Josué Martínez de la Puente

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.

On the specificity of avian blood parasites: revealing specific and generalist relationships between haemosporidians and biting midges

The study of host–parasite relationships involving vector‐borne parasites requires understanding interactions between parasites and vectors. The capacity of haemosporidians to infect insects has clear evolutionary consequences for the transmission of diseases. Here, we investigated (i) the associations between blood parasites, biting midges and birds and (ii) the potential specificity between biting midge and haemosporidian haplotypes. A total of 629 parous biting midges Culicoides and 224 wild birds (belonging to seven species) from a locality of central Spain were individually examined for the presence of Haemoproteus and Plasmodium parasites by sequencing a fragment of cytochrome B. Biting midges were identified morphologically and characterized on the basis of a fragment of the cytochrome c oxidase (COI) gene. Overall, 12 Haemoproteus and three Plasmodium haplotypes were isolated and sequenced. Among them, 10 haplotypes were exclusively isolated from biting midges, three haplotypes only from birds and two haplotypes from both biting midges and birds. Biting midge haplotypes showed both specific and generalist relationships with Haemoproteus haplotypes but only generalist relationships with Plasmodium haplotypes. Several C. festivipennis and C. kibunesis haplotypes established significant coevolutionary links with Haemoproteus haplotypes. These results shed light on the specificity of interactions between vectors and blood parasites.

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.

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.

Nest-Dwelling Ectoparasites of Two Sympatric Hole-Nesting Passerines in Relation to Nest Composition: An Experimental Study

Abstract: 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. Nomenclature: Moss, 1978; Cramp, Perrins & Brooks, 1993.

Avian Plasmodium in Culex and Ochlerotatus Mosquitoes from Southern Spain: Effects of Season and Host-Feeding Source on Parasite Dynamics

Haemosporidians, a group of vector-borne parasites that include Plasmodium, infect vertebrates including birds. Although mosquitoes are crucial elements in the transmission of avian malaria parasites, little is known of their ecology as vectors. We examined the presence of Plasmodium and Haemoproteus lineages in five mosquito species belonging to the genera Culex and Ochlerotatus to test for the effect of vector species, season and host-feeding source on the transmission dynamics of these pathogens. We analyzed 166 blood-fed individually and 5,579 unfed mosquitoes (grouped in 197 pools) from a locality in southern Spain. In all, 15 Plasmodium and two Haemoproteus lineages were identified on the basis of a fragment of 478 bp of the mitochondrial cytochrome b gene. Infection prevalence of blood parasites in unfed mosquitoes varied between species (range: 0–3.2%) and seasons. The feeding source was identified in 91 mosquitoes where 78% were identified as bird. We found that i) several Plasmodium lineages are shared among different Culex species and one Plasmodium lineage is shared between Culex and Ochlerotatus genera; ii) mosquitoes harboured Haemoproteus parasites; iii) pools of unfed females of mostly ornithophilic Culex species had a higher Plasmodium prevalence than the only mammophylic Culex species studied. However, the mammophylic Ochlerotatus caspius had in pool samples the greatest Plasmodium prevalence. This relative high prevalence may be determined by inter-specific differences in vector survival, susceptibility to infection but also the possibility that this species feeds on birds more frequently than previously thought. Finally, iv) infection rate of mosquitoes varies between seasons and reaches its maximum prevalence during autumn and minimum prevalence in spring.

Effect of blood meal digestion and DNA extraction protocol on the success of blood meal source determination in the malaria vector Anopheles atroparvus

