Experimentally altering pre‐breeding sex steroids reduces extra‐pair paternity in female tree swallows

Abstract

Extra‐pair paternity commonly occurs in many socially monogamous animal species, yet the reasons why females mate with males outside the social pair bond remain poorly understood. Because sex steroids mediate aggressive and sexual behaviors that peak prior to egg laying in female birds, we tested whether they also influence the rate of extra‐pair paternity in nests of female tree swallows (Tachycineta bicolor). In one treatment, we experimentally elevated testosterone (T) using implants containing exogenous T, and in another treatment, we blocked the estrogenic and androgenic actions of T using implants containing 1,4,6‐androstatrien‐3,17‐dione in combination with flutamide (ATD+F). Females with empty implants served as controls. Nests of females treated with T and those with ATD+F were less likely to contain extra‐pair offspring and had a lower proportion of extra‐pair offspring, compared to control females. Treating females with T also delayed clutch initiation dates and disrupted incubation behavior, as found in previous studies, but clutch sizes of T females did not differ from controls. Females treated with ATD+F tended to lay larger clutches than control and T‐treated females, which may be due to their clutch sizes not declining with later initiation dates as found in the T and control treatments; however, after controlling for brood size, ATD+F females produced nestlings that were lighter at day 16 than control females. Although the effect of T on extra‐pair paternity that we observed in tree swallows is consistent with previous studies in female birds, our results demonstrate that blocking the estrogenic and/or androgenic actions of T during pre‐breeding also lowers extra‐pair paternity. Overall, our study suggests that extra‐pair paternity in tree swallows is mediated by sex steroids of females.

Does experimentally altered plumage ornamentation of female passerines influence nestling quality? A test in female tree swallows

“…Sex of offspring was determined using DNA extracted from blood and tissue samples (see Berzins et al, ), and the P2 and P8 primers following Whittingham and Dunn (). DNA extracted from blood samples was amplified using polymerase chain reaction (PCR) performed in a 10 µl final volume containing 1× PCR buffer, 0.2 mM of each dNTP (Invitrogen, Carlsbad, CA, USA; #10297‐018), 2.5 mM MgCl 2 , 0.4 μg/μl bovine serum albumin (New England Biolabs, Ipswich, MA, USA; #B90015), 0.3 μM of P2 and P8 primers, 0.05 U/μl Taq polymerase (Invitrogen; #10342‐020), and 20–50 ng of genomic DNA.…”

“…PCR was performed in a MJ Research Peltier Thermal Cycler under the following PCR cycling protocol: an initial denaturing step at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 47°C for 60 s, and 72°C for 60 s. A final extension cycle at 72°C for 5 min completed the PCR. For DNA extracted from tissue samples of unhatched eggs and offspring that died before blood sampling occurred, PCRs were performed using a Qiagen Multiplex PCR kit (Qiagen; #206143) following the manufacturer’s instructions, except that we used a final volume of 10 and 5 μl of Multiplex PCR Master Mix (Berzins et al, ); primer concentrations and annealing temperatures remained the same as described above.…”

“…On day 12, a small blood sample (20 μl) was collected from the brachial vein of nestlings with non-heparinized capillary tubes for molecular sexing; blood samples were stored in Queen's lysis buffer (Seutin, White, & Boag, 1991) at −20°C until DNA extraction (details below). Any eggs that failed to hatch or nestlings found dead prior to day 12 were collected and stored at −20°C until tissue collection and DNA extraction (see Berzins, Shrimpton, & Dawson, 2018). Nests were visited on day 22 to record the number of young that successfully fledged.…”