Surveillance on ESBL-Escherichia coli and Indicator ARG in Wastewater and Reclaimed Water of Four Regions of Spain: Impact of Different Disinfection Treatments.

In the present study, the occurrence of indicator antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) both in the influent and the effluent of four Spanish wastewater treatment plants (WWTPs) was monitored for 12 months, and the susceptibility profiles of 89 recovered extended spectrum ß-lactamase (ESBL)-producing Escherichia coli isolates were obtained against a wide range of antimicrobials. The aim of the study was to better understand whether the current wastewater treatment practices allow us to obtain safe reclaimed water mitigating the spread of ARB and ARGs to the environment. Results showed high concentrations of ESBL-producing E. coli as well as a high prevalence of a range of ARGs in the influent samples. The reclamation treatments implemented in the WWTPs were effective in reducing both the occurrence of ESBL E. coli and ARGs, although significant differences were observed among WWTPs. Despite these reductions in occurrence observed upon wastewater treatment, our findings suggest that WWTP effluents may represent an important source of ARGs, which could be transferred among environmental bacteria and disseminate antimicrobial resistance through the food chain. Remarkably, no major differences were observed in the susceptibility profiles of the ESBL E. coli isolated from influent and effluent waters, indicating that water treatments do not give rise to the emergence of new resistance phenotypes.

Functional explanation of extreme hatching asynchrony: Male Manipulation Hypothesis.

Hatching asynchrony in birds is considered an adaptation to facilitate brood reduction because under conditions of food scarcity, the smallest nestling usually dies soon after hatching, thereby minimizing parental effort. However, in species with extreme hatching asynchrony, the last hatchlings paradoxically experience a very low probability of survival and death can take so long that it can hardly be considered an adaptation. Here, we propose and experimentally tested a new adaptive hypothesis explaining the brood reduction paradox, namely the "Male Manipulation Hypothesis". Our hypothesis suggests that by inducing asynchronous hatching, females increase the feeding requirements of the brood, which will induce males to increase provisioning effort. In addition, females may extend the period of male manipulation by feeding the smallest nestling just enough to sustain life. Our study showed that male common blackbirds ( Turdus merula) increased their effort (i.e., number of food items per hour) in experimental asynchronous broods compared to synchronous broods, while females reduced their contribution, as predicted by the hypothesis.

Physiological stress responses to nonmimetic model brood parasite eggs: Leukocyte profiles and heat-shock protein Hsp70 levels.

Obligate avian brood parasites lay their eggs in the nest of other bird species, known as hosts. Brood parasitism often imposes severe fitness costs on hosts, selecting for the evolution of effective antiparasitic defences, such as recognition and rejection of brood parasite eggs. Glucocorticoids have been recently found to mediate host physiological and behavioral adjustments in response to brood parasite eggs; however, it remains unclear whether brood parasitism triggers a general response involving multiple physiological elements. In this study, we experimentally investigated whether a salient brood parasitic stimulus (the presence of a nonmimetic model egg in the nest) causes physiological adjustments in adult Eurasian blackbirds (Turdus merula) at immune (leukocyte profiles) and cellular (heat-shock protein Hsp70 synthesis) level. Also, we explored whether these physiological changes are mediated by variations in corticosterone (CORT) levels. We found that experimental brood parasitism caused an increase in heterophils and a decrease in lymphocytes, leading to higher heterophils and lymphocytes ratios in parasitized birds. Nevertheless, we did not find tradeoffs between immune function and CORT levels. Hsp70 synthesis was not affected by our experimental manipulation. Our findings provide evidence that brood parasite eggs trigger a general stress response in egg-rejecter hosts, including changes in cellular immune profiles.

Strategies for mitigating chlorinated disinfection byproducts in wastewater treatment plants.

