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 eVect of multiple infections by blood parasites on plumage colour. By comparing the yellow colour in the breast plumCommunicated by Heli Siitari. age of blue tits, Cyanistes caeruleus, between birds infected by diVerent 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 Wnd any signiWcant association between colour and body condition or immunoglobulin levels. In addition, we found a positive signiWcant 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 eVects of parasitism on this character.

Avian Plasmodium and Haemoproteus parasites are easily detected by DNA analyses of infected samples but only correctly assigned to each genus by sequencing and use of a phylogenetic approach. Here, we present a restriction site to differentiate between both parasite genera avoiding the use of those analyses. Alignments of 820 sequences currently listed in GenBank encoding a particular cytochrome B region of avian Plasmodium and Haemoproteus show a shared restriction site for both genera using the endonuclease Hpy CH4III. An additional restriction site is present in Plasmodium sequences that would initially allow differentiation of both genera by differential migration of digested products on gels. Overall 9 out of 326 sequences containing both potential restriction sites do not fit to the general rule. We used this differentiation of parasite genera based on Hpy CH4III restriction sites to evaluate the efficacy of 2 sets of general primers in detecting mixed infections. To do so, we used samples from hosts infected by parasites of both genera. The use of general primers was only able to detect 25% or less of the mixed infections. Therefore, parasite DNA amplification using general primers to determine the species composition of haemosporidian infections in individual hosts is not recommended. Specific primers for each species and study area should be designed until a new method can efficiently discriminate both parasites.

There are often marked differences in the incidence of nest-dwelling ectoparasite species on different coexisting and similar avian host species. This has been shown especially for fleas (Siphonaptera), larvae of flies (Diptera), and mites (Acarina) in nests of tits and flycatchers breeding in nest-boxes in close proximity to each other. One of the possible reasons for these differences is the marked differences in nest composition between avian species. We show here differences in ectoparasite presence and nest composition for blue tits (Cyanistes caeruleus) and pied flycatchers (Ficedula hypoleuca) coexisting in oak forests in central Spain. There also may be intraspecific differences in local preferences for nest-building materials according to availability of plant materials, which could be due to arthropod repellent properties of different potential nest components. We show here a local difference in nest composition between 2 pied flycatcher populations in relation to availability of a preferred material associated with higher ectoparasite presence in the locality without the preferred nest material. We test the hypotheses that differential incidence of ectoparasites at the interspecific and intraspecific levels may depend on nest composition. We conducted a nest exchange experiment in one study locality in 2007 placing in nest-boxes occupied by flycatchers 1) nests constructed by blue tits in 2006 in another locality and removed before use and stored frozen, 2) nests constructed by flycatchers in 2006 in another locality with different nest composition and removed before use and stored frozen, and 3) nests constructed by other flycatcher pairs in the same study locality in 2007. Another group of flycatcher nests was kept as pure control, while a few blue tit nests constructed in 2007 and taken over by flycatchers were used for comparison with nests of treatment 1. No effect on presence of mites and blowflies of either nest-constructing species or locality of construction was observed for pied flycatcher nests, and for fleas only an effect of locality but not of nest constructing species was detected. On the other hand, presence of mites and blowflies differed between nests constructed by blue tits and occupied by either blue tits or pied flycatchers. Nest composition does not explain the differential incidence of nest-dwelling ectoparasites on coexisting avian host species.

Environmental factors may strongly affect avian‐biting fly interactions in different ways because insects are heterothermic organisms that depend greatly on environmental variables to activate their metabolism and behaviour. We studied the effects of weather on both blackfly (Simuliidae) and biting midge Culicoides (Ceratopogonidae) abundance in nests of three passerine species: blue tits Cyanistes caeruleus, great tits Parus major and pied flycatchers Ficedula hypoleuca, breeding in the same area. We controlled for different host‐related factors (hatching date, brood size and host species). Blackfly abundance was negatively related to minimum temperature. In addition, blackfly and biting midge abundances were negatively affected by wind speed measured at 07.00 h, but blackfly abundance was positively associated to wind speed at 18.00 h. We found higher blackfly and biting midge abundances in nests with larger broods breeding later in the season, and significantly higher biting midge abundance in pied flycatcher nests as compared to tit nests. These results represent, to our knowledge, the first report of both environmental and host‐related effects on haematophagous fly abundance in the nests of wild hole‐nesting birds.

