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.

• MeSH
• Biological Evolution MeSH
• Nesting Behavior * MeSH
• Ovum growth & development   MeSH
• Birds MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Brood parasitic birds lay their eggs in other birds' nests, leaving hosts to raise their offspring. To understand parasite-host coevolutionary arms races, many studies have examined host responses to experimentally introduced eggs. However, attending parents often need to be flushed from their nests to add experimental eggs. If these birds witness parasitism events, they may recognize and reject foreign eggs more readily than parents who did not. We found that, after being flushed, female blackbirds, Turdus merula, remained close to their nests. Flushed females were more likely to eject foreign eggs and did so more quickly than females that were not flushed during experimentation. In contrast, flushing did not predict responses and latency to responses to parasitism by song thrush, Turdus philomelos, which flew farther from their nests and likely did not witness experimental parasitism. When statistically considering flushing, previously published conclusions regarding both species' response to experimental parasitism did not change. Nevertheless, we recommend that researchers record and statistically control for whether hosts were flushed prior to experimental parasitism. Our results have broad implications because more vigilant and/or bolder parents can gain more information about parasitism events and therefore have better chances of successfully defending against brood parasitism.

• MeSH
• Behavior, Animal MeSH
• Host-Parasite Interactions * MeSH
• Birds parasitology   MeSH
• Eggs MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH