Differences in intestinal microbiota between avian brood parasites and their hosts

The intestinal microbiota determines the effectiveness of digestion in vertebrates, and is influenced by the external environment (mainly the diet), gut characteristics, and phylogeny. Avian brood-parasitic nestlings of the sub-family Cuculinae develop in nests of phylogenetically distant passerines and can be fed with the host diet. If the shaping of bacterial communities is dominated by phylogenetic constraints, and therefore the microbiota of parasitic nestlings differs from that of host nestlings, the energy and micronutrients that parasites and hosts obtain from a similar amount of food would be different. In this case, the bacterial communities of parasitic and host nestlings would have important consequences with respect to brood parasite development. By experimentally creating mixed broods of magpies ( Pica pica ) and great spotted cuckoos ( Clamator glandarius ), we investigated their cloacal microbiota using ribosomal intergenic spacer analysis. We found significant differences in bacterial assemblages of the parasitic and host nestlings, although none of the phylotypes were specific in either great spotted cuckoos or magpies. Cuckoos presented more complex communities, which could help the brood parasitic life style and allow the digestion of food provided by different potential hosts. Moreover, the intestinal morphology is different between the two species due to phylogenetic differences in the two taxa, which would influence the dissimilar bacterial assemblages. The detected differences in microbiota of great spotted cuckoo and magpie nestlings, which might occur in other brood parasite–host systems, may imply a lower digestion efficiency in parasites. Thus, the higher level requirements of cuckoo nestlings may be explained, at least in part, by cuckoos having a suboptimal bacterial community for processing the host diet.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 406–414.     

Comparison of digestive efficiency in the parasitic great spotted cuckoo and its magpie host nestlings

Altricial nestlings are under strong selection pressures to optimize digestive efficiency because this is one of the main factors affecting nestling growth and survival. Bird species vary in their ability to assimilate different nutrients and current theory predicts that nestlings should also be able to adjust their nutritional physiology to feeding frequency. Variation in parental provisioning to nestlings would select for flexibility in nestling digestive physiology, which would allow maximization of nutrient assimilation. In the present study, by making use of a brood parasite–host study system in which great spotted cuckoo nestlings (Clamator glandarius) are reared by magpie (Pica pica) host foster parents when sharing the nest with host nestlings, we tested several predictions of the adaptive digestive efficiency paradigm. A hand‐feeding experiment was employed in which we fed both great spotted cuckoo and magpie nestlings with exactly the same diet simulating one food abundance period and one food deprivation period. The results obtained show that cuckoo nestlings ingested more food, gained significantly more weight during the abundance period, and assimilated a higher proportion of the ingested food than magpie nestlings. These results demonstrate for the first time that cuckoo nestlings enjoy digestive adaptations that favour a rapid processing of the ingested food, thereby maximizing their intake rate but without decreasing digestive efficiency. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 280–289.     

Great Spotted Cuckoo Nestlings but not Magpie Nestlings Starve in Experimental Age‐Matched Broods

Nestlings of non‐evicting avian brood‐parasites have to compete for food with foster parents' own nestlings. The outcome of these competitive contests is determined mainly by body size differences between parasitic and host nestlings. As part of the coevolutionary arms race between brood parasites and their hosts at the nestling stage, it has been reported that some host foster parents discriminate against parasitic chicks and are reluctant to feed them. Here, by experimentally creating size‐matched broods of different composition (only magpie Pica pica chicks, only great spotted cuckoo Clamator glandarius chicks or mixed broods), we show that great spotted cuckoo chicks starved in 20.2 per cent (17 of 84) of the parasitized magpie nests even in absence of size asymmetries, while in none (0 of 72) of the nests a magpie chick starved. As far as we know, this is the first record of non‐evictor brood parasitic nestlings starving without being smaller than their host nestmates in a frequently used host species. Nest composition had no effect on chick starvation. The cuckoo nestling starved even in two of the nests occupied by only one cuckoo chick. Our results could be explained by (1) magpies being reluctant to feed cuckoo chicks; (2) parasitic chicks receiving lower‐quality food items or cuckoo nestlings being sensitive to some particular component of the diet (e.g. cereal grains); and (3) the existence of cuckoo chick discrimination ability by magpie foster parents.     