A case study of 15 wastewater treatment plants (WWTPs) at a full-scale was assessed for the risks of disinfection byproduct (DBP) formation, mainly the regulated trihalomethanes (THMs) and haloacetic acids (HAAs) and chlorate as an inorganic byproduct regulated recently in the EU. Raw wastewater from large, medium/small urban areas were treated with single or combined disinfection processes (i.e., chlorine, peracetic acid (PAA) and ultraviolet (UV) radiation). Sampling was executed once a month over seven months for the medium/small WWTPs and twice a month for the large ones. Due to the potential risk of SARS-CoV-2 contaminated wastewater, several inactivation methods were examined before the DBP analysis. Due to the inactivation step, the stability of THM4 and HAA9 suffered reductions, monitoring their presence only in the effluents after the disinfection treatments. In contrast, chlorate levels remained unchanged after the inactivation treatment; thus both raw wastewater and effluents were examined for their occurrence before disinfection treatments. Results showed that chlorate residues in the raw wastewater varied greatly from undetected levels to as high as 42.2 mg L-1. As the continuous monitoring of DBPs was performed, a positive correlation with chlorine or chlorine/UV was found. Changes in the physicochemical parameters indicated that the quality of the raw wastewater varied considerably depending on the WWTPs, and it influenced byproduct formation. In all WWTPs, chlorine alone or combined with UV significantly increased the presence of THMs, HAAs, and chlorate levels in the treated effluents. When the same WWTPs changed to PAA or PAA/UV, DBPs were diminished completely. This study highlights the risk of chlorate residues in raw wastewater during the pandemic. It also showed how the chemical risks of DBP formation could be reduced by changing the chlorinated disinfection technologies to PAA or PAA/UV, particularly if reclaimed water is intended for agricultural irrigation to minimize DBP residues.

Prolactin mediates behavioural rejection responses to avian brood parasitism.

Adaptations resulting from co-evolutionary interactions between avian brood parasites and their hosts have been extensively studied, yet the physiological mechanisms underlying antiparasitic host defences remain little known. Prolactin, one of the main hormones involved in the regulation of avian parental behaviour, might play a key role in the orchestration of the host responses to avian brood parasitism. Given the positive association between prolactin and parental behaviour during incubation, decreasing prolactin levels are expected to facilitate egg-rejection decisions. We tested this prediction by implanting Eurasian blackbird (Turdus merula) females with an inhibitor of prolactin secretion, bromocriptine mesylate, to experimentally decrease their plasma prolactin levels. Bromocriptine mesylate-implanted individuals ejected mimetic model eggs at higher rates, and showed shorter latency to egg ejection, than placebo-treated birds. To our knowledge, this is the first experimental evidence that behavioural host defences against avian brood parasitism are mediated by prolactin.

Replication of the mirror mark test experiment in the magpie (Pica pica) does not provide evidence of self-recognition.

Self-recognition in animals is demonstrated when individuals pass the mark test. Formerly, it was thought that self-recognition was restricted to humans, great apes, and certain mammals with large brains and highly evolved social cognition. However, 1 study showed that 2 out of 5 magpies (Pica pica) passed the mark test, suggesting that magpies have a similar level of cognitive abilities to great apes. The scientific advancement depends on confidence in published science, and this confidence can be reached only after rigorous replication of published studies. Here, we present a close replication of the magpie study but using a larger sample size while following a very similar experimental protocol. Like the previous study, in our experiment, magpies showed both social and self-directed behavior more frequently in front of the mirror versus a control cardboard stimulus. However, during the mark test, self-directed behavior proved more frequent in front of the cardboard than in the mirror. Thus, our replication failed to confirm the previous results. Close replications, while not disproving an earlier study, identify results that should be considered with caution. Therefore, more replication studies and additional experimental work is needed to unambiguously demonstrate that magpies are consistently able to pass the mark test. The existence of compelling evidence of self-recognition in other corvid species is discussed in depth. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Signal detection and optimal acceptance thresholds in avian brood parasite-host systems: implications for egg rejection.

Signal detection theory addresses the challenge of successfully identifying informative signals in noisy information contexts, allowing optimal behavioural decisions in diverse ecological contexts. The optimal acceptance threshold hypothesis proposed by Reeve (Reeve 1989 Am. Nat.133, 407-435. (doi:10.1086/284926)) is an elegant theoretical model to predict the flexibility of acceptance thresholds for conspecific discrimination. This model has provided a robust framework used to explore recognition systems in a broad range of contexts such as animal communication, nest-mate discrimination or anti-parasitic host responses. In this review, we discuss key concepts related to the optimal acceptance threshold hypothesis applied to egg rejection decisions in avian brood parasite-host interactions. We explore those factors determining signal detectability in parasitized nests and how hosts adjust their rejection decisions to both the risk of parasitism and the potential costs associated with egg rejection. In addition, we discuss recent results that challenge some traditional assumptions of the optimal acceptance threshold hypothesis and provide a novel perspective to explore rejection decisions, such as the existence of single-threshold decision rules or acceptance decisions. An integrative view combining current evidence with traditional theory is needed to further advance the comprehension of optimal acceptance thresholds. This article is part of the theme issue 'Signal detection theory in recognition systems: from evolving models to experimental tests'.

