewaldt, L.R., 1962. Some effects of castration on a migratory sparrow. Physiol. Zool. 35, 237–247.

Norris, D.O., 1997. Vertebrate Endocrinology, 3rd ed. Academic Press, London. Owen, J.C., Garvin, M.C., Moore, F.R., 2014. Elevated testosterone advances onset of

migratory restlessness in a nearctic-neotropical landbird. Behav. Ecol. Sociobiol. 68, 561–569. http://dx.doi.org/10.1007/s00265-013-1671-x.

Partecke, J., Van Hof, T.J., Gwinner, E., 2005. Underlying Physiological Control of Reproduction in Urban and Forest-dwelling European Blackbirds Turdus merula. 4. pp. 295–305.

Ramenofsky, M., 2011. Hormones in migration and reproductive cycles of birds. In: Norris, D., Lopez, K. (Eds.), Hormones and Reproduction in Vertebrates Volume 8. Academic Press, pp. 205–236.

Ramenofsky, M., Németh, Z., 2014. Regulatory mechanisms for the development of the migratory phenotype: roles for photoperiod and the gonad. Horm. Behav. 66, 148–158. http://dx.doi.org/10.1016/j.yhbeh.2014.04.012.

Ramenofsky, M., Wingfield, J.C., 2006. Behavioral and physiological conflicts in mi- grants: the transition between migration and breeding. J. Ornithol. 147, 135–145. http://dx.doi.org/10.1007/s10336-005-0050-4.

Ramenofsky, A.M., Gray, J.M., Johnson, R.B., 1992. Behavioral and physiological ad- justments of birds living in winter flocks. Ornis Scand. 23, 371–380.

Ramenofsky, M., Savard, R., Greenwood, M.R.C., 1999. Seasonal and diel transitions in physiology and behavior in the migratory dark-eyed junco. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 122, 385–397. http://dx.doi.org/10.1016/S1095-6433(99) 00013-6.

Rosvall, K.A., 2013. Proximate perspectives on the evolution of female aggression: good for the gander, good for the goose? Philos. Trans. R. Soc. B Biol. Sci. 368, 20130083. http://dx.doi.org/10.1098/rstb.2013.0083.

Rowan, W., 1925. Relation of light to bird migration and developmental changes. Nature 115, 494–495.

Rowan, W., 1929. Experiments in bird migration. I. Manipulation of the reproductive cycle: seasonal histological changes in the gonads. Proc. Bost. Soc. Nat. Hist. 39, 151–208.

Schaper, S.V., Dawson, A., Sharp, P.J., Caro, S.P., Visser, M.E., 2012. General and Comparative Endocrinology Individual variation in avian reproductive physiology does not reliably predict variation in laying date. Gen. Comp. Endocrinol. 179, 53–62. http://dx.doi.org/10.1016/j.ygcen.2012.07.021.

Schoech, S.J., Mumme, R.L., Wingfield, J.C., 1996. Delayed breeding in the cooperatively breeding Florida scrub-jay (Aphelocoma coerulescens): inhibition or the absence of stimulation? Behav. Ecol. Sociobiol. 39, 77–90. http://dx.doi.org/10.1007/ s002650050269.

Schwabl, H., Farner, D.S., 1989. Dependency on testosterone of photoperiodically-in- duced vernal fat deposition in female White-crowned Sparrows. Condor 91, 108–112.

Schwabl, H., Schwabl-Benzinger, I., Goldsmith, A.R., Farner, D.S., 1988. Effects of ovariectomy on long-day-induced premigratory fat deposition, plasma levels of lu- teinizing hormone and prolactin, and molt in white-crowned sparrows, Zonotrichia leucophrys gambelii. Gen. Comp. Endocrinol. 71, 398–405. http://dx.doi.org/10. 1016/0016-6480(88)90268-7.

Smith, R.J., Moore, F.R., 2005. Arrival timing and seasonal reproductive performance in a long-distance migratory landbird. Behav. Ecol. Sociobiol. 57, 231–239.

Soma, K.K., 2006. Testosterone and aggression: berthold, birds and beyond. J. Neuroendocrinol. 18, 543–551. http://dx.doi.org/10.1210/jc.2006-0178.

Staub, N.L., De Beer, M., 1997. The role of androgens in female vertebrates. Gen. Comp. Endocrinol. 108, 1–24. http://dx.doi.org/10.1006/gcen.1997.6962.

Stetson, M.H., Erickson, J.E., 1972. Hormonal control of photoperiodically induced fat deposition in White-crowned Sparrows. Gen. Comp. Endocrinol. 19, 355–362.