Bacterial diversity at the cloaca relates to an immune response in magpie Pica pica and to body condition of great spotted cuckoo Clamator glandarius nestlings

Diversity of the gut bacterial community is of prime importance for optimal food digestion and, therefore, for nutritional condition of avian nestlings. Consequently, bacterial community should be considered as a predictor of the future survival and recruitment of young birds. To explore this hypothesis, we studied the cloacal microbiota, by using RISA procedure, in two avian species sharing environmental conditions during growth, the avian brood parasitic great spotted cuckoo Clamator glandarius , and their main host in Europe, the magpie Pica pica . As estimates of phenotypic condition of nestlings we studied two nutrition-dependent traits, the immune response to an innocuous antigen (phytohemagglutinin), and the residuals of body mass on tarsus and wing length of nestlings. According to the hypothesis, we found significant relationships between microbial diversity and nestling phenotypic traits related to probability of recruitment. Briefly, both magpie and cuckoo nestlings having more similar microbial diversity were also those with similar immune response and body condition index respectively. Our results show a possible association between bacterial communities and variables related to the probability of post-fledging survival and recruitment of birds, as well as possible reasons explaining magpie-cuckoo differences in the nutritionally conditioned variables better associated with their bacterial diversity.     

Corticosterone levels in host and parasite nestlings: Is brood parasitism a hormonal stressor?

Parasite chicks from non-evictor species usually try to monopolize host parental care, thereby increasing considerably the level of food competition in the nest. Here, we propose that brood parasitism is an important stressor for host and parasite nestlings and explore this hypothesis in the non-evictor great spotted cuckoo (Clamator glandarius) and its main hosts, the same-sized black-billed magpie (Pica pica) and the larger carrion crow (Corvus corone). We experimentally created 3-nestling broods of different brood compositions (only cuckoo chicks, only host chicks, or cuckoo and host chicks together) and measured baseline corticosterone levels of nestlings along their developmental period (early, middle and late). We found that brood parasitism increased corticosterone levels in magpie nestlings in the mid and late nestling period compared to those raised in unparasitized nests. Interestingly, carrion crow nestlings from parasitized nests only increased their corticosterone levels in the mid nestling period, when the competition for food with the cuckoo nestling was highest. Our results suggest that brood parasitism could be a potential physiological stressor for host nestlings, especially during the developmental stages where food requirements are highest. Conversely, cuckoo nestlings could be physiologically adapted to high competition levels since they did not show significant differences in corticosterone levels in relation to brood composition.     

Complex feeding behaviour by magpies in nests with great spotted cuckoo nestlings

Parent decisions about food allocation are usually based on simple time‐saving rules that optimize their own fitness; however, they can sometimes vary depending on the prevailing ecological conditions both outside and inside the nest. Parent–offspring interactions also become more complex when parents suffer from brood parasitism, which implies that they care for the parasite's eggs and unrelated young. The great spotted cuckoo Clamator glandarius is a specialist brood parasite that uses the magpie Pica pica as its primary host. Here, by filming food allocation by magpie parents in natural non‐parasitized and experimentally parasitized and non‐parasitized magpie nests, we have found that magpie provisioning behaviour is highly complex including two types of feedings apart from normal ones. First, false feedings, when the parent touched the chick's beak but did not leave any food, occurred more frequently when feeding a cuckoo than when feeding magpie nestlings. Second, two types of what we have called coax feedings: 2a) when magpie parents induce a nestling to beg by waking it up by touching it softly with the beak, and 2b) when parents disregard begging signals (always from brood parasitic great spotted cuckoos) while coaxing one non‐begging nestling (always one of their own) to feed it. We suggest that brood parasitism, involving selfish excessively begging nestlings, could have acted as a selective pressure for both false and coax feedings to evolve, as both imply ignoring nestlings that beg too much. We also discuss that these parental responses could have evolved either by a discrimination without recognition mechanism, or, more probably, by a recognition‐based discrimination mechanism.     

Eavesdropping cuckoos: further insights on great spotted cuckoo preference by magpie nests and egg colour

Reproductive success of brood parasites largely depends on appropriate host selection and, although the use of inadvertent social information emitted by hosts may be of selective advantage for cuckoos, this possibility has rarely been experimentally tested. Here, we manipulated nest size and clutch colouration of magpies (Pica pica), the main host of great spotted cuckoos (Clamator glandarius). These phenotypic traits may potentially reveal information about magpie territory and/or parental quality and could hence influence the cuckoo’s choice of host nests. Experimentally reduced magpie nests suffered higher predation rate, and prevalence of cuckoo parasitism was higher in magpie nests with the densest roofs, which suggests a direct advantage for great spotted cuckoos choosing this type of magpie nest. Colouration of magpie clutches was manipulated by adding one artificial egg (blue or cream colouration) at the beginning of the egg-laying period. We found that host nests holding an experimental cream egg experienced a higher prevalence of cuckoo parasitism than those holding a blue-coloured egg. Results from these two experiments suggest that great spotted cuckoos cue on magpie nest characteristics and the appearance of eggs to decide parasitism, and confirm, for the first time, the ability of cuckoos to distinguish between eggs of different colours within the nest of their hosts. Several hypothetical scenarios explaining these results are discussed.     

