Samuel Perret

Filiz Karadas

Jiguet Frédéric

Frédéric Angellier

Blandine Boligez

Adrien Frantz

François Sarrazin

Alicia Munoz

Arnaud Begue

Benjamin Burguet

Camille Bloch

Caroline Vickridge

Céline Legoux

Clément Dubois

Manon Munoz

Manon Niviere

Marie Le Brazidec

Nicolas Richard

Sophie Dupont

En

Mélanie

Anaïs, Yoze, Fanny, Solène, Pierre, Seb, Ewen, Thomas, Marion, Clémentine, Floriane, Marta, Claudia, Mélissa, Lisa Alix Bertrand

Marie Vaugoyeau
email: marie.vaugoyeau@gmail.com

Beatriz Decencière

Sandrine Meylan
email: sandrine.meylan@upmc.fr

Clotilde Biard
email: clotilde.biard@upmc.fr

Frédéric Jiguet

Is oxidative status influenced by dietary carotenoid and physical activity after moult in the great tit (Parus major)?

Keywords: antioxidants, carotenoid-based colour, physiological stress, immunity Expérience de maternal effects, parental investment, reproductive success, urban heat island effect Sexual selection, Urbanization, Colouration, pigment, bird Birds, Eggshell pigmentation, Maternal effects, Protoporphyrin

In the context of sexual and natural selection, an allocation trade-off for carotenoid pigments may exist because of their obligate dietary origin and their role both in the antioxidant and immune systems and in the production of coloured signals in various taxa, particularly birds. MANUSCRIT 1 oxidative stress outside the moult period, and their combined potential influence on attractiveness and reproductive investment later during the breeding season.

I. Introduction

A. Caractérisation des changements globaux

Avec la hausse des températures, de la pluviométrie et de la fréquence des phénomènes extrêmes tels que les crues, les tsunamis ou les ouragans, les changements globaux sont [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013). Les changements globaux vironnement et les écosystèmes [START_REF] Walther | Ecological responses to recent climate change[END_REF][START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF]Stocker et al. 2013) et peuvent-être dus à des processus naturels internes comme la saisonnalité du climat alternance jour-nuit, externes tels que les éruptions volcaniques ou les raz-de-marée ou à des changements anthropiques -à-dire la destruction suite utilisation des sols. Les changements globaux sont influencés par et induisent des changements urbanis exploitation intensive des milieux acidification des milieux ou le déplacement massif de matière et augmentant le déplacement fortuit ou . Tous ces changements vont, selon leurs natures et leurs origines, influencer sol naturel à un état anthropique [START_REF] Gaston | Urban Ecology[END_REF]Pickett et al. 2011). Les hausses de la température, de la pluviométrie et de la luminosité induisent un glissement spatio-temporel de la niche (Alberti 2007;[START_REF] Grimm | Global change and the ecology of cities[END_REF]Pickett et al. 2011;[START_REF] Gil | Avian Urban Ecology[END_REF]. Les influences des changements globaux se retrouvent à toutes les échelles, , et il s mécanismes associés et leurs conséquences sur les dynamiques des populations Tableau 1 : Processus (A) physiques, (B) chimiques et (C) biologiques liés aux changements globaux

A) Caractéristiques Physiques

Occupation des sols taille de la population urbaine mondiale et aux changements des pratiques agricoles, croissance démographique urbaine [START_REF] Grimm | Global change and the ecology of cities[END_REF].

Les constructions vont influencer les caractéristiques physiques augmentation du ruissellement ou la réduction de la perméabilité, et chimiques telles que la pollution ou la diminution du stockage des nutriments, du sol (Pickett et al. 2011) et peuvent jouer le rôle de barrières à la dispersion des espèces et à la communication inter et intraspécifique [START_REF] Slabbekoorn | Birdsong and anthropogenic noise: implications and applications for conservation[END_REF][START_REF] Slabbekoorn | Songs of the city: noise-dependent spectral plasticity in the acoustic phenotype of urban birds[END_REF].

Température

Depuis les années 80, les températures mondiales annuelles sont supérieurs à celles [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013). Les températures en villes sont plus hautes que celles des campagnes avoisinantes et (Escourrou 1990;[START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013). La hausse des températures s terrestres comme aquatique en zone tempérée en avançant les comportements saisonniers des espèces [START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF]Caro et al. 2013;Charmantier and Gienapp 2014) et/ou en induisant un glissement spatio-temporel de la niche écologique et (Alberti 2007;[START_REF] Grimm | Global change and the ecology of cities[END_REF]Pickett et al. 2011;[START_REF] Gil | Avian Urban Ecology[END_REF].

Luminosité

La pollution lumineuse engendrée par les villes mais aussi les routes, les zones industrielles et portuaires, est et de la densité humaine [START_REF] Small | Mapping Decadal Change in Anthropogenic Night Light. Spatial Statistics[END_REF] et peut perturber le comportement des animaux et la distribution des espèces [START_REF] Small | Mapping Decadal Change in Anthropogenic Night Light. Spatial Statistics[END_REF][START_REF] Dominoni | Individual-based measurements of light intensity provide new insights into the effects of artificial light at night on daily rhythms of urban-dwelling songbirds[END_REF][START_REF] Fonken | The Effects of Light at Night on Circadian Clocks and Metabolism[END_REF].

Champ ÉlectroMagnétique (CEM)

Les CEM produits par les lignes à haute tension varient entre les pays en fonction du voltage et du type de courant [START_REF] Tomás | Clutch size and egg volume in great tits (Parus major) increase under low intensity electromagnetic fields: A long-term field study[END_REF].

s ont montré une influence sur les organismes même si ils perturbent la perméabilité des membranes cellulaires [START_REF] Miller | Electric-field induced in chicken eggs by 60-Hz magnetic-fields and the dosimetric importance of biological membranes[END_REF][START_REF] Juutilainen | Developmental effects of extremely low frequency electric and magnetic fields[END_REF].

Fond sonore

La pollution sonore est comme la pollution lumineuse et associée aux centres urbains, ions (voies ferrées, routes, couloirs aériens et nautiques) les reliant. Le [START_REF] Slabbekoorn | Songs of the city: noise-dependent spectral plasticity in the acoustic phenotype of urban birds[END_REF]) qui influence la dispersion [START_REF] Sun | Anthropogenic sounds differentially affect amphibian call rate[END_REF][START_REF] Barber | The costs of chronic noise exposure for terrestrial organisms[END_REF][START_REF] Cunnington | Plasticity in the vocalizations of anurans in response to traffic noise[END_REF][START_REF] Francis | Noise Pollution Filters Bird Communities Based on Vocal Frequency[END_REF] et la communication inter et intraspécifique diminuant ainsi la détectabilité des partenaires sexuels éventuels et des prédateurs [START_REF] Brumm | The impact of environmental noise on song amplitude in a territorial bird[END_REF][START_REF] Barber | The costs of chronic noise exposure for terrestrial organisms[END_REF][START_REF] Snell-Rood | The effect of climate on acoustic signals: Does atmospheric sound absorption matter for bird song and bat echolocation?[END_REF].

Pluviométrie

La fréquence et la puissance des phénomènes pluviométriques ont augmenté dans les zones tempérées et équatoriales mais ont diminué dans les zones désertiques [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013).

Evènement extrême

La fréquence des processus extrêmes naturels tels que les tsunamis, les crues ou les ouragans a augmenté mais le mécanisme sous-jacent est encore inconnu [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013). Cette augmentation de fréquence diminue la prédictibilité de [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013).

B) Caractéristiques Chimiques

Cycle de matière perturbés localement par une modification de la perméabilité des sols ou du fonctionnement des écosystèmes (Pickett et al. 2011). Les concentrations atmosphériques de dioxyde de carbone, méthane et oxyde de nitrate ne 000 ans [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013), et plus particulièrement le , ce qui a acidifié les océans qui en ont absorbé une grande partie [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013).

Pesticides

Les pesticides, comme les organochlorés, sont principalement retrouvés autour des terres cultivées même si les concentrations de certains (biphenyl polychloré et polybromé) sont plus grandes en zone u agricole, résidentielle ou naturelle [START_REF] Van Den Steen | Variation, levels and profiles of organochlorines and brominated flame retardants in great tit (Parus major) eggs from different types of sampling locations in Flanders (Belgium)[END_REF][START_REF] Van Den Steen | Brominated flame retardants and organochlorines in the European environment using great tit eggs as a biomonitoring tool[END_REF]; [START_REF] Van Den Steen | The use of blue tit eggs as a biomonitoring tool for organohalogenated pollutants in the European environment[END_REF]. Les pesticides organohalogénés comme les biphenyls polychlorés (PCBs) ou les organochlorés (OCPs) persistent qui contrôlent la différentiation sexuelle des gonades ou le comportement reproducteur des animaux [START_REF] Vos | Health Effects of Endocrine-Disrupting Chemicals on Wildlife, with Special Reference to the European Situation[END_REF].

