Viability Is Associated with Melanin-Based Coloration in the Barn Swallow (Hirundo rustica)

Pigmentation of body surface in animals can have multiple determinants and accomplish diverse functions. Eumelanin and pheomelanin are the main animal pigments, being responsible of yellow, brownish-red and black hues, and have partly common biosynthetic pathways. Many populations of vertebrates show individual variation in melanism, putatively with large heritable component. Genes responsible for eu- or pheomelanogenesis have pleiotropic but contrasting effects on life-history traits, explaining the patterns of covariation observed between melanization and physiology (e.g. immunity and stress response), sexual behavior and other characters in diverse taxa. Yet, very few studies in the wild have investigated if eu- and pheomelanization predict major fitness traits like viability or fecundity. In this correlative study, by contrasting adult barn swallows (Hirundo rustica) matched for age, sex, breeding site, and year and date of sampling, we show that males but not females that survived until the next year had paler, relatively more eu- than pheomelanic pigmentation of ventral body feathers. Better performance of individuals that allocate relatively more to eumelanogenesis was expected based on previous evidence on covariation between eumelanic pigmentation and specific traits related to immunity and susceptibility to stress. However, together with the evidence of no covariation between viability and melanization among females, this finding raises the question of the mechanisms that maintain variation in genes for melanogenesis. We discuss the possibility that eu- and pheomelanization are under contrasting viability and sexual selection, as suggested by larger breeding and sperm competition success of darker males from other barn swallow subspecies.     

Sexual Dimorphism in Melanin Pigmentation,Feather Coloration and Its Heritability in the Barn Swallow (Hirundo rustica)

Melanin is the main pigment in animal coloration and considerable variation in the concentrations of the two melanin forms (pheo- and eumlanin) in pigmented tissues exists among populations and individuals. Melanin-based coloration is receiving increasing attention particularly in socio-sexual communication contexts because the melanocortin system has been hypothesized to provide a mechanistic basis for covariation between coloration and fitness traits. However, with few notable exceptions, little detailed information is available on inter-individual and inter-population variation in melanin pigmentation and on its environmental, genetic and ontogenetic components. Here, we investigate melanin-based coloration in an Italian population of a passerine bird, the barn swallow (Hirundo rustica rustica), its sex- and age-related variation, and heritability. The concentrations of eu- and pheomelanin in the throat (brown) and belly (white-to-brownish) feathers differed between sexes but not according to age. The relative concentration of either melanin (Pheo:Eu) differed between sexes in throat but not in belly feathers, and the concentrations in males compared to females were larger in belly than in throat feathers. There were weak correlations between the concentrations of melanins within as well as among plumage regions. Coloration of belly feathers was predicted by the concentration of both melanins whereas coloration of throat feathers was only predicted by pheomelanin in females. In addition, Pheo:Eu predicted coloration of throat feathers in females and that of belly feathers in males. Finally, we found high heritability of color of throat feathers. Melanization was found to differ from that recorded in Hirundo rustica rustica from Scotland or from H. r. erythrogaster from North America. Hence, present results show that pigmentation strategies vary in a complex manner according to sex and plumage region, and also among geographical populations, potentially reflecting adaptation to different natural and sexual selection regimes, and that some coloration components seem to be highly heritable.     

Parent Barn Swallow fluctuating asymmetry and offspring quality

Fluctuating asymmetry may impair locomotion but may also reflect intrinsic phenotypic quality. I tested whether fluctuating asymmetry of adult Barn Swallows Hirundo rustica negatively influenced offspring quality, by estimating the relationship between parental asymmetry and offspring size, condition and immunocompetence during three breeding seasons. Controlling for timing of breeding, brood size and the size of a secondary sexual character (tail length), wing and outermost tail feather asymmetry of male and female parents was not significantly correlated with offspring size, condition and immunocompetence. This was the case in spite of clear differences in nestling quality among years. In addition, parents with extreme asymmetries due to tail feather damage (not representing fluctuating asymmetry) did not have nestlings of lower quality than parents with undamaged tail feathers. These results indicate that there is only a weak relation between parental asymmetry and offspring quality.     

