Assessing behavioral sex differences to chemical cues of predation risk while provisioning nestlings in a hole-nesting bird.

Birds can assess nest predation risk and adjust their parental activity accordingly. Risk taking behavior should be related to investment in reproduction as well as to confidence in parenthood that often differ between sexes. In those cases, sexual differences in risk taking behavior may be expected. For example, in blue tits, females invest more time and energy than males in nest-building, egg laying and incubation. Furthermore, confidence in parenthood is supposed to be higher for females, as extrapair paternity is common in this species. Therefore, the reproductive value of nestlings may be higher for females than for males and the former may assume greater risks to ensure nestling growth and maximize their reproductive success. We examined potential sexual differences in the risk assumed by parents in relation to perceived risk of predation inside the nest cavity, where predation risk perception may be higher. We increased perceived predation risk by adding predator chemical cues inside blue tit (Cyanistes caeruleus) nest-boxes, and we tested whether female and male parents differed in the risk assumed when taking care of nestlings. Females and males did not differ in the risk assumed in response to perceived predation risk. However, females reduced time devoted to nest sanitation activities when predator chemical cues were detected inside the nest-box, likely as an anti-predatory strategy to minimize their own risk of predation. Therefore, these results add to the evidence that birds can detect chemical cues of predators inside the nest cavity and suggest that the behavioral response to an increase in risk of predation perceived through olfactory cues is not sex-dependent in blue tit.

Microbially Mediated Chemical Ecology of Animals: A Review of Its Role in Conspecific Communication, Parasitism and Predation.

Microbial symbionts are nowadays considered of pivotal importance for animal life. Among the many processes where microorganisms are involved, an emerging research avenue focuses on their major role in driving the evolution of chemical communication in their hosts. Volatiles of bacterial origin may underlie chemical communication and the transfer of social information through signals, as well as inadvertent social information. We reviewed the role of microorganisms in animal communication between conspecifics, and, because the microbiome may cause beneficial as well as deleterious effects on their animal hosts, we also reviewed its role in determining the outcome of the interactions with parasites and predators. Finally, we paid special attention to the hypothetical role of predation and parasitism in driving the evolution of the animal microbiome. We highlighted the novelty of the theoretical framework derived from considering the microbiota of animals in scenarios of communication, parasitism, and predation. We aimed to encourage research in these areas, suggesting key predictions that need to be tested to better understand what is one of the main roles of bacteria in animal biology.

Antimicrobial activity of nest-lining feathers is enhanced by breeding activity in avian nests.

The use of feathers as nest material has been proposed as a kind of self-medication strategy because antimicrobial-producing microorganisms living on feathers may defend offspring against pathogenic infections. In this case, it is expected that density of antimicrobial-producing bacteria, and their antimicrobial effects, are higher in feathers that line the nests than in eggshells. Moreover, we know that feather pigmentation and breeding activity may influence density and antimicrobial production of bacteria. To test these predictions, we analyzed bacterial densities and antimicrobial activity of bacterial colonies isolated from bird eggshells and nest-lining feathers against bacterial strains comprising potential pathogens. Samples were collected from spotless starling (Sturnus unicolor) nests, and from artificial nests to isolate the effects of breeding activity on bacterial communities. The composition of feathers lining the nests was experimentally manipulated to create groups of nests with pigmented feathers, with unpigmented feathers, with both types of feathers or without feathers. Although we did not detect an effect of experimental feather treatments, we found that bacterial colonies isolated from feathers were more active against the tested bacterial strains than those isolated from eggshells. Moreover, bacterial density on feathers, keratinolytic bacteria on eggshells and antimicrobial activity of colonies isolated were higher in starling nests than in artificial nests. These results suggest that antimicrobial activity of bacteria growing on nest-lining feathers would be one of the mechanisms explaining the previously detected antimicrobial effects of this material in avian nests, and that breeding activity results in nest bacterial communities with higher antimicrobial activity.

Wild great and blue tits do not avoid chemical cues of predators when selecting cavities for roosting.