Diversity of HLA Class I and Class II blocks and conserved extended haplotypes in Lacandon Mayans.

Here we studied HLA blocks and haplotypes in a group of 218 Lacandon Maya Native American using a high-resolution next generation sequencing (NGS) method. We assessed the genetic diversity of HLA class I and class II in this population, and determined the most probable ancestry of Lacandon Maya HLA class I and class II haplotypes. Importantly, this Native American group showed a high degree of both HLA homozygosity and linkage disequilibrium across the HLA region and also lower class II HLA allelic diversity than most previously reported populations (including other Native American groups). Distinctive alleles present in the Lacandon population include HLA-A*24:14 and HLA-B*40:08. Furthermore, in Lacandons we observed a high frequency of haplotypes containing the allele HLA-DRB1*04:11, a relatively frequent allele in comparison with other neighboring indigenous groups. The specific demographic history of the Lacandon population including inbreeding, as well as pathogen selection, may have elevated the frequencies of a small number of HLA class II alleles and DNA blocks. To assess the possible role of different selective pressures in determining Native American HLA diversity, we evaluated the relationship between genetic diversity at HLA-A, HLA-B and HLA-DRB1 and pathogen richness for a global dataset and for Native American populations alone. In keeping with previous studies of such relationships we included distance from Africa as a covariate. After correction for multiple comparisons we did not find any significant relationship between pathogen diversity and HLA genetic diversity (as measured by polymorphism information content) in either our global dataset or the Native American subset of the dataset. We found the expected negative relationship between genetic diversity and distance from Africa in the global dataset, but no relationship between HLA genetic diversity and distance from Africa when Native American populations were considered alone.

Territoriality and variation in home range size through the entire annual range of migratory great spotted cuckoos (Clamator glandarius).

Variation in home range size throughout the year and its causes are not well understood yet. Migratory brood parasites offer a unique opportunity to incorporate this spatio-temporal dimension into the study of the factors regulating home range dynamics. Using satellite transmitters, we tracked sixteen migratory great spotted cuckoos (Clamator glandarius) of both sexes for up to three years. We constructed home ranges in all major staging areas, from the Spanish breeding areas to the African wintering grounds, analyzed their temporal and geographical variation and investigated their main potential determinants (e.g. food and host availability). We found that home ranges were significantly larger in the breeding area compared to non-breeding areas. Using NDVI as a proxy for food availability, we showed that breeding area home ranges have significantly lower food availability per km2 than home ranges elsewhere which could explain why cuckoos use alternative areas with higher food availability before initiating migration. We also found some evidence for sex differences. Additionally, we found no indications of territoriality in this species, providing novel information into the current debate on brood parasite territoriality. Overall, food availability seems to be an important factor regulating home range dynamics and influencing migratory patterns throughout the year in great spotted cuckoos.

Migration behavior and performance of the great spotted cuckoo (Clamator glandarius).

The study of brood parasitism has traditionally been focused on the breeding period, but recent evidence suggests that it urgently needs a new spatio-temporal perspective to explore novel avenues on brood parasite-host co-evolutionary interactions. Many brood parasites are migrants, but their ecology outside their short breeding season is poorly known. The great spotted cuckoo (Clamator glandarius) is one of the classical models in the study of brood parasitism, however, there is very little information on its migratory strategy, route and wintering grounds. Furthermore, there is no previous information on the geographical distribution of mortality and its causes in this species; information that is critical to understand the fluctuations in cuckoo populations and detect potential conservation risks. Using satellite tracking technology, we provide novel insight into the migratory behavior and performance of the great spotted cuckoo. We found individuals from southern Spain to be long-distance nocturnal migrants that use the East Atlantic Flyway for both post and pre-breeding migration, and that winter in the western Sahel. We found evidence of individual variation in their migration route, particularly regarding their post-breeding behavior in Spain. Our study also suggests that the south of Morocco is the most dangerous area due to a large number of deaths during the post-breeding migratory period. Furthermore, we found that natural predation seems to be the main cause of death, probably due to raptors, although human activities (i.e. hunting) could also played a role in the southern Mediterranean shore. Our study offers novel findings and challenges traditional ideas on the ecology of this species providing a good example of how the new spatio-temporal perspective can expand our knowledge on brood parasites.