Métaux lourds

Les métaux lourds (Argent, Arsenic, Cadmium, Cuivre, Mercure, Nickel, Plomb et Zinc) sont produits entre (Pickett et al. 2011) et induisent une acidification du milieu [START_REF] Eeva | Pollution-related variation in food supply and breeding success in two hole-nesting passerines[END_REF]. (Roux and Marra 2007;Fritsch et al. 2012) [START_REF] Dauwe | Relationships between metal concentrations in great tit nestlings and their environment and food[END_REF]). Les métaux lourds provoquent la production de radicaux libres (Koivula and Eeva 2010) qui vont détériorer les cellules.

C) Caractéristiques Biologiques

Pression socio-politico-économique dispersion (volontaire ou involontaire) des espèces et conduit à une homogénéisation des milieux urbains et agricoles [START_REF] Gaston | Urban Ecology[END_REF]Pickett et al. 2011). es [START_REF] Gaston | Urban Ecology[END_REF][START_REF] Faeth | Urban biodiversity: patterns and mechanisms[END_REF]Pickett et al. 2011).

B. Influence des changements globaux sur les populations et les écosystèmes

Certaines composantes des changements globaux modifient le fonctionnement des écosystèmes (Figure 1). Par exemple, asives augmente par rapport aux espèces natives en relation avec et de la pression socio-politico-économique imposé aux écosystèmes [START_REF] Gaston | Urban Ecology[END_REF][START_REF] Faeth | Urban biodiversity: patterns and mechanisms[END_REF]Pickett et al. 2011). Par les mêmes processus, les communautés animales et végétales spécifique [START_REF] Bol'shakov | Specific features of the formation of animal species communities in technogenic and urbanized landscapes[END_REF][START_REF] Clergeau | Avifauna homogenisation by urbanisation: Analysis at different European latitudes[END_REF][START_REF] Devictor | Functional biotic homogenization of bird communities in disturbed landscapes[END_REF][START_REF] Shwartz | How do habitat variability and management regime shape the spatial heterogeneity of birds within a large Mediterranean urban park?[END_REF][START_REF] Huste | Determinants of bird community composition on patches in the suburbs of Paris, France[END_REF]Pickett et al. 2011;[START_REF] Jokimaki | Residential areas support overwintering possibilities of most bird species[END_REF][START_REF] Johnson | Urbanization and wetland communities: applying metacommunity theory to understand the local and landscape effects[END_REF][START_REF] Vittoz | Climate change impacts on biodiversity in Switzerland: A review[END_REF]) ou fonctionnelle [START_REF] Aronson | A global analysis of the impacts of urbanization on bird and plant diversity reveals key anthropogenic drivers[END_REF].

L homogénéisation et la grande densité de populations en milieu anthropique comme les champs cultivés ou les villes peut augmenter la propagation des pathogènes et des maladies (Bradley and Altizer 2007). Les changements globaux influencent toutes les composantes des dynamiques des populations (interaction intra et interspécifique, survie, reproduction, Figure 1) et vont perturber les compromis physiologiques ou évolutifs associés en modifiant la balance entre les caractères qui ne peuvent pas être optimisés simultanément.

modifie les signaux acoustiques et visuels ainsi que les sélections sexuelles et naturelles associées. En effet, ces dernières années au niveau des basses fréquences liée à [START_REF] Brumm | The impact of environmental noise on song amplitude in a territorial bird[END_REF][START_REF] Slabbekoorn | Birdsong and anthropogenic noise: implications and applications for conservation[END_REF].

Un environnement bruyant peut influencer la distribution des espèces animales [START_REF] Barber | The costs of chronic noise exposure for terrestrial organisms[END_REF][START_REF] Francis | Noise Pollution Filters Bird Communities Based on Vocal Frequency[END_REF]) ainsi que diminuer leur survie [START_REF] Threlfall | Ecological processes in urban landscapes: mechanisms influencing the distribution and activity of insectivorous bats[END_REF][START_REF] Snell-Rood | The effect of climate on acoustic signals: Does atmospheric sound absorption matter for bird song and bat echolocation?[END_REF][START_REF] Slabbekoorn | Songs of the city: noise-dependent spectral plasticity in the acoustic phenotype of urban birds[END_REF]) en diminuant la détectabilité des prédateurs [START_REF] Slabbekoorn | Songs of the city: noise-dependent spectral plasticity in the acoustic phenotype of urban birds[END_REF]) ou [START_REF] Threlfall | Ecological processes in urban landscapes: mechanisms influencing the distribution and activity of insectivorous bats[END_REF][START_REF] Snell-Rood | The effect of climate on acoustic signals: Does atmospheric sound absorption matter for bird song and bat echolocation?[END_REF]. Un fond sonore bruyant peut aussi diminuer le succès de reproduction directement [START_REF] Halfwerk | Negative impact of traffic noise on avian reproductive success[END_REF];

Snell-Rood 2012) ou indirectement en diminuant la détectabilité des partenaires sexuelles [START_REF] Slabbekoorn | Songs of the city: noise-dependent spectral plasticity in the acoustic phenotype of urban birds[END_REF]. Pour peuvent diminuer la distance entre eux [START_REF] Halfwerk | Male great tit song perch selection in response to noise-dependent female feedback[END_REF][START_REF] Owens | The effects of extended exposure to traffic noise on parid social and risk-taking behavior[END_REF], attendre une période de silence [START_REF] Sun | Anthropogenic sounds differentially affect amphibian call rate[END_REF] ou augmenter la fréquence minimale ou le volume sonore de leurs ne soient plus recouverts par le bruit anthropique (mesuré chez les oiseaux [START_REF] Slabbekoorn | Ecology: Birds sing at a higher pitch in urban noise -Great tits hit the high notes to ensure that their mating calls are heard above the city's din[END_REF][START_REF] Brumm | The impact of environmental noise on song amplitude in a territorial bird[END_REF][START_REF] Dowling | Comparative effects of urban development and anthropogenic noise on bird songs[END_REF][START_REF] Nemeth | Bird song and anthropogenic noise: vocal constraints may explain why birds sing higherfrequency songs in cities[END_REF][START_REF] Slabbekoorn | Songs of the city: noise-dependent spectral plasticity in the acoustic phenotype of urban birds[END_REF] La communication inter et intra-spécifique passe aussi par les signaux colorés lié à la valeur adaptative (fitness) des individus [START_REF] Danchin | Ecologie comportementale: cours et questions de réflexion[END_REF]Hill and McGraw 2006;[START_REF] Krebs | Behavioural Ecology: An Evolutionary Approach[END_REF]. La couleur d un tégument varie en fonction des pigments utilisés comme la mélanine ou les caroténoïdes (coloration pigmentaire) et de la réflexion de la lumière sur les nanostructures du tégument (coloration structurale) [START_REF] Danchin | Ecologie comportementale: cours et questions de réflexion[END_REF]Hill and McGraw 2006;[START_REF] Krebs | Behavioural Ecology: An Evolutionary Approach[END_REF]. La variation de la nanostructure des plumes influence la couleur réfléchie dans les UV (Griffith et al. 2003;Limbourg et al. 2004;Limbourg et al. 2013). Les pigments utilisés pour la coloration peuvent être soit produits comme la mélanine pour les champignons, les plantes et les animaux ou les caroténoïdes pour les plantes ou les champignons, soit apportés par la nourriture comme les caroténoïdes pour les animaux. Dans les deux cas, la quantité de pigments allouée à la coloration dépend de la disponibilité et de état de santé . Par exemple, face à un challenge murailles (Podarcis muralis) diminue (Martin et al. 2008;Lopez et al. 2009;Calsbeek et al. 2010), le bec des mâles de merles noirs est moins jaune (Faivre et al. 2003) et le bec des diamants mandarins (Taeniopygia guttata) est moins rouge (Cote et al. 2010) ou les plumes des jeunes de mésanges charbonnières sont moins jaunes (Romero-Diaz et al. 2013). Les caroténoïdes, induisant des couleurs du jaune au rouge (Hill and McGraw 2006) sont des précurseur de la vitamine A qui joue un rôle important dans les défenses immunitaires (Hill and Johnson 2012). des caroténoïdes entre la réponse immunitaire et la coloration jaune-rouge A -être le signe mésanges charbonnières mâles est plus grande au sein des zones polluées en métaux lourds (Dauwe and Eens 2008) et les pigeons mélaniques (Columba livia) fixent plus de zinc dans leurs plumes [START_REF] Chatelain | The adaptive function of melanin-based plumage coloration to trace metals[END_REF]. Les métaux lourds peuvent aussi accélérer la synthèse de la mélanine [START_REF] Mcgraw | Melanins, metals, and mate quality[END_REF] qui serait capable de fixer dans les plumes les radicaux libres produits par la pollution [START_REF] Mcgraw | Melanins, metals, and mate quality[END_REF] sous contrôle de la testostérone (McGraw 2008).

Donc les changements globaux vont influencer, de manière plus ou moins direct, la coloration pigmentaire des organismes. Chez de nombreuses espèces animales, les mâles les plus colorés sont préférés par les femelles, ont un meilleur succès de reproduction et gagnent plus souvent les compétitions mâle-mâle (Møller et al. 2000;Hill and McGraw 2006). Ainsi les changements globaux vont influencer le phénotype et donc le choix du partenaire. De manière générale, la reproduction des espèces est fortement influencée par les changements globaux.