Small birds use cavities for roosting to decrease the thermoregulatory costs during the winter nights. The ability of birds to detect and escape from an approaching predator is impaired during roosting and thus the selection of such cavities should take into account the risk that a predator will find the cavity. Previous evidence suggested that birds in captivity are able to detect predator scent and avoid roosting in nest-boxes containing such predator chemical cues. Here, we tested whether birds also show this avoidance response under natural conditions. We performed three studies in three populations of blue and great tits. We added predator scent, a pungency scent or an odourless control to nest-boxes and compared the use of these nest-boxes for roosting. We found no differences between the scent treatments in the use of nest-boxes. Therefore, chemical cues indicating the potential presence of a predator are not enough for birds to avoid roosting in nest-boxes under natural conditions.

Nest Material Shapes Eggs Bacterial Environment.

Selective pressures imposed by pathogenic microorganisms to embryos have selected in hosts for a battery of antimicrobial lines of defenses that includes physical and chemical barriers. Due to the antimicrobial properties of volatile compounds of green plants and of chemicals of feather degrading bacteria, the use of aromatic plants and feathers for nest building has been suggested as one of these barriers. However, experimental evidence suggesting such effects is scarce in the literature. During two consecutive years, we explored experimentally the effects of these nest materials on loads of different groups of bacteria (mesophilic bacteria, Enterobacteriaceae, Staphylococcus and Enterococcus) of eggshells in nests of spotless starlings (Sturnus unicolor) at the beginning and at the end of the incubation period. This was also explored in artificial nests without incubation activity. We also experimentally increased bacterial density of eggs in natural and artificial nests and explored the effects of nest lining treatments on eggshell bacterial load. Support for the hypothetical antimicrobial function of nest materials was mainly detected for the year and location with larger average values of eggshell bacterial density. The beneficial effects of feathers and plants were more easily detected in artificial nests with no incubation activity, suggesting an active role of incubation against bacterial colonization of eggshells. Pigmented and unpigmented feathers reduced eggshell bacterial load in starling nests and artificial nest boxes. Results from artificial nests allowed us to discuss and discard alternative scenarios explaining the detected association, particularly those related to the possible sexual role of feathers and aromatic plants in starling nests. All these results considered together confirm the antimicrobial functionality mainly of feathers but also of plants used as nest materials, and highlight the importance of temporally and geographically environmental variation associated with risk of bacterial proliferation determining the strength of such effects. Because of costs associated to nest building, birds should adjust nest building effort to expected bacterial environments during incubation, a prediction that should be further explored.

Laying date, incubation and egg breakage as determinants of bacterial load on bird eggshells: experimental evidence.

Exploring factors guiding interactions of bacterial communities with animals has become of primary importance for ecologists and evolutionary biologists during the last years because of their likely central role in the evolution of animal life history traits. We explored the association between laying date and eggshell bacterial load (mesophilic bacteria, Enterobacteriaceae, Staphylococci, and Enterococci) in natural and artificial magpie (Pica pica) nests containing fresh commercial quail (Coturnix coturnix) eggs. We manipulated hygiene conditions by spilling egg contents on magpie and artificial nests and explored experimental effects during the breeding season. Egg breakage is a common outcome of brood parasitism by great spotted cuckoos (Clamator glandarius) on the nests of magpie, one of its main hosts. We found that the treatment increased eggshell bacterial load in artificial nests, but not in magpie nests with incubating females, which suggests that parental activity prevents the proliferation of bacteria on the eggshells in relation to egg breakage. Moreover, laying date was positively related to eggshell bacterial load in active magpie nests, but negatively in artificial nests. The results suggest that variation in parental characteristics of magpies rather than climatic variation during the breeding season explained the detected positive association. Because the eggshell bacterial load is a proxy of hatching success, the detected positive association between eggshell bacterial loads and laying date in natural, but not in artificial nests, suggests that the generalized negative association between laying date and avian breeding success can be, at least partially, explained by differential bacterial effects.

Are female starlings able to recognize the scent of their offspring?

