Coevolutionary Biology Research Articles

Rearing zombie flies: Laboratory culturing of the behaviourally manipulating fungal pathogen Entomophthora muscae

Insect pathogenic fungi (IPF) and insects have ubiquitous interactions in nature. The extent of these interkingdom host-pathogen interactions are both complex and diverse. Some IPF, notably of the order Entomophthorales, manipulate their species-specific host before death. The fungus-induced altered insect behaviours are sequential and can accurately be repeatedly characterised temporally, making them a valuable model for understanding the molecular and chemical underpinnings of behaviour and host-pathogen co-evolutionary biology. Here, we present methods for the isolation and laboratory culturing of the emerging behaviourally manipulating model IPF Entomophthora muscae for experimentation.•E. muscae isolation and culturing in vitro.•Establishing and maintaining an E. muscae culture in vivo in houseflies (Musca domestica).•Controlled E. muscae infections for virulence experiments and quantification of conidia discharge per cadaver.

Assessing symbiont extinction risk using cophylogenetic data

Symbionts have a unique mode of life that has attracted the attention of ecologists and evolutionary biologists for centuries. As a result of this attention, these disciplines have produced a mature body of literature on host-symbiont interactions. In contrast, the discipline of symbiont conservation is still in a foundational stage. Here, we aim to integrate methodologies for symbiont coevolutionary biology with symbiont conservation. We focus on host-symbiont cophylogenies, because they have been widely used to study symbiont diversification history and contain information on symbiont extinction. However, cophylogenetic information has never been used nor adapted to the perspective of conservation. Here, we propose a new statistic, “cophylogenetic extinction rate” (Ec), which is based on coevolutionary knowledge from event-based cophylogenetic analyses and could be informative to assess relative symbiont extinction risks. Finally, we propose potential future research to further develop methods to estimate symbiont extinction risk from cophylogenetic analyses, and to continue the integration of this existing knowledge of coevolutionary biology and cophylogenetics into future symbiont conservation studies and practices.

Vive la difference! Self/non-self recognition and the evolution of signatures of identity in arms races with parasites.

In arms races with parasites, hosts can evolve defences exhibiting extensive variability within populations, which signals individual identity ('signatures'). However, few such systems have evolved, suggesting that the conditions for their evolution are uncommon. We review (a) polymorphic egg markings that allow hosts of brood-parasitic birds to recognize and reject parasitic eggs, and (b) polymorphic tissue antigens encoded in the major histocompatibility complex (MHC), which present self- and pathogen-derived peptides to T cells of the immune system. Despite the profound differences between these systems, they share analogous features: (i) self/non-self discrimination by a highly specific recognition system (bird eyes and T-cell antigen receptor, respectively), which antagonists may escape by evolving evasion or mimicry; (ii) a self substrate upon which diversifying selection can act (eggs, and MHC molecules); (iii) acquired knowledge of self (resulting in acceptance of own eggs, and immune tolerance); and (iv) fitness costs associated with attack on self or lack of parasite detection. We suggest that these features comprise a set of requirements for parasites to drive the evolution of identity signatures in hosts, which diminish the likelihood of recognition errors. This may help to explain the variety of trajectories arising from arms races in different antagonistic contexts. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Variation in multicomponent recognition cues alters egg rejection decisions: a test of the optimal acceptance threshold hypothesis.

The optimal acceptance threshold hypothesis provides a general predictive framework for testing behavioural responses to discrimination challenges. Decision-makers should respond to a stimulus when the perceived difference between that stimulus and a comparison template surpasses an acceptance threshold. We tested how individual components of a relevant recognition cue (experimental eggs) contributed to behavioural responses of chalk-browed mockingbirds, Mimus saturninus, a frequent host of the parasitic shiny cowbird, Molothrus bonariensis. To do this, we recorded responses to eggs that varied with respect to two components: colour, ranging from bluer to browner than the hosts' own eggs, and spotting, either spotted like their own or unspotted. Although tests of this hypothesis typically assume that decisions are based on perceived colour dissimilarity between own and foreign eggs, we found that decisions were biased toward rejecting browner eggs. However, as predicted, hosts tolerated spotted eggs more than unspotted eggs, irrespective of colour. These results uncover how a single component of a multicomponent cue can shift a host's discrimination threshold and illustrate how the optimal acceptance threshold hypothesis can be used as a framework to quantify the direction and amount of the shift (in avian perceptual units) of the response curve across relevant phenotypic ranges. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

The brood parasite's guide to inclusive fitness theory.