Some avian species incorporate aromatic plants to their nests. The "nest protection hypothesis", which posits that volatile secondary compounds contained in these plants may have antiparasite properties, has not received full support. All previous tests of this hypothesis have only considered effects on nestdwelling ectoparasites, but not on blood-sucking flies. The "drug hypothesis" posits that aromatic plants may stimulate nestling immune system, development, or condition. We tested these hypotheses experimentally in wild blue tits Cyanistes caeruleus, a species that adds aromatic plants to their nests. We supplemented aromatic plants to half of a sample of nests, while adding grass to the other half of nests. We quantified abundance of two groups of blood-sucking flies (blackflies and biting midges) at two different stages of the reproductive period, and abundance of three nest-dwelling ectoparasites (fleas, mites, and blowflies). Experimental supplementation of aromatic plants reduced abundance of fleas only in nests of yearling females and not in nests of older females. Blackflies and biting midges were both more numerous in nests of yearling females than in nests of older females. Mass of aromatic plants added by females was negatively related with abundance of fleas in control nests but not in experimental nests supplied with aromatic plants. Mass of plants added by females was also positively related with abundance of blackflies during the nestling stage. Finally, aromatic plants did not affect nestling growth or immune responses. We conclude that several factors such as female experience and their ability to add plants to the nest interact to explain effects of aromatic plants on different parasites. .

Previous studies have found strong relationships between calcium availability and eggshell pigmentation in the Great Tit (Parus major). According to the ''structural function hypothesis'', protoporphyrins, the pigments responsible for reddish spots on speckled eggs, are deposited in those areas of the shell where calcium deposition is less intense. In the study reported here, which was carried out in three Blue Tit (Cyanistes caeruleus) populations in central Spain, we provide partial experimental support for the association between protoporphyrin eggshell pigmentation and shell thinning. Contrary to our expectations, we did not observe a decrease in the size and intensity of pigment spots for those eggs from calciumsupplemented nests. However, we did find that the provision of calcium-rich material during the egg-laying period led to a more wide distribution of pigment spots and reduced the proportion of eggs with defective shells (deviant pigmentation, dull and rough surface). When only the supplemented nests were considered in the analysis, within the same clutch we also detected differences in the spotting distribution between those eggs laid by female Blue Tits observed to have consumed calcium consumption on the day prior to laying and those observed not to have consumed calcium on the day prior to laying. Clutch size was not affected by the calcium supplementation. Female Blue Tits experimentally supplied with calcium-rich material had a shorter incubation period than control females, and they laid eggs with thicker shells. Eggshell thickness markedly affects the probability of hatching and could explain the lower proportion of unhatched late eggs found in supplemented nests in comparison with control ones. This study highlights the role of calcium in eggshell maculation and its effects on breeding performance of small passerines. We found the spotting distribution to be a good predictor of calcium deficiency. However, our results provide only mixed support for the ''structural function hypothesis'': in our study populations, the thickness of the eggshell was intimately associated with calcium availability, but the relationship between calcium and protoporphyrin deposition remains far from clear.

The first step in the establishment of a hostebiting fly relationship is host location. While a number of studies highlight the role of host emitted products as important cues affecting host location by biting flies, the role of host temperature is far from clear. We investigated the role of different nest microclimatic variables affecting the interaction between pied flycatchers and two biting flies: black flies and biting midges. Biting midge abundances increased with temperature inside the nest, supporting the potential importance of nest temperature as a cue used by insects to localize their hosts. The possibility that biting fly infestations were associated to ecological conditions in the vicinity of the nests is also discussed. Furthermore, we found a negative association between nestling weight (including tarsus length as a covariate in the analyses) and the interaction between the abundance of biting midges and the presence/ absence of black flies in nests. The potential negative effect of these ectoparasites on nestling weight (condition index) and potential differences in the bird phenotypic/genetic quality associated with nest site choice and parasite infestations are considered..

The effect of insect vectors on avian exposure to infection by pathogens remains poorly studied. Here, we used an insect repellent treatment to reduce the number of blood-sucking flying insects in blue tit Cyanistes caeruleus nests and examined its effect on nestling health status measured as body mass, nestling phytohaemagglutinin (PHA) response and blood parasite prevalence. We found that (i) the insect repellent treatment significantly reduced the number of blood-sucking flying insects in nests and (ii) the number of blood-sucking flying insects had a significant effect on the prevalence of the blood parasite Trypanosoma independently of the treatment. In addition, we found support for an adverse effect of parasite infections on nestling PHA response. Nestlings infected by Trypanosoma mounted a weaker response against PHA than non-parasitized ones. In addition, the number of blowflies in the nest was negatively associated with nestling PHA response. Overall, we found support for the hypothesis that blood-sucking flying insects attacking nestlings increase their exposure to parasite infections. Our results further substantiate the adverse effect of parasites on nestling condition.