Name: probing the limits of egg recognition using egg rejection experiments along phenotypic gradients
Uploaded: 2018-08-22T13:00:00
Description: Watch this Scientific Journal Video about Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients at JoVE.com

MEH was funded by the HJ Van Cleave Professorship at the University of Illinois, Urbana-Champaign. In addition, for funding we thank the Human Frontier Science Program (to M.E.H. and T.G.) and the European Social Fund and the state budget of the Czech Republic, project no. CZ.1.07/2.3.00/30.0041 (to T.G.). We thank Ocean Optics for covering publication costs.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Long Island University-Post.
1. Mixing Acrylic Paints
<ol>
	<li>Mix the eggshell ground coloration, which is the color that will uniformly cover the entire eggshell surface. The following recipe will make 50 g of paint, which will fill a little more than two 22 mL aluminum paint tubes.
	<ol>
		<li>Generate a blue-green color, representing a blue-green eggshell (e.g., an Americ...

Although egg rejection experiments are the most common method to study brood parasite-host coevolution70, concerted efforts to standardize materials, techniques, or protocols are lacking. This is especially problematic for meta-analyses. No meta-analysis, to our knowledge, of host egg rejection so far has controlled for methodological discrepancies among studies71,72, including what is considered mimetic or non-mimetic. This represents a m...

Brood parasites lay their eggs in the nests of other species that may then raise their young and pay the costs associated with parental care1,2,3. This act of deception to outwit the host on the part of the parasite and sleuthing to detect the parasite on the part of the host provides strong selective pressures on both actors. In some cases of avian brood parasitism, the host&#39;s recognition of disparate parasitic eggs selects for parasites that mimic host eggs, which produces an evolutionary arms race between host and parasite4. Studying brood parasitism is important because it is a model system for investigating coevolutionary dynamics and decision-making in the wild5. Egg rejection experiments are one of the most common methods used for studying avian brood parasitism in the field and an important tool that ecologists use to investigate interspecific interactions6.
During the course of egg rejection experiments, researchers typically introduce natural or model eggs and assess the host&#39;s response to these experimental eggs over a standardized period. Such experiments can involve swapping real eggs (that vary in appearance) between nests7, or dyeing or painting the surfaces of real eggs (optionally adding patterns) and returning them to their original nests8, or generating model eggs that have manipulated traits such as color9, spotting10, size11, and/or shape12. The host response to eggs of varying appearance can provide valuable insight into the information content they use to reach an egg rejection decision13 and just how different that egg needs to be to elicit a response14. Optimal acceptance threshold theory15 states that hosts should balance the risks of mistakenly accepting a parasitic egg (acceptance error) or mistakenly removing their own egg (rejection error) by examining the difference between their own eggs (or an internal template of those eggs) and the parasitic eggs. As such, an acceptance threshold exists beyond which hosts decide a stimulus is too different to tolerate. When parasitism risk is low, the risk of acceptance errors is lower than when the risk of parasitism is high; thus, decisions are context specific and will shift appropriately as perceived risks change14,16,17.
Optimal acceptance threshold theory assumes that hosts base decisions upon continuous variation in host and parasite phenotypes. Therefore, measuring host responses to varying parasite phenotypes is necessary to establish how tolerant a host population (with its own phenotypic variation) is to a range of parasitic phenotypes. However, virtually all prior studies have relied on categorical egg color and maculation treatments (e.g., mimetic/non-mimetic). Only if host eggshell phenotypes do not vary, which is not a biologically practical expectation, would all responses be directly comparable (regardless of the degree of mimicry). Otherwise, a &#34;mimetic&#34; egg model will vary in how similar it is to host eggs within and between populations, which could potentially lead to confusion when comparing findings18. Theory suggests that host decisions are based upon the difference between the parasitic egg and their own14, not necessarily a particular parasitic egg color. Therefore, using a single egg model type is not an ideal approach to test hypotheses on host decision thresholds or discrimination abilities, unless the just noticeable difference (hereafter JND) between the egg model type and individual host egg color is the variable of interest. This also applies to experimental studies that swap or add natural eggs to test host responses to a natural range of colors19. However, while these studies do allow for variation in host and parasite phenotypes, they are limited by natural variation found in traits6, particularly when using conspecific eggs7.
By contrast, researchers that make artificial eggs of varied colors are free from the constraints of natural variation (e.g., they can investigate responses to superstimuli20), allowing them to probe the limits of host perception6. Recent research has used novel techniques to measure host responses across a phenotypic range, by painting experimental eggs designed to match and surpass the natural range of variation in eggshell9 and spot colors21. Studying host responses to eggs with colors along gradients can uncover underlying cognitive processes because theoretical predictions, such as acceptance thresholds15 or coevolved mimicry4, are based on continuous differences between traits. For example, by using this approach, Dainson et al.21 established that when chromatic contrast between eggshell ground coloration and spot coloration is higher, the American Robin Turdus migratorius tends to reject eggs more strongly. This finding provides valuable insights on how this host processes information, in this case through spotting, to decide whether to remove a parasitic egg. By customizing paint mixtures, researchers can precisely manipulate the similarity between an experimental egg&#39;s color and host&#39;s egg color, while standardizing other confounding factors such as spotting patterns10, egg size22 and egg shape23.
To encourage further replication and metareplication24 of classic and recent egg rejection work, it is important that scientists use methodologies that are standardized across phylogeny (different host species)7,22, space (different host populations)7,22,25,26 and time (different breeding seasons)7,22,25,26,27, which was done only rarely. Methodologies that were not standardized28 were later shown to lead to artefactual results29,30. This paper serves as a set of guidelines for researchers seeking to replicate this type of egg rejection experiment that examines responses to continuous variation and highlights a number of important methodological concepts: the importance of control nests, a priori hypotheses, metareplication, pseudoreplication, and color and spectral analysis. Despite egg rejection experiments dominating the field of avian host-parasite coevolution, no comprehensive protocol exists yet. Therefore, these guidelines will be a valuable resource to increase inter- and intra-lab repeatability as the true test of any hypothesis lies in metareplication, i.e., repeating whole studies across phylogeny, space and time24, which can only be meaningfully done when using consistent methods29,30,31.