Hamilton's theory of inclusive fitness provides a framework for understanding the evolution of social behaviour between kin, including parental and alloparental care. Brood parasitism is a reproductive tactic in which parasites exploit the care of other individuals of the same species (conspecific parasitism) or different species (interspecific parasitism) to rear their brood. Here, drawing from examples in birds and social insects, we identify two insights into brood parasitism that stem from inclusive fitness theory. First, the kin structure within nests, or between neighbouring nests, can create a niche space favouring the evolution of conspecific parasitism. For example, low average relatedness within social insect nests can increase selection for reproductive cheats. Likewise, high average relatedness between adjacent nests of some birds can increase a female's tolerance of parasitism by her neighbour. Second, intrabrood conflict will be high in parasitized broods, from the perspective of both parasite and host young, relative to unparasitized broods. We also discuss offspring recognition by hosts as an example of discrimination in a kin-selected social behaviour. We conclude that the inclusive fitness framework is instructive for understanding aspects of brood parasite and host evolution. In turn, brood parasites present some unique opportunities to test the predictions of inclusive fitness theory. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Early life-history features associated with brood parasitism in the cuckoo catfish, Synodontis multipunctatus (Siluriformes: Mochokidae).

The cuckoo catfish, Synodontis multipunctatus, is the only known obligate brood parasite among fishes, exploiting the parental care of mouthbrooding cichlids endemic to Lake Tanganyika. Comparisons of this system to brood parasitism in birds may reveal broader principles that underlie the evolution of this life-history strategy in vertebrates. However, little is known about the features of the cuckoo catfish that enable this species to successfully parasitize cichlids. Here, we examine early ontogeny of the cuckoo catfish and compare it to that of its cichlid hosts as well as a non-parasitic congener. We found that cuckoo catfish embryos develop and hatch in advance of host embryos, and begin feeding on cichlid young just as they start to hatch. Overall timing of ontogeny in the cuckoo catfish was found to be similar to that of the substrate-spawning congener Synodontis lucipinnis, suggesting that more rapid development of the cuckoo catfish relative to cichlids is not a unique adaptation to brood parasitism. However, we found that cuckoo catfish progeny exhibit extensive morphological differences from S. lucipinnis, which may represent adaptations to brood parasitism. These life-history observations reveal both similarities and differences between the cuckoo catfish system and brood parasitism in other lineages. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Brood parasitism in eusocial insects (Hymenoptera): role of host geographical range size and phylogeny.

Interspecific brood parasitism is common in many animal systems. Brood parasites enter the nests of other species and divert host resources for producing their own offspring, which can lead to strong antagonistic parasite-host coevolution. Here, we look at commonalities among social insect species that are victims of brood parasites, and use phylogenetic data and information on geographical range size to predict which species are most probably to fall victims to brood parasites in the future. In our analyses, we focus on three eusocial hymenopteran groups and their brood parasites: (i) bumblebees, (ii) Myrmica ants, and (iii) vespine and polistine wasps. In these groups, some, but not all, species are parasitized by obligate workerless inquilines that only produce reproductive-caste descendants. We find phylogenetic signals for geographical range size and the presence of parasites in bumblebees, but not in ants and wasps. Phylogenetic logistic regressions indicate that the probability of being attacked by one or more brood parasite species increases with the size of the geographical range in bumblebees, but the effect is statistically only marginally significant in ants. However, non-phylogenetic logistic regressions suggest that bumblebee species with the largest geographical range sizes may have a lower likelihood of harbouring social parasites than do hosts with medium-sized ranges. Our results provide new insights into the ecology and evolution of host-social parasite systems, and indicate that host phylogeny and geographical range size can be used to predict threats posed by social parasites, as well to design efficient conservation measures for both hosts and their parasites. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Higher-level pattern features provide additional information to birds when recognizing and rejecting parasitic eggs.