A Trade-Off between Reproduction and Feather Growth in the Barn Swallow (Hirundo rustica)

Physiological trade-offs mediated by limiting energy, resources or time constrain the simultaneous expression of major functions and can lead to the evolution of temporal separation between demanding activities. In birds, plumage renewal is a demanding activity, which accomplishes fundamental functions, such as allowing thermal insulation, aerodynamics and socio-sexual signaling. Feather renewal is a very expensive and disabling process, and molt is often partitioned from breeding and migration. However, trade-offs between feather renewal and breeding have been only sparsely studied. In barn swallows (Hirundo rustica) breeding in Italy and undergoing molt during wintering in sub-Saharan Africa, we studied this trade-off by removing a tail feather from a large sample of individuals and analyzing growth bar width, reflecting feather growth rate, and length of the growing replacement feather in relation to the stage in the breeding cycle at removal and clutch size. Growth bar width of females and length of the growing replacement feather of both sexes were smaller when the original feather had been removed after clutch initiation. Importantly, in females both growth bar width and replacement feather length were negatively predicted by clutch size, and more strongly so for large clutches and when feather removal occurred immediately after clutch completion. Hence, we found strong, coherent evidence for a trade-off between reproduction, and laying effort in particular, and the ability to generate new feathers. These results support the hypothesis that the derived condition of molting during wintering in long-distance migrants is maintained by the costs of overlapping breeding and molt.     

Haematocrit correlates with tail ornament size in three populations of the Barn Swallow (Hirundo rustica)

1. Handicap models of sexual selection propose that male ornaments are indicators of male quality and that honesty is enforced by the costs imposed by the exaggerated ornamental traits. In long-distance migratory birds that feed on the wing, the aerodynamic cost of exaggerated ornamental characters should be particularly high because the size of the ornaments deviates from the natural selection optimum. During migration, birds are expected to raise their oxygen consumption in relation to the energetic demands imposed by their morphology. An increase of haematocrit is an adaptive response to enhance oxygen uptake and efficiency of transfer to the muscular tissues during spells of intense muscular activity.
2. The change of haematocrit of Barn Swallows ( Hirundo rustica ) after their arrival to the breeding sites, and the relationships between haematocrit values recorded after migration and the size of ordinary and sexually selected morphological characters in three Barn Swallow populations were analysed.
3. Males had higher haematocrit values than females. Individual haematocrit values declined after arrival to the breeding sites. Haematocrit values of males were significantly and positively correlated with the size of their ornamental tail but not correlated with other characters, thus suggesting that well-ornamented males, in order to arrive early, have to raise their haematocrit above the level of short-tailed males.
4. Males and females of similar tail length did not differ in their haematocrit, thus suggesting that sexual dimorphism in haematocrit might be functionally related to dimorphism in tail length.
5. Our results are consistent with the handicap principle because long-tailed males experience lower mortality and larger seasonal reproductive success compared with short-tailed males.     

Early-Life Telomere Dynamics Differ between the Sexes and Predict Growth in the Barn Swallow (Hirundo rustica)

Telomeres are conserved DNA-protein structures at the termini of eukaryotic chromosomes which contribute to maintenance of genome integrity, and their shortening leads to cell senescence, with negative consequences for organismal functions. Because telomere erosion is influenced by extrinsic and endogenous factors, telomere dynamics may provide a mechanistic basis for evolutionary and physiological trade-offs. Yet, knowledge of fundamental aspects of telomere biology under natural selection regimes, including sex- and context-dependent variation in early-life, and the covariation between telomere dynamics and growth, is scant. In this study of barn swallows (Hirundo rustica) we investigated the sex-dependent telomere erosion during nestling period, and the covariation between relative telomere length and body and plumage growth. Finally, we tested whether any covariation between growth traits and relative telomere length depends on the social environment, as influenced by sibling sex ratio. Relative telomere length declined on average over the period of nestling maximal growth rate (between 7 and 16 days of age) and differently covaried with initial relative telomere length in either sex. The frequency distribution of changes in relative telomere length was bimodal, with most nestlings decreasing and some increasing relative telomere length, but none of the offspring traits predicted the a posteriori identified group to which individual nestlings belonged. Tail and wing length increased with relative telomere length, but more steeply in males than females, and this relationship held both at the within- and among-broods levels. Moreover, the increase in plumage phenotypic values was steeper when the sex ratio of an individual’s siblings was female-biased. Our study provides evidence for telomere shortening during early life according to subtly different dynamics in either sex. Furthermore, it shows that the positive covariation between growth and relative telomere length depends on sex as well as social environment, in terms of sibling sex ratio.     