Nest predation risk modifies nestlings' immune function depending on the level of threat.

Predation risk is thought to modify the physiology of prey mainly through the stress response. However, little is known about its potential effects on the immunity of animals, particularly in young individuals, despite the importance of overcoming wounding and pathogen aggression following a predator attack. We investigated the effect of four progressive levels of nest predation risk on several components of the immune system in common blackbird (Turdus merula) nestlings by presenting them with four different calls during 1 h: non-predator calls, predator calls, parental alarm calls and conspecific distress calls to induce a null, moderate, high and extreme level of risk, respectively. Nest predation risk induced an increase in ovotransferrin, immunoglobulin and the number of lymphocytes and eosinophils. Thus, the perception of a potential predator per se could stimulate the mobilization of a nestling's immune function and enable the organism to rapidly respond to the immune stimuli imposed by a predator attack. Interestingly, only high and extreme levels of risk caused immunological changes, suggesting that different immunological parameters are modulated according to the perceived level of threat. We also found a mediator role of parasites (i.e. Leucocytozoon) and the current health status of the individual, as only nestlings not parasitized or in good body condition were able to modify their immune system. This study highlights a previously unknown link between predation risk and immunity, emphasizing the complex relationship among different selective pressures (predation, parasitism) in developing organisms and accentuating the importance of studying predation from a physiological point of view.

Great spotted cuckoo nestlings have no antipredatory effect on magpie or carrion crow host nests in southern Spain.

Host defences against cuckoo parasitism and cuckoo trickeries to overcome them are a classic example of antagonistic coevolution. Recently it has been reported that this relationship may turn to be mutualistic in the case of the carrion crow (Corvus corone) and its brood parasite, the great spotted cuckoo (Clamator glandarius), given that experimentally and naturally parasitized nests were depredated at a lower rate than non-parasitized nests. This result was interpreted as a consequence of the antipredatory properties of a fetid cloacal secretion produced by cuckoo nestlings, which presumably deters predators from parasitized host nests. This potential defensive mechanism would therefore explain the detected higher fledgling success of parasitized nests during breeding seasons with high predation risk. Here, in a different study population, we explored the expected benefits in terms of reduced nest predation in naturally and experimentally parasitized nests of two different host species, carrion crows and magpies (Pica pica). During the incubation phase non-parasitized nests were depredated more frequently than parasitized nests. However, during the nestling phase, parasitized nests were not depredated at a lower rate than non-parasitized nests, neither in magpie nor in carrion crow nests, and experimental translocation of great spotted cuckoo hatchlings did not reveal causal effects between parasitism state and predation rate of host nests. Therefore, our results do not fit expectations and, thus, do not support the fascinating possibility that great spotted cuckoo nestlings could have an antipredatory effect for host nestlings, at least in our study area. We also discuss different possibilities that may conciliate these with previous results, but also several alternative explanations, including the lack of generalizability of the previously documented mutualistic association.

Relationships between egg-recognition and egg-ejection in a grasp-ejector species.