Despite a recent explosion of research on pattern recognition, in both neuroscience and computer vision, we lack a basic understanding of how most animals perceive and respond to patterns in the wild. Avian brood parasites and their hosts provide an ideal study system for investigating the mechanisms of pattern recognition. The cuckoo finch, Anomalospiza imberbis, and its host the tawny-flanked prinia, Prinia subflava, lay highly polymorphic eggs with a great deal of variation in colour and patterning, with the cuckoo finch capable of close egg mimicry. Behavioural experiments in Zambia have previously shown that prinias use colour and multiple 'low-level' (occurring in early stages of visual processing) pattern attributes, derived from spatial frequency analysis, when rejecting foreign eggs. Here, we explore the extent to which host birds might also use 'higher-level' pattern attributes, derived from a feature detection algorithm, to make rejection decisions. Using a SIFT-based pattern recognition algorithm, NaturePatternMatch, we show that hosts are more likely to reject a foreign egg if its higher-level pattern features-which capture information about the shape and orientation of markings-differ from those of the host eggs. A revised statistical model explains about 37% variance in egg rejection behaviour, and differences in colour, low-level and higher-level pattern features all predict rejection, accounting for 42, 44 and 14% of the explained variance, respectively. Thus, higher-level pattern features provide a small but measurable improvement to the original model and may be especially useful when colour and low-level pattern features provide hosts with little information. Understanding the relative importance of low- and higher-level pattern features is a valuable goal for future work on animal coloration, especially in the contexts of mimicry, camouflage and individual recognition. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Host density predicts the probability of parasitism by avian brood parasites.

The spatial distribution of hosts can be a determining factor in the reproductive success of parasites. Highly aggregated hosts may offer more opportunities for reproduction but can have better defences than isolated hosts. Here we connect macro- and micro-evolutionary processes to understand the link between host density and parasitism, using avian brood parasites as a model system. We analyse data across more than 200 host species using phylogenetic comparative analyses and quantify parasitism rate and host reproductive success in relation to spatial distribution using field data collected on one host species over 6 years. Our comparative analysis reveals that hosts occurring at intermediate densities are more likely to be parasitized than colonial or widely dispersed hosts. Correspondingly, our intraspecific field data show that individuals living at moderate densities experience higher parasitism rates than individuals at either low or high densities. Moreover, we show for the first time that the effect of host density on host reproductive success varies according to the intensity of parasitism; hosts have greater reproductive success when living at high densities if parasitism rates are high, but fare better at low densities when parasitism rates are low. We provide the first evidence of the trade-off between host density and parasitism at both macro- and micro-evolutionary scales in brood parasites. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Defences against brood parasites from a social immunity perspective.

Parasitic interactions are so ubiquitous that all multicellular organisms have evolved a system of defences to reduce their costs, whether the parasites they encounter are the classic parasites which feed on the individual, or brood parasites which usurp parental care. Many parallels have been drawn between defences deployed against both types of parasite, but typically, while defences against classic parasites have been selected to protect survival, those against brood parasites have been selected to protect the parent's inclusive fitness, suggesting that the selection pressures they impose are fundamentally different. However, there is another class of defences against classic parasites that have specifically been selected to protect an individual's inclusive fitness, known as social immunity. Social immune responses include the anti-parasite defences typically provided for others in kin-structured groups, such as the antifungal secretions produced by termite workers to protect the brood. Defences against brood parasites, therefore, are more closely aligned with social immune responses. Much like social immunity, host defences against brood parasitism are employed by a donor (a parent) for the benefit of one or more recipients (typically kin), and as with social defences against classic parasites, defences have therefore evolved to protect the donor's inclusive fitness, not the survival or ultimately the fitness of individual recipients This can lead to severe conflicts between the different parties, whose interests are not always aligned. Here, we consider defences against brood parasitism in the light of social immunity, at different stages of parasite encounter, addressing where conflicts occur and how they might be resolved. We finish with considering how this approach could help us to address longstanding questions in our understanding of brood parasitism. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Patterns of host use by brood parasitic Maculinea butterflies across Europe.