Par exemple, des températures basses (Kluen et al. 2011;Nord and Nilsson 2011;Naef-Daenzer et al. 2012), la présence de métaux lourds [START_REF] Janssens | Effects of heavy metal exposure on aggressive behavior in a small territorial songbird[END_REF]) ou de voies de circulation [START_REF] Halfwerk | Negative impact of traffic noise on avian reproductive success[END_REF][START_REF] Snell-Rood | The effect of climate on acoustic signals: Does atmospheric sound absorption matter for bird song and bat echolocation?[END_REF] diminuent le succès de reproduction des oiseaux et/ou des chauves-souris.

Les fluctuations environnementales influence tels que la reproduction [START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF][START_REF] Walther | Ecological responses to recent climate change[END_REF][START_REF] Both | Large-scale geographical variation confirms that climate change causes birds to lay earlier[END_REF]Charmantier et al. 2008;[START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF]Visser et al. 2009;Naef-Daenzer et al. 2012;Schaper et al. 2012;[START_REF] Vedder | Individual birds advance offspring hatching in response to increased temperature after the start of laying[END_REF]), la migration [START_REF] Both | Climate change and population declines in a long-distance migratory bird[END_REF][START_REF] Both | Climate change and timing of avian breeding and migration throughout Europe[END_REF][START_REF] Pulido | Phenotypic changes in spring arrival: evolution, phenotypic plasticity, effects of weather and condition[END_REF]Charmantier and Gienapp 2014) ou la dispersion [START_REF] Massot | Climate warming, dispersal inhibition and extinction risk[END_REF]. Lorsque la périodicité du comportement , la valeur adaptative des individus diminue [START_REF] Parmesan | Ecological and evolutionary responses to recent climate change[END_REF][START_REF] Visser | Shifts in caterpillar biomass phenology due to climate change and its impact on the breeding biology of an insectivorous bird[END_REF][START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF][START_REF] Reed | Phenological mismatch strongly affects individual fitness but not population demography in a woodland passerine[END_REF].

, le succès de reproduction diminue (Figure 2). Les organismes utilisent des indices environnementaux [START_REF] Parmesan | Ecological and evolutionary responses to recent climate change[END_REF][START_REF] Visser | Keeping up with a warming world; assessing the rate of adaptation to climate change[END_REF][START_REF] Sih | Evolution and behavioural responses to humaninduced rapid environmental change[END_REF]) tels que la température [START_REF] Both | Large-scale geographical variation confirms that climate change causes birds to lay earlier[END_REF]Charmantier et al. 2008;Naef-Daenzer et al. 2012), la luminosité [START_REF] Dominoni | Artificial light at night advances avian reproductive physiology[END_REF]) ou la date de débourrage des arbres [START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF]) pour se synchroniser .

C. Effet maternel et changements environnementaux

des informations génétiques transmises (Figure 3) [START_REF] Mousseau | The adaptive significance of maternal effects[END_REF]. Historiquement paternel était ignoré mais le mâle a aussi une influence, on parle donc maintenant

Oncorhynchus tshawytscha de deux ans engendrent des jeunes plus agressifs que les mâles de quatre ans [START_REF] Henkel | Effects of Paternal Reproductive Tactic on Juvenile Behaviour and Kin Recognition in Chinook Salmon (Oncorhynchus tshawytscha)[END_REF]. Les effets parentaux ne sont pas nécessairement adaptatif et peuvent conduire à une diminution de la valeur adaptative des adultes et des jeunes [START_REF] Mousseau | The adaptive significance of maternal effects[END_REF]Lindström 1999). En effet, la transmission de polluants ou de molécules toxiques (Marshall and Uller 2007) comme augmente le nombre de fausses- [START_REF] Warren | Alcohol-related birth defects: an update[END_REF].

Les effets parentaux influencent le jeune et le futur adulte [START_REF] Mousseau | The adaptive significance of maternal effects[END_REF]Lindström 1999;[START_REF] Dufty | Hormones, developmental plasticity and adaptation[END_REF] entre la conception et la fin du développement [START_REF] Henry | Long-term Consequences of Early Environment: Growth, Development and the Lifespan Developmental Perspective[END_REF]. Dans le contexte des changements globaux, augmentation de fréquence des phénomènes extrêmes tels que les inondations ou les ouragans et des caractéristiques météorologiques associées (hausse de la pluviométrie et de la température) [START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013) va diminuer la pré des adultes dans la reproduction. Par exemple, la date de ponte des oiseaux varie en fonction des populations et des années [START_REF] Both | Large-scale geographical variation confirms that climate change causes birds to lay earlier[END_REF][START_REF] Both | Climate change and timing of avian breeding and migration throughout Europe[END_REF]Charmantier et al. 2008;[START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF]Naef-Daenzer et al. 2012;[START_REF] Gienapp | Predicting demographically sustainable rates of adaptation: can great tit breeding time keep pace with climate change?[END_REF][START_REF] Reed | Phenological mismatch strongly affects individual fitness but not population demography in a woodland passerine[END_REF]. Ce décalage peut-être adaptatif pour permettre une meilleure synchronisation avec le pic annuel [START_REF] Van Noordwijk | Selection for the timing of Great Tit breeding in relation to caterpillar growth and temperature[END_REF][START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF][START_REF] Visser | Shifts in caterpillar biomass phenology due to climate change and its impact on the breeding biology of an insectivorous bird[END_REF][START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF]) ou subi et orienté par les stimuli environnementaux [START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF]) tels que la lumière [START_REF] Dominoni | Artificial light at night advances avian reproductive physiology[END_REF]), la date de débourrage des arbres [START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF][START_REF] Schaper | Spring phenology does not affect timing of reproduction in the great tit (Parus major)[END_REF]) ou la température (Charmantier et al. 2008;Visser et al. 2009;Kluen et al. 2011;Nord and Nilsson 2011;Mainwaring et al. 2012;Naef-Daenzer et al. 2012;Schaper et al. 2012;Chmielewski et al. 2013). té beaucoup étudiée et a permis de mieux modéliser les réponses des populations comme la variation de la date de ponte aux futurs changements (Naef-Daenzer et al. 2012;[START_REF] Gienapp | Predicting demographically sustainable rates of adaptation: can great tit breeding time keep pace with climate change?[END_REF]. Malgré le fait que le milieu urbain devienne de plus en plus courant y ont été réalisées.

En effet, si l reste deux fois plus rapide que la croissance démographique urbaine alors qu s [START_REF] Grimm | Global change and the ecology of cities[END_REF], le milieu urbain sera plus courant que les milieux naturels ou agricoles. Comme la majorité de la population mondiale vie en zone urbaine, un changement de comportement dans les villes aura un fort impact dans le futur, si les contraintes majeures sont identifiées de des études pour comprendre comment les populations répondent aux caractéristiques environnementales propres aux zones urbaines.

Si les insectes, les reptiles et les mammifères sont des modèles biologiques très étudiés dans le cadre des changements globaux, il reste beaucoup à apprendre sur les conséquences de due à leur position haute dans la chaine alimentaire et à leur facilité de manipulation. Grâce aux ét reproduction des oiseaux peut-être estimée (Figure 4).

bien dû ponte en zone urbaine sera plus précoce que celles des zones rurales proches. Si le délai entre la date de fin de et le seront us si les températures hautes réduisent [START_REF] Whitehouse | Preferred habitat of breeding birds may be compromised by climate change: Unexpected effects of an exceptionally cold, wet Spring[END_REF]. De la même manière, les températures plus hautes en zones urbaines peuvent contribuer à diminuer la durée de (Nord and Nilsson 2011).

La femelle peut transmettre des molécules du système immunitaire [START_REF] Hõrak | Fat-soluble antioxidants in the eggs of great tits Parus major in relation to breeding habitat and laying sequence[END_REF][START_REF] Saino | Early maternal effects and antibacterial immune factors in the eggs, nestlings and adults of the barn swallow[END_REF]Holveck et al. 2012) ainsi que des caroténoïdes ou des vitamines [START_REF] Hõrak | Fat-soluble antioxidants in the eggs of great tits Parus major in relation to breeding habitat and laying sequence[END_REF]Biard et al. 2005;Biard et al. 2006;Isaksson et al. 2008) mais elle peut aussi transmettre des polluants (Jagannath et al. 2008;Tsipoura et al. 2008;[START_REF] Van Den Steen | Variation, levels and profiles of organochlorines and brominated flame retardants in great tit (Parus major) eggs from different types of sampling locations in Flanders (Belgium)[END_REF][START_REF] Van Den Steen | Brominated flame retardants and organochlorines in the European environment using great tit eggs as a biomonitoring tool[END_REF]; [START_REF] Van Den Steen | The use of blue tit eggs as a biomonitoring tool for organohalogenated pollutants in the European environment[END_REF]) qui diminueront le succès des jeunes (Jagannath et al. 2008;[START_REF] Koivula | Metal pollution indirectly increases oxidative stress in great tit (Parus major) nestlings[END_REF], donc un succès de reproduction plus faible est attendu en milieu urbain.