Tonra, C.M., Marra, P.P., Holberton, R.L., 2011. Early elevation of testosterone advances migratory preparation in a songbird. J. Exp. Biol. 214, 2761–2767. http://dx.doi.org/ 10.1242/jeb.054734.

Tonra, C.M., Marra, P.P., Holberton, R.L., 2013. Experimental and observational studies of seasonal interactions between overlapping life history stages in a migratory bird. Horm. Behav. 64, 825–832. http://dx.doi.org/10.1016/j.yhbeh.2013.10.004.

Wang, G., Ramenofsky, M., Wingfield, J.C., 2013. Apparent Dissociation of Photoperiodic Time Measurement between Vernal Migration and Breeding under Dim Green Light Conditions in Gambel’s White-crowned Sparrow Zonotrichia leucophrys gambelii. 59. pp. 349–359.

Weise, C.M., 1967. Castration and spring migration in the White-throated Sparrow. Condor 69, 49–68.

Wingfield, J.C., 1984. Environmental and endocrine control of reproduction in the song

K.M. Covino et al. Hormones and Behavior 97 (2018) 112–120

119

http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0040
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0040
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0040
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0040
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0040
http://dx.doi.org/10.1111/jeb.13005
http://dx.doi.org/10.1111/jeb.13005
http://dx.doi.org/10.1016/j.yhbeh.2006.03.001
http://dx.doi.org/10.1016/j.yhbeh.2006.03.001
http://dx.doi.org/10.1016/j.ygcen.2015.08.012
http://dx.doi.org/10.1007/s10336-016-1385-8
http://dx.doi.org/10.1016/j.ygcen.2015.05.005
http://dx.doi.org/10.1177/074873001129002079
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0075
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0075
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0075
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0075
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0075
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0080
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0080
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0085
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0085
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0085
http://dx.doi.org/10.1016/j.ygcen.2006.06.004
http://dx.doi.org/10.1016/j.ygcen.2006.06.004
http://dx.doi.org/10.1016/j.ygcen.2011.09.016
http://dx.doi.org/10.1016/j.ygcen.2011.09.016
http://dx.doi.org/10.1016/j.yhbeh.2012.05.008
http://dx.doi.org/10.1016/j.yhbeh.2012.05.008
http://dx.doi.org/10.1016/j.ygcen.2015.05.016
http://dx.doi.org/10.1016/j.anbehav.2003.12.012
http://dx.doi.org/10.1016/j.anbehav.2003.12.012
http://dx.doi.org/10.1093/beheco/aru019
http://dx.doi.org/10.1093/beheco/aru019
http://dx.doi.org/10.1016/j.yhbeh.2007.01.007
http://dx.doi.org/10.1016/j.yhbeh.2007.01.007
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0125
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0125
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0130
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0130
http://dx.doi.org/10.1093/icb/35.3.259
http://dx.doi.org/10.1242/jeb.025411
http://dx.doi.org/10.1650/0010-5422(2000)102[0035:ECOLCS]2.0.CO;2
http://dx.doi.org/10.1650/0010-5422(2000)102[0035:ECOLCS]2.0.CO;2
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0150
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0150
http://dx.doi.org/10.1016/j.ygcen.2006.05.013
http://dx.doi.org/10.1111/j.1365-2435.2007.01280.x
http://dx.doi.org/10.1016/0018-506X(91)90016-B
http://dx.doi.org/10.1086/444602
http://dx.doi.org/10.1016/j.cobeha.2015.09.001
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0180
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0180
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0185
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0185
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0190
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0190
http://dx.doi.org/10.1111/j.1420-9101.2007.01471.x
http://dx.doi.org/10.1111/j.1420-9101.2007.01471.x
http://dx.doi.org/10.1086/652469
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0205
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0205
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0210
http://dx.doi.org/10.1007/s00265-013-1671-x
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0220
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0220
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0220
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0225
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0225
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0225
http://dx.doi.org/10.1016/j.yhbeh.2014.04.012
http://dx.doi.org/10.1007/s10336-005-0050-4
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0240
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0240
http://dx.doi.org/10.1016/S1095-6433(99)00013-6
http://dx.doi.org/10.1016/S1095-6433(99)00013-6
http://dx.doi.org/10.1098/rstb.2013.0083
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0255
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0255
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0260
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0260
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0260
http://dx.doi.org/10.1016/j.ygcen.2012.07.021
http://dx.doi.org/10.1007/s002650050269
http://dx.doi.org/10.1007/s002650050269
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0275
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0275
http://dx.doi.org/10.1016/0016-6480(88)90268-7
http://dx.doi.org/10.1016/0016-6480(88)90268-7
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0285
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0285
http://dx.doi.org/10.1210/jc.2006-0178
http://dx.doi.org/10.1006/gcen.1997.6962
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0300
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0300
http://dx.doi.org/10.1242/jeb.054734
http://dx.doi.org/10.1242/jeb.054734
http://dx.doi.org/10.1016/j.yhbeh.2013.10.004
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0315
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0315
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0315
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0315
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0320
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0320
sparrow, Melospiza melodia. I. Temporal organization of the breeding cycle. Gen. Comp. Endocrinol. 56, 417–424. http://dx.doi.org/10.1016/0016-6480(84)90084-4.