BACKGROUND: Although there is growing evidence that birds may have individual chemical profiles that can function in several social contexts, offspring recognition based on olfactory cues has never been explored. This ability should be more likely evolved in colonial birds and/or species suffering brood parasitism, in which the risk of being engaged in costly misdirected parental care is high. METHODOLOGY/PRINCIPAL FINDINGS: We performed a choice experiment to examine whether females of the spotless starling, Sturnus unicolor, a species that is colonial, and where a fraction of the population is exposed to intraspecific brood parasitism, can discriminate between the scent of their offspring and that of unrelated nestlings. We also explored whether the development of the uropygial gland secretion may play a role in such olfactory discrimination by performing the choice experiments to females rearing nestlings of two different ages, that is, without and with developed uropygial glands. Results showed that female starlings did not preferentially choose the scent of their offspring, independently of whether the gland of nestlings was developed or not. CONCLUSIONS/SIGNIFICANCE: Our results suggest that female starlings do not have or do not show the ability to distinguish their offspring based on olfaction, at least up to 12-14 days of nestling age. Further research is needed to examine whether odour-based discrimination may function when fledgling starlings leave the nest and the risk of costly misidentification is likely to increase.

Manipulating individual decisions and environmental conditions reveal individual quality in decision-making and non-lethal costs of predation risk.

Habitat selection is a crucial decision for any organism. Selecting a high quality site will positively impact survival and reproductive output. Predation risk is an important component of habitat quality that is known to impact reproductive success and individual condition. However, separating the breeding consequences of decision-making of wild animals from individual quality is difficult. Individuals face reproductive decisions that often vary with quality such that low quality individuals invest less. This reduced reproductive performance could appear a cost of increased risk but may simply reflect lower quality. Thus, teasing apart the effects of individual quality and the effect of predation risk is vital to understand the physiological and reproductive costs of predation risk alone on breeding animals. In this study we alter the actual territory location decisions of pied flycatchers by moving active nests relative to breeding sparrowhawks, the main predators of adult flycatchers. We experimentally measure the non-lethal effects of predation on adults and offspring while controlling for effects of parental quality, individual territory choice and initiation of breeding. We found that chicks from high predation risk nests (<50 m of hawk) were significantly smaller than chicks from low risk nests (>200 m from hawk). However, in contrast to correlative results, females in manipulated high risk nests did not suffer decreased body condition or increased stress response (HSP60 and HSP70). Our results suggest that territory location decisions relative to breeding avian predators cause spatial gradients in individual quality. Small adjustments in territory location decisions have crucial consequences and our results confirm non-lethal costs of predation risk that were expressed in terms of smaller offspring produced. However, females did not show costs in physiological condition which suggests that part of the costs incurred by adults exposed to predation risk are quality determined.

Clutch size and egg volume in great tits (Parus major) increase under low intensity electromagnetic fields: a long-term field study.

Exposure to electromagnetic fields (EMFs) can affect a wide range of biological processes, including reproduction, growth and development. Experiments aimed at investigating the biological effects of EMFs, focused on potential harmful effects on humans, have been mostly carried out in vitro or with animal models in laboratory conditions. By contrast, studies performed on wild animals are scarce. The effects of EMFs created by an electric power line on reproductive traits of a wild great tit (Parus major) population were explored by analysing data gathered during nine breeding seasons. EMF exposure significantly increased clutch size (7%) and egg volume (3%), implying a 10% increase in clutch volume. This indicates an increase in reproductive investment from parent birds exposed to EMFs as compared to the adjacent reference area. These results cannot be attributed to habitat or adult quality differences between the exposed and reference group. Nevertheless, no differences in hatching success or final productivity (fledging and reproductive success or nestling body mass) could be detected. Our study clearly shows that EMFs created by power lines can have biological consequences in wild organisms that live intimately with them. To our knowledge, this is the first study showing an increase in clutch size, and one of the few reporting an increase in egg size, associated with EMF exposure. The possible mechanisms by which great tits invest more under EMF exposure are discussed, and future research directions to evaluate the effect of EMFs on avian reproduction in the wild are suggested.

Sex recognition by odour and variation in the uropygial gland secretion in starlings.