Les pesticides et métaux lourds sont aussi transmis par la nourriture apportée au nid (Isaksson and Andersson 2007;[START_REF] Sillanpaa | Carotenoids in a food chain along a pollution gradient[END_REF]) même si le nourrissage des nichées peut-être [START_REF] Eeva | Pollution-related changes in diets of two insectivorous passerines[END_REF]Isaksson and Andersson 2007). La nourriture est un pivot central (Figure 1). En effet, la disponibilité en nourriture est contrainte par ou la température, au même titre que la qualité (disponibilité en élément essentiels comme les caroténoïdes ou le calcium). En effet, les chenilles consommées par les mésanges sont moins présentes en ville et elles sont aussi moins riches en caroténoïdes (Isaksson and Andersson 2007) s [START_REF] Lososova | Diversity of Central European urban biota: effects of human-made habitat types on plants and land snails[END_REF], première source de calcium des mésanges (Sturkie 1986;Solomon 1997;Nys 2010), (Graveland 1996;Trammell et al. 2011). La [START_REF] Andersson | Ultraviolet sexual dimorphism and assortative mating in blue tits[END_REF]. Enfin les femelles nourrissent moins fréquemment les jeunes quand les mâles sont pâles (Limbourg et al. 2004;Limbourg et al. 2013). Comme la mésange charbonnière (Figure 7B), la mésange bleue a des joues blanches et un ventre jaune. La coloration blanche est elle aussi une couleur structurale mais le jaune des plumes est produit par des caroténoïdes. Les caroténoïdes sont des pigments uniquement produits par les plantes qui colorent les téguments de couleurs allant du jaune au rouge et qui ont des propriétés antioxydantes de par leurs structures (Hill and McGraw 2006) (Partali et al. 1987;Hill and McGraw 2006;Isaksson 2009). Le fait que les femelles sélectionnent les mâles les plus colorés (Møller et al. 2000;Hill and McGraw 2006) compromis des caroténoïdes entre la réponse immunitaire e individu. Les femelles de mésanges charbonnières sélectionnent aussi selon la coloration basée sur la mélanine en choisissant les mâles avec de larges bandes ventrales. En effet, la mésange charbonnière présente en plus du ventre jaune une bande ventrale noire, plus large chez les mâles que chez les femelles. Son dos est verdâtre alors que ses ailes et sa queue sont grisâtres avec une bande alaire blanche et les rectrices externes blanches (Figure 3B). Les mélanines sont des pigments produits par les animaux qui colorent les téguments en noir, gris ou roux selon les espèces (Hill and McGraw 2006). Les mélanines ont la propriété de fixer les radicaux libres produits par les polluants dans les téguments [START_REF] Mcgraw | Melanins, metals, and mate quality[END_REF]. Les mésanges bleues et charbonnières se reproduisent à la même période et utilisent le même type de cavités pour nidifier mais ne semblent pas être en compétition. Ces deux espèces sont territoriales pendant la saison de reproduction.

Pour les deux espèces les mêmes données ont été récoltés lors des deux pontes (Figure 8)

Seules les données de premières ponte ont été exploitées ici. couleurs au sein des couples selon leurs habitats a permis de mettre en évidence que (Box 1).

Les études sur la phénologie de la reproduction ont montré que déterminants majeurs du succès de reproduction était la synchronicité entre la période de pic des chenilles et de nourrissage des jeunes [START_REF] Van Noordwijk | Selection for the timing of Great Tit breeding in relation to caterpillar growth and temperature[END_REF][START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF][START_REF] Visser | Shifts in caterpillar biomass phenology due to climate change and its impact on the breeding biology of an insectivorous bird[END_REF][START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF][START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF]. caractéristiques physiques des dates de débourrage et pic de chenilles, amène à supposer que la phénologie de reproduction des oiseaux sera avancée en zone urbaine par rapport aux zones rurales voisines (Pickett et al. 2011;[START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF]Stocker et al. 2013). Les premières études chez les mésanges charbonnières et bleues le long du gradient urbain-rural (Isaksson and Andersson 2007;Geens et al. 2009;[START_REF] Brahmia | Breeding performance of blue tits (Cyanistes caeruleus ultramarinus) in relation to lead pollution and nest failure rates in rural, intermediate, and urban sites in Algeria[END_REF]

II. Variation phénotypiques et phénologiques entre et au sein des populations de mésanges bleues et charbonnières

A. e phénotype coloré des mésanges charbonnières

La coloration des oiseaux est un compromis entre la résistance aux rayonnements solaires, la capacité à se dissimuler des prédateurs et à être vu par les partenaires sexuels éventuels (Endler 1992;[START_REF] Stuart-Fox | Sexual selection, natural selection and the evolution of dimorphic coloration and ornamentation in agamid lizards[END_REF][START_REF] Danchin | Ecologie comportementale: cours et questions de réflexion[END_REF]Hill and McGraw 2006). La couleur est non-absorbées par les pigments et réfléchie par sa microstructure (Endler 1992;[START_REF] Danchin | Ecologie comportementale: cours et questions de réflexion[END_REF]Hill and McGraw 2006).

Les mésanges charbonnières produire la couleur jaune des plumes du ventre et des mélanines, des pigments produis par les animaux eux-mêmes pour la coloration de la bande ventrale noire (Hill and McGraw 2006).

La disponibilité des différents caroténoïdes (Isaksson and Andersson 2007;Isaksson 2009) et la production de mélanine [START_REF] Mcgraw | Melanins, metals, and mate quality[END_REF]McGraw 2008) sont perturbées p La variation de couleurs des mésanges charbonnières a été étudiée selon l bitat en interaction avec la condition corporelle, le stress physiologique et immunitaire .

De plus, la corrélation entre la couleur du couple et son succès de reproduction entre les différents habitats a aussi été analysée.

Variation de la couleur entre les sites

Great tits reveal carotenoid-based and not melanin-based colouration variation in urban polluted environments (Manuscrit 3), contrairement à ce qui était attendu, aucune Ceci est surprenant car les métaux lourds, fortement présent en zone urbaine (atmosphère plus concentrée (Pickett et al. 2011;[START_REF] Gil | Avian Urban Ecology[END_REF])), sont connus pour stimuler la production de mélanine [START_REF] Mcgraw | Melanins, metals, and mate quality[END_REF]. Il est possible que la contraire [START_REF] Dauwe | Relationships between metal concentrations in great tit nestlings and their environment and food[END_REF]Dauwe and Eens 2008;Geens et al. 2009). D , une étude récente a montré que contrairement à ce qui se passe en milieu rural, les mâles urbains avec une grande bande ventrale noire ont une survie plus faible que ceux avec une petite bande ventrale (Senar et al. 2014). êche de confirmer cette inversion par contre, si cette hypothèse est vraie, cela expliquerait pourquoi la taille de la bande ventrale

Le manque de différence entre les populations pourrait-être aussi lié à la présence de flux migratoires entre les populations urbaines et rurales, ce qui a déjà été mesuré chez différentes espèces (merle noir, Turdus merula, [START_REF] Evans | Independent colonization of multiple urban centres by a formerly forest specialist bird species[END_REF], moineau domestique, Passer domesticus, [START_REF] Vangestel | Spatial heterogeneity in genetic relatedness among house sparrows along an urban rural gradient as revealed by individual-based analysis[END_REF], mésanges charbonnières [START_REF] Bjorklund | Genetic differentiation in the urban habitat: the great tits (Parus major) of the parks of Barcelona city[END_REF]) et bruant chanteur, Melospiza melodia, (Unfried et al.

2013))

. Les données de capture-marquage-recapture ne sont pas suffisantes pour confirmer ou non une connexion entre les populations mais pourrait le faire.

La coloration des plumes du torse des mésanges charbonnières est plus jaune dans la population parisienne rurale (Manuscrit 3). Plus précisément (Box 1), les femelles urbaines ont un jaune légèrement plus intense, par contre les mâles ne montrent pas de différence de coloration. La variation de couleur va dans le sens inverse des prédictions. En effet, comme la quantité des caroténoïdes est plus faible mais plus diversifiée en milieu urbain que rurale (Isaksson 2009;[START_REF] Gil | Avian Urban Ecology[END_REF] urbaines moins jaunes que les mésanges rurales (Hõrak et al. 2001;Isaksson et al. 2005).

Cette différence de coloration est peut-être dû à la nature et/ou à la quantité de caroténoïdes disponible dans les parcs parisiens (Isaksson 2009) ou simplement à une différence populations. En effet, les caroténoïdes sont des pigments avec une capacité antioxydante (Surai and Speake 1998;Møller et al. 2000;Blount et al. 2002) mais ce sont aussi des précurseurs de la vitamine A, molécule centrale de la réponse immunitaire (Møller et al. 2000;Biard et al. 2010;Romero-Diaz et al. 2013).

