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Simultaneous treatment with an aromatase inhibitor and an anti-androgen decreases the likelihood of dawn song in free-living male great tits, Parus major
Hormones and Behavior 48 (2005) 243 – 251 www.elsevier.com/locate/yhbeh
Simultaneous treatment with an aromatase inhibitor and an anti-androgen decreases the likelihood of dawn song in free-living male great tits, Parus major Els Van Duyse, Rianne Pinxten, Tinne Snoeijs, Marcel Eens * Behaviour and Ecology Research Group, Department of Biology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium Received 7 May 2004; revised 4 February 2005; accepted 7 February 2005 Available online 5 May 2005
Abstract Gonadal steroids, most importantly testosterone (T), are considered to be a major factor in the expression of adult song behavior in temperate-zone songbirds. The action of T within specific brain regions involved in the regulation of song may occur either directly, or through its androgenic or estrogenic metabolites. In the present study, we tested steroid-dependence of great tit dawn song by blocking both known pathways of T action by simultaneous implantation of flutamide, an anti-androgen, and ATD, an aromatase inhibitor. By our knowledge, this is the first study investigating the effects of androgen inhibitors on dawn song in free-living birds. Male great tits were implanted during their mate’s egg laying stage, being the time of maximal male song activity at dawn. Treatment with ATD and flutamide significantly increased plasma T levels, probably because feedback mechanisms on T secretion were inhibited. The treatment decreased the likelihood of showing dawn song, which is in line with the hypothesis that sex steroids are involved in the endocrine control of song behavior. In males that did show dawn song, we found no evidence for a treatment effect on song quality. Although the implants were present for the larger part of the breeding season, males were able to maintain control of a territory and mate and to complete their brood cycle as successful as control males. D 2005 Elsevier Inc. All rights reserved. Keywords: Song; Dawn chorus; Parus major; Testosterone; ATD; Flutamide; Androgen inhibitors
Introduction In temperate-zone songbirds, gonadal steroids, primarily testosterone (T), are considered to play a key role in the activation of song behavior in adult males (Balthazart, 1983; Brenowitz and Kroodsma, 1996; Schlinger, 1997). For instance, castration typically reduces or eliminates song production, and treatment of castrated males with exogenous T can reinstate song (e.g., Arnold, 1975; Harding et al., 1988). Furthermore, implanting intact males with T generally increases song activity (e.g., De Ridder et al., 2000; Hegner and Wingfield, 1987; Ketterson et al., 1992; Silverin, 1980). Also, seasonal changes in circulating T levels and
* Corresponding author. Fax: +32 3 820 22 71. E-mail address: [email protected] (M. Eens). 0018-506X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2005.02.013
testes size are closely correlated with changes in song activity (Ball, 1999). The action of T, or its active androgenic and estrogenic metabolites, within specific brain regions is thought to be critical for the regulation of song behavior. Several aspects of song such as production, learning, and memory are regulated by a specialized group of interconnected nuclei known as the ‘‘song control system’’ (Gil and Gahr, 2002; Nottebohm et al., 1976; Schlinger, 1997). Androgen and estrogen receptors and the mRNA coding for these proteins are found within several of these song control nuclei, and it is generally assumed that the activity of T or its metabolites, through its metabolic conversion into 17h-estradiol (E2, via aromatization) or 5a-DHT (via 5a-reduction), within the song control system is critical for song expression. However, recent studies in European starlings, Sturnus vulgaris, suggest that aromatase activity in the medial preoptic
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nucleus (POM), a nucleus outside these brain areas that is known to regulate male sexual arousal, might be involved in the seasonal expression of courtship song, thus in the motivation to sing in a reproductive context (see Ball et al., 2002; Riters and Ball, 1999; Riters et al., 2000). So far, only few studies have examined how blocking both pathways of T action affects male song behavior, and results were inconsistent. Combined treatment with an aromatase inhibitor and an anti-androgen decreased song activity, both spontaneously and towards a female, in captive spotted antbirds, Hylophylax naevioides, a tropical species (Hau et al., 2000), and in response to a simulated territorial intrusion in free-living song sparrows, Melospiza melodia, outside the breeding season (Soma et al., 1999). By contrast, similar treatment increased the duration males spent vocalizing in free-living red-winged blackbirds, Agelaius phoeniceus, during the breeding season (Beletsky et al., 1990), and no effect was found on song activity during a simulated territorial intrusion in free-living tropical rufous-collared sparrows, Zonotrichia capensis, in the prebreeding season (Moore et al., 2004). We used the great tit, Parus major, a small passerine, to study the effects of blocking T action on male singing behavior. There are several indications that T modulates song performance in male great tits. For instance, a strong correlation was found between the annual course of plasma T levels and day-time song activity, both peaking early in the breeding season (Van Duyse et al., 2003). Also, in two independent studies, T-implanted males showed an increased day-time song activity during the breeding season (Van Duyse et al., 2000, 2002). The presence of telencephalic aromatase activity in the great tit brain indicates that T might need to be converted into E2 for an effect on song behavior to occur (Silverin et al., 2000). Furthermore, in unmanipulated great tits, the level of plasma T was positively related to the length of their songs during the dawn chorus (Van Duyse, 2004), suggesting that, besides an activational role for T on song behavior, the level of plasma T might also affect the way in which song is produced. In the present study, we focused on singing behavior at dawn because, in contrast to song during the day, male great tits sing continuously with a very high performance at this time of the day (Mace, 1986, 1987). During the egg laying period, males sing almost uninterruptedly at dawn until their female has laid her egg and leaves the nest to join the male (Mace, 1986, 1987, but also see Gammon, 2004 for similar behavior in North-American black-capped chickadees, Poecile atricapillus). In contrast to daytime singing, dawn song is less or not affected by possibly confounding variables such as foraging and male– male interactions (Gammon, 2004; Slagsvold et al., 1994). To block both the androgenic and estrogenic pathway of T action, free-living male great tits were implanted simultaneously with the anti-androgen flutamide, which inhibits the action of T and 5a-DHT through
their receptors, and the aromatase inhibitor ATD, which inhibits the synthesis of estrogens from androgens (Soma et al., 1999 and references therein). Furthermore, implanted males were monitored for breeding performance, because manipulating a male’s hormonal levels may affect his breeding success for instance through an effect on his fertility or territorial or reproductive behavior (e.g., Ketterson et al., 2001; Wingfield et al., 2001). To the best of our knowledge, our study is the first to investigate the effects of blocking T action on dawn song behavior instead of on diurnal song.
Materials and methods Study population and study species All data were collected during the breeding season of 2001 in a nest box breeding great tit population inhabiting the semi-rural grounds surrounding the University of Antwerp at Wilrijk, Belgium. Since 1997, all birds in the population were systematically marked with an individual combination of plastic color rings and a numbered aluminum ring to allow identification in the field. Great tits are socially monogamous (Cramp and Perrins, 1993; Gosler, 1993). Normally, 1 egg is laid daily at dawn, until the clutch contains on average 8 to 9 eggs. We defined the day on which the first egg is laid in a clutch Fday 1_ of the egg laying stage and we numbered all following days accordingly. In the study population, laying of first clutches started between March 30 and April 30 in the year of study. The population is mainly single-brooded. Procedures in this study have been approved by the Ethical Advisory Committee of the University of Antwerp and are in agreement with Belgian and Flemish legislation concerning the protection of animal welfare. Experimental design During the weeks preceding the experiment, censuses were made to identify breeding pairs at each nest box in the study area and to situate territory boundaries by observing territorial disputes, foraging areas by pairs, and positions of singing males. Also, nest boxes were checked regularly to determine the start of egg laying. The experimental procedure was to (i) record a male’s entire dawn chorus early in the egg laying stage of its mate (days 2 –3), (ii) implant the male as soon as possible after the recording (usually later on the same day) with androgen inhibitors (N = 7) or empty silastic tubes (N = 6), and (iii) record the male’s entire dawn chorus again during the same egg laying stage (2– 5 days after the day of implantation). As additional controls, 4 males were recorded twice during the egg laying stage of their mate without treatment (intact males), with the first recording corresponding to (i) and the second to (iii).
