Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients

This method can help answer key questions in the field of evolutionary ecology, such as how hosts can discriminate between their own eggs and those of their parasites. The main advantage of this technique is that it provides a reproducible technique for conducting egg rejection experiments. To begin, paint an even coat of titanium white over a 3D printed, or otherwise sculpted, model egg.

Use a hairdryer on the cool setting to expedite drying. Once dry, apply high quality acrylic paints to the egg to give it a unique look. Spots can first be added using a brush by hand and then by splattering paint with a toothbrush.

To quantify the color, use a spectrometer. First, insert an SD card into the spectrometer. To link the SD card into the system, from the controls, select File System, and then Find SD cards.

Complete this by pressing the Home button. Next, attach the fiber optic cables to the spectrometer and light source. Follow the cables to secure the correct ends to the correct connector.

Then, insert a customized probe tip into the end of the fiber optic probe. Before proceeding, wash the probe tip with 95%ethanol. Next, turn on the light source by pressing the down button three times.

First, select PX Lamp, then select Setup, and lastly, select Free Running from timing controls. Then, wait at least 15 minutes before taking any measurements to let the instrument warm up. While the machine warms up, take note to measure color using a standardized distance between the egg and the probe that has the best signal to noise ratio.

Begin at about five millimeters and adjust from there. Once the machine is warmed up, calibrate it. Begin with pressing the Home button to select Tools.

Then, from the menu, select Manual control and then select Acquire Parameters. Now, set the box car smoothing. Move the cursor right twice and then increase the box car setting to five using the Up button.

Save the change using the green accept button. Next, navigate by pressing down, right, right, to set the averages to 10. Scroll through the 10s digits using the up button, then right one position and set the position of the ones digit.

Press the green accept button once complete. Next, press the Home button and select the Reflectants option. Then, place the probe firmly on the white standard and store the saved value by pressing the Up button.

Now, place the probe in a dark area away from light. Then, store a dark standard by pressing the scroll right button and view the reflectants by pressing the Menu Down button. Now, measure each eggshell's reflectants six times.

Twice near the blunt pole, twice at the equator, and twice at the sharp pole. Be sure to report if the spots are avoided or not. Perform these measures on both the experimental eggs and the natural hosts eggs, which the experimental eggs are intended to replicate.

Approach the monitored nest with the usual caution. Then, gently add an experimental egg to a host's nest by sliding it into the side of the nests cup. Do not drop the experimental models, as it can damage the hosts eggs.

It's very important that birds don't watch the egg introduction, or if unavoidable, the presence at the time of the experiment controlled for statistically. We found evidence in a number of species that this can help explain their rejection behavior. Always remain at each nest for the same amount of time before retreating.

Within a few hours, and periodically thereafter, revisit all the nests to determine the response of the host bird to the experimental egg. When checking on the nests, use a telescopic mirror to avoid the direct contact with either the nest or the clutch. Paint mixtures used in brood parasitism studies should closely correspond with natural reflectants measurements in terms of their spectral shape and magnitude.

If that is achieved across both the ultraviolet and visible range, the color of an experimental egg should be perceived by the host as a natural color egg. Avian color perception is accomplished using four photo receptors. The quantum catch from these four photo receptors can be transformed into coordinates within a tetrahedral color space, where each vertex represents the relative stimulation of a specific photo receptor.

Into this color space, the coordinates of painted eggs can be plotted. The color of the dot is the color of the egg as humans see it. Most studies have assumed that the hosts respond to trait dissimilarity, such as the difference between a parasitic egg and their own in an absolute or symmetrical fashion.

However, most traits vary along multiple dimensions and there is no a priori reason to assume their responses should be similar across the phenotypic space. Research that manipulates traits across their full phenotypic range contests this assumption. When American robins are presented with eggs that were increasingly dissimilar from the bright blue color of their own eggs, they rejected them more frequently than eggs bluer than their own.

American robins were also found to be more likely to reject spotted experimental eggs when they perceived those spots as browner then the blue-green color of the eggshell. Robins lay unspotted eggs, but their parasite, the brown-headed cow bird lays eggs with brown spots, such that their eggs appear browner than the American robin's eggs, thus, the response seems adaptive. After watching this video, you should have a good understanding of how to run an egg rejection experiment.

Once mastered, this technique can produce comparable results. While attempting this procedure, it's important to collect data from control nests. This will help you determine whether or not hosts abandon their nests in response to the experimental treatment.

Don't forget to exercise caution while working with wild animals. It's important to observe precautions, such as visiting nests infrequently, not marking nests, and approaching and leavings nests from different locations, whenever possible. Thank you for watching and good luck with your experiments.