Lien entre la coloration et santé des mésanges charbonnières

investissement et celui de son partenaire dans la reproduction. La coloration basée sur les pigments varie avec les contraintes environnementales (Hõrak et al. 2001;Isaksson et al. 2005;Dauwe and Eens 2008;McGraw 2008) et comme la coloration strucutrale, varie aussi Par example, la variation de couleur dans les UV est due à une variation de la microstructure de la plume et en particulier, les plumes les plus usées brillent plus dans les UV [START_REF] Ornborg | Seasonal changes in a ultraviolet structural colour signal in blue tits, Parus caeruleus[END_REF]. De même, les individus parasités (Biard et al. 2010;Kilgas et al. 2012;Romero-Diaz et al. 2013) ou ceux dont le système immunitaire est activé (Biard et al. 2010;Romero-Diaz et al. 2013) [START_REF] Andersson | Ultraviolet sexual dimorphism and assortative mating in blue tits[END_REF]Møller et al. 2000;Hill and McGraw 2006).

mâles alors que la coloration des femelles influen couple plus coloré aura globalement un meilleur succès de reproduction quel que soit habitat.

lorés produisent des qualité avec hormones et/ou moins de polluants dû caractéristiques.

nourrissent peut-être plus fréquemment ou en plus grande quantité (Limbourg et al. 2004;Limbourg et al. 2013), malheureusement ces hypothèses ne peuvent pas être confirmées par nos données. M changements globaux, la coloration jaune des mésanges charbonnières apportent des informations sur leurs succès de reproduction. Les mâles les plus jaunes ont un meilleur succès éclosion alors que les femelles les plus jaunes ont un meilleur succès

B. mésanges bleues et charbonnières

La date de ponte et plus particulièrement la synchronicité entre le pic des chenilles et le pic de nourrissage des jeunes ont une influence très forte sur le succès de reproduction des petits passereaux en zone rurale [START_REF] Van Noordwijk | Selection for the timing of Great Tit breeding in relation to caterpillar growth and temperature[END_REF][START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF][START_REF] Visser | Shifts in caterpillar biomass phenology due to climate change and its impact on the breeding biology of an insectivorous bird[END_REF][START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF][START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF] on globale des températures en hiver qui global de la date de ponte (Charmantier et al. 2008;Visser et al. 2009;Kluen et al. 2011;Nord and Nilsson 2011;Mainwaring et al. 2012;Naef-Daenzer et al. 2012;Schaper et al. 2012;Chmielewski et al. 2013).

luminosité avance aussi la reproduction des oiseaux [START_REF] Small | Mapping Decadal Change in Anthropogenic Night Light. Spatial Statistics[END_REF][START_REF] Dominoni | Individual-based measurements of light intensity provide new insights into the effects of artificial light at night on daily rhythms of urban-dwelling songbirds[END_REF][START_REF] Fonken | The Effects of Light at Night on Circadian Clocks and Metabolism[END_REF]. Or les températures en ville sont supérieures à celles des campagnes avoisinantes (Escourrou 1990;[START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013) et la luminosité artificielle est hautement corrélée à la densité humaine [START_REF] Small | Mapping Decadal Change in Anthropogenic Night Light. Spatial Statistics[END_REF].

plus, les caractéristiques environnementales associées à la zone urbaine comme s températures (Escourrou 1990;[START_REF] Pachauri | Bilan 2007 des changements climatiques : Rapport de synthèse du climat Genève[END_REF]Stocker et al. 2013), à la pollution chimique (Roux and Marra 2007;Fritsch et al. 2012) [START_REF] Gaston | Urban Ecology[END_REF][START_REF] Faeth | Urban biodiversity: patterns and mechanisms[END_REF]Pickett et al. 2011). Par exemple, les escargots sont moins présents en zone urbaine par rapport aux zones rurales voisines [START_REF] Lososova | Diversity of Central European urban biota: effects of human-made habitat types on plants and land snails[END_REF] alimen (Sturkie 1986;Solomon 1997;Nys 2010) coquilles des mésanges bleues et charbonnières sont plus pigmentées (Graveland 1996;Gosler et al. 2005;Jubb et al. 2006). La pigmentation serait due à la protoporphyrine produit lors de la dégradation des hématies (Baird et al. 1975). La protoporphyrine pourrait par sa structure (Gosler et al. 2005; Martínez-de la [START_REF] Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF][START_REF] García-Navas | Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus[END_REF]Gosler et al. 2011;Holveck et al. 2012;Magi et al. 2012;Hargitai et al. 2013) -ànt osseux (Sturkie 1986;Solomon 1997 La taille de ponte ne varie pas entre les populations pour la mésange charbonnière par contre, les pontes urbaines de mésanges bleues sont toujours plus petites que les pontes rurales. Les jeunes de mésanges bleues de Paris ont une condition corporelle similaire à celle des jeunes ruraux, par

le succès de reproduction pport expérimental

La température influence les organismes tout au long de leurs vies et peut même influencer la saisonnalité des comportements (Caro et al. 2013). Par exemple, la température semble jouer un rôle prédominant dans le déclenchement de la période de reproduction (Charmantier et al. 2008;Visser et al. 2009;Kluen et al. 2011;Nord and Nilsson 2011;Mainwaring et al. 2012;Naef-Daenzer et al. 2012;Schaper et al. 2012;Chmielewski et al. 2013). Les femelles de mésanges bleues et charbonnières assurent seules une température stable et suffisante pour le (Gosler 1993;Del Hoyo et al. 2007) par une femelle pour chauffer sa pont : taille et propriété thermique de la cavité et du nid, taille de la ponte, condition corporelle et investissement du mâle dans le nourrissage de la femelle, température extérieure. Lorsque la température augmente, la dépense énergétique pour incuber l diminue (pipit spioncelle (Anthus spinoletta) (Rauter and Reyer 2000), diamant mandarin (Nord et al. 2010) et mésange charbonnière (Bryan and Bryant 1999)). Comme la température est plus haute en ville (Stocker et al. 2013) et plus particulièrement pendant la nuit (Escourrou 1990 9A) correspondant au spectre de visibilité des mésanges (300 700 nm) (Endler 1990;Hart et al. 2000)). Afin de comparer les spectres ou entre individus, deux valeurs sont calculées sur chaque spectre : caroténoïdes chroma (Figure 9B) et la réflectance dans les UV (Figure 9C). Plus la valeur de caroténoïdes chroma augmente, plus la plume est jaune (Isaksson et al. 2005;Hill and McGraw 2006;Peters et al. 2007) et q [START_REF] Ornborg | Seasonal changes in a ultraviolet structural colour signal in blue tits, Parus caeruleus[END_REF]. La mesure de caroténoïdes chroma a été sélectionné lutéine (Hõrak et al. 2001) caroténoïdes des plumes (Hill and McGraw 2006) comparaison de populations urbaines et rurales (Isaksson et al. 2005;Isaksson et al. 2007 prédit que la date de ponte sera plus précoce en milieu urbain par rapport au milieu rural voisin, luminosité [START_REF] Dominoni | Artificial light at night advances avian reproductive physiology[END_REF]), de températures plus hautes (Charmantier et al. 2008;Visser et al. 2009;Kluen et al. 2011;Nord and Nilsson 2011;Mainwaring et al. 2012;Naef-Daenzer et al. 2012;Schaper et al. 2012;Chmielewski et al. 2013) et un avancement de la date de débourrage [START_REF] Møller | Climate change and micro-geographic variation in laying date[END_REF][START_REF] Schaper | Spring phenology does not affect timing of reproduction in the great tit (Parus major)[END_REF]. Les analyses de date de ponte ne montrent pas de différences en phénologie de reproduction pour les deux espèces entre (Figure 10) mais la phénologie varie entre les années. Le Figure

Accepté à Journal of Experimental Biology

When birds have expended large amounts of carotenoids to feather growth such as after autumn moult, bird health and oxidative status might be more constrained. We tested this hypothesis in a bird species with carotenoid-based plumage colour, by manipulating dietary carotenoids and physical activity, which can decrease antioxidant capacity and increase reactive oxygen metabolites (ROM) concentration. Great tits were captured after moult and kept in aviaries, under three treatments: physical handicap and dietary supplementation with carotenoids, physical handicap and control diet, and no handicap and control diet. We measured plasma composition (antioxidant capacity, ROM concentration, vitamin A, E and total carotenoid concentrations), immune system activation (blood sedimentation) and stress response (heterophil/lymphocyte ratio) and predicted that handicap treatment should influence these negatively and carotenoid supplementation positively. Colouration of yellow feathers was also measured. Carotenoid supplementation increased total plasma carotenoid concentrations, decreased feather carotenoid chroma and marginally increased ROM concentration. Handicap increased blood sedimentation only in males but had no clear influence on oxidative stress, which contradicted previous studies. Further studies are needed to investigate how physical activity and carotenoid availability might interact and influence

Introduction

Resource limitations and energetic costs of biological activities do not allow simultaneous maximization of all life history traits, and this leads to physiological trade-offs [START_REF] Stearns | The evolution of life histories[END_REF]. For example, the expression of carotenoid-based coloured signals may involve an allocation trade-off of these pigments between signalling, immune defence and antioxidant activity (Faivre et al., 2003). Indeed, carotenoids are plant pigments that typically produce red, orange and yellow animal colouration (Hill and McGraw, 2006) in different species notably in birds (Martin et al., 2008;Lopez et al., 2009;Calsbeek et al., 2010). Vertebrates cannot produce these molecules, and must acquire them from food (Hill and McGraw, 2006;Isaksson, 2009). In addition to their role in pigmentation, carotenoids have multiple physiological roles (Møller et al., 2000;Hill and McGraw, 2006) including regulating and stimulating immune function (Hõrak et al., 2004;[START_REF] Biard | Dynamics of PHA-induced immune response and plasma carotenoids in birds: should we have a closer look?[END_REF]Cote et al., 2010;Hill and Johnson, 2012) and participating in maintaining oxidative balance through their antioxidant properties (Surai and Speake, 1998;[START_REF] Blount | Why egg yolk is yellow[END_REF]Isaksson, 2009).