1. Although a growing body of evidence supports that olfaction based on chemical compounds emitted by birds may play a role in individual recognition, the possible role of chemical cues in sexual selection of birds has been only preliminarily studied. 2. We investigated for the first time whether a passerine bird, the spotless starling Sturnus unicolor, was able to discriminate the sex of conspecifics by using olfactory cues and whether the size and secretion composition of the uropygial gland convey information on sex, age and reproductive status in this species. 3. We performed a blind choice experiment during mating, and we found that starlings were able to discriminate the sex of conspecifics by using chemical cues alone. Both male and female starlings preferred male scents. Furthermore, the analysis of the chemical composition of the uropygial gland secretion by using gas chromatography-mass spectrometry (GC-MS) revealed differences between sexes, ages and reproductive status. 4. In conclusion, our study reveals for first time that a passerine species can discriminate the sex of conspecifics by relying on chemical cues and suggests that the uropygial gland secretion may potentially function as a chemical signal used in mate choice and/or intrasexual competition in this species.

Nest ectoparasites increase physiological stress in breeding birds: an experiment.

Parasites are undoubtedly a biotic factor that produces stress. Heat shock proteins (HSPs) are important molecules buffering cellular damage under adverse conditions. During the breeding season, blue tit Cyanistes caeruleus (L.) adults are affected by blood parasites, nest-dwelling parasites and biting flies, potentially affecting their HSP-mediated responses. Here, we treated females with primaquine to reduce blood parasites and fumigated nests with permethrin to reduce nest-dwelling parasites to test whether these treatments affect HSP60 level during the breeding season. Medicated females, but not controls, had a significant reduction of the intensity of infection by Haemoproteus spp. blood parasites. However, final intensity of infection did not differ significantly between groups, and we did not find an effect of medication on change in HSP60 level. Fumigation reduced the abundance of nest-dwelling parasites (mites, fleas and blowfly larvae) and engorged biting midges in nests. Females breeding in non-fumigated nests increased HSP60 levels during the season more than those breeding in fumigated nests. Furthermore, the change in HSP60 level was positively correlated with the abundance of biting midges. These results show how infections by nest ectoparasites during the breeding period can increase the level of HSPs and suggest that biting midges impose physiological costs on breeding female blue tits. Although plausible, the alternative that biting midges prefer to feed on more stressed birds is poorly supported by previous studies.

Predator proximity as a stressor in breeding flycatchers: mass loss, stress protein induction, and elevated provisioning.

We investigated the physiological and behavioral consequences for prey breeding at different distances from a nesting predator. In a natural setting, Pied Flycatchers (Ficedula hypoleuca) made territory location decisions relative to established Sparrowhawk (Accipiter nisus) nests. From female flycatchers attending nests at different distances from Sparrowhawk nests, we measured body mass, blood stress protein (HSP60 and HSP70), and plasma immunoglobulin levels at the beginning (initial) and end (final) of the flycatcher breeding cycle, and provisioning rates during the nestling phase. We found that individuals breeding in closer proximity to Sparrowhawk nests, under higher perceived predation risk, showed significantly lower body mass, higher stress protein and immunoglobulin levels, and higher nestling provisioning rates compared to those individuals breeding farther away. Across the range of distances investigated (30-610 m), final stress protein levels decreased linearly with distance, whereas the final measures of the other variables showed unimodal trends, increasing or decreasing until an intermediate distance (approximately 350 m) and reversing the direction of the trend. Within 300 m, however, all measures showed significant linear associations with distance from the Sparrowhawk nest. Body mass and stress protein associations with distance from Sparrowhawk nests were only present during late breeding, and not in early incubation. Spatial proximity to Sparrowhawk nests consistently explained significant variation in both physiological and behavioral measures, despite the multitude of potential sources of variation for these measures in a natural setting. This suggests that predictable spatial patterns in these measures in avian communities are determined by the sites of breeding predators. Habitat selection decisions of migrant prey that vary only slightly spatially have consequences even at the cellular level, which plausibly have impacts on individual survival. In addition, this study suggests that predation risk is an important factor affecting physiological condition of prey, including stress protein induction in terrestrial vertebrates.

The blood parasite Haemoproteus reduces survival in a wild bird: a medication experiment.