<table>
	<tbody>
		<tr>
			<td>Replicator Mini +</td>
			<td>Makerbot</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Cobalt Turquoise Light</td>
			<td>Winsor &#38; Newton</td>
			<td>28382</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Titanium White</td>
			<td>Winsor &#38; Newton</td>
			<td>28489</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Cobalt Green</td>
			<td>Winsor &#38; Newton</td>
			<td>28381</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Cobalt Turquoise</td>
			<td>Winsor &#38; Newton</td>
			<td>28449</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Burnt Umber</td>
			<td>Winsor &#38; Newton</td>
			<td>28433</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Red Iron Oxide</td>
			<td>Winsor &#38; Newton</td>
			<td>28486</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Cadmium Orange</td>
			<td>Winsor &#38; Newton</td>
			<td>28437</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Raw Umber Light</td>
			<td>Winsor &#38; Newton</td>
			<td>28391</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Yellow Ochre</td>
			<td>Winsor &#38; Newton</td>
			<td>28491</td>
			<td></td>
		</tr>
		<tr>
			<td>Professional Acrylic Paint Mars Black</td>
			<td>Winsor &#38; Newton</td>
			<td>28460</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint Brush</td>
			<td>Utrecht</td>
			<td>206-FB</td>
			<td>Filbert brush</td>
		</tr>
		<tr>
			<td>Paint Brush</td>
			<td>Utrecht</td>
			<td>206-F</td>
			<td>Flat brush</td>
		</tr>
		<tr>
			<td>Hair Dryer</td>
			<td>Oster</td>
			<td>202</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiber optic cables</td>
			<td>Ocean Optics Inc.</td>
			<td>OCF-103813</td>
			<td>1 m custom bifurcating fiber optic assembly with blue zip tube (PVDF), 3.8mm nominal OD jeacketing and 2 legs</td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics Inc.</td>
			<td>Jaz</td>
			<td>Spectrometer unit with a 50 um slit width, installed with a 200-850 nm detector (DET2B-200-850), and grating option # 2.</td>
		</tr>
		<tr>
			<td>Battery and SD card module for spectrometer</td>
			<td>Ocean Optics Inc.</td>
			<td>Jaz-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Ocean Optics Inc.</td>
			<td>Jaz-PX</td>
			<td>A pulsed xenon light source</td>
		</tr>
		<tr>
			<td>White standard</td>
			<td>Ocean Optics Inc.</td>
			<td>WS-1-SL</td>
			<td>made from Spectralon</td>
		</tr>
		<tr>
			<td>OHAUS Adventurer Pro Scale</td>
			<td>OHAUS</td>
			<td>AV114C</td>
			<td>A precision microbalance</td>
		</tr>
		<tr>
			<td>Gemini-20 portable scale</td>
			<td>AWS</td>
			<td>Gemini-20</td>
			<td>A standard scale</td>
		</tr>
		<tr>
			<td>Empty Aluminum Paint Tubes (22 ml)</td>
			<td>Creative Mark</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Telescopic mirror</td>
			<td>SE</td>
			<td>8014TM</td>
			<td></td>
		</tr>
		<tr>
			<td>GPS</td>
			<td>Garmin</td>
			<td>Oregon 600</td>
			<td></td>
		</tr>
		<tr>
			<td>220-grit sandpaper</td>
			<td>3M</td>
			<td>21220-SBP-15</td>
			<td>very fine sandpaper</td>
		</tr>
		<tr>
			<td>400-grit sandpaper</td>
			<td>3M</td>
			<td>20400-SBP-5</td>
			<td>very fine sandpaper</td>
		</tr>
		<tr>
			<td>color analysis software: &#8216;pavo&#8217;, an R package</td>
			<td>for use in, R: A language and environment for statistical computing</td>
			<td>v 1.3.1</td>
			<td>https://cran.r-project.org/web/packages/pavo/index.html</td>
		</tr>
		<tr>
			<td>UV clear transparent</td>
			<td>Flock off!</td>
			<td>UV-001</td>
			<td>A transparent ultraviolet paint</td>
		</tr>
		<tr>
			<td>Plastic sandwich bags</td>
			<td>Ziploc</td>
			<td></td>
			<td>Regular plastic sandwich bags from Ziploc that can be purchased at the supermarket.</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>34120</td>
			<td>11 x 21 cm kimwipes</td>
		</tr>
		<tr>
			<td>Toothbrush</td>
			<td>Colgate</td>
			<td></td>
			<td>Toothbrush</td>
		</tr>
	</tbody>
</table>