The range of hosts exploited by a parasite is determined by several factors, including host availability, infectivity and exploitability. Each of these can be the target of natural selection on both host and parasite, which will determine the local outcome of interactions, and potentially lead to coevolution. However, geographical variation in host use and specificity has rarely been investigated. Maculinea (= Phengaris) butterflies are brood parasites of Myrmica ants that are patchily distributed across the Palæarctic and have been studied extensively in Europe. Here, we review the published records of ant host use by the European Maculinea species, as well as providing new host ant records for more than 100 sites across Europe. This comprehensive survey demonstrates that while all but one of the Myrmica species found on Maculinea sites have been recorded as hosts, the most common is often disproportionately highly exploited. Host sharing and host switching are both relatively common, but there is evidence of specialization at many sites, which varies among Maculinea species. We show that most Maculinea display the features expected for coevolution to occur in a geographic mosaic, which has probably allowed these rare butterflies to persist in Europe. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Monogamy without parental care? Social and genetic mating systems of avian brood parasites.

Classic evolutionary theory predicts that monogamy should be intimately linked with parental care. It has long been assumed, therefore, that avian brood parasites-which lay their eggs in the nests of 'host' species and provide little, if any, parental care-should be overwhelmingly promiscuous. However, recent studies have revealed that the social mating systems of brood parasites are surprisingly diverse, encompassing lek polygyny, monogamy, polygamy and promiscuity. What ecological or phylogenetic factors explain this variation, and why are some brood parasites apparently monogamous? Here we review the social and genetic mating systems of all 75 brood parasitic species for which data are available and evaluate several hypotheses that may help explain these patterns. We find that social monogamy is widespread, often co-occurring with territoriality and cooperative behaviour by the mated pair. Comparative studies, though preliminary, suggest that in some species, monogamy is associated with low host density and polygamy with higher host density. Interestingly, molecular data show that genetic and social mating systems can be entirely decoupled: genetic monogamy can occur in parasitic species that lack behavioural pair-bonds, possibly as a by-product of territoriality; conversely, social monogamy has been reported in parasites that are genetically polygamous. This synthesis suggests that social and genetic monogamy may result from very different selective pressures, and that male-female cooperative behaviours, population density and territoriality may all interact to favour the evolution of monogamous mating in brood parasites. Given that detailed descriptive data of social, and especially genetic, mating systems are still lacking for the majority of brood parasitic species, definitive tests of these hypotheses await future work. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

The coevolutionary biology of brood parasitism: a call for integration.

Obligate brood-parasitic cheats have fascinated natural historians since ancient times. Passing on the costs of parental care to others occurs widely in birds, insects and fish, and often exerts selection pressure on hosts that in turn evolve defences. Brood parasites have therefore provided an illuminating system for researching coevolution. Nevertheless, much remains unknown about how ecology and evolutionary history constrain or facilitate brood parasitism, or the mechanisms that shape or respond to selection. In this special issue, we bring together examples from across the animal kingdom to illustrate the diverse ways in which recent research is addressing these gaps. This special issue also considers how research on brood parasitism may benefit from, and in turn inform, related fields such as social evolution and immunity. Here, we argue that progress in our understanding of coevolution would benefit from the increased integration of ideas across taxonomic boundaries and across Tinbergen's Four Questions: mechanism, ontogeny, function and phylogeny of brood parasitism. We also encourage renewed vigour in uncovering the natural history of the majority of the world's brood parasites that remain little-known. Indeed, it seems very likely that some of nature's brood parasites remain entirely unknown, because otherwise we are left with a puzzle: if parental care is so costly, why is brood parasitism not more common? This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Ant behaviour and brain gene expression of defending hosts depend on the ecological success of the intruding social parasite.