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Implantation procedure Androgen inhibitors used were 1-4-6-androstatriene3,17-dione (ATD), an aromatization inhibitor, and flutamide (a-a-a-trifluoro-2-methyl-4’-nitro-m-propionotoluidid), an androgen receptor antagonist (see Soma et al., 1999, and references therein). Implants were 13-mm-long silastic tubes (Degania Silicone, i.d. 1.47 mm, o.d. 1.96 mm), which remained empty or were packed with crystalline ATD or flutamide over a length of 10 mm and were sealed at both ends with silastic glue (Dow Corning). To ascertain rapid diffusion, implants were kept in a physiological solution for 15 –20 h preceding implantation. We captured males in mist nets as they approached a life male decoy and conspecific song playback installed close to the nest box. Implants were inserted subcutaneously through a small incision of the skin upward along the pectoral muscle while the birds were under local anesthesia (Xylocaine, 10% spray). A male either received 2 ATD implants and 2 flutamide implants (one of each kind at either side, AF males), or 2 2 empty tubes (empty-implanted males). The whole treatment took about 5 min. Size and number of implants were selected on the basis of previous behavioral studies on similar-sized male birds of other songbird species (Schwabl and Kriner, 1991; Soma et al., 1999). Dawn song recording and analysis Great tits are discontinuous singers (Krebs, 1976). The basic unit of great tit song is the phrase (Fig. 1). A phrase (usually 0.2 –0.5 s) is a combination of 1 –5 notes (Krebs, 1976; McGregor and Krebs, 1982), with a note being a continuous trace on the spectrogram. An individual great tit male has a song repertoire of 1 to 8 distinct types of phrases, called song types (McGregor et al., 1981). A phrase is repeated in a stereotyped way during 1 – 5 s to form a strophe (Lambrechts and Dhondt, 1986; McGregor and Krebs, 1982). Successive strophes are separated by periods of silence, called interstrophe pauses. The combination of a strophe and the following interstrophe pause is referred to as a song unit (Lambrechts and Dhondt, 1988). Great tits typically sing strophes of the same song type for several minutes before they switch to the next song type (Krebs, 1976). A song bout is a group of strophes of the same song type (Krebs, 1976). Dawn song occurs between the male’s wakening time, which depends largely on light intensity, and the first emergence of the female from her roost-hole (male and female roost apart, Mace, 1986). Female emergence typically
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coincides with a sharp drop in male singing activity as the pair engages in copulation (Mace, 1986). In general, great tit males sing at dawn from about 3 weeks before egg laying until a variable number of days after clutch completion (Mace, 1986). Although much debated, the exact function of great tit dawn song is still not understood, but probably relates to mate guarding, mate attraction, and territoriality (Gorissen and Eens, 2004; Krebs, 1977; Krebs et al., 1981; Mace, 1987; Slagsvold et al., 1994). Song recordings were made using a Sony WM-D3 tape recorder and a Sennheiser ME 67 directional microphone. Care was taken by the observer to be present on the territory before the start of the dawn chorus. First emergence of the female from the nest box, typically around sunrise, was considered as the end of the dawn chorus (see above), unless the male had stopped singing before female emergence. The song recordings were submitted to spectrographic analysis using the computer program Avisoft SASlab Pro (Specht, 1993). We measured the duration (in seconds) of each strophe (i.e., strophe length) and interstrophe pause (i.e., pause length) in each dawn chorus, and assigned each strophe to a song type through description of the phrase (number of notes, note frequency, note length) constituting it. A male’s dawn chorus was characterized by the following song features: (1) Total song time, calculated by summing all strophe lengths (in seconds) in the dawn chorus. (2) Repertoire size, defined as the number of song types sung during the dawn chorus. (3) Song rate, defined as the average number of strophes per minute of dawn chorus. (4) Mean strophe length, obtained by averaging the mean strophe lengths of song types that were sung both before and after implantation by the respective male. This way, mean strophe length was not confounded by the number of notes in a phrase (Lambrechts and Dhondt, 1987). (5) Drift, being the phenomenon where singing rate systematically changes in the course of a song bout (Lambrechts and Dhondt, 1988). Drift is attributed to neuromuscular exhaustion of the respiratory and song system (Lambrechts and Dhondt, 1988) and a decreasing motivation (Weary et al., 1988). Although drift generally occurs in the great tit (Lambrechts and Dhondt, 1988), there is large inter-male variability in
Fig. 1. Sound spectrogram of a song unit of a two-note song type of a great tit male.