According to Sies (1997) oxidative stress is caused by an imbalance between oxidants and antioxidants in favour of the oxidants , potentially leading to cellular or tissue damage.

Reactive oxygen metabolites (ROM) i.e. oxidant molecules, are produced by cellular activity or exposure to radiation or pollutants (Finkel and Holbrook, 2000) and may cause a disruption of redox signalling and control and/or molecular damage [START_REF] Nikolaidis | Redox biology of exercise: an integrative and comparative consideration of some overlooked issues[END_REF]. Exogenous molecules with antioxidant capacity include some vitamins (e.g. vitamin E) and carotenoid pigments (Sies, 1997). A link between carotenoids and organism antioxidant capacity has been shown in birds (Surai and Speake, 1998;[START_REF] Blount | Why egg yolk is yellow[END_REF]Isaksson, 2009). Physical and chemical structure of carotenoids allow them to scavenge free radicals (oxidant molecules) by electron absorption and electron transfer reactions (Hill and McGraw, 2006). Hill and Johnson (2012) demonstrated that carotenoids in the endoplasmic reticulum stimulate redox reactions thereby protecting against oxidative stress. For example in laying hens or gulls carotenoid supplementation reduced levels of lipid peroxidation (i.e. oxidative damage) in egg yolk (Surai and Speake, 1998;Blount et al., 2002) and in passerine adults and nestlings plasma carotenoid concentrations were positively correlated with plasma vitamin A and E concentrations (Biard et al., 2005[START_REF] Biard | An analysis of pre-and post-hatching maternal effects mediated by carotenoids in the blue tit[END_REF] which are important lipid-soluble antioxidants [START_REF] Surai | Vitamin E in avian reproduction[END_REF][START_REF] Surai | Natural Antioxidants in Avian Nutrition and Reproduction[END_REF]. After an immune stimulation, plasma carotenoid concentration decreased in blackbirds Turdus merula [START_REF] Biard | Dynamics of PHA-induced immune response and plasma carotenoids in birds: should we have a closer look?[END_REF] and in zebra finches Taeniopygia guttata [START_REF] Alonso Alvarez | An Experimental Test of the Dose-Dependent Effect of Carotenoids and Immune Activation on Sexual Signals and Antioxidant Activity[END_REF]. Moreover, plasma carotenoid concentration was 4 MANUSCRIT 1 positively correlated with concentrations of lymphocytes and eosinophils in the great tit (Hõrak et al., 2004). Finally carotenoids also influence body condition. Indeed, in a dietary supplementation experiment, carotenoid-fed blue tit Cyanistes caeruleus nestlings were heavier at fledging than control-fed nestlings and in great tits the influence of carotenoid supplementation on fledging body mass depended on tarsus length (Biard et al., 2006).

It has been shown in several bird species that individuals that ingest more carotenoids or absorb and metabolise them better have brighter yellow plumage (Hill and McGraw, 2006). In the great tit, plasma carotenoid concentration peaks during moult [START_REF] Del Val | Seasonal variation of circulating plasma carotenoids in Mediterranean Great Tit males Parus major[END_REF], which lasts three weeks from mid-June to September in Europe (Gosler, 1993;Del Hoyo et al., 2007), and increased yellow feather pigmentation (Isaksson et al., 2007a). Furthermore, carotenoid-based plumage colour in birds has relatively low heritability [START_REF] Roulin | Genetics of colouration in birds[END_REF] and depends more on individual health and environment than genetic constitution. Indeed, immune challenge (parasitism or infection) decreases brightness of the yellow throat and ventral colour in wall lizards (Martin et al., 2008;Lopez et al., 2009;Calsbeek et al., 2010). Similarly in birds, immune challenge decreases carotenoid-based yellow bill colour in blackbirds (Faivre et al., 2003), red bill colour of zebra finches (Cote et al., 2010) and feather colour of great tit nestlings before fledging (Romero-Diaz et al., 2013). Thus, colouration may be a reliable signal of physiological or immune condition. However, although many studies document the positive relationship between colouration and body condition or immune system, not all confirm these findings (Hõrak et al., 2004;Isaksson et al., 2005;Geens et al., 2009). Mechanisms underlying seasonal variation in colouration are not yet well understood, but some hypotheses have been advanced, such as UV bleaching, bacterial or mechanical degradation [START_REF] Delhey | Seasonal Changes in Colour: A Comparison of Structural, Melanin-and Carotenoid-Based Plumage Colours[END_REF]. In great tits, brightness of the yellow colouration has been shown to increase with the size of the uropygial gland [START_REF] Galvan | Feather mite abundance increases with uropygial gland size and plumage yellowness in Great Tits Parus major[END_REF] which produces an oil that birds deposit on their feathers, its size and oil composition varying with microbiome composition [START_REF] Jacob | Uropygial gland size and composition varies according to experimentally modified microbiome in Great tits[END_REF]. Moreover, activation of the immune system may induce changes in antioxidant status. For example immune challenged nestlings of Falco tinnunculus showed higher ROM concentration and lower antioxidant capacity [START_REF] Costantini | Effects of T-cell-mediated immune response on avian oxidative stress[END_REF], and in old zebra finches total antioxidant activity decreased more for immune-activated birds than for control birds whereas the opposite pattern was observed for young birds (Cote et al., 2010). The potential signalling function of carotenoid-based colouration with regards to oxidative stress is still unclear. Indeed, while many studies did not find a significant correlation between oxidative status and colouration (Isaksson et al., 2007b;Isaksson and Andersson, 2008;Cote et al., 2010;[START_REF] Hõrak | Oxidative stress and information content of black and yellow plumage coloration: an experiment with greenfinches[END_REF], others documented a MANUSCRIT 1 negative correlation between colouration and oxidative damage [START_REF] Slagsvold | Influence of male and female quality on clutch size in Tits (Parus spp)[END_REF][START_REF] Sanz | Differential response by males and females to manipulation of partner contribution in the great tit (Parus major)[END_REF][START_REF] Leclaire | Behavioral and physiological responses to male handicap in chickrearing black-legged kittiwakes[END_REF][START_REF] Patterson | Glucocorticoids, individual quality and reproductive investment in a passerine bird[END_REF] due to the antioxidant activity of carotenoids (Møller et al., 2000).

We investigated the influence of dietary carotenoids on the antioxidant system, using the great tit, a small passerine common in Europe. This species displays carotenoid-based yellow ventral plumage (Partali et al., 1987) and is insectivorous in spring, caterpillars being the major source of these pigments during the reproductive season and just before moult (Isaksson, 2009), and mainly granivorous (seeds containing few carotenoids) during the rest of the year (Del Hoyo et al., 2007). In order to investigate the role of carotenoids in regulating oxidative stress, we manipulated carotenoid availability through dietary supplementation and measured plasma total antioxidant capacity, ROM concentration as a measure of potential oxidative damages, and concentrations of plasma carotenoids, vitamin A and vitamin E. To be able to examine the effect of carotenoids on oxidative stress, our study was conducted after moult because at this time there are few available carotenoids due to low insect abundance [START_REF] Sehhatisabet | Food diversity and niche-overlap of sympatric tits (Great Tit, Parus major, Blue Tit, Cyanistes caeruleus and Coal Tit Periparus ater) in the Hyrcanian Plain forests[END_REF][START_REF] Vel'ky | Food composition of wintering great tits (Parus major): habitat and seasonal aspects[END_REF], and any stored pigments should have been used for producing yellow feather colouration. Previous studies in species with carotenoid-based plumage colour showed that plasma carotenoid concentration was lower in winter (after moult) than before or during moult, which could be due to lower carotenoid availability and/or to depletion of carotenoid stores during feather growth (Isaksson et al., 2007a;[START_REF] Del Val | Seasonal variation of circulating plasma carotenoids in Mediterranean Great Tit males Parus major[END_REF]. Moreover birds cannot remobilise carotenoids that have already been deposited in feathers, unlike those used for bill or soft tissue colouration (Hill and McGraw, 2006). At the same time we manipulated physical activity by plucking (or not) three flight feathers from both wings, which should increase physical effort and thereby physiological stress [START_REF] Slagsvold | Influence of male and female quality on clutch size in Tits (Parus spp)[END_REF][START_REF] Sanz | Differential response by males and females to manipulation of partner contribution in the great tit (Parus major)[END_REF][START_REF] Leclaire | Behavioral and physiological responses to male handicap in chickrearing black-legged kittiwakes[END_REF][START_REF] Patterson | Glucocorticoids, individual quality and reproductive investment in a passerine bird[END_REF] and thus modify antioxidant balance. Indeed, intense activity is known to generate oxidative stress [START_REF] Nikolaidis | Redox biology of exercise: an integrative and comparative consideration of some overlooked issues[END_REF]. A non-linear increase in metabolic rate caused by intense physical activity has been shown to increase ROM concentration by increasing activity in electrontransfer chains and hydrogen escape (Sies, 1997;Metcalfe and Alonso-Alvarez, 2010;Isaksson et al., 2011). Physiological stress is a sign of disturbance of homeostasis [START_REF] Bradshaw | Vertebrate Ecophysiology: An Introduction to Its Principles and Applications[END_REF] and is often reflected by an increase in plasma corticosterone and heterophil concentrations and a decrease in plasma lymphocyte concentration (Ots et al., 1998;Davis et al., 2008). Here, the ratio of heterophils to lymphocytes (hereafter H/Ly) was used as an index of physiological stress (Ots et al., 1998;Davis et al., 2008), and blood sedimentation rate was used as an index of immune system activation (Glick, 2000). Finally, because plumage colour 6 MANUSCRIT 1 has recently been shown to be a dynamic trait [START_REF] Galvan | Feather mite abundance increases with uropygial gland size and plumage yellowness in Great Tits Parus major[END_REF][START_REF] Delhey | Seasonal Changes in Colour: A Comparison of Structural, Melanin-and Carotenoid-Based Plumage Colours[END_REF], in order to test for any potential effect of our treatment, we decided to measure feather colour at the beginning and at the end of the experiment. Since metabolism is linked with body size [START_REF] Peters | The Ecological Implications of Body Size[END_REF] and physical activity modifies body condition (Verspoor et al., 2007), body mass and tarsus length were used as covariates.