While avian chronic haemoparasite infections induce reproductive costs, infection has not previously been shown to affect survival. Here, we experimentally reduced, through medication, the intensity of infection by Haemoproteus parasites in wild-breeding female blue tits Cyanistes caeruleus. However, this treatment did not reduce the intensity of infection in males or the intensity of infection by Leucocytozoon. Medicated females, but not males, showed increased local survival until the next breeding season compared with control birds. To our knowledge, this is the first empirical evidence showing long-term direct survival costs of chronic Haemoproteus infections in wild birds.

Carotenoid-based plumage colouration is associated with blood parasite richness and stress protein levels in blue tits (Cyanistes caeruleus).

Carotenoids are molecules that birds are not able to synthesize and therefore, must be acquired through their diet. These pigments, besides their function of giving birds red and yellow colouration when deposited in feathers, seem to act as immune-stimulators and antioxidants in the organism. Hence, only the healthiest individuals would be able to express carotenoid-based ornaments to a larger extent without compromising the physiological functions of carotenoids. Various studies have reported that birds infected by parasites are paler than those uninfected, but, to our knowledge, none of them has assessed the possible effect of multiple infections by blood parasites on plumage colour. By comparing the yellow colour in the breast plumage of blue tits, Cyanistes caeruleus, between birds infected by different numbers of blood parasite genera, we found that those birds infected by more than one genus were paler than those parasitized just by one. In addition, we examined the potential role of carotenoid-based plumage colour of blue tits as a long-term indicator of other parameters of health status, such as body condition and immunoglobulin and heat shock protein (HSP) levels. Our results indicate that more brightly coloured birds had lower HSP70 levels than paler birds, but we did not find any significant association between colour and body condition or immunoglobulin levels. In addition, we found a positive significant association between Haemoproteus density of infection and HSP60 levels. Overall, these results support the role of carotenoid-based colours as indicators of health status in blue tits and show detrimental effects of parasitism on this character.

Pre-laying nutrition mediates maternal effects on offspring immune capacity and growth in the pied flycatcher.

We have aimed at detecting prelaying maternal effects on nestling antibody defences and growth through experimental food supplementation of female pied flycatchers Ficedula hypoleuca and subsequent exchange of whole clutches with control nests. The levels of immunoglobulins and the mass and size of chicks at 12 days of age were ascertained. This is the first study controlling for maternal incubation effects by exchanging eggs rather than nestlings. Our prediction is that the females' availability of pre-laying nutritional resources affects offspring immune capacity and growth through maternal effects in the eggs when conditions during incubation and rearing are controlled for. Nestling immunoglobulin Y (IgY) levels and tarsus length were indeed positively associated with maternal food supplementation at laying. The only rearing environmental effect detected was that of mite infestation which affected both IgY levels and growth of nestlings. Nestlings that recruited to the population in the subsequent 2 years had higher IgY levels than those that did not. Maternal adaptations for allocating resources to eggs play an important role in moulding offspring phenotypes and may affect their survival prospects.

Determinants of abundance and effects of blood-sucking flying insects in the nest of a hole-nesting bird.

Compared to non-flying nest-dwelling ectoparasites, the biology of most species of flying ectoparasites and its potential impact on avian hosts is poorly known and rarely, if ever, reported. In this study we explore for the first time the factors that may affect biting midge (Diptera: Ceratopogonidae) and black fly (Diptera: Simuliidae) abundances in the nest cavity of a bird, the hole-nesting blue tit Cyanistes caeruleus, and report their effects on adults and nestlings during reproduction. The abundance of biting midges was positively associated with nest mass, parental provisioning effort and abundance of blowflies and black flies, while negatively associated with nestling condition. Furthermore, a medication treatment to reduce blood parasitaemias in adult birds revealed that biting midges were more abundant in nests of females whose blood parasitaemias were experimentally reduced. This finding would be in accordance with these insect vectors attacking preferentially uninfected or less infected hosts to increase their own survival. The abundance of black flies in the population was lower than that of biting midges and increased in nests with later hatching dates. No significant effect of black fly abundance on adult or nestling condition was detected. Blood-sucking flying insects may impose specific, particular selection pressures on their hosts and more research is needed to better understand these host-parasite associations.

Corticosterone metabolites in blue tit and pied flycatcher droppings: effects of brood size, ectoparasites and temperature.

