Evolution of nestling faeces removal in avian phylogeny

Nest sanitation-related traits have often been explained at the intraspecific level as reducing the probability of infection or detection by predators and parasites, but its evolution within the avian phylogeny is still poorly understood. We compiled detailed information of such traits for more than 400 bird species and, by means of modern comparative methodologies, we reconstructed the evolution of adults' contribution to removing their offspring's faeces and the production of faecal sacs by nestlings. Furthermore, because the functional hypotheses used to explain nest sanitation behaviour assume potential effects of brood size, body mass, nestling period and diet, we explored the association between these traits and those related to nest sanitation in a phylogenetically controlled framework. Our results suggest that parental removal of nestling faeces has driven the evolution of faecal sacs, while the ancestral states involved birds with faecal sacs removed by parents. These results support the long-held idea that faecal sacs facilitate the removal of faeces by parents. Moreover, we found that animal diets and small body sizes have favoured the evolution of faecal sacs suggesting the existence of some chemical and physical constraints in relation to the evolution of the mucous covering. Our results highlight the importance of nest sanitation in the evolution of birds and their life history characteristicsJ.D.I. was financed by a postdoctoral contract (TAHUB-104) from the program ‘Andalucía Talent Hub’ (cofunded by the European Union's Seventh Framework Program Marie Skłodowska-Curie actions (COFUND) and the regional Government of Andalucía). Funding was partially provided by the Spanish Ministerio de Economía y Competitividad (European funds (FEDER)) (CGL2013-48193-C3-1-P).Peer reviewe

Egg-recognition abilities in non-incubating males:Implications for the evolution of anti-parasitic host defenses

In the field of brood parasitism, it has been traditionally assumed that only the incubating sex rejects parasitic eggs, but this assumption has been rarely explored despite its important implications for the evolutionary relationship between brood parasites and hosts. Here, we used information on previous egg-rejection experiments to explore the recognition abilities of both males and females of Eurasian blackbirds Turdus merula towards experimental eggs with a variable degree of mimicry. We found that both sexes recognized non-mimetic eggs, supporting the idea that visits to the nest can favor the evolution of rejection abilities. In contrast, only females recognized mimetic eggs, indicating that although recognition abilities can evolve in both sexes, they are subsequently refined in females probably due to their more frequent interaction with parasitic eggs. Clutch size affected nest attendance since females, but not males, spent more time at the nest and visited it more frequently in larger clutches. Finally, our recordings showed that blackbird males are able not only to recognize, but also to eject parasitic eggs. Our results provide new insights into the main anti-parasitic defense in birds, egg rejection, and highlight the need of considering the role of the non-incubating sex in egg-rejection studies.Significance statementGiven the high costs associated to avian brood parasitism, both sexes are expected to evolve anti-parasitic defenses. However, in those species in which only females incubate, females have traditionally been assumed to be the responsible for egg rejection. Here, using the Eurasian blackbird (Turdus merula), we investigated the existence of egg-rejection abilities in non-incubating males and compared them to those exhibited by females. We found that males can recognize non-mimetic eggs, although their recognition abilities were less fine-tuned compared to females, who also recognized mimetic eggs. Even though females were the responsible for most documented egg-ejection events, recordings confirmed that males could also be involved in egg ejection, which could have important implication for the evolution of anti-parasitic defenses in host populations

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 hypothesisPeer reviewe

Fecal sacs do not increase nest predation in a ground nester

Most altricial birds remove their nestlings' feces from the nest, but the evolutionary forces driving this behavior are poorly understood. A possible adaptive explanation for this could be that birds avoid the attraction of nest predators to their nests due to the visual or olfactory cues produced by feces (nest predation hypothesis). This hypothesis has received contrasting support indicating that additional experimental studies are needed, particularly with respect to the visual component of fecal sacs. To test this hypothesis, we conducted an experiment manipulating the presence of fecal sacs on inactive Woodlark (Lullula arborea) nests. This ground nester has highly cryptic nests that are mainly depredated by visually oriented nest predators (i.e., corvids) in our study population, making it an excellent system to test for the nest predation hypothesis. Our results showed that the presence of fecal sacs in the nest does not seem to be an important factor explaining nest predation. Interestingly, the effect of nest concealment, the most important factor explaining nest predation in Woodlark nests, depended on whether the nest was depredated the previous year or not, supporting the importance of using different nesting sites between years. Our findings indicate that this important nest sanitation behavior is not likely motivated by nest predation and highlight the need to explore alternative selective pressures in this context

Physiological stress does not increase with urbanization in European blackbirds:Evidence from hormonal, immunological and cellular indicators

Urbanization changes the landscape structure and ecological processes of natural habitats. While urban areas expose animal communities to novel challenges, they may also provide more stable environments in which environmental fluctuations are buffered. Species´ ecology and physiology may determine their capacity to cope with the city life. However, the physiological mechanisms underlying organismal responses to urbanization, and whether different physiological systems are equally affected by urban environments remain poorly understood. This severely limits our capacity to predict the impact of anthropogenic habitats on wild populations. In this study, we measured indicators of physiological stress at the endocrine, immune and cellular level (feather corticosterone levels, heterophil to lymphocyte ratio, and heat-shock proteins) in urban and non-urban European blackbirds (Turdus merula) across 10 European populations. Among the three variables, we found consistent differences in feather corticosterone, which was higher in non-urban habitats. This effect seems to bedependent on sex, being greater in males. In contrast, we found no significant differences between urban and non-urban habitats in the two other physiological indicators. The discrepancy between these different measurements of physiological stress highlights the importance of including multiple physiological variables to understand the impact of urbanization on species' physiology. Overall, our findings suggest that adult European blackbirds living in urban and non-urban habitats do not differ in terms of physiological stress at an organismal level. Furthermore, we found large differences among populations on the strength and direction of the urbanization effect, which illustrates the relevance of spatial replication when investigating urban-induced physiological responses

Breeding biology of Western Bonelli's Warblers Phylloscopus bonelli in the Mediterranean region

Capsule: The Western Bonelli's Warbler Phylloscopus bonelli has a nest success of only 25% in the core of its range in western Europe.Aims: To investigate the breeding biology of Western Bonelli's Warbler P. bonelli, focusing on possible altitude effects and potential reproductive problems.Methods: Three Western Bonelli's Warbler populations were monitored during the 2012 and 2013 breeding seasons in the massif range of Sierra Nevada, Spain. We determined all the breeding parameters and calculated daily survival and success rates for each reproductive period.Results: The three studied populations did not differ in any breeding parameters. Altitude showed a positive relationship with clutch size and duration of incubation period, but a negative relationship with nestling tarsus growth and body mass gain. Daily survival rates during incubation and nestling periods were similar to those of common warblers, but the species presented a low breeding success of 25%.Conclusion: The absence of differences among the three populations suggests that the information provided here could be representative of its distribution in the woodlands of Sierra Nevada. The novel and detailed information reported is crucial not only for expanding our understanding of this species but also to draw attention to the potential risks that it might face in the near future, considering the reduction that this species has suffered in Sierra Nevada during recent decades

Predation risk affects egg-ejection but not recognition in blackbirds

Predation and brood parasitism have critical effects on the fitness of animals, but few studies have focused on the potential interactions between these two important selective forces. For instance, the egg-rejection process, one of the most important defensive responses of hosts against brood parasites, may be affected by variation in predation risk, which might divert their focus from the nest (present reproduction) to self-maintenance (future reproduction). In this study we explore the effect of predation risk on the first two stages of the egg-rejection process (judgment and decision) and if this potential effect changes according to the target of predation (adults vs offspring). To do so, we experimentally parasitized nests of common blackbirds (Turdus merula) with mimetic model eggs simultaneously exposing them to different predation-risk situations: adult predator, egg predator, and control. We found that predation risk did not affect egg recognition. Nevertheless, blackbirds exposed to the adult predation risk showed a significant reduction in the ejection rate, particularly at the end of the breeding season. We discuss our results in relation to the egg-rejection process and life-history theory. Our findings suggest that a predation risk directed to adults of parasitized nests, but not to their offspring, can play an important role in the blackbirds' decision-making influencing the ejection of parasitic eggs, consequently affecting the outcome of the evolutionary relationship between brood parasites and their hosts. Significance statement Brood parasitism and predation are two important selective forces in nature, which play a crucial role in the evolutionary process in birds. Despite this, few studies have explored the possible relationships between these two selective pressures. In particular, the possibility that predation risk affects host defenses against brood parasites has usually been neglected. Predation risk could influence the egg-rejection process, which is the main defensive measure adopted by hosts once they have been parasitized. In this study we showed that predation risk seems to modulate host defenses against brood parasites in common blackbirds, but depending on the threat posed by predators. In particular, adult predation risk affects the second stage of the egg-rejection process reducing the ejection rate of parasitic eggs. Our results open a new line of research in brood parasitism studies, demonstrating that external stimuli to brood parasite-host systems can influence egg-rejection decisions

Hormonal responses to non-mimetic eggs: Is brood parasitism a physiological stressor during incubation?

Many host species have evolved sophisticated defences to mitigate the high fitness costs imposed by brood parasitism. Even though the physiological mechanisms behind such defences can offer important insights into the evolutionary relationship between brood parasites and hosts, they have received little attention so fat Hormones play a critical role in the regulation of bird reproduction, which make them a key element when investigating the physiological effects of brood parasitism on hosts. Here, we experimentally parasitized Eurasian blackbird (Turdus merula) nests with non-mimetic eggs to study its impact on the hormonal levels (corticosterone and prolactin) of females during incubation, as well as the magnitude of the response to the standardized stress protocol in parasitized and non-parasitized individuals. Parasitized females had higher baseline corticosterone levels and showed a poorer body condition than non-parasitized birds, while we found no differences for prolactin levels. Both parasitized and non-parasitized females responded to the standardized-stress protocol with a significant increase in corticosterone levels. However, the decrease in prolactin after the standardized stress protocol was significantly more pronounced in parasitized individuals. Our results suggest that the presence of a non-mimetic parasitic egg involves a stressful situation for hosts, negatively affecting the physical state of parasitized females. Unaffected prolactin levels of parasitized individuals could explain the absence of nest desertion found in this species in response to parasitism. Finally, both hormones were not correlated in blackbirds, confirming that their combined study provides valuable pieces of information on the endocrine mechanisms underlying behavioural responses in animals, including hosts of brood parasites. Significance statement Physiological mechanisms behind avian brood parasitism remain unclear. In this study, we assessed the effect of experimental parasitism on the hormonal profiles of hosts. We found that the presence of a non-mimetic egg in the nest modified baseline corticosterone levels, but not prolactin levels, of parasitized females and negatively impacted their body condition. Moreover, experimental parasitism affected the prolactin response to stress. These results expand previous information on the endocrine consequences of brood parasitism at other stages of the breeding cycle (nestling and fledgling stage) and might shed light on the hormonal mechanisms that underlie the host response against parasitic eggs

Immunological changes in nestlings growing under predation risk

Predation is one of the most relevant selective forces in nature. However, the physiological mechanisms behind anti-predator strategies have been overlooked, despite their importance to understand predator-prey interactions. In this context, the immune system could be especially revealing due to its relationship with other critical functions and its ability to enhance prey's probabilities of survival to a predator's attack. Developing organisms (e.g. nestlings) are excellent models to study this topic because they suffer a high predation pressure while undergoing the majority of their development, which maximizes potential trade-offs between immunity and other biological functions. Using common blackbirds Turdus merula as model species, we experimentally investigated whether an elevated nest predation risk during the nestling period affects nestlings' immunity and its possible interactions with developmental conditions (i.e. body condition and growth). Experimental nestlings modified some components of their immunity, but only when considering body condition and growth rate, indicating a multifaceted immunological response to predation risk and an important mediator role of nestlings' developmental conditions. Predation risk induced a suppression of IgY but an increase in lymphocytes in nestlings with poor body condition. In addition, experimental but not control nestlings showed a negative correlation between growth and heterophils, demonstrating that nest predation risk can affect the interaction between growth and immunity. This study highlights the importance of immunity in anti-predator response in nestlings and shows the relevance of including physiological components to the study of predation risk

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