Final Report Summary - MELANIN EVOLUTION (The role of oxidative stress for the evolution of traits generated by melanins) The first objective of the project was the determination of the role of endogenous oxidative stress for the evolution of signals generated by melanins. As previous studies had shown that the production of the pigment melanins depends, amongst other factors, on the levels of the amino acid cysteine and its main physiological reservoir (i.e. the key intracellular antioxidant glutathione (GSH)), we hypothesised that this could explain why many melanin-based traits in animals are used as honest signals of quality in intraspecific communication. In particular, the production of eumelanin, which is one of the two main types of melanins and is responsible for black-grey colourations, requires that the levels of GSH are low because the presence of cysteine shifts the process toward the production of pheomelanin, the other main type of pheomelanin which is responsible for reddish-chesnut colourations. We performed an experiment with house sparrows Passer domesticus, a bird species that exhibits a eumelanic bib patch that is a well-known honest signal of quality. During the period of development of new feathers, the levels of GSH were experimentally decreased in some birds while others were controls. The results show that only high-quality birds can develop larger eumelanic plumage patches after experimentally inducing an antioxidant cost, thus confirming the hypothesis that GSH mediates the honesty of eumelanic signals of quality. The knowledge developed during the investigations carried out for this objective also led us to reveal a positive association between eumelanin deposition in testes and mitochondrial deoxyribonucleic acid (mtDNA) mutation rates in several species of birds, which is the first report of melanisation patterns in animals evolving in response to historical mutation rates. We could also conclude that the expression of eumelanin-based plumage traits is not only determined by the process of melanogenesis but also by particular elements of feather microstructure. The second objective was the determination of the role of the interaction between exogenous oxidative stress and melanin precursors for the evolution of honest signals generated by melanins. With this aim, another experiment was made with house sparrows to investigate the interaction of endougenous oxidative stress (i.e. cysteine / GSH levels) and exogenous oxidative stress in the production of traits generated by eumelanin. Some birds were treated with cysteine only, some birds were treated with diquat dibromide (a substance that generated free radicals and thus constitutes a source of exogenous oxidative stress) only, some birds were treated with both cysteine and diquat, and some birds received water only as controls. All planned measurements could be taken and we are currently analysing the data from the experiment. The third objective was the determination of the effect of radioactive contamination on the synthesis of eumelanin and pheomelanin in birds. Ionising radiation produces free radical and thus causes oxidative stress. Therefore, radioactive contamination is a potential factor affecting the expression and the evolution of traits generated by melanins. In particular, the production of pheomelanin should be especially costly under radioactive contamination because pheomelanogenesis requires cysteine / GSH and thus consumes this antioxidant resource. To test this hypothesis, we captured several species of birds in sites with different levels of radioactivity levels around Chernobyl (Ukraine) and obtained feather and blood samples from them. We have measured the levels of eumelanin and pheomelanin in feathers and antioxidant and oxidative damage levels in blood, hypothesising the levels of oxidative damage should be higher in the species producing greater levels of pheomelanin, after controling for GSH levels. We are currently analysing these data. Our investigation for this objective has also allowed us to: (1) describe for the first time that natural radioactivity levels can explain clinal variations in the expression of pheomelanin-based colouration in wild birds; (2) propose a new hypothesis for the evolution of pheomelanin; and (3) discover that birds covered by greater proportions of pheomelanic plumage have higher rates of cataracts but higher survival rates if exposed to relatively low levels of environmental oxidative stress. The fourth objective was the determination of senescence effects on melanin production. For this, we took feather and blood samples from barn swallows Hirundo rustica of known age so that we can investigate if age-related variations of antioxidants (particularly GSH) and melanin levels are associated. We have also analysed information on this pattern in wild boars Sus scrofa, concluding that, although there were no senescence effects on melanisation, individuals producing lower amounts of pheomelanin and exhibiting hair graying had higher levels of oxidative damage. We have also found that vitamin E, but not the proportion of plumage covered by eumelanic or pheomelanic colour, is a good predictor of long life span across species of birds. We also had the opportunity to address new questions related to the evolution of melanins that arised during the development of the main objectives. In particular, we were able to obtain and exceptional large number of feather samples from a wild population of goshawks Accipiter gentilis in Denmark, which we are currently using to investigate how plumage melanisation varies with age. With these samples we have already found for the first time that bacteria is associated with important fitness components in wild birds. Additionally, we have described for the first time the Raman spectrum of pheomelanin, opening the possibility that Raman spectroscopy can be applied as a non-invasive tool to identify and quantify melanins. Lastly, we had the opportunity to participate in three comparative analysis in birds showing that: (1) mate-feeding has evolved as a compensatory energetic strategy in birds; (2) feather mites exert weak effects on the body condition of their avian hosts; and (3) uropygial gland tuft size is positively associated with olfactory ability.