The geographical mosaic theory of coevolution predicts that species interactions vary between locales. Depending on who leads the coevolutionary arms race, the effectivity of parasite attack or host defence strategies will explain parasite prevalence. Here, we compare behaviour and brain transcriptomes of Temnothorax longispinosus ant workers when defending their nest against an invading social parasite, the slavemaking ant Temnothorax americanus. A full-factorial design allowed us to test whether behaviour and gene expression are linked to parasite pressure on host populations or to the ecological success of parasite populations. Albeit host defences had been shown before to covary with local parasite pressure, we found parasite success to be much more important. Our chemical and behavioural analyses revealed that parasites from high prevalence sites carry lower concentrations of recognition cues and are less often attacked by hosts. This link was further supported by gene expression analysis. Our study reveals that host-parasite interactions are strongly influenced by social parasite strategies, so that variation in parasite prevalence is determined by parasite traits rather than the efficacy of host defence. Gene functions associated with parasite success indicated strong neuronal responses in hosts, including long-term changes in gene regulation, indicating an enduring impact of parasites on host behaviour. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Convergent evolution of reduced eggshell conductance in avian brood parasites.

Brood parasitism has evolved independently in several bird lineages, giving rise to strikingly similar behavioural adaptations that suggest convergent evolution. By comparison, convergence of physiological traits that optimize this breeding strategy has received much less attention, yet these species share many similar physiological traits that optimize this breeding strategy. Eggshell structure is important for embryonic development as it controls the flux of metabolic gases, such as O2, CO2 and H2O, into and out of the egg; in particular, water vapour conductance ( GH2O) is an essential process for optimal development of the embryo. Previous work has shown that common cuckoos ( Cuculus canorus) have a lower than expected eggshell GH2O compared with their hosts. Here, we sought to test whether this is a trait found in other independently evolved avian brood parasites, and therefore reflects a general adaptation to a parasitic lifestyle. We analysed GH2O for seven species of brood parasites from four unique lineages as well as for their hosts, and combined this with species from the literature. We found lower than expected GH2O among all our observed brood parasites both compared with hosts (except for brown-headed cowbirds ( Molothrus ater)) and compared with the expected rates given their phylogenetic positions. These findings suggest that a lowered GH2O may be a general adaptation for brood parasitism, perhaps helping the parasite nestling to develop greater aerobic fitness. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Under the radar: detection avoidance in brood parasitic bees.

Brood parasitism is a specialized form of parasitism in which the offspring of a parasite develops on the food provisions gathered by a host species for its own young. Obligate brood parasitic lineages have lost the ability to acquire provisions for their young and thus rely entirely on the location of an appropriate host to serve as a food-provider. Solitary bees provide some of the most fascinating examples of brood parasitism in animals. Most solitary bees build and provision their own nests. Some, however, usurp the nests of other species of bees. These brood parasites, or 'cuckoo' bees, deposit their eggs on the pollen provisions collected by a host bee for her own offspring. The provisions stored by the host bee are not sufficient to sustain the development of both the host's larva and that of the brood parasite and the parasite must kill the offspring of its host in order to obtain enough nourishment to complete its development. As a consequence, there is fierce competition between the host bee seeking to protect her nest from attack and the brood parasite seeking to avoid detection by the host in order to successfully deposit her eggs in an appropriate nest. In this paper, I review the behaviours that allow brood parasitic bees to escape detection by their hosts. Identifying these behaviours, and placing them within the general context of strategies employed by brood parasitic bees to parasitize the nests of their hosts, is key to understanding how brood parasitic lineages may have evolved from nest-building ancestors, decrypting the selective pressures that drive evolutionary transitions from one strategy to another and, more broadly, revealing how similar selective pressures in widely divergent lineages of animals have given rise to remarkably convergent behaviours. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Egg retrieval versus egg rejection in cuckoo hosts.