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the proportion of bouts in a male’s dawn chorus that show drift. As a measure of drift in a bout, we used the slope of the linear regression, expressing the percentage performance time in a song unit (strophe length divided by the sum of strophe and pause length) against strophe number (following Lambrechts and Dhondt, 1988). We called Fbouts with negative drift_ those bouts for which the slope of the regression had a negative sign and differed significantly from zero, i.e., those bouts with a significant reduction in singing rate throughout the bout (Lambrechts and Dhondt, 1988). To assess the occurrence of drift in the dawn chorus of an individual, we calculated the percentage of bouts with negative drift. This percentage will be referred to as Fdrift_. For each individual, we also determined the Faverage bout length_ as Faverage number of strophes in a bout_ to be used in the statistical analysis (see below). Throughout these calculations, we considered all song bouts of a male’s dawn chorus that consisted of at least 10 song units. Blood sampling and testosterone analysis Both ATD and flutamide have been shown to increase plasma T levels, probably because feedback mechanisms on T secretion were inhibited (Robaire, 1999; Saldanha and Schlinger, 1999). To examine the effect of treatment on plasma T levels, we took a blood sample at the time of implantation and during the nestling stage. During the nestling stage, we succeeded in recapturing 5 AF males and 5 empty-implanted males on the nest while feeding young (on average respectively 30.4 (SE 0.9) and 31.8 (SE 3.5) days after implantation). None of the recaptured males had lost its implants. After blood sampling, the implants were removed under local anesthesia. Blood was obtained by puncturing a wing vein with a sterile needle (Terumo, 0.4 20 mm), and approximately 100 – 180 Al (less than 1% of a bird’s weight) was transferred into an Eppendorf tube using heparinized microhematocrit capillaries. Samples were centrifuged for 14 min at 2500 g in an Eppendorf centrifuge. The plasma fraction was removed and stored below 25-C until assayed for T. Plasma T concentrations were measured using a commercial 125I RIA kit purchased from ICN Biomedicals, Inc. (Costa Mesa, CA) following the manufacturer’s protocol (see Van Duyse et al., 2003, for more information concerning the extraction procedure). The specification sheets provided by the company indicate that the primary antibody used in this assay does not cross-react significantly with other steroids beside T (5a-dihydrotestosterone: 3.4%; 5a-androstane-3h,17h-diol: 2.2%; 11-oxo-testosterone: 2%; all other steroids: <1%). All samples were processed within a single assay. Intra-assay variation ranged from 6.0% (low concentrations) to 9.1% (high concentrations). After implantation, samples of 3 AF males contained T levels above the
maximum detection limit (33.3 ng/ml). For statistical purposes, these samples were assigned a value equivalent to this maximum detection limit. Breeding success A male’s breeding success may have been affected in several ways. For example, because males were implanted early in the egg laying stage and pairs continue copulating throughout the egg laying stage (Mace, 1987), interfering with a male’s endocrine state may affect fertilization success and thus hatching success. Further, fledging success and fledging age may have been affected through an effect on male feeding behavior (Moreno et al., 1999). Also, loss of territory or mate could have resulted in nest failure. To collect data on breeding performance, nest boxes of implanted males were inspected regularly throughout the breeding season. We only used data of the brood during which implantation took place (one AF male fathered a second clutch). Nests of AF and empty-implanted males were compared for clutch size (the total number of eggs in the clutch), hatching success (defined as the proportion of eggs that hatched successfully), fledging success (defined as the proportion of hatchlings that fledged successfully), and fledging age (in number of days with day 1 = the day the first eggs hatched). Sample size was smaller for fledging age (see Results) because two AF nests and one control nest were deserted during the feeding period for unknown reasons. Statistical analysis In our design, each male, assigned to either of three treatments (AF, empty-implanted, intact), was recorded in two periods (before and after treatment). Accordingly, we performed two-way repeated-measures analyses of variance (ANOVAR) with the respective song variable (total song time, strophe length, song rate, or repertoire size) as response variable and treatment and period as fixed factors. For drift, we carried out a two-way repeated-measures analysis of covariance (ANCOVAR) including average bout length as a covariate since the number of song units in a bout may affect the occurrence of drift in a bout (Lambrechts and Dhondt, 1988). In all analyses, the test of interest was the period treatment interaction since it indicates whether the change in the respective song variable between the two periods depends on the treatment given, i.e., the treatment effect. Therefore, tests of main effects are not reported in the Results section. Tukey HSD post hoc tests are given where the period treatment interaction was significant. For all song variables, the change in dawn song between the two recordings did not differ significantly between empty-implanted and intact males. Therefore, we considered it justified to combine both groups into one control group (C males) and the results are reported as such.
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Similarly, an ANOVAR with treatment (AF, emptyimplanted) and period as factors was used to test the effect of the treatment on plasma T levels. To meet assumptions required for parametric testing, drift was subjected to an arcsine square-root transformation, and T was log transformed. Proportions of males showing dawn song were compared using Fisher’s exact probability tests. Components of breeding success were compared between nests of C males and AF males using t tests or Mann –Whitney U tests. All tests were two-tailed, and a was set at 0.05. Untransformed data are given as means T SE when they followed a normal distribution, or as medians with quartile ranges when they could not be normalized. Transformed data are presented as back-transformed means with 95% confidence intervals. Analyses were conducted using Statistica (Statsoft, 1994).
Results Plasma testosterone Treatment with ATD and flutamide (AF treatment) significantly affected plasma T levels (ANOVAR, period treatment interaction, F 1,8 = 73.11, P = 0.000027; Fig. 2). While plasma T levels did not differ significantly between the two treatment groups at the time of implantation, AF males had significantly higher plasma T levels than C males at recapture during the feeding period (Tukey, P = 0.00023). The plasma T increase in AF males between implantation and recapture was highly significant (Tukey, P = 0.00029), whereas plasma T levels in C males tended to decrease between the two captures (Tukey, P = 0.052). These results indicate that the implants worked effectively. Note that ATD and flutamide inhibit the high plasma T levels in AF males to exert a biological effect.
Fig. 2. Plasma T levels at the time of implantation during the egg laying stage (before treatment) and 1 month later during the nestling stage (after treatment) for empty-implanted males (C males) and males that were implanted simultaneously with ATD and flutamide (AF males). Numbers above bars indicate sample sizes. Data shown are means T SE.
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Dawn song While all 10 C males sang a dawn chorus after implantation, 2 of the 7 AF males did not show dawn song after implantation, which is not significantly different. However, of the 60 intact males we set out to record during the egg laying stage in the same study population during 1999, 2000, and 2001, none failed to produce dawn song. In comparison to this proportion (60 out of 60), the proportion of AF males that did not sing at dawn (2 out of 7) was significantly larger than would be expected by chance (Fisher’s exact test, P = 0.0095). We are certain that these two silent AF males had not died after implantation, since they were recaptured during feeding. We are also confident that we would have found them if they had been singing. Based on their song repertoire and position, surrounding singing males could all be assigned unambiguously to neighboring territories. Furthermore, because of the heterogeneous landscape, the birds could be located accurately. Also, one of the two silent males was visited a second morning, during which it also did not sing. Finally, there were no other obvious reasons, such as for example cold weather or heavy rain, that could explain the lack of dawn singing in these two males. Before implantation, total song time ranged 273s – 1075s for the C males and 358s – 1814s for the AF males (see Fig. 3). Thus, the two silent AF males (358s and 525s) were initially situated at the lower end of their range, with only one AF male in between (430s). Yet, they did not sing unusually little before implantation, because two C males had an even shorter total singing time (both 273s, see Fig. 3). When comparing total song time, a two-way repeatedmeasures ANOVA showed a significant period treatment interaction ( F 1,15 = 5.14, P = 0.038). AF males sang significantly more before implantation (847.4 T 192.8s) than after implantation (308.3 T 90.5s, Tukey, P = 0.0024), while total song time in C males did not differ between both song recordings (606.4 T 77.3s versus 423.9 T 65.5s, see Fig. 3). There was no significant difference between treatments in total song time before or after implantation. There was still a clear trend for an effect on total song time when the two AF males that did not sing after implantation were not included in the analysis (see Fig. 3; ANOVAR, period treatment interaction, F 1,13 = 4.55, P = 0.053). For comparison of the other song characteristics between AF and C males, we did not consider the two AF males that did not sing (Table 1). Using a two-way repeated-measures ANOVA, we found no significant effect of the treatment on strophe length, song rate, or repertoire size. Furthermore, an ANOVAR showed that drift was not significantly affected by treatment while controlling for average bout length (Table 1). Summarized, the results suggest that blocking both androgenic and estrogenic action decreased the likelihood
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Fig. 3. Comparison of total song time during the dawn chorus before and after treatment between individual males that were treated simultaneously with ATD and flutamide (AF males: black dots, N = 7) and control males (C males: empty dots, N = 10). The two AF males with dashed lines did not show song activity after treatment.
of showing dawn song. However, we found no evidence for a treatment effect on song quality in males that did show dawn song. Breeding success AF treatment did not significantly affect male breeding success in terms of clutch size, hatching success, fledging success, or fledging age (Table 2). Table 2 shows fledging success of all nests, including nests that were deserted during the feeding stage. In nests that were not deserted, all nestlings fledged, except for two nestlings in one control nest.
Discussion Dawn song In the present study, combined treatment with an aromatase inhibitor (ATD) and an anti-androgen (flutamide), impairing both estrogenic and androgenic actions, decreased the likelihood of showing dawn song in male great tits. Thus, dawn song activity appears to be modulated by androgens, their estrogenic metabolites, or both in this species. This result is in line with the hypothesis that sex steroids are involved in the activation
of song behavior in temperate-zone male songbirds (Balthazart, 1983; Brenowitz and Kroodsma, 1996; Schlinger, 1997; Pinxten et al., 2002). On the other hand, we found no evidence for a treatment effect on song quality in treated males that did show dawn song. While two treated males (AF males) showed no dawn song at all, the five AF males that did sing at dawn did not perform significantly different from the control males. Perhaps these results indicate a threshold mechanism of action of steroid hormones on dawn singing. If so, ATD and flutamide were successful in suppressing steroid action below the threshold level in the silent males but not in singing males. This difference among males could have been caused for example by individual differences in threshold level, in responsiveness to the inhibitors, or in diffusion rate of the inhibitors from the implants. A difference in initial plasma T levels is not supported by our data, because the two silent AF males had T levels close to the average value. Yet, the relatively short total song times before implantation in these silent males (see Fig. 3) may explain why they were most affected by the treatment. In line with this threshold-hypothesis, Pinxten et al. (2003) recently found evidence for a threshold level of T action on mate attraction behavior, including singing behavior, in European starlings, S. vulgaris. Also, Fusani and Hutchison (2003) report indications for a threshold model of action of steroid hormones on the structure of courtship behavior in male ring doves, Streptopelia risoria. Alternatively, we cannot exclude completely that the treatment may have made these two silent males slightly sick such that they stopped singing. In a previous study in the same population, we were unable to demonstrate an effect of experimental T elevation on dawn song performance in the great tit (Van Duyse, 2004). One explanation for that result could be that T is not causally related with dawn song performance. On the other hand, we suggested that dawn song performance could not be enhanced by T implantation because of some kind of constraint, for example, because males were already singing on the limits of their ability or because receptors were already saturated by naturally occurring steroid levels. The suppressive effect of androgen inhibitors on dawn song activity in the current study makes this constraint hypothesis more plausible.
Table 1 Dawn song performance before and after treatment for control males (C males) and males that were treated simultaneously with ATD and flutamide (AF males) Treatment
Strophe length (s) Song rate (no./min) Repertoire size Drift (%)
C males (N = 10)
AF males (N = 5)
Before
After
Before
After
2.31 T 0.17 6.69 T 0.47 3.9 T 0.57 52.3 (25.6 – 78.3)
2.11 T 0.17 5.36 T 0.71 3.6 T 0.56 38.1 (26.3 – 50.6)
2.39 T 0.24 7.18 T 0.88 4.2 T 0.58 47.8 (30.9 – 64.9)
2.14 T 0.19 5.41 T 0.61 3.8 T 0.37 50 (32.8 – 67.2)
Only males that showed dawn song are included. Note. Values are shown as means T SE or as back-transformed means and 95% confidence intervals.
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Table 2 Comparison of components of breeding success between nests of control males (C nests) and nests of males that were treated with ATD and flutamide early in the egg laying stage (AF nests)
Clutch size Hatching success (%) Fledging success (%) Fledging age (days)
C nests
AF nests
Statistical comparison
8 T 1.71 (6) 73 (50 – 91) (6) 100 (78 – 100) (6) 18.8 T 0.66 (5)
7.7 T 0.6 (7) 90 (0 – 100) (7) 100 (0 – 100) (7) 19 T 0.71 (5)
t test, t 11 = 0.17, P = 0.87 U test, Z = 0.66, P = 0.51 U test, Z = 0.0, P = 1 t test, t 8 = 0.21, P = 0.84
Note. Values are shown as mean T SE (sample size) or as median (quartile range) (sample size).
Breeding success A bird’s internal endocrine environment may be currently adaptive. If so, experimentally induced deviations from the natural situation may negatively affect male fitness (Ketterson and Nolan, 1999). In this study, the elevated plasma T levels during the feeding stage clearly indicate that treatment with ATD and flutamide interfered with the internal endocrine environment. Yet, AF males were able to maintain control of a territory and mate, and to complete their brood cycle as successful as control males. One AF male even reared a second brood, while no control male did so. The effect of blocking T action on breeding success is likely to be dependent on the importance of T action. Indeed, androgen inhibitors have been found to suppress Tdependent correlates of reproductive performance. For instance, treatment with the anti-androgen cyproterone acetate (CA) has decreased copulation behavior in Japanese quail, Coturnix c. japonica (Adkins and Mason, 1974), and courtship behavior and testes size in male ring doves (Silver, 1977). Furthermore, combined treatment with ATD and flutamide has been found to decrease territorial behavior in European stonechats, Saxicola torquata (Canoine and Gwinner, 2002), and in non-breeding song sparrows (Soma et al., 1999) and territory size in red-winged blackbirds (Beletsky et al., 1990). On the other hand, in house sparrows, Passer domesticus (Hegner and Wingfield, 1987), and spotless starlings, Sturnus unicolor (Moreno et al., 1999), there are indications that treatment with, respectively, flutamide or cyproterone acetate during the feeding stage can actually increase the number of fledglings, probably through an increase in male feeding rates. This is in line with the finding in several species that experimental T elevation is incompatible with full paternal care (Ketterson and Nolan, 1999). We know of no previous studies examining male breeding success after simultaneous inhibition of androgenic and estrogenic actions. By contrast to other species, T treatment does not depress male nestling feeding behavior in the great tit (Van Duyse et al., 2000, 2002), which possibly explains why blocking the action of T did not affect male breeding success in the current study. Further, some effects could have been left unnoticed. For example, we may have been unable to demonstrate a positive effect on fledging success, because fledging success was already high in control nests. Also, because sample sizes
were rather small, statistical power was limited. In monogamous temperate-zone passerines, including the great tit (Van Duyse et al., 2003), T secretion is highest during territorial establishment and egg laying and lowest in parental phases, probably reflecting the changing importance of T action throughout the breeding season (Wingfield et al., 1990). In our study, great tits were implanted with androgen inhibitors for the larger part of the breeding season. Hence, a net effect on breeding success might have been absent because positive effects in some stages were counteracting negative effects in other stages. Furthermore, we did not consider all possible fitness correlates that are likely affected by T metabolism (e.g., territory size, male survival, paternity loss through extra-pair behavior of the female), leaving the possibility of an overall negative effect on male fitness as a consequence of blocking the action of T in this study. Clearly, discriminating between these possible explanations is not feasible with the present data. However, the observation that interference with the action of T in free-living birds does not necessarily result in abandonment of breeding may offer opportunities for future studies. In particular, suppression of song activity using androgen inhibitors may be the basis of studies on the function of song behavior in general and dawn song in particular.