Rejection of parasitic eggs in relation to egg appearance in magpies

The coevolutionary process between avian brood parasites and their hosts predicts that low intraclutch variation in egg colour appearance favours egg discrimination of parasite eggs by hosts. Low intraclutch variation would also result in high interclutch variation, which would increase the difficulty of evolution of mimicry by the cuckoo, because many host colour patterns might coexist in the same host population. We explored this possibility using an experimental approach in the common magpie, Pica pica, and great spotted cuckoo, Clamator glandarius, system. We artificially parasitized magpie nests with great spotted cuckoo model eggs to assess host response in two populations in Spain (Guadix and Doñana) in relation to intraclutch variation in egg appearance, measured by ultraviolet-visible reflectance spectrophotometry. Individuals that rejected model cuckoo eggs had higher intraclutch variation than accepters, suggesting that an increase, rather than a decrease, in intraclutch variation in magpie egg appearance was advantageous for cuckoo egg discrimination.     

Brood parasitism is associated with increased bacterial contamination of host eggs: bacterial loads of host and parasitic eggs

Factors related to bacterial environment of nests are of primary interest for understanding the causes of embryo infection and the evolution of antimicrobial defensive traits in birds. Nest visitors such as parasites could act as vectors for bacteria and/or affect the hygienic conditions of nests and hence influence the nest bacterial environment. In the present study, we explored some predictions of this hypothetical scenario in the great spotted cuckoo (Clamator glandarius)–magpie (Pica pica) system of brood parasitism. Great spotted cuckoos visit the nests of their magpie hosts and frequently damage some of the host eggs when laying eggs or on subsequent visits. Therefore, it represents a good system for testing the effect of nest visitors on the bacterial environment of nests. In accordance with this hypothesis, we found that the bacterial load of magpie eggshells was greater in parasitized nests, which may suggest that brood parasitism increases the probability of bacterial infection of magpie eggs. Moreover, comparisons of bacterial loads of cuckoo and magpie eggs revealed that: (1) cuckoo eggshells harboured lower bacterial densities than those of their magpie hosts in the same nests and (2) the prevalence of bacteria inside unhatched eggs was higher for magpies than for great spotted cuckoos. These interspecific differences were predicted because brood parasitic eggs (but not host eggs) always experience the bacterial environments of parasitized nests. Therefore, the results obtained in the present study suggest that parasitic eggs are better adapted to environments with a high risk of bacterial contamination than those of their magpie hosts. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 836–848.     

Great Spotted Cuckoo Fledglings Often Receive Feedings from Other Magpie Adults than Their Foster Parents: Which Magpies Accept to Feed Foreign Cuckoo Fledglings?

Natural selection penalizes individuals that provide costly parental care to non-relatives. However, feedings to brood-parasitic fledglings by individuals other than their foster parents, although anecdotic, have been commonly observed, also in the great spotted cuckoo (Clamator glandarius) – magpie (Pica pica) system, but this behaviour has never been studied in depth. In a first experiment, we here show that great spotted cuckoo fledglings that were translocated to a distant territory managed to survive. This implies that obtaining food from foreign magpies is a frequent and efficient strategy used by great spotted cuckoo fledglings. A second experiment, in which we presented a stuffed-cuckoo fledgling in magpie territories, showed that adult magpies caring for magpie fledglings responded aggressively in most of the trials and never tried to feed the stuffed cuckoo, whereas magpies that were caring for cuckoo fledglings reacted rarely with aggressive behavior and were sometimes disposed to feed the stuffed cuckoo. In a third experiment we observed feedings to post-fledgling cuckoos by marked adult magpies belonging to four different possibilities with respect to breeding status (i.e. composition of the brood: only cuckoos, only magpies, mixed, or failed breeding attempt). All non-parental feeding events to cuckoos were provided by magpies that were caring only for cuckoo fledglings. These results strongly support the conclusion that cuckoo fledglings that abandon their foster parents get fed by other adult magpies that are currently caring for other cuckoo fledglings. These findings are crucial to understand the co-evolutionary arms race between brood parasites and their hosts because they show that the presence of the host''s own nestlings for comparison is likely a key clue to favour the evolution of fledgling discrimination and provide new insights on several relevant points such as learning mechanisms and multiparasitism.     