Before complex nests evolved, birds laid eggs on the ground, and egg retrieval evolved as an adaptation against accidental displacement of eggs outside the nest. Therefore, egg retrieval is an ancient, and likely ancestral, widespread behaviour in birds. However, it has received little attention in studies of avian brood parasitism, perhaps because most parasitism occurs in species with complex nests, a context in which egg retrieval seems irrelevant. However, for cavity-nesting hosts of avian brood parasites, egg retrieval may still play an important role in the coevolutionary interactions between obligate brood parasites and hosts, because egg retrieval can be considered to be antagonistic to egg rejection behaviour in hosts, yet both may involve cognition to recognize eggs. We hypothesized that (1) cavity-nesting hosts should retrieve misplaced eggs from outside the nest cup, (2) brood parasitism has modulated egg retrieval behaviour in cavity-nesting hosts and (3) hosts use the same visual cues for decision-making during egg recognition in both egg retrieval and egg rejection actions. To test these hypotheses, we performed a series of experiments in a cavity-nesting host, the green-backed tit ( Parus monticolus). Foreign eggs with different levels of mimicry were placed within or outside nest cups of hosts to test their responses. We found that host decisions about whether to retrieve or reject an egg both depended on the degree of mimicry. However, hosts sometimes first retrieved poorly mimetic foreign eggs and then rejected them. Alternatively, hosts sometimes failed to retrieve highly mimetic conspecific eggs. We suggest that egg retrieval in hosts is likely to be a result of the interaction between ancient retrieval behaviour and subsequent adaptation against brood parasitism. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Parasitic cuckoo catfish exploit parental responses to stray offspring.

Interspecific brood parasitism occurs in several independent lineages of birds and social insects, putatively evolving from intraspecific brood parasitism. The cuckoo catfish, Synodontis multipunctatus, the only known obligatory non-avian brood parasite, exploits mouthbrooding cichlid fishes in Lake Tanganyika, despite the absence of parental care in its evolutionary lineage (family Mochokidae). Cuckoo catfish participate in host spawning events, with their eggs subsequently collected and brooded by parental cichlids, though they can later be selectively rejected by the host. One scenario for the origin of brood parasitism in cuckoo catfish is through predation of cichlid eggs during spawning, eventually resulting in a spatial and temporal match in oviposition by host and parasite. Here we demonstrate experimentally that, uniquely among all known brood parasites, cuckoo catfish have the capacity to re-infect their hosts at a late developmental stage following egg rejection. We show that cuckoo catfish offspring can survive outside the host buccal cavity and re-infect parental hosts at a later incubation phase by exploiting the strong parental instinct of hosts to collect stray offspring. This finding implies an alternative evolutionary origin for cuckoo catfish brood parasitism, with the parental response of host cichlids facilitating its evolution. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Inquiline social parasites as tools to unlock the secrets of insect sociality.

Insect societies play a crucial role in the functioning of most ecosystems and have fascinated both scientists and the lay public for centuries. Despite the long history of study, we are still far from understanding how insect societies have evolved and how social cohesion in their colonies is maintained. Here we suggest inquiline social parasites of insect societies as an under-exploited experimental tool for understanding sociality. We draw on examples from obligate inquiline (permanent) social parasites in wasps, ants and bees to illustrate how these parasites may allow us to better understand societies and learn more about the evolution and functioning of insect societies. We highlight three main features of these social parasite-host systems-namely, close phylogenetic relationships, strong selective pressures arising from coevolution and multiple independent origins-that make inquiline social parasites particularly suited for this aim; we propose a conceptual comparative framework that considers trait losses, gains and modifications in social parasite-host systems. We give examples of how this framework can reveal the more elusive secrets of sociality by focusing on two cornerstones of sociality: communication and reproductive division of labour. Together with social parasites in other taxonomic groups, such as cuckoos in birds, social parasitism has a great potential to reveal the mechanisms and evolution of complex social groups. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

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.