Acknowledgments We are indebted to Jacques Balthazart and Philippe Absil for providing us with ATD and flutamide. Thanks are also extended to Lieve Geenen for hormone analyses and to all field assistants for their help with nestbox inspections. E.V.D, R.P., and T.S. were supported by the Fund for Scientific Research Flanders, Belgium (F.W.O.-Flanders). This study was also made possible through financial support from the University of Antwerp and by Research Projects (G.0075.98 and G.0420.02) of the F.W.O.-Flanders. References Adkins, E.K., Mason, P., 1974. Effects of cyproterone acetate in the male Japanese quail. Horm. Behav. 5, 1 – 6. Arnold, A.P., 1975. The effects of castration and androgen replacement on song, courtship and aggression in zebra finches (Poephila guttata). J. Exp. Zool. 191, 309 – 326. Ball, G.F., 1999. The neuroendocrine basis of seasonal changes in vocal behavior among songbirds. In: Hauser, M.D., Konishi, M. (Eds.),
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Decline in song output by great tits: exhaustion or motivation. Anim. Behav. 36, 1242 – 1244. Wingfield, J.C., Hegner, R.E., Duffy Jr., A.M., Ball, G.F., 1990. The Fchallenge hypothesis_: theoretical implications for patterns of testosterone secretion, mating systems and breeding strategies. Am. Nat. 136, 829 – 846. Wingfield, J.C., Lynn, S.E., Soma, K.K., 2001. Avoiding the Fcosts_ of testosterone: ecological bases of hormone-behavior interactions. Brain Behav. Evol. 57, 239 – 251.
Simultaneous treatment with an aromatase inhibitor and an anti-androgen decreases the likelihood of dawn song in free-living male great tits, Parus major Els Van Duyse, Rianne Pinxten, Tinne Snoeijs, Marcel Eens * Behaviour and Ecology Research Group, Department of Biology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium Received 7 May 2004; revised 4 February 2005; accepted 7 February 2005 Available online 5 May 2005
Abstract Gonadal steroids, most importantly testosterone (T), are considered to be a major factor in the expression of adult song behavior in temperate-zone songbirds. The action of T within specific brain regions involved in the regulation of song may occur either directly, or through its androgenic or estrogenic metabolites. In the present study, we tested steroid-dependence of great tit dawn song by blocking both known pathways of T action by simultaneous implantation of flutamide, an anti-androgen, and ATD, an aromatase inhibitor. By our knowledge, this is the first study investigating the effects of androgen inhibitors on dawn song in free-living birds. Male great tits were implanted during their mate’s egg laying stage, being the time of maximal male song activity at dawn. Treatment with ATD and flutamide significantly increased plasma T levels, probably because feedback mechanisms on T secretion were inhibited. The treatment decreased the likelihood of showing dawn song, which is in line with the hypothesis that sex steroids are involved in the endocrine control of song behavior. In males that did show dawn song, we found no evidence for a treatment effect on song quality. Although the implants were present for the larger part of the breeding season, males were able to maintain control of a territory and mate and to complete their brood cycle as successful as control males. D 2005 Elsevier Inc. All rights reserved. Keywords: Song; Dawn chorus; Parus major; Testosterone; ATD; Flutamide; Androgen inhibitors
Introduction In temperate-zone songbirds, gonadal steroids, primarily testosterone (T), are considered to play a key role in the activation of song behavior in adult males (Balthazart, 1983; Brenowitz and Kroodsma, 1996; Schlinger, 1997). For instance, castration typically reduces or eliminates song production, and treatment of castrated males with exogenous T can reinstate song (e.g., Arnold, 1975; Harding et al., 1988). Furthermore, implanting intact males with T generally increases song activity (e.g., De Ridder et al., 2000; Hegner and Wingfield, 1987; Ketterson et al., 1992; Silverin, 1980). Also, seasonal changes in circulating T levels and
* Corresponding author. Fax: +32 3 820 22 71. E-mail address: [email protected] (M. Eens). 0018-506X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2005.02.013
testes size are closely correlated with changes in song activity (Ball, 1999). The action of T, or its active androgenic and estrogenic metabolites, within specific brain regions is thought to be critical for the regulation of song behavior. Several aspects of song such as production, learning, and memory are regulated by a specialized group of interconnected nuclei known as the ‘‘song control system’’ (Gil and Gahr, 2002; Nottebohm et al., 1976; Schlinger, 1997). Androgen and estrogen receptors and the mRNA coding for these proteins are found within several of these song control nuclei, and it is generally assumed that the activity of T or its metabolites, through its metabolic conversion into 17h-estradiol (E2, via aromatization) or 5a-DHT (via 5a-reduction), within the song control system is critical for song expression. However, recent studies in European starlings, Sturnus vulgaris, suggest that aromatase activity in the medial preoptic
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nucleus (POM), a nucleus outside these brain areas that is known to regulate male sexual arousal, might be involved in the seasonal expression of courtship song, thus in the motivation to sing in a reproductive context (see Ball et al., 2002; Riters and Ball, 1999; Riters et al., 2000). So far, only few studies have examined how blocking both pathways of T action affects male song behavior, and results were inconsistent. Combined treatment with an aromatase inhibitor and an anti-androgen decreased song activity, both spontaneously and towards a female, in captive spotted antbirds, Hylophylax naevioides, a tropical species (Hau et al., 2000), and in response to a simulated territorial intrusion in free-living song sparrows, Melospiza melodia, outside the breeding season (Soma et al., 1999). By contrast, similar treatment increased the duration males spent vocalizing in free-living red-winged blackbirds, Agelaius phoeniceus, during the breeding season (Beletsky et al., 1990), and no effect was found on song activity during a simulated territorial intrusion in free-living tropical rufous-collared sparrows, Zonotrichia capensis, in the prebreeding season (Moore et al., 2004). We used the great tit, Parus major, a small passerine, to study the effects of blocking T action on male singing behavior. There are several indications that T modulates song performance in male great tits. For instance, a strong correlation was found between the annual course of plasma T levels and day-time song activity, both peaking early in the breeding season (Van Duyse et al., 2003). Also, in two independent studies, T-implanted males showed an increased day-time song activity during the breeding season (Van Duyse et al., 2000, 2002). The presence of telencephalic aromatase activity in the great tit brain indicates that T might need to be converted into E2 for an effect on song behavior to occur (Silverin et al., 2000). Furthermore, in unmanipulated great tits, the level of plasma T was positively related to the length of their songs during the dawn chorus (Van Duyse, 2004), suggesting that, besides an activational role for T on song behavior, the level of plasma T might also affect the way in which song is produced. In the present study, we focused on singing behavior at dawn because, in contrast to song during the day, male great tits sing continuously with a very high performance at this time of the day (Mace, 1986, 1987). During the egg laying period, males sing almost uninterruptedly at dawn until their female has laid her egg and leaves the nest to join the male (Mace, 1986, 1987, but also see Gammon, 2004 for similar behavior in North-American black-capped chickadees, Poecile atricapillus). In contrast to daytime singing, dawn song is less or not affected by possibly confounding variables such as foraging and male– male interactions (Gammon, 2004; Slagsvold et al., 1994). To block both the androgenic and estrogenic pathway of T action, free-living male great tits were implanted simultaneously with the anti-androgen flutamide, which inhibits the action of T and 5a-DHT through
their receptors, and the aromatase inhibitor ATD, which inhibits the synthesis of estrogens from androgens (Soma et al., 1999 and references therein). Furthermore, implanted males were monitored for breeding performance, because manipulating a male’s hormonal levels may affect his breeding success for instance through an effect on his fertility or territorial or reproductive behavior (e.g., Ketterson et al., 2001; Wingfield et al., 2001). To the best of our knowledge, our study is the first to investigate the effects of blocking T action on dawn song behavior instead of on diurnal song.