MAGPIE HOST MANIPULATION BY GREAT SPOTTED CUCKOOS: EVIDENCE FOR AN AVIAN MAFIA?

Why should the hosts of brood parasites accept and raise parasitic offspring that differ dramatically in appearance from their own? There are two solutions to this evolutionary enigma. (1) Hosts may not yet have evolved the capability to discriminate against the parasite, or (2) parasite-host systems have reached an evolutionary equilibrium. Avian brood parasites may either gain renesting opportunities or force their hosts to raise parasitic offspring by destroying or preying upon host eggs or nestlings following host ejection of parasite offspring. These hypotheses may explain why hosts do not remove parasite offspring because only then will hosts avoid clutch destruction by the cuckoo. Here we show experimentally that if the egg of the parasitic great spotted cuckoo Clamator glandarius is removed from nests of its magpie Pica pica host, nests suffer significantly higher predation rates than control nests in which parasite eggs have not been removed. Using plasticine model eggs resembling those of magpies and observations of parasites, we also confirm that great spotted cuckoos that have laid an ejected egg are indeed responsible for destruction of magpie nests with experimentally ejected parasite eggs. Cuckoos benefit from destroying host offspring because they thereby induce some magpies to renest and subsequently accept a cuckoo egg.     

Egg rejection by Iberian azure-winged magpies Cyanopica cyanus in the absence of brood parasitism

The Iberian azure-winged magpie Cyanopica cyanus shows a remarkable ability to discriminate against great spotted cuckoo Clamator glandarius eggs. Here, I studied whether egg recognition in this species could be a derived feature resulting from intra-specific brood parasitism. Azure-winged magpies showed a very high level of discrimination and rejection of great spotted cuckoo models (73.7%), and of conspecific eggs (42.8%), even when no evidence of great spotted cuckoo or conspecific brood parasitism has been found in the population. Azure-winged magpie discriminated more readily than magpies, the current favourite host of the great spotted cuckoo. The high rejection rate of conspecific eggs by the azure-winged magpie suggests that it is quite possible that egg discrimination in this species evolved in response to conspecific brood parasitism rather than to cuckoo parasitism.     

Great spotted cuckoos respond earlier to the arrival of feeding foster parents and perform less erroneous begging when hungry than their magpie host nest‐mates

In many bird species, parents usually feed the first nestling that starts to beg before its nest‐mates. The pressure to avoid missed feeds could trigger nestlings to perform in erroneous begging in absence of parents, which has the same costs as begging in the presence of parents but without any reward. So, nestlings should try to minimize both erroneous begging and missed feeds simultaneously. The threshold to start begging is predicted to be lower for hungry nestlings and for nestlings that are unrelated to their nest‐mates, because they suffer lower inclusive fitness costs when depriving nest‐mates of food. In line with this idea, we found that brood parasitic great spotted cuckoo nestlings responded sooner than their magpie nest‐mates when an adult arrived to the nest. Under laboratory conditions, nestlings of both species rarely incurred in erroneous begging when food was abundant, but under conditions of restricted food, magpie nestlings increased erroneous begging while cuckoo nestlings did not. Highly conspicuous begging in cuckoos results in an increased predation risk, which could have resulted in stronger selection pressures on cuckoos to avoid erroneous begging, probably resulting in better developed perceptual abilities, allowing cuckoos to perform better than their host nest‐mates.     

Telomere dynamics in parasitic great spotted cuckoos and their magpie hosts

Although little is known on the impact of environment on telomere length dynamics, it has been suggested to be affected by stress, lifestyle and/or life‐history strategies of animals. We here compared telomere dynamics in erythrocytes of hatchlings and fledglings of the brood parasite great spotted cuckoos (Clamator glandarius) and of magpies (Pica pica), their main host in Europe. In magpie chicks, telomere length decreased from hatching to fledging, whereas no significant change in telomere length of great spotted cuckoo chicks was found. Moreover, we found interspecific differences in the association between laying date and telomere shortening. Interspecific differences in telomere shortening were interpreted as a consequence of differences in lifestyle and life‐history characteristics of magpies and great spotted cuckoos. In comparison with magpies, cuckoos experience reduced sibling competition and higher access to resources and, consequently, lower stressful environmental conditions during the nestling phase. These characteristics also explain the associations between telomere attrition and environmental conditions (i.e. laying date) for magpies and the absence of association for great spotted cuckoos. These results therefore fit expectations on telomere dynamics derived from interspecific differences in lifestyle and life history of brood parasites and their bird hosts.     

