Heart rate analysis
In 2018 we exposed an additional set of robin females (n=14) to the same
binary model egg treatments and measured their heart rate during
incubation immediately following the experimental parasitism. We
measured heart rate by adopting an approach from Arnold et al.(2011) where a microphone-fitted model egg is used to record audio
signatures of heartbeats in incubating females through contact with the
featherless brood patch. We 3D-printed a custom model eggshell
resembling American robin eggs in size and shape using MakerBot
Replicator Mini+ 3D printer (MakerBot, Brooklyn, NY, USA). The model
shell consisted of two halves, which connected along the long axis of
symmetry. The two halves each had an opening on the egg equator – one
half had an opening on for a 6 mm microphone, the other had an opening
for a headphone cord (Fig. S1a). After printing, each model egg’s
surface was smoothed using sandpaper. Each egg was then fitted with a
unidirectional microphone (PUM-3546L-R, PUI Audio Inc, Dayton, OH, USA).
The microphone was soldered to a headphone cable and inserted into the
model egg (“top” side of the egg), with the headphone cable leaving
the model egg on the opposite (“bottom”) side of the egg (Fig. S1a). A
white rubber balloon (Walmart, Bentonville, AR, USA) was then stretched
over the egg and tied with a fishing line around the headphone cable.
The egg was then painted in mimetic robin-blue following the methods
outlined above.
To insert the model egg in the nest, we first removed all the robin eggs
to prevent damage to them. We then poked a small hole in the mud-lined
bottom of the robin’s nest cup through which we passed the headphone
cable, leaving the model egg resting on the bottom of the nest with the
microphone side facing up (Fig. S1b). The robin eggs were then returned
to the nest, typically within 1 min of their removal. One robin egg was
removed to avoid changes to the focal subjects’ apparent own
(robin-sized egg) clutch size. The headphone cord was then connected to
a digital sound recorder (Olympus Digital Voice Recorded WS-852,
Olympus, Tokyo, Japan).
We first placed the microphone-egg into the focal nests a day before the
experimental model egg addition to minimize the disturbance at the nest.
We recorded 2 hours of heartbeat audio signatures to check that the
model egg was contacting the brood patch and recording audio signatures
of heart beats (Fig. S2). One day following the insertion of the
microphone egg, we returned to the nest and inserted either mimetic blue
or non-mimetic beige cowbird-sized solid nylon model eggs (as described
above) in the focal nest and started to record the heart rate again. We
returned to the nest 2 hours following the experimental parasitism,
removed the experimental model egg and replaced it with the opposite
treatment. For example, in nests where a female was experimentally
parasitized with a non-mimetic beige egg, the beige egg was removed and
a mimetic blue egg was added in its place (and vice versa). Each female
was therefore sequentially exposed to both mimetic and non-mimetic
treatments, but the order of the treatments was randomized among
subjects. Two hours after the addition of the second experimental egg we
removed both the experimental and the microphone egg.
We used loud rustling sounds to determine the arrival of the female at
the nest cup. We then filtered the heart rate audio recordings to remove
sounds above 1 KHz frequencies using Adobe Audition (Adobe, San Jose,
CA, USA). We then manually scored the heartbeats using R programming
environment using seewave (Sueur et al. 2008) and tuneR (Liggeset al. 2018) packages following published protocols (Sueur 2018).
Heartbeats were then transformed into instantaneous heart rate
(beats/sec) using the following formula: 1/i, where i is the interval
(sec) between two successive heart beats. Unlike endocrine responses,
heart rate is labile and can reflect instantaneous responses to stimuli
(Wascher 2021) therefore we limited our analyses to the first 10 minutes
following the arrival of the focal female.