probing the limits of egg recognition using egg rejection experiments along phenotypic gradients

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate hosts into raising their young and how hosts detect parasitism provide important insights in the field of coevolutionary biology. Brood parasites, such as cuckoos and cowbirds, gain an evolutionary advantage because they do not have to pay the costs of rearing their own young. However, these costs select for host defenses against all developmental stages of parasites, including eggs, their young, and adults. Egg rejection experiments are the most common method used to study host defenses. During these experiments, a researcher places an experimental egg in a host nest and monitors how hosts respond. Color is often manipulated, and the expectation is that the likelihood of egg discrimination and the degree of dissimilarity between the host and experimental egg are positively related. This paper serves as a guide for conducting egg rejection experiments from describing methods for creating consistent egg colors to analyzing the findings of such experiments. Special attention is given to a new method involving uniquely colored eggs along color gradients that has the potential to explore color biases in host recognition. Without standardization, it is not possible to compare findings between studies in a meaningful way; a standard protocol within this field will allow for increasingly accurate and comparable results for further experiments.

Generating colorful egg models
Reflectance spectra of custom paint mixtures and natural eggs are shown in Figure&#160;1A-1D. Paint mixtures used in brood parasitism studies should closely correspond with natural reflectance measurements in terms of spectral shape (color) and magnitude (brightness). If that is achieved, the color of the experimental egg should be per...

Watch this Scientific Journal Video about Probing the Limits of Egg Recognition Using Egg Rejection Experiments Along Phenotypic Gradients at JoVE.com

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

This protocol provides guidelines for running egg rejection experiments: outlining techniques for painting experimental egg models to emulate the colors of natural bird eggs, conducting fieldwork, and analyzing the collected data. This protocol provides a uniform method for conducting comparable egg rejection experiments.

Brood parasites lay their eggs in other females' nests, leaving the host parents to hatch and rear their young. Studying how brood parasites manipulate ...

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