COMPARATIVE POPULATION STRUCTURE AND GENE FLOW OF A BROOD PARASITE,THE GREAT SPOTTED CUCKOO (CLAMATOR GLANDARIUS), AND ITS PRIMARY HOST,THE MAGPIE (PICA PICA)

The amount of gene flow is an important determinant of population structure and therefore of central importance for understanding coevolutionary processes. We used microsatellite markers to estimate population structure and gene flow rates of the great spotted cuckoo (Clamator glandarius) and its main host in Europe, the magpie (Pica pica), in a number of populations (seven and 15, respectively) across their distribution range in Europe. The genetic analysis shows that there exists a pattern of isolation by distance in both species, although the cuckoo data are only indicative due to a small sample size. Gene flow seems to be extensive between nearby populations, higher for magpies than cuckoos, and especially high for magpie populations within the area of distribution of the great spotted cuckoo. There is no correlation between genetic distances between magpie populations and genetic distances between cuckoo populations. We discuss the implications of extensive gene flow between magpie populations in sympatry with cuckoos for the population dynamics of hosts, in particular for the occurrence of egg rejection behavior in host populations and how the different rates of migration for both species can affect the dynamics of coevolutionary processes.     

Gut Microbiota Dynamics during Dietary Shift in Eastern African Cichlid Fishes

The gut microbiota structure reflects both a host phylogenetic history and a signature of adaptation to the host ecological, mainly trophic niches. African cichlid fishes, with their array of closely related species that underwent a rapid dietary niche radiation, offer a particularly interesting system to explore the relative contribution of these two factors in nature. Here we surveyed the host intra- and interspecific natural variation of the gut microbiota of five cichlid species from the monophyletic tribe Perissodini of lake Tanganyika, whose members transitioned from being zooplanktivorous to feeding primarily on fish scales. The outgroup riverine species Astatotilapia burtoni, largely omnivorous, was also included in the study. Fusobacteria, Firmicutes and Proteobacteria represented the dominant components in the gut microbiota of all 30 specimens analysed according to two distinct 16S rRNA markers. All members of the Perissodini tribe showed a homogenous pattern of microbial alpha and beta diversities, with no significant qualitative differences, despite changes in diet. The recent diet shift between zooplantkon- and scale-eaters simply reflects on a significant enrichment of Clostridium taxa in scale-eaters where they might be involved in the scale metabolism. Comparison with the omnivorous species A. burtoni suggests that, with increased host phylogenetic distance and/or increasing herbivory, the gut microbiota begins differentiating also at qualitative level. The cichlids show presence of a large conserved core of taxa and a small set of core OTUs (average 13–15%), remarkably stable also in captivity, and putatively favoured by both restricted microbial transmission among related hosts (putatively enhanced by mouthbrooding behavior) and common host constraints. This study sets the basis for a future large-scale investigation of the gut microbiota of cichlids and its adaptation in the process of the host adaptive radiation.     

Spatial patterns of egg laying and multiple parasitism in a brood parasite: a non-territorial system in the great spotted cuckoo (Clamator glandarius)

We analysed the spatial and temporal pattern of egg laying in great spotted cuckoo females using microsatellite typing to determine parentage of the eggs and nestlings found in host (magpie) nests. The results showed that there were no exclusive laying territories in the study area. Cases of multiparasitism could be due to single females laying two or more eggs in a nest, or to several females using the same nest. In the latter case multiparasitism was due to a shortage of available host nests. We argue that the need for very large laying areas and the likely small cost of sharing parental care for chicks make the costs of defending territories higher than the benefits, which has constrained the evolution of territoriality in this species. Received: 16 March 1998 / Accepted: 15 June 1998     

Change in host rejection behavior mediated by the predatory behavior of its brood parasite