Brood parasitism frequently leads to a total loss of host fitness, which selects for the evolution of defensive traits in host species. Experimental studies have demonstrated that recognition and rejection of the parasite egg is the most common and efficient defence used by host species. Egg-recognition experiments have advanced our knowledge of the evolutionary and coevolutionary implications of egg recognition and rejection. However, our understanding of the proximate mechanisms underlying both processes remains poor. Egg rejection is a complex behavioural process consisting of three stages: egg recognition, the decision whether or not to reject the putative parasitic egg and the act of ejection itself. We have used the blackbird (Turdus merula) as a model species to explore the relationship between egg recognition and the act of egg ejection. We have manipulated the two main characteristics of parasitic eggs affecting egg ejection in this grasp-ejector species: the degree of colour mimicry (mimetic and non-mimetic, which mainly affects the egg-recognition stage of the egg-rejection process) and egg size (small, medium and large, which affects the decision to eject), while maintaining a control group of non-parasitized nests. The behaviour of the female when confronted with an experimental egg was filmed using a video camera. Our results show that egg touching is an indication of egg recognition and demonstrate that blackbirds recognized (i.e., touched) non-mimetic experimental eggs significantly more than mimetic eggs. However, twenty per cent of the experimental eggs were touched but not subsequently ejected, which confirms that egg recognition does not necessarily mean egg ejection and that accepting parasitic eggs, at least sometimes, is the consequence of acceptance decisions. Regarding proximate mechanisms, our results show that the delay in egg ejection is not only due to recognition problems as usually suggested, given that experimental eggs are not touched significantly more often. Thus, the delay in egg ejection is mainly the consequence of a delay in the decision to eject, probably triggered by mechanical constraints imposed by eggs that are harder to eject (i.e. larger). Our results offer important information on the relationships between recognition and ejection and contribute to a better understanding of host defences against brood parasites.

Egg rejection in blackbirds Turdus merula: a by-product of conspecific parasitism or successful resistance against interspecific brood parasites?

BACKGROUND: Traditional theory assumes that egg recognition and rejection abilities arise as a response against interspecific brood parasitism (IBP). However, rejection also appears in some species that are currently not exploited by interspecific parasites, such as Turdus thrushes. Recent evidences suggest that rejection abilities evolved in these species as a response to conspecific brood parasitism (CBP). To test these two alternative hypotheses, we performed an experimental study by parasitizing nests of the common blackbird (Turdus merula) with conspecifics or heterospecific eggs under different risk of parasitism (presence of interspecific or conspecific parasites near the nest). Common blackbird is a potential host of the common cuckoo (Cuculus canorus) but suffers low levels of CBP too. RESULTS: We found that blackbirds were able to recognize and eject heterospecific eggs at high rates whereas most of conspecifics eggs were not recognized and, therefore, accepted. Ejection rates of conspecific eggs did not exceed 13 %, even in situations of high risk of CBP (blackbird female placed near the nest), which contradict the main prediction derived from the CBP hypothesis. Conversely, ejection rates of experimental eggs simulating IBP were much higher (80-100 %). Furthermore, female blackbirds were more aggressive towards cuckoos than towards blackbird dummies. CONCLUSIONS: Our results considered together support the IBP hypothesis, indicating that recognition and rejection of parasitic eggs in blackbirds have probably evolved due to previous cuckoo parasitism. The current absence of IBP in blackbirds may be due to the highly efficient rejection abilities in this species. Thus, these abilities have been retained in absence of brood parasitism as a consequence of the low costs involved for blackbirds, resulting in a successful resistance against interspecific brood parasitism.

Fecal sacs attract insects to the nest and provoke an activation of the immune system of nestlings.

BACKGROUND: Nest sanitation is a widespread but rarely studied behavior in birds. The most common form of nest sanitation behavior, the removal of nestling feces, has focused the discussion about which selective pressures determine this behavior. The parasitism hypothesis, which states that nestling fecal sacs attract parasites that negatively affect breeding birds, was proposed 40 years ago and is frequently cited as a demonstrated fact. But, to our knowledge, there is no previous experimental test of this hypothesis. RESULTS: We carried out three different experiments to investigate the parasitism hypothesis. First, we used commercial McPhail traps to test for the potential attraction effect of nestling feces alone on flying insects. We found that traps with fecal sacs attracted significantly more flies (Order Diptera), but not ectoparasites, than the two control situations. Second, we used artificial blackbird (Turdus merula) nests to investigate the combined attraction effect of feces and nest materials on arthropods (not only flying insects). Flies, again, were the only group of arthropods significantly attracted by fecal sacs. We did not detect an effect on ectoparasites. Third, we used active blackbird nests to investigate the potential effect of nestling feces in ecto- and endoparasite loads in real nestlings. The presence of fecal sacs near blackbird nestlings did not increase the number of louse flies or chewing lice, and unexpectedly reduced the number of nests infested with mites. The endoparasite prevalence was also not affected. In contrast, feces provoked an activation of the immune system as the H/L ratio of nestlings living near excrements was significantly higher than those kept under the two control treatments. CONCLUSIONS: Surprisingly, our findings do not support the parasitism hypothesis, which suggests that parasites are not the main reason for fecal sac removal. In contrast, the attraction of flies to nestling feces, the elevation of the immune response of chicks, and the recently described antimicrobial function of the mucous covering of fecal sacs suggest that microorganisms could be responsible of this important form of parental care behavior (microbial hypothesis).