Wingfield, J.C., Farner, D.S., 1978a. The endocrinology of a natural breeding population of the White-crowned Sparrow (Zonotrichia leucophrys pugetensis). Physiol. Zool. 51, 188–205.

Wingfield, J.C., Farner, D.S., 1978b. The annual cycle of plasma irLH and steroid hor- mones in feral populations of the White-crowned Sparrow, Zonotrichia leucophrys gambelii. Biol. Reprod. 19, 1046–1056.

Wingfield, J.C., Ramenofsky, M., 2011. Hormone-behavior interrelationships of birds in response to weather. Adv. Study Behav. 43, 93–188. http://dx.doi.org/10.1016/ B978-0-12-380896-7.00003-4.

Wingfield, J.C., Silverin, B., 2009. Ecophysiological studies of hormone – behavior rela- tions in birds. Horm. Brain Behav. 817–855.

Wingfield, J.C., Crim, J.W., Mattocks, P.W.J., Farner, D.S., 1979. Responses of photo- sensitive and photorefractory male White-crowned Sparrows (Zonotrichia leucophrys gambelii) to synthetic mammalian luteinizing hormone releasing hormone (Syn- LHRH). Biol. Reprod. 21, 801–806.

Wingfield, J.C., Hegner, R.E., Dufty, A., Ball, G., 1990. The “Challenge Hypothesis”: theoretical implications for patterns of testosterone secretion, mating systems, and breeding strategies. Am. Nat. 136, 829–846.

Wingfield, J.C., Hegner, R.E., Lewis, D.M., 1991. Circulating levels of luteinizing hormone and steroid hormones in relation to social status in the cooperatively breeding white- browed sparrow weaver, Plocepasser mahali. J. Zool. 225, 43–58. http://dx.doi.org/ 10.1111/j.1469-7998.1991.tb03800.x.

Wingfield, J.C., Hahn, T.P., Wada, M., Astheimer, L.B., 1996. Interrelationship of day length and temperature on the control of gonadal development, body mass, and fat score in White-Crowned Sparrows, Zonotrichia leucophrys gambelii. Gen. Comp. Endocrinol. 255, 242–255.

Wingfield, J.C., Hahn, T.P., Wada, M., Schoech, S.J., 1997. Effects of day length and temperature on gonadal development, body mass, and fat depots in white-crowned sparrows, Zonotrichia leucophrys pugetensis. Gen. Comp. Endocrinol. 107, 44–62. http://dx.doi.org/10.1006/gcen.1997.6894.

Wingfield, J.C., Lynn, E., Soma, K.K., 2001. Avoiding the “Costs” of testosterone: ecolo- gical bases of hormone-behavior interactions. Brain Behav. Evol. 57, 239–251.

K.M. Covino et al. Hormones and Behavior 97 (2018) 112–120

120

http://dx.doi.org/10.1016/0016-6480(84)90084-4
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0330
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0330
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0330
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0335
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0335
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0335
http://dx.doi.org/10.1016/B978-0-12-380896-7.00003-4
http://dx.doi.org/10.1016/B978-0-12-380896-7.00003-4
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0345
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0345
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0350
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0350
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0350
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0350
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0355
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0355
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0355
http://dx.doi.org/10.1111/j.1469-7998.1991.tb03800.x
http://dx.doi.org/10.1111/j.1469-7998.1991.tb03800.x
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0365
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0365
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0365
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0365
http://dx.doi.org/10.1006/gcen.1997.6894
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0375
http://refhub.elsevier.com/S0018-506X(17)30006-5/rf0375
Sex-specific hypothalamic-pituitary-gonadal axis sensitivity in migrating songbirds
Introduction
Materials and methods
Study species and study sites
Capture and sampling
Testosterone assays
Statistical analyses
Results
Controls and time series trials
Seasonal variation in GnRH response
Temporal variation
Discussion
Conclusions
Acknowledgements
References

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