Passerine hosts of parasitic cuckoos usually vary in their abilityto discriminate and reject cuckoo eggs. Costs of discriminationand rejection errors have been invoked to explain the maintenanceof this within-population variability. Recently, enforcementof acceptance by parasites has been identified as a rejectioncost in the magpie (Pica pica) and its brood parasite, the greatspotted cuckoo (Clamator glandarius). Previous experimentalwork has shown that rejecter magpies suffer from increased nestpredation by the great spotted cuckoo. Cuckoo predatory behavioris supposed to confer a selective advantage to the parasitebecause magpies experiencing a reproductive failure may providea second opportunity for the cuckoo to parasitize a replacementclutch. This hypothesis implicitly assumes that magpies modulatetheir propensity to reject parasite eggs as a function of previousexperience. We tested this hypothesis in a magpie populationbreeding in study plots varying in parasitism rate. Magpie pairs thatwere experimentally parasitized and had their nests depredated,after their rejection behavior had been assessed, changed theirbehavior from rejection to acceptance. The change in host behaviorwas prominent in study plots with high levels of parasitism,but not in plots with rare or no cuckoo parasitism. We discussthree possible explanations for these differences, concludingthat in study plots with a high density of cuckoos, the probability fora rejecter magpie nest of being revisited and depredated bya cuckoo is high, particularly for replacement clutches, and,therefore, the cost for magpies of rejecting a cuckoo egg ina replacement clutch is increased. Moreover, in areas with highlevels of host defense (low parasitism rate), the probabilityof parasitism and predation of rejecter-magpie nests by thecuckoo is reduced in both first and replacement clutches. Therefore,rejecter magpies in such areas should not change their rejectionbehavior in replacement clutches.     

Climatic effects and phenological mismatch in cuckoo–host interactions: a role for host phenotypic plasticity in laying date?

Climatic effects on breeding phenology vary across organisms and therefore might promote a phenological mismatch in ecologically interacting species, including those engaged in coevolutionary interactions such as brood parasites and their hosts. Recent studies suggest that climatic induced changes in migration phenology may have mismatched cuckoos and their hosts in Europe. However, it is currently unknown whether cuckoo–host phenological mismatch results from different degrees of phenotypic plasticity or to different speeds of microevolutionary processes affecting hosts and parasites. Here we performed 1) cross‐sectional correlations between climate conditions and population level of phenological mismatch between the migratory brood parasite great spotted cuckoo Clamator glandarius and its main resident host in Europe, the magpie Pica pica; and 2) a longitudinal analysis to study within‐individual variation in breeding phenology for individual hosts experiencing different climate conditions over a period of nine years (2005–2013). Cross‐sectional analyses revealed independent and contrary effects of winter and spring temperature on magpie phenology: magpie hosts tend to breed earlier those years with lower February temperatures, however, high temperature in the first half of April spur individuals to lay eggs. Breeding phenology of cuckoos was tuned to that of their magpie host in time and duration. However, annual phenological mismatch between cuckoos and magpie hosts increased with NAO index and January temperature. Longitudinal analyses revealed high individual consistency in magpie host phenology, but a low influence of climate, suggesting that the climatic‐driven phenological mismatch between cuckoos and magpies at the population‐level cannot be explained by a host plastic response to climatic conditions.     

Is egg-damaging behavior by great spotted cuckoos an accident or an adaptation?

Adult great spotted cuckoos Clamator glandarius damage the eggsof their magpie Pica pica host without removing them from thenest or eating them but by producing the death of the embryo.Observations as well as experiments were used to test severalpredictions of two different possibilities: great spotted cuckooegg-damaging behavior is a parasitic tactic resulting froma direct selection process (the adaptation hypothesis), oregg damage is caused by thick-shelled cuckoo eggs which evolvedto avoid breakage during rapid laying (the nonadaptation hypothesis).Previously, we provided experimental evidence that egg damageincreased the breeding success of cuckoos when they laid lateduring the laying sequence of the magpie. However, when theylaid early, egg-damaging behavior did not increase cuckoo breedingsuccess, contrary to the adaptation hypothesis. In an experimental study, when we simulated laying behavior by the great spottedcuckoo, we found that (1) the number of damaged magpie eggswas significantly lower than in natural parasitism, and (2)whereas in the experimental manipulations the number of damagedeggs did not depend on the number of magpie eggs, in natural parasitism, the number of damaged eggs increased with clutchsize of the magpie. These results support the predictions ofthe adaptation hypothesis, implying that egg damage is notan incidental consequence of rapid egg laying, but an adaptation.