Materials and methods Study population and study species All data were collected during the breeding season of 2001 in a nest box breeding great tit population inhabiting the semi-rural grounds surrounding the University of Antwerp at Wilrijk, Belgium. Since 1997, all birds in the population were systematically marked with an individual combination of plastic color rings and a numbered aluminum ring to allow identification in the field. Great tits are socially monogamous (Cramp and Perrins, 1993; Gosler, 1993). Normally, 1 egg is laid daily at dawn, until the clutch contains on average 8 to 9 eggs. We defined the day on which the first egg is laid in a clutch Fday 1_ of the egg laying stage and we numbered all following days accordingly. In the study population, laying of first clutches started between March 30 and April 30 in the year of study. The population is mainly single-brooded. Procedures in this study have been approved by the Ethical Advisory Committee of the University of Antwerp and are in agreement with Belgian and Flemish legislation concerning the protection of animal welfare. Experimental design During the weeks preceding the experiment, censuses were made to identify breeding pairs at each nest box in the study area and to situate territory boundaries by observing territorial disputes, foraging areas by pairs, and positions of singing males. Also, nest boxes were checked regularly to determine the start of egg laying. The experimental procedure was to (i) record a male’s entire dawn chorus early in the egg laying stage of its mate (days 2 –3), (ii) implant the male as soon as possible after the recording (usually later on the same day) with androgen inhibitors (N = 7) or empty silastic tubes (N = 6), and (iii) record the male’s entire dawn chorus again during the same egg laying stage (2– 5 days after the day of implantation). As additional controls, 4 males were recorded twice during the egg laying stage of their mate without treatment (intact males), with the first recording corresponding to (i) and the second to (iii).
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Implantation procedure Androgen inhibitors used were 1-4-6-androstatriene3,17-dione (ATD), an aromatization inhibitor, and flutamide (a-a-a-trifluoro-2-methyl-4’-nitro-m-propionotoluidid), an androgen receptor antagonist (see Soma et al., 1999, and references therein). Implants were 13-mm-long silastic tubes (Degania Silicone, i.d. 1.47 mm, o.d. 1.96 mm), which remained empty or were packed with crystalline ATD or flutamide over a length of 10 mm and were sealed at both ends with silastic glue (Dow Corning). To ascertain rapid diffusion, implants were kept in a physiological solution for 15 –20 h preceding implantation. We captured males in mist nets as they approached a life male decoy and conspecific song playback installed close to the nest box. Implants were inserted subcutaneously through a small incision of the skin upward along the pectoral muscle while the birds were under local anesthesia (Xylocaine, 10% spray). A male either received 2 ATD implants and 2 flutamide implants (one of each kind at either side, AF males), or 2 2 empty tubes (empty-implanted males). The whole treatment took about 5 min. Size and number of implants were selected on the basis of previous behavioral studies on similar-sized male birds of other songbird species (Schwabl and Kriner, 1991; Soma et al., 1999). Dawn song recording and analysis Great tits are discontinuous singers (Krebs, 1976). The basic unit of great tit song is the phrase (Fig. 1). A phrase (usually 0.2 –0.5 s) is a combination of 1 –5 notes (Krebs, 1976; McGregor and Krebs, 1982), with a note being a continuous trace on the spectrogram. An individual great tit male has a song repertoire of 1 to 8 distinct types of phrases, called song types (McGregor et al., 1981). A phrase is repeated in a stereotyped way during 1 – 5 s to form a strophe (Lambrechts and Dhondt, 1986; McGregor and Krebs, 1982). Successive strophes are separated by periods of silence, called interstrophe pauses. The combination of a strophe and the following interstrophe pause is referred to as a song unit (Lambrechts and Dhondt, 1988). Great tits typically sing strophes of the same song type for several minutes before they switch to the next song type (Krebs, 1976). A song bout is a group of strophes of the same song type (Krebs, 1976). Dawn song occurs between the male’s wakening time, which depends largely on light intensity, and the first emergence of the female from her roost-hole (male and female roost apart, Mace, 1986). Female emergence typically
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coincides with a sharp drop in male singing activity as the pair engages in copulation (Mace, 1986). In general, great tit males sing at dawn from about 3 weeks before egg laying until a variable number of days after clutch completion (Mace, 1986). Although much debated, the exact function of great tit dawn song is still not understood, but probably relates to mate guarding, mate attraction, and territoriality (Gorissen and Eens, 2004; Krebs, 1977; Krebs et al., 1981; Mace, 1987; Slagsvold et al., 1994). Song recordings were made using a Sony WM-D3 tape recorder and a Sennheiser ME 67 directional microphone. Care was taken by the observer to be present on the territory before the start of the dawn chorus. First emergence of the female from the nest box, typically around sunrise, was considered as the end of the dawn chorus (see above), unless the male had stopped singing before female emergence. The song recordings were submitted to spectrographic analysis using the computer program Avisoft SASlab Pro (Specht, 1993). We measured the duration (in seconds) of each strophe (i.e., strophe length) and interstrophe pause (i.e., pause length) in each dawn chorus, and assigned each strophe to a song type through description of the phrase (number of notes, note frequency, note length) constituting it. A male’s dawn chorus was characterized by the following song features: (1) Total song time, calculated by summing all strophe lengths (in seconds) in the dawn chorus. (2) Repertoire size, defined as the number of song types sung during the dawn chorus. (3) Song rate, defined as the average number of strophes per minute of dawn chorus. (4) Mean strophe length, obtained by averaging the mean strophe lengths of song types that were sung both before and after implantation by the respective male. This way, mean strophe length was not confounded by the number of notes in a phrase (Lambrechts and Dhondt, 1987). (5) Drift, being the phenomenon where singing rate systematically changes in the course of a song bout (Lambrechts and Dhondt, 1988). Drift is attributed to neuromuscular exhaustion of the respiratory and song system (Lambrechts and Dhondt, 1988) and a decreasing motivation (Weary et al., 1988). Although drift generally occurs in the great tit (Lambrechts and Dhondt, 1988), there is large inter-male variability in
Fig. 1. Sound spectrogram of a song unit of a two-note song type of a great tit male.