Glycemia and Immune Responses

The intensity of experimental and clinical immune responses was correlated with the degree of glycemia of the reacting subject. Hyperglycemias resulting from overdosage with sugars, cortisol, adrenaline, or from diabetes inhibit the anaphylactoid reactions; anaphylaxis, and the tuberculin reaction; but potentiate infections. Hypoglycemias resulting from fasting, insulin and adrenalectomy potentiate the anaphylactoid reactions, anaphylaxis, and the tuberculin reaction; but inhibit infections. The hypothesis is proposed that hyperglycemia inhibits certain antigen-antibody combinations; this results in an inhibition of hypersensitivity, but an aggravation of infection.     

Sexual Dimorphism and Population Differences in Structural Properties of Barn Swallow (Hirundo rustica) Wing and Tail Feathers

Sexual selection and aerodynamic forces affecting structural properties of the flight feathers of birds are poorly understood. Here, we compared the structural features of the innermost primary wing feather (P1) and the sexually dimorphic outermost (Ta6) and monomorphic second outermost (Ta5) tail feathers of barn swallows (Hirundo rustica) from a Romanian population to investigate how sexual selection and resistance to aerodynamic forces affect structural differences among these feathers. Furthermore, we compared structural properties of Ta6 of barn swallows from six European populations. Finally, we determined the relationship between feather growth bars width (GBW) and the structural properties of tail feathers. The structure of P1 indicates strong resistance against aerodynamic forces, while the narrow rachis, low vane density and low bending stiffness of tail feathers suggest reduced resistance against airflow. The highly elongated Ta6 is characterized by structural modifications such as large rachis width and increased barbule density in relation to the less elongated Ta5, which can be explained by increased length and/or high aerodynamic forces acting at the leading tail edge. However, these changes in Ta6 structure do not allow for full compensation of elongation, as reflected by the reduced bending stiffness of Ta6. Ta6 elongation in males resulted in feathers with reduced resistance, as shown by the low barb density and reduced bending stiffness compared to females. The inconsistency in sexual dimorphism and in change in quality traits of Ta6 among six European populations shows that multiple factors may contribute to shaping population differences. In general, the difference in quality traits between tail feathers cannot be explained by the GBW of feathers. Our results show that the material and structural properties of wing and tail feathers of barn swallows change as a result of aerodynamic forces and sexual selection, although the result of these changes can be contrasting.     

Parasite Infestation and Parental Care in the Barn Swallow Hirundo rustical a Test of the Resource-provisioning Model of Parasite-mediated Sexual Selection

Barn swallows, Hirundo rustica, are commonly infested by the haematophagous tropical fowl mite Omithonyssus bursa (Macronyssidae, Gamasida), which severely reduces various measures of reproductive success among the barn swallow hosts. Food provisioning rate by parent barn swallows, measured in terms of absolute feeding rate by males and females and relative feeding rate by males (percentage of food provided by the male parent), was not significantly related to natural levels of infestation of nests. Experimental manipulation of mite loads in nests during the egg-laying period of the first clutch, which also affected mite loads of parent barn swallows, significantly affected food-provisioning rates of single-brooded, but not of double-brooded barn swallows. These results suggest that effects of mites on the parenting ability of barn swallow hosts depend on host resistance towards parasites. This is consistent with the resource-provisioning hypothesis of parasite-mediated sexual selection, suggesting that females prefer parasite-free males because they are efficient parents, but also with the hypothesis that females prefer males with traits signalling genetic resistance to parasites.     