We predicted that (1) physical activity would increase ROM concentration and physiological stress and decrease antioxidant capacity (2) an increase in dietary carotenoids would increase carotenoid concentration in plasma, immune activation and antioxidant capacity and decrease ROM concentration.

Results

Table 1 presents all mean ± SE values separately for males, females and treatment groups at the start and at the end of the experiment.

1-Plasma composition

Total carotenoid concentration was significantly influenced by the interaction between time and dietary carotenoid supplementation (F 1,33 = 14.61, p < 0.01, supplementary Table 1, Figure 1) and by the interaction between time and handicap (F 1,33 = 4.67, p = 0.03, supplementary Table 1). Post-hoc tests showed that carotenoid supplementation increased total plasma carotenoid concentration (all t 1,71 < 3.17, p < 0.01) and that plasma carotenoid concentration in handicapped and non-handicapped birds did not significantly differ at the end of the experiment (t 1,71 = -2.29, p = 11). However, non-handicapped birds had higher total plasma carotenoid concentration at the end than other groups at the start of the experiment (t 1,71 < -3.23, p < 0.01, H-End > H-Start and H+ Start, H+ End not being significantly different from other groups).

Variation in plasma total vitamin E and retinol concentrations were not significantly explained by any variable (supplementary Table 1).

Plasma antioxidant capacity did not differ between carotenoid supplementation groups but differed between sexes (F 1,28 = 17.72, p < 0.01, supplementary Table 2) and between handicapped and control birds in interaction with time (F 1,39 = 4.97, p = 0.03, supplementary Table 2). Total plasma antioxidant capacity decreased more over time for the nonhandicapped group (post-hoc test: t 1,77 = 5.59, p < 0.01) than for the handicapped group (posthoc test: t 1,77 = 3.94, p = 0.01). We found no significant difference between handicap MANUSCRIT 1 treatments at the start (post-hoc test: t 1,77 = -0.16, p = 0.99) and a marginal difference at the end of the experiment (post-hoc test: t 1,77 = 2.37, p = 0.09).

ROM concentration did not significantly differ between handicap treatments, but its variation was explained by the interaction between carotenoid supplementation and time (F 1,39 = 3.80, p = 0.05, supplementary Table 2,Figure 2). At the end of the experiment, birds supplemented with carotenoids had marginally higher ROM concentration than non-supplemented birds (post-hoc test: t 1,39 = 1.95, p = 0.06).

At the start of the experiment, plasma carotenoid concentration was positively correlated with antioxidant capacity (r Pearson = 0.47, t 1,37 = 3.22, p < 0.01) and was not correlated with ROM concentration (r Pearson = 0.26, t 1,37 = 1.62, p = 0.11), whereas antioxidant capacity was positively correlated with ROM concentration (r Pearson = 0.50, t 1,39 = 3.65, p < 0.01). Plasma total vitamin E and retinol concentrations were not correlated with antioxidant capacity or ROM concentration (-0.15 < r Pearson < 0.04, t 1,39 < 0.30, p > 0.30).

At the end of the experiment, however, total antioxidant capacity and carotenoid concentration were no longer significantly correlated (r Pearson = 0.25, t 1,36 = 1.55, p = 0.13), ROM concentration and carotenoid concentration were positively correlated (r Pearson = 0.32, t 1,36 = 1.99, p = 0.05 n.s. for table-wide significance after Bonferroni correction) and antioxidant capacity was no longer significantly correlated with ROM concentration (r Pearson = 0.17, t 1,39 = 1.06, p = 0.29). Plasma total vitamin E and retinol concentrations were not correlated with antioxidant capacity or ROM concentration (-0.07 < r Pearson < 0.13, t 1,38 < 0.80, p > 0.40).

2-Immune system activation and stress response

Blood sedimentation rate significantly increased with body mass (F 1,38 = 3.71, p = 0.05, estimate = 0.02 ± 0.01), was lower at the end of the experiment (F 1,38 = 20.20, p < 0.01, Start = 0.78 ± 0.01 versus End = 0.71 ± 0.01) and was influenced by the interaction between sex and handicap (F 1,27 = 8.83, p < 0.01, supplementary Table 3). Handicapped males had higher blood sedimentation rate than non-handicapped males (Table 1, post-hoc test: t 1,75 = -3.26, p = 0.01) whereas females did not significantly differ between groups (post-hoc test: t 1,75 = 0.95, p = 0.78). Non-handicapped males had lower blood sedimentation rates than non-handicapped females (post-hoc test: t 1,75 = 3.43, p < 0.01) and handicapped females (post-hoc test: t 1,75 = -2.59, p = 0.06).

H/Ly ratio was not influenced by handicap but varied significantly with the interaction between sex and carotenoid supplementation (F 1,27 = 5.36, p = 0.02, , Table 1 andMANUSCRIT 1 supplementary Table 3). Post-hoc tests revealed that males had marginally lower H/Ly than females in the supplemented group (post-hoc test: t 1,73 = 1.95, p = 0.06) but no significant difference between sexes in the other group (post-hoc test: 0.90 > t 1,73 > -1.69, p > 0.10).

3-Colour parameters

Variation in lutein absorbance was significantly explained by the interaction between sex and time (F 1,39 = 4.67, p = 0.03, supplementary Table 4), while carotenoid supplementation and handicap were not retained in the final model. At the end of the experiment, lutein absorbance ratio was significantly higher for males than for females (supplementary Table 4, post-hoc test: t 1,78 = -3.12, p = 0.01), i.e. males were more yellow than females, whereas at the start of experiment values for males and females did not significantly differ (post-hoc tests: t 1,78 > -1.87, p > 0.25).

Larger birds had higher carotenoid chroma (F 1,28 = 7.72, p < 0.01, estimate = 0.03 ± 0.01, supplementary Table 4) which also varied with the interaction between dietary carotenoid supplementation and time (F 1,37 = 4.36, p = 0.04). Surprisingly, breast feathers of supplemented birds had lower carotenoid chroma at the end of the experiment than those of non-supplemented birds (Figure 3, post-hoc test: t 1,74 = 2.66, p = 0.04), i.e. supplemented tits were less yellow than controls.

In the final model investigating variation in UV brightness, both the interaction between handicap and time (F 1,39 = 6.50, p = 0.01) and that between handicap and sex (F 1,27 = 6.80, p < 0.01) were significant. Handicapped birds had lower UV brightness at the end of experiment than at the start (post-hoc test: t 1,79 = 4.54, p < 0.01, Figure 4) but there being no difference in non-handicapped individuals between beginning and end of the experiment (post-hoc tests: t 1,26 < 1.40, p > 0.50). Among non-handicapped birds, males had greater UV brightness than females (Table 1, post-hoc test: t 1,76 = -2.93, p = 0.02) but there was no difference between males and females in the handicapped group (post-hoc test: t 1,76 = 0.39, p = 0.98).

Discussion

Carotenoids are known to regulate and stimulate immune function (Hõrak et al., 2004;[START_REF] Biard | Dynamics of PHA-induced immune response and plasma carotenoids in birds: should we have a closer look?[END_REF]Cote et al., 2010;Hill and Johnson, 2012) and to participate in reducing oxidative stress (Surai and Speake, 1998;[START_REF] Blount | Why egg yolk is yellow[END_REF]Isaksson, 2009). We experimentally investigated the influence of dietary carotenoids on oxidative stress in the great tit after moult, by simultaneously manipulating carotenoid availability and physical activity, the latter being MANUSCRIT 1 an attempt to increase oxidative stress as a result of an increase in metabolism. Our study revealed several results in contradiction with our initial predictions. First, increasing physical activity did not affect ROM concentration but increased total antioxidant capacity, prevented the increase in total carotenoid concentration during the experiment and decreased UV brightness (Table 2). Second, although carotenoid supplementation increased total carotenoid concentration as expected, it did not increase antioxidant capacity but slightly increased ROM concentration and decreased a parameter of feather colour, carotenoid chroma (Table 2).