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the proportion of bouts in a male’s dawn chorus that show drift. As a measure of drift in a bout, we used the slope of the linear regression, expressing the percentage performance time in a song unit (strophe length divided by the sum of strophe and pause length) against strophe number (following Lambrechts and Dhondt, 1988). We called Fbouts with negative drift_ those bouts for which the slope of the regression had a negative sign and differed significantly from zero, i.e., those bouts with a significant reduction in singing rate throughout the bout (Lambrechts and Dhondt, 1988). To assess the occurrence of drift in the dawn chorus of an individual, we calculated the percentage of bouts with negative drift. This percentage will be referred to as Fdrift_. For each individual, we also determined the Faverage bout length_ as Faverage number of strophes in a bout_ to be used in the statistical analysis (see below). Throughout these calculations, we considered all song bouts of a male’s dawn chorus that consisted of at least 10 song units. Blood sampling and testosterone analysis Both ATD and flutamide have been shown to increase plasma T levels, probably because feedback mechanisms on T secretion were inhibited (Robaire, 1999; Saldanha and Schlinger, 1999). To examine the effect of treatment on plasma T levels, we took a blood sample at the time of implantation and during the nestling stage. During the nestling stage, we succeeded in recapturing 5 AF males and 5 empty-implanted males on the nest while feeding young (on average respectively 30.4 (SE 0.9) and 31.8 (SE 3.5) days after implantation). None of the recaptured males had lost its implants. After blood sampling, the implants were removed under local anesthesia. Blood was obtained by puncturing a wing vein with a sterile needle (Terumo, 0.4 20 mm), and approximately 100 – 180 Al (less than 1% of a bird’s weight) was transferred into an Eppendorf tube using heparinized microhematocrit capillaries. Samples were centrifuged for 14 min at 2500 g in an Eppendorf centrifuge. The plasma fraction was removed and stored below 25-C until assayed for T. Plasma T concentrations were measured using a commercial 125I RIA kit purchased from ICN Biomedicals, Inc. (Costa Mesa, CA) following the manufacturer’s protocol (see Van Duyse et al., 2003, for more information concerning the extraction procedure). The specification sheets provided by the company indicate that the primary antibody used in this assay does not cross-react significantly with other steroids beside T (5a-dihydrotestosterone: 3.4%; 5a-androstane-3h,17h-diol: 2.2%; 11-oxo-testosterone: 2%; all other steroids: <1%). All samples were processed within a single assay. Intra-assay variation ranged from 6.0% (low concentrations) to 9.1% (high concentrations). After implantation, samples of 3 AF males contained T levels above the
maximum detection limit (33.3 ng/ml). For statistical purposes, these samples were assigned a value equivalent to this maximum detection limit. Breeding success A male’s breeding success may have been affected in several ways. For example, because males were implanted early in the egg laying stage and pairs continue copulating throughout the egg laying stage (Mace, 1987), interfering with a male’s endocrine state may affect fertilization success and thus hatching success. Further, fledging success and fledging age may have been affected through an effect on male feeding behavior (Moreno et al., 1999). Also, loss of territory or mate could have resulted in nest failure. To collect data on breeding performance, nest boxes of implanted males were inspected regularly throughout the breeding season. We only used data of the brood during which implantation took place (one AF male fathered a second clutch). Nests of AF and empty-implanted males were compared for clutch size (the total number of eggs in the clutch), hatching success (defined as the proportion of eggs that hatched successfully), fledging success (defined as the proportion of hatchlings that fledged successfully), and fledging age (in number of days with day 1 = the day the first eggs hatched). Sample size was smaller for fledging age (see Results) because two AF nests and one control nest were deserted during the feeding period for unknown reasons. Statistical analysis In our design, each male, assigned to either of three treatments (AF, empty-implanted, intact), was recorded in two periods (before and after treatment). Accordingly, we performed two-way repeated-measures analyses of variance (ANOVAR) with the respective song variable (total song time, strophe length, song rate, or repertoire size) as response variable and treatment and period as fixed factors. For drift, we carried out a two-way repeated-measures analysis of covariance (ANCOVAR) including average bout length as a covariate since the number of song units in a bout may affect the occurrence of drift in a bout (Lambrechts and Dhondt, 1988). In all analyses, the test of interest was the period treatment interaction since it indicates whether the change in the respective song variable between the two periods depends on the treatment given, i.e., the treatment effect. Therefore, tests of main effects are not reported in the Results section. Tukey HSD post hoc tests are given where the period treatment interaction was significant. For all song variables, the change in dawn song between the two recordings did not differ significantly between empty-implanted and intact males. Therefore, we considered it justified to combine both groups into one control group (C males) and the results are reported as such.
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Similarly, an ANOVAR with treatment (AF, emptyimplanted) and period as factors was used to test the effect of the treatment on plasma T levels. To meet assumptions required for parametric testing, drift was subjected to an arcsine square-root transformation, and T was log transformed. Proportions of males showing dawn song were compared using Fisher’s exact probability tests. Components of breeding success were compared between nests of C males and AF males using t tests or Mann –Whitney U tests. All tests were two-tailed, and a was set at 0.05. Untransformed data are given as means T SE when they followed a normal distribution, or as medians with quartile ranges when they could not be normalized. Transformed data are presented as back-transformed means with 95% confidence intervals. Analyses were conducted using Statistica (Statsoft, 1994).
Results Plasma testosterone Treatment with ATD and flutamide (AF treatment) significantly affected plasma T levels (ANOVAR, period treatment interaction, F 1,8 = 73.11, P = 0.000027; Fig. 2). While plasma T levels did not differ significantly between the two treatment groups at the time of implantation, AF males had significantly higher plasma T levels than C males at recapture during the feeding period (Tukey, P = 0.00023). The plasma T increase in AF males between implantation and recapture was highly significant (Tukey, P = 0.00029), whereas plasma T levels in C males tended to decrease between the two captures (Tukey, P = 0.052). These results indicate that the implants worked effectively. Note that ATD and flutamide inhibit the high plasma T levels in AF males to exert a biological effect.
Fig. 2. Plasma T levels at the time of implantation during the egg laying stage (before treatment) and 1 month later during the nestling stage (after treatment) for empty-implanted males (C males) and males that were implanted simultaneously with ATD and flutamide (AF males). Numbers above bars indicate sample sizes. Data shown are means T SE.
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Dawn song While all 10 C males sang a dawn chorus after implantation, 2 of the 7 AF males did not show dawn song after implantation, which is not significantly different. However, of the 60 intact males we set out to record during the egg laying stage in the same study population during 1999, 2000, and 2001, none failed to produce dawn song. In comparison to this proportion (60 out of 60), the proportion of AF males that did not sing at dawn (2 out of 7) was significantly larger than would be expected by chance (Fisher’s exact test, P = 0.0095). We are certain that these two silent AF males had not died after implantation, since they were recaptured during feeding. We are also confident that we would have found them if they had been singing. Based on their song repertoire and position, surrounding singing males could all be assigned unambiguously to neighboring territories. Furthermore, because of the heterogeneous landscape, the birds could be located accurately. Also, one of the two silent males was visited a second morning, during which it also did not sing. Finally, there were no other obvious reasons, such as for example cold weather or heavy rain, that could explain the lack of dawn singing in these two males. Before implantation, total song time ranged 273s – 1075s for the C males and 358s – 1814s for the AF males (see Fig. 3). Thus, the two silent AF males (358s and 525s) were initially situated at the lower end of their range, with only one AF male in between (430s). Yet, they did not sing unusually little before implantation, because two C males had an even shorter total singing time (both 273s, see Fig. 3). When comparing total song time, a two-way repeatedmeasures ANOVA showed a significant period treatment interaction ( F 1,15 = 5.14, P = 0.038). AF males sang significantly more before implantation (847.4 T 192.8s) than after implantation (308.3 T 90.5s, Tukey, P = 0.0024), while total song time in C males did not differ between both song recordings (606.4 T 77.3s versus 423.9 T 65.5s, see Fig. 3). There was no significant difference between treatments in total song time before or after implantation. There was still a clear trend for an effect on total song time when the two AF males that did not sing after implantation were not included in the analysis (see Fig. 3; ANOVAR, period treatment interaction, F 1,13 = 4.55, P = 0.053). For comparison of the other song characteristics between AF and C males, we did not consider the two AF males that did not sing (Table 1). Using a two-way repeated-measures ANOVA, we found no significant effect of the treatment on strophe length, song rate, or repertoire size. Furthermore, an ANOVAR showed that drift was not significantly affected by treatment while controlling for average bout length (Table 1). Summarized, the results suggest that blocking both androgenic and estrogenic action decreased the likelihood
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Fig. 3. Comparison of total song time during the dawn chorus before and after treatment between individual males that were treated simultaneously with ATD and flutamide (AF males: black dots, N = 7) and control males (C males: empty dots, N = 10). The two AF males with dashed lines did not show song activity after treatment.
of showing dawn song. However, we found no evidence for a treatment effect on song quality in males that did show dawn song. Breeding success AF treatment did not significantly affect male breeding success in terms of clutch size, hatching success, fledging success, or fledging age (Table 2). Table 2 shows fledging success of all nests, including nests that were deserted during the feeding stage. In nests that were not deserted, all nestlings fledged, except for two nestlings in one control nest.
