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.