The hypothalamic-pituitary-adrenal (HPA) axis of birds induces the secretion of corticosterone (CORT) as a response to different ecological variables. In this study we tested experimentally if manipulations of brood size or ectoparasitism led to subsequent differences in the concentration of excreted CORT metabolites of adult and nestling blue tits (Cyanistes caeruleus). No significant effect of the manipulation of brood size was detected in adults or nestlings. No significant effect of ectoparasitism was detected in males or nestlings, although females from uninfested nests showed lower concentrations of excreted CORT metabolites. In addition, we analysed if weather conditions had an influence on the concentration of excreted CORT metabolites of blue tits and pied flycatchers (Ficedula hypoleuca) breeding in the same forest. We detected no effect of weather conditions on adults, but nestlings of both species showed a negative correlation between their excreted CORT metabolites and the average mean temperatures they were subjected to during their growth. This effect was not found in blue tits in a colder year, suggesting that the sensitivity of the HPA axis to ambient temperature may be subjected to interannual variation. Moreover, we found a positive effect of the maximum temperature on the day of sampling on the concentration of CORT metabolites of blue tit nestlings in one of the years. These results suggest that weather conditions may act as environmental stressors to which the HPA axis of blue tit and pied flycatcher nestlings may be sensitive.

Effects of parental effort on blood stress protein HSP60 and immunoglobulins in female blue tits: a brood size manipulation experiment.

1. Physiological stress in animals may impose a limit for investment in current reproduction in the wild. A brood manipulation experiment was conducted in a population of blue tits Cyanistes caeruleus to study the effect of parental effort on changes in two types of proteins related with stress: the blood stress protein HSP60 and the plasma immunoglobulins. 2. Levels of HSP60 were reduced across the experiment for females attending reduced broods, and females attending enlarged broods experienced a reduction of immunoglobulin levels. Moreover, the overall changes in the levels of both proteins were positively related. 3. By controlling for the change in immunoglobulin levels we found an increase in HSP60 for females in the enlarged treatment, presumably to offset deleterious effects derived from increased effort. 4. Maternal effort was able to partially compensate for the effect of treatment as nestlings did not differ in mass and levels of immunoglobulins and HSP60 among treatments. 5. Physiological stress as reflected in stress and immunoglobulin proteins may limit maternal effort in breeding blue tits.

Molecular characterization of the 18S rDNA gene of an avian Hepatozoon reveals that it is closely related to Lankesterella.

As a part of intensive study of blood parasite infections in a population of the passerine bird blue tit (Cyanistes caeruleus, Paridae), we detected a parasite species that, based on its morphological similarity, was tentatively identified as Hepatozoon parus, the only species of this parasite genus described from birds of this family. However, morphological measurements show that H. parus is slightly larger than the parasite detected in our population. A molecular characterization of the parasite species was conducted by amplification of the 18S rDNA gene, using primers that were reported previously to amplify in Hepatozoon sp. of water pythons. Additional primers were developed based on the new sequence obtained. The 1,484-bp fragment amplified reveals that the parasite from our bird population is more closely related to Lankesterella minima than to Hepatozoon species from other vertebrates according to analysis using the BLAST comparison method (93% identity). In addition, phylogenetic analyses using parsimony and Kimura procedures unequivocally related the parasite species detected by PCR with L. minima. The bootstrap values obtained were 97% and 100%, respectively. These results imply that this parasite is a species of a lankesterellid instead of Hepatozoon.

Double gametocyte infections in apicomplexan parasites of birds and reptiles.

The simultaneous occurrence of male and female gametocytes inside a single host blood cell has been suggested to enhance apicomplexan transmission ["double gametocyte infection (DGI) hypothesis"]. We did a bibliographic search and a direct screen of blood smears from wild birds and reptiles to answer, for the first time, how common are these infections in the wild. Taking these two approaches together, we report here cases of DGIs in Plasmodium, Haemoproteus, Leucocytozoon and Hepatozoon, and cases of male-female DGIs in Haemoproteus of birds and reptiles and in Leucocytozoon of birds. Thus, we suggest that DGIs and male female DGIs are more widespread than previously thought, opening a new research avenue on apicomplexan transmission.