Could a Factor That Does Not Affect Egg Recognition Influence the Decision of Rejection?

Rejection of the parasitic egg is the most important defence of hosts against brood parasites. However, this response is variable among and within species, and egg discrimination is not always followed by egg rejection. Low risk of parasitism and high risk of rejection costs may lead to the acceptance of the parasitic egg even if it has been previously recognized. The main aim of this paper is to answer a relevant question: can a single egg trait provoke the acceptance of an experimental egg previously recognized as foreign? Increased egg mass should hamper the ejection of an egg that has been discriminated because ejection of a heavy egg may imply higher rejection costs for hosts. We have tested this prediction by experimentally parasitizing natural nests of Common Blackbirds (Turdus merula) with non-mimetic model eggs of different mass (heavy, normal-weight, and light) while controlling for potential confounding factors such as egg size and colour. Our results showed that blackbirds more frequently accepted heavy eggs, even when previously recognized. This differential acceptance may be related to insufficient motivation to assume the higher costs that the ejection of a heavy egg could impose.

A long-term experimental study demonstrates the costs of begging that were not found over the short term.

Parent-offspring conflict theory predicts that begging behaviour could escalate continuously over evolutionary time if it is not prevented by costliness of begging displays. Three main potential physiological costs have been proposed: growth, immunological and metabolic costs. However, empirical evidence on this subject remains elusive because published results are often contradictory. In this study, we test for the existence of these three potential physiological costs of begging in house sparrow (Passer domesticus) nestlings by stimulating a group of nestlings to beg for longer and another group for shorter periods than in natural conditions. All nestlings were fed with the same quantity of food. Our study involves a long-term experimental treatment for begging studies (five consecutive days). Long-term studies frequently provide clearer results than short-term studies and, sometimes, relevant information not reported by the latter ones. Our long-term experiment shows (i) a clear effect on the immune response even since the first measurement (6 hours), but it was higher during the second (long-term) than during the first (short-term) test; (ii) evidence of a growth cost of begging in house sparrow nestlings not previously found by other studies; (iii) body condition was affected by our experimental manipulation only after 48 hour; (iv) a metabolic cost of begging never previously shown in any species, and (v) for the first time, it has shown a simultaneous effect of the three potential physiological costs of begging: immunocompetence, growth, and metabolism. This implies first, that a multilevel trade-off can occur between begging and all physiological costs and, second, that a lack of support in a short-term experiment for the existence of a tested cost of begging does not mean absence of that cost, because it can be found in a long-term experiment.

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.

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.

Mirror-mark tests performed on jackdaws reveal potential methodological problems in the use of stickers in avian mark-test studies.

Some animals are capable of recognizing themselves in a mirror, which is considered to be demonstrated by passing the mark test. Mirror self-recognition capacity has been found in just a few mammals having very large brains and only in one bird, the magpie (Pica pica). The results obtained in magpies have enormous biological and cognitive implications because the fact that magpies were able to pass the mark test meant that this species is at the same cognitive level with great apes, that mirror self-recognition has evolved independently in the magpie and great apes (which diverged 300 million years ago), and that the neocortex (which is not present in the bird's brains) is not a prerequisite for mirror self-recognition as previously believed. Here, we have replicated the experimental design used on magpies to determine whether jackdaws (Corvus monedula) are also capable of mirror self-recognition by passing the mark test. We found that our nine jackdaws showed a very high interest towards the mirror and exhibited self-contingent behavior as soon as mirrors were introduced. However, jackdaws were not able to pass the mark test: both sticker-directed actions and sticker removal were performed with a similar frequency in both the cardboard (control) and the mirror conditions. We conclude that our jackdaws' behaviour raises non-trivial questions about the methodology used in the avian mark test. Our study suggests that the use of self-adhesive stickers on sensitive throat feathers may open the way to artefactual results because birds might perceive the stickers tactilely.