BackgroundHost identification is an essential step in studies on the transmission dynamics of vector-borne disease. Nowadays, molecular tools allow the identification of vertebrate hosts to the species level. However, the proportion of successful identifications is variable and may be affected by the quality of the samples and the laboratory protocols. Here, the effect of two of these factors, namely the digestion status of mosquito blood meal and the DNA extraction procedure, on the success of host identification by amplification and sequencing of a fragment of the cytochrome oxidase 1 gene were tested.MethodsMosquitoes collected both outdoors and indoors during 2012 in southern Spain were identified to species level and their blood meal digestion status recorded using the Sella score, a visual estimation of the digestion status of mosquito blood meals. Each mosquito was assigned randomly to one of two DNA extraction procedures: the quick and cheap HotSHOT procedure or the QIAGEN DNeasy Blood and Tissue® kit and their hosts identified by a molecular method.ResultsThree hundred and forty-seven blood-fed mosquitoes belonging to Anopheles atroparvus (n=171), Culex perexiguus (n=84), Culex pipiens (n=43), Culex theileri (n=39), Culex modestus (n=5), Ochlerotatus caspius (n=4), Culiseta sp. (n=1) were included in this study. Overall, hosts were identified from 234 blood meals compromising at least 25 species including mammals, birds and a single reptile. The success of host identification was lower in mosquitoes with an advanced stage of blood meal digestion and for blood meals extracted using the HotSHOT procedure.ConclusionsThe success of host identification decreases with the advanced stage of mosquito blood meal digestion, from 84.5% for recent blood meals to 25.0% for more digested ones. Using the QIAGEN kit, the identification success improved by 17.6%, with larger increases at more advanced stages of blood meal digestion. Availability of blood-fed females used to be very limited for studies of vector ecology, and these results may help to increase the efficiency of blood meal analyses. In addition, results obtained in this study clearly support that the potential malaria vector An. atroparvus feeds on animals located outdoors but use human-made shelters for resting after feeding.

Nest Construction Rate and Stress in Female Pied Flycatchers Ficedula hypoleuca

Abstract. Nest building effort has received scant attention in the literature although it may involve costs which can be detected as physiological stress. We prolonged nest construction effort in a population of Spanish Pied Flycatchers by removing nests from nest-boxes and forcing females to build a second nest. In comparison with control nests, the experimental females had to work for longer periods and accumulate more nest material, but nest construction rates (g of nest material per day of construction) were not affected. There was a positive association of clutch mass with nest construction rate. To measure physiological stress, we captured females shortly after laying to obtain blood samples for heat-shock protein quantification. Heat-shock proteins quantify stress at cell level. The level of HSP60 in peripheral blood was positively associated with total nest construction rate (including second nests for experimental females), but not with laying date, clutch mass or experimental treatment. A third of the variation in the HSP60 level was explained by the nest construction rate. Fast nest builders are physiologically stressed, suggesting that the nest construction rate may constitute an index of female physiological performance.

Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus

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 calcium-supplemented 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.

Extraordinary MHC class II B diversity in a non-passerine, wild bird: the Eurasian Coot Fulica atra (Aves: Rallidae).

The major histocompatibility complex (MHC) hosts the most polymorphic genes ever described in vertebrates. The MHC triggers the adaptive branch of the immune response, and its extraordinary variability is considered an evolutionary consequence of pathogen pressure. The last few years have witnessed the characterization of the MHC multigene family in a large diversity of bird species, unraveling important differences in its polymorphism, complexity, and evolution. Here, we characterize the first MHC class II B sequences isolated from a Rallidae species, the Eurasian Coot Fulica atra. A next-generation sequencing approach revealed up to 265 alleles that translated into 251 different amino acid sequences (β chain, exon 2) in 902 individuals. Bayesian inference identified up to 19 codons within the presumptive peptide-binding region showing pervasive evidence of positive, diversifying selection. Our analyses also detected a significant excess of high-frequency segregating sites (average Tajimas D = 2.36, P < 0.05), indicative of balancing selection. We found one to six different alleles per individual, consistent with the occurrence of at least three MHC class II B gene duplicates. However, the genotypes comprised of three alleles were by far the most abundant in the population investigated (49.4%), followed by those with two (29.6%) and four (17.5%) alleles. We suggest that these proportions are in agreement with the segregation of MHC haplotypes differing in gene copy number. The most widespread segregating haplotypes, according to our findings, would contain one single gene or two genes. The MHC class II of the Eurasian Coot is a valuable system to investigate the evolutionary implications of gene copy variation and extensive variability, the greatest ever found, to the best of our knowledge, in a wild population of a non-passerine bird.