Discussion Dawn song In the present study, combined treatment with an aromatase inhibitor (ATD) and an anti-androgen (flutamide), impairing both estrogenic and androgenic actions, decreased the likelihood of showing dawn song in male great tits. Thus, dawn song activity appears to be modulated by androgens, their estrogenic metabolites, or both in this species. This result is in line with the hypothesis that sex steroids are involved in the activation
of song behavior in temperate-zone male songbirds (Balthazart, 1983; Brenowitz and Kroodsma, 1996; Schlinger, 1997; Pinxten et al., 2002). On the other hand, we found no evidence for a treatment effect on song quality in treated males that did show dawn song. While two treated males (AF males) showed no dawn song at all, the five AF males that did sing at dawn did not perform significantly different from the control males. Perhaps these results indicate a threshold mechanism of action of steroid hormones on dawn singing. If so, ATD and flutamide were successful in suppressing steroid action below the threshold level in the silent males but not in singing males. This difference among males could have been caused for example by individual differences in threshold level, in responsiveness to the inhibitors, or in diffusion rate of the inhibitors from the implants. A difference in initial plasma T levels is not supported by our data, because the two silent AF males had T levels close to the average value. Yet, the relatively short total song times before implantation in these silent males (see Fig. 3) may explain why they were most affected by the treatment. In line with this threshold-hypothesis, Pinxten et al. (2003) recently found evidence for a threshold level of T action on mate attraction behavior, including singing behavior, in European starlings, S. vulgaris. Also, Fusani and Hutchison (2003) report indications for a threshold model of action of steroid hormones on the structure of courtship behavior in male ring doves, Streptopelia risoria. Alternatively, we cannot exclude completely that the treatment may have made these two silent males slightly sick such that they stopped singing. In a previous study in the same population, we were unable to demonstrate an effect of experimental T elevation on dawn song performance in the great tit (Van Duyse, 2004). One explanation for that result could be that T is not causally related with dawn song performance. On the other hand, we suggested that dawn song performance could not be enhanced by T implantation because of some kind of constraint, for example, because males were already singing on the limits of their ability or because receptors were already saturated by naturally occurring steroid levels. The suppressive effect of androgen inhibitors on dawn song activity in the current study makes this constraint hypothesis more plausible.
Table 1 Dawn song performance before and after treatment for control males (C males) and males that were treated simultaneously with ATD and flutamide (AF males) Treatment
Strophe length (s) Song rate (no./min) Repertoire size Drift (%)
C males (N = 10)
AF males (N = 5)
Before
After
Before
After
2.31 T 0.17 6.69 T 0.47 3.9 T 0.57 52.3 (25.6 – 78.3)
2.11 T 0.17 5.36 T 0.71 3.6 T 0.56 38.1 (26.3 – 50.6)
2.39 T 0.24 7.18 T 0.88 4.2 T 0.58 47.8 (30.9 – 64.9)
2.14 T 0.19 5.41 T 0.61 3.8 T 0.37 50 (32.8 – 67.2)
Only males that showed dawn song are included. Note. Values are shown as means T SE or as back-transformed means and 95% confidence intervals.
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Table 2 Comparison of components of breeding success between nests of control males (C nests) and nests of males that were treated with ATD and flutamide early in the egg laying stage (AF nests)
Clutch size Hatching success (%) Fledging success (%) Fledging age (days)
C nests
AF nests
Statistical comparison
8 T 1.71 (6) 73 (50 – 91) (6) 100 (78 – 100) (6) 18.8 T 0.66 (5)
7.7 T 0.6 (7) 90 (0 – 100) (7) 100 (0 – 100) (7) 19 T 0.71 (5)
t test, t 11 = 0.17, P = 0.87 U test, Z = 0.66, P = 0.51 U test, Z = 0.0, P = 1 t test, t 8 = 0.21, P = 0.84
Note. Values are shown as mean T SE (sample size) or as median (quartile range) (sample size).
Breeding success A bird’s internal endocrine environment may be currently adaptive. If so, experimentally induced deviations from the natural situation may negatively affect male fitness (Ketterson and Nolan, 1999). In this study, the elevated plasma T levels during the feeding stage clearly indicate that treatment with ATD and flutamide interfered with the internal endocrine environment. Yet, AF males were able to maintain control of a territory and mate, and to complete their brood cycle as successful as control males. One AF male even reared a second brood, while no control male did so. The effect of blocking T action on breeding success is likely to be dependent on the importance of T action. Indeed, androgen inhibitors have been found to suppress Tdependent correlates of reproductive performance. For instance, treatment with the anti-androgen cyproterone acetate (CA) has decreased copulation behavior in Japanese quail, Coturnix c. japonica (Adkins and Mason, 1974), and courtship behavior and testes size in male ring doves (Silver, 1977). Furthermore, combined treatment with ATD and flutamide has been found to decrease territorial behavior in European stonechats, Saxicola torquata (Canoine and Gwinner, 2002), and in non-breeding song sparrows (Soma et al., 1999) and territory size in red-winged blackbirds (Beletsky et al., 1990). On the other hand, in house sparrows, Passer domesticus (Hegner and Wingfield, 1987), and spotless starlings, Sturnus unicolor (Moreno et al., 1999), there are indications that treatment with, respectively, flutamide or cyproterone acetate during the feeding stage can actually increase the number of fledglings, probably through an increase in male feeding rates. This is in line with the finding in several species that experimental T elevation is incompatible with full paternal care (Ketterson and Nolan, 1999). We know of no previous studies examining male breeding success after simultaneous inhibition of androgenic and estrogenic actions. By contrast to other species, T treatment does not depress male nestling feeding behavior in the great tit (Van Duyse et al., 2000, 2002), which possibly explains why blocking the action of T did not affect male breeding success in the current study. Further, some effects could have been left unnoticed. For example, we may have been unable to demonstrate a positive effect on fledging success, because fledging success was already high in control nests. Also, because sample sizes
were rather small, statistical power was limited. In monogamous temperate-zone passerines, including the great tit (Van Duyse et al., 2003), T secretion is highest during territorial establishment and egg laying and lowest in parental phases, probably reflecting the changing importance of T action throughout the breeding season (Wingfield et al., 1990). In our study, great tits were implanted with androgen inhibitors for the larger part of the breeding season. Hence, a net effect on breeding success might have been absent because positive effects in some stages were counteracting negative effects in other stages. Furthermore, we did not consider all possible fitness correlates that are likely affected by T metabolism (e.g., territory size, male survival, paternity loss through extra-pair behavior of the female), leaving the possibility of an overall negative effect on male fitness as a consequence of blocking the action of T in this study. Clearly, discriminating between these possible explanations is not feasible with the present data. However, the observation that interference with the action of T in free-living birds does not necessarily result in abandonment of breeding may offer opportunities for future studies. In particular, suppression of song activity using androgen inhibitors may be the basis of studies on the function of song behavior in general and dawn song in particular.
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