Capture
To assess circulating corticosterone (n=43) and pituitary gene
expression for a subset of subjects (n=13), we captured experimentally
parasitized females 2 hours after the addition of a model egg. The 2 h
time point was chosen because plasma corticosterone in birds typically
increases rapidly in the first 30 min to an hour following an exposure
to an acute stressor (such as handling or nest disturbance), after which
plasma corticosterone titer remains near its maximum if the stressor
persists (Abolins-Abols et al. 2016; Romero et al. 1998;
Meddle et al. 2003; Breuner et al. 2006). For logistical
reasons we were not able to remain near the nest to determine the return
time of each female, but in this population, females typically arrive
back to their nests on average 12 min after being flushed (pers. obs.
n=31, standard deviation (SD) = 7.37, range = 2 to 28). Most of the
females (86%: 38 out of 43) were flushed from their nests prior the
insertion of the model egg (the proportion of flushed females did not
differ between the treatments (Fisher’s exact test, p=1.0)). The 2-hour
time window therefore provided most of the females with an opportunity
for a prolonged interaction with the model egg before capture, allowing
any corticosterone increase in response to the model eggs to reach its
maximum.
Previous research also showed that most female robins’ decisions to
reject or accept a model egg are not immediate but occur on the
timescale of hours rather than minutes (Hauber et al. 2019;
Scharf et al. 2019). Importantly, the only other study we are
aware of in Turdus thrushes investigating the effect of
experimental parasitism on plasma corticosterone detected elevated
corticosterone 24+ hours after the addition of the model egg (Ruiz-Rayaet al. 2018). However, sampling robins at 24 hours following the
experimental parasitism without tethering the model eggs would have
meant that nearly all the females would have rejected the non-mimetic
parasitic eggs (e.g. Hauber et al., 2020b) and that they would therefore
have been unstimulated for several hours before capture. The 2-hour time
window thus allowed us to catch females during or closely following
stimulation by model eggs, and, according to our estimate, allowed
sufficient time for any changes in corticosterone in most females reach
or remain near peak levels.
We captured females with a 6 m mist net, arranged in a V-shape around
the nest tree. The poles and a rolled-up closed mist net were set up
immediately before the addition of the model eggs. Two hours after the
initial net setup and the addition of the model egg, we unfurled the
mist net and captured the female either by flushing it into the net or
passively capturing it as it attempted to land on the nest. Setting up
the net prior to experimental parasitism enabled rapid set up and
capture following the 2-hour mark (n = 42, mean capture time = 15.33
minutes, min = 0, max = 36, SD = 9.19) and allowed us to minimize any
changes to corticosterone levels due to nest disturbance. Importantly,
we found no association between baseline plasma corticosterone
concentration (see below for corticosterone assay methods) and the time
it took to catch the female (Pearson’s r =-0.16, df=40, p=0.30) or
whether the female was flushed from the nest (two-tailed t-test, t=0.15,
df=4.64, p=0.88). Therefore, we did not account for these metrics in the
statistical models testing the effect of egg type on corticosterone
levels (see section below). At the time of capture, we also noted
whether the model egg was rejected or still present (“accepted”).