Searching for potential wintering and migration areas of a Danish Barn Swallow population in South Africa by correlating NDVI with survival estimates

On the basis of correlation analyses between annual Normalised Difference Vegetation Index (NDVI) values in Africa and the annual survival rate estimated for a breeding population of barn swallows Hirundo rustica from Denmark, we identified potential wintering and migration areas in South Africa during December–February and March–May, when barn swallows commonly occur in South Africa. During December–February we identified potential wintering areas only in the western part of South Africa, in the Karoo. Potential areas in the central and eastern parts of the country were only identified during March–May. NDVI values in the Karoo during March–May explained most of the variance in annual adult survival rate of the population. The high ratio of European ringed barn swallows among controlled individuals in the Karoo was similar to the ratio that would be expected based on the number of ringed barn swallows and the population sizes of barn swallows in north-western European breeding populations. The level of this ratio in the Karoo was higher than in any other locality in the central and eastern parts of South Africa and Botswana, indicating that ringed birds from the eastern flyway are absent to a much smaller extent than ringed birds from the western flyway. This approach shows that the NDVI and survival method can focus ringing efforts to regions and areas that are likely to harbour specific breeding populations, thereby helping to identify potential wintering and migration areas for breeding populations of migratory birds.     

Encephalomyocarditis Virus Disrupts Stress Granules,the Critical Platform for Triggering Antiviral Innate Immune Responses

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.     

Anti-Insulin Immune Responses Are Detectable in Dogs with Spontaneous Diabetes

Diabetes mellitus occurs spontaneously in dogs. Although canine diabetes shares many features with human type-1 diabetes, there are differences that have cast doubt on the immunologic origin of the canine disease. In this study, we examined whether peripheral immune responses directed against islet antigens were present in dogs with diabetes. Routine diagnostics were used to confirm diabetic status, and serum samples from dogs with (N = 15) and without (N = 15) diabetes were analyzed for the presence of antibodies against islet antigens (insulin, glutamic acid decarboxylase, insulinoma-associated protein tyrosine phosphatase, and islet beta-cell zinc cation efflux transporter) using standard radioassays. Interferon-γ production from peripheral blood T cells stimulated by porcine insulin and by human insulin was tested using Elispot assays. Anti-insulin antibodies were detectable in a subset of diabetic dogs receiving insulin therapy. Pre-activated T cells and incipient insulin-reactive T cells in response to porcine or human insulin were identified in non-diabetic dogs and in dogs with diabetes. The data show that humoral and cellular anti-insulin immune responses are detectable in dogs with diabetes. This in turn provides support for the potential to ethically use dogs with diabetes to study the therapeutic potential of antigen-specific tolerance.     

Role of the Melanocyte in Epidermal Inflammatory and Immune Responses

The epidermis is composed of four major cell types in the mouse. In addition to keratinocytes and Langerhans cells, there are also melanocytes and Thyl⁺ lymphocytes. We propose that for the epidermis to maintain homeostasis, all four cell types must interact together in an integrated fashion. Vitiligo is a form of depigmentation that affects human subjects. Depigmentation is common in the animal kingdom. In at least several animal species, depigmentation is accompanied by loss of responsiveness of the epidermis to potent contact allergens like dinitrofluorobenzene. The C57Bl/Ler vit/vit mouse spontaneously depigments and is phenotypically similar to humans with vitiligo. We have studied this species extensively and have found that it has an isolated immune defect to contact allergens. In this report, we document that there is loss of epidermal melanocytes. Although there is a loss of epidermal contact sensitivity in this animal, other immune parameters such as dermal delayed-type hypersensitivity are normal. We attribute this loss of immune sensitivity in the epidermis to be associated with loss of the melanocyte. We report and review in detail the many peptides and other types of inflammatory mediators that affect both melanocyte function as well as immune responsiveness.     

Stress Responses in Avian Embryos

The day 13–14 chicken embryo is a useful model for studieson prenatal stress responses. Free dopamine, norepinephrineand epinephrine in its plasma, amniotic and allantoic fluidrespond to a variety of stresses. The allantoic fluid also containsconjugated catecholamines and conjugated steroids. However,a blood/allantois barrier excludes free thyroid hormones andfree steroids, and insulin. On the other hand, the allantoicfluid contains at least 40 amino acids (including six excitatoryamino acids) and related compounds. Most, possibly all, componentsof the allantoic fluid are regulated at specific blood/allantoisand amnion/allantois barriers, and they respond to ethanol stressand metabolite loading differentially. The avian allantois isa depot for important metabolites and messenger substances whichseems to be controlled by as yet unidentified hormones     

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen¹. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection². After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8α⁺ dendritic cells and Kupffer cells^3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells⁵. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8⁺ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection⁶.Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses⁶ . There is a broad range of sensitivities amongst inbred mouse strains^7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection^6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain¹⁵ that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to β-hemolysis¹ (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.