A previous study conducted on birds showed that flight activity increases ROM concentration [START_REF] Costantini | Loss of integration is associated with reduced resistance to oxidative stress[END_REF]. Indeed, in zebra finches, when flight activity in a test cage was increased by moving perches during two hours, immediate ROM concentration increased but there was no change in antioxidant capacity, as measured by the concentration of antioxidant enzymes [START_REF] Costantini | Loss of integration is associated with reduced resistance to oxidative stress[END_REF]. In the present study, flight activity was increased by plucking three primary feathers from each wing. This did not influence ROM concentration but did influence total antioxidant capacity. Indeed there was a general decrease in total antioxidant capacity over the course of the experiment, and this decrease was greater for control than for handicapped birds. This suggests that the handicap treatment up-regulated the antioxidant system. However, the absence of a detectable effect on ROM concentration could be due to the time scale. Our study was conducted over three weeks and thus measured long term, and not immediate, variation in ROM concentration (as in [START_REF] Costantini | Loss of integration is associated with reduced resistance to oxidative stress[END_REF]).

Moreover, food was given ad libitum and might be more caloric and/or richer in antioxidants, than that found in natural conditions at that time of year [START_REF] Sanz | Differential response by males and females to manipulation of partner contribution in the great tit (Parus major)[END_REF][START_REF] Patterson | Glucocorticoids, individual quality and reproductive investment in a passerine bird[END_REF], which could have buffered the expected effect of plucking some wing feathers on ROM concentration. Birds may not have been significantly constrained with regards to the availability of energy or antioxidants that are needed to activate repair pathways (Finkel and Holbrook, 2000;[START_REF] Larcombe | The effects of short-term antioxidant supplementation on oxidative stress and flight performance in adult budgerigars Melopsittacus undulatus[END_REF][START_REF] Larcombe | Impacts of dietary antioxidants and flight training on post-exercise oxidative damage in adult parrots[END_REF] and may have thus overcome the physical handicap without increasing ROM concentration. However, the effects of physical activity on ROM concentration may also depend on other factors such as age (Finkel and Holbrook, 2000;[START_REF] Costantini | Long flights and age affect oxidative status of homing pigeons (Columba livia)[END_REF], or pollutants (Koivula and Eeva, 2010). Moreover, we found that in males but, not in females, physical handicap increased blood sedimentation rate. It has been reported that in birds, females have a higher blood sedimentation rate than males ( (Glick, 2000) but see [START_REF] Svensson | Molt and migratory condition in Blue Tits: A serological study[END_REF]), which was indeed the case at the start of the experiment. The higher female blood sedimentation rate may be related to a higher haematocrit of female than male great tits ( (Hõrak et al., 1998;MANUSCRIT 1 Glick, 2000;[START_REF] Kilgas | Hematological parameters in broodrearing great tits in relation to habitat, multiple breeding and sex[END_REF] but see for other species [START_REF] Fair | Sources of variation in haematocrit in birds[END_REF]). This may have rendered males more sensitive to the handicap treatment than females, whose blood sedimentation rate was not significantly affected over the course of the experiment.

Physical handicap decreased breast feather UV brightness during the experiment. Variation in UV colour is determined by feather structure and quality (Griffith et al., 2003;Limbourg et al., 2004;Limbourg et al., 2013) but also by the presence or composition of uropygial gland secretions or the community of feather bacteria or mites (Hill and McGraw, 2006;[START_REF] Pérez-Rodríguez | The effects of preen oils and soiling on the UV visible reflectance of carotenoid-pigmented feathers[END_REF]. For example, when preen oil was removed from feathers of Bohemian waxwings (Bombycilla garrulus), UV chroma increased, suggesting a direct effect of preen oil on structural colour [START_REF] Pérez-Rodríguez | The effects of preen oils and soiling on the UV visible reflectance of carotenoid-pigmented feathers[END_REF]. Feather plucking may thus have modified uropygial gland functioning and/or induced an infection, as feather mite and microbiome abundance have been shown to influence uropygial gland size and functioning [START_REF] Galvan | Feather mite abundance increases with uropygial gland size and plumage yellowness in Great Tits Parus major[END_REF][START_REF] Jacob | Uropygial gland size and composition varies according to experimentally modified microbiome in Great tits[END_REF]) and, handicapped males showed higher blood sedimentation rate. However, the effect of the handicap treatment on UV chroma affected females similarly to males, though the former showed no parallel effect on blood sedimentation. This suggests the effect of the handicap treatment may have been mediated by a change in preening behaviour and increase in the deposition of uropygial secretions to feathers.

Surprisingly, dietary carotenoid supplementation directly or indirectly decreased an aspect of yellow plumage colouration, carotenoid chroma, during the experiment. Feathers are passive structures and variation in their colouration is due to pigment composition and concentration which reflects the type and quantity of carotenoids ingested during moult (Hill and McGraw, 2006;Peters et al., 2007). Feather colouration may also change after moult [START_REF] Galvan | Feather mite abundance increases with uropygial gland size and plumage yellowness in Great Tits Parus major[END_REF][START_REF] Delhey | Seasonal Changes in Colour: A Comparison of Structural, Melanin-and Carotenoid-Based Plumage Colours[END_REF]. Few studies explored variation in carotenoid-based colour after moult (Hill and McGraw, 2006;[START_REF] Peters | Condition-dependence of multiple carotenoid-based plumage traits: an experimental study[END_REF][START_REF] Delhey | Seasonal Changes in Colour: A Comparison of Structural, Melanin-and Carotenoid-Based Plumage Colours[END_REF]. Previous studies have experimentally shown that feathers exposed to sunlight became less yellow [START_REF] Surmacki | Preen waxes do not protect carotenoid plumage from bleaching by sunlight[END_REF][START_REF] Surmacki | Effects of sunlight exposure on carotenoid-based and structural coloration of the blue-tailed bee-eater[END_REF] probably due to an effect of pigment oxidation caused by photon absorption. In our experiment, carotenoid supplementation increased ROM concentration in plasma and may also have influenced other parameters that were not measured here, such as uropygial secretions, which may have indirectly induced the observed decrease in carotenoid chroma. Our results showed that dietary carotenoid availability affected yellow plumage coloration also after moult, which was never shown to our knowledge. Although the mechanism is yet unknown, as females may select more colourful males (Gosler, 1993;Hill MANUSCRIT 1 and McGraw, 2006), dietary carotenoids availability after moult may also influence partner choice in the next reproductive season.

Previous studies on birds have shown that carotenoid-supplemented females produced eggs with higher carotenoid concentration and lower lipid peroxidation in yolk (Surai and Speake, 1998;Blount et al., 2002) and a negative correlation between carotenoid concentration and lipid peroxidation in plasma (Woodall et al., 1996;[START_REF] Hõrak | Do Dietary Antioxidants Alleviate the Cost of Immune Activation? An Experiment with Greenfinches[END_REF]. However we did not find the expected increase in total antioxidant capacity or decrease in ROM concentration with carotenoid supplementation. We even found a slight ROM concentration increase. There are two possible explanations for these counterintuitive results. First the marginal increase in ROM concentration might be explained by inappropriately high dose of supplementary antioxidant (Hill and McGraw, 2006;Hill and Johnson, 2012). We can reject this hypothesis because blood assays revealed that total carotenoid concentration in the plasma of supplemented birds was within the natural range and lower than previously reported in the same and/or closely related species during the breeding period (Hõrak et al., 2004;Biard et al., 2005;Isaksson et al., 2007a) and in autumn (Isaksson et al., 2007a). Another possibility could be that increased availability of carotenoids in the diet might promote short-term benefits for the antioxidant system through decreased ROM concentration [START_REF] Larcombe | The effects of short-term antioxidant supplementation on oxidative stress and flight performance in adult budgerigars Melopsittacus undulatus[END_REF] but might also induce long-term effect on ROM concentration by lowering the stimulation of antioxidant capacity (Metcalfe and Alonso-Alvarez, 2010). Metcalfe and Alonso-Alvarez (2010) proposed that reactive oxygen species could be regulating agents that continually stimulate antioxidant defences to allow an efficient response in the case of an important oxidative challenge.

In summary, physical activity did not increase ROM concentration but increased antioxidant capacity and decreased breast feather UV reflectance, while carotenoid supplementation decreased breast feather carotenoid chroma and slightly increased ROM concentration.

Complex influences of dietary carotenoid and physical activity on ROM concentration were uncovered, and further research should seek to elucidate how physical activity may or may not increase ROM concentration depending on diet quality. Moreover, how diet quality affects change in yellow colour once feathers are fully grown and whether this could be mediated by oxidative stress is still unclear and would deserve further studies. Finally, our results showed that the availability of carotenoids in the diet is not only essential during moult for the development of coloured signals, but may also play a role outside this period, with potential consequences for sexual attractiveness and reproductive investment, few months later.

Materials and Methods

1-Biological model and rearing conditions

The great tit is a small common insectivorous passerine found in Europe except at high altitudes and latitudes. This species breeds in artificial cavities such as nestboxes, so its demography, life-history and behaviour have been intensively studied in natural populations (Hõrak et al., 2004;Biard et al., 2006;Quesada and Senar, 2007;Dauwe and Eens, 2008;Isaksson, 2009;[START_REF] Evans | Pigments versus structure: examining the mechanism of age-dependent change in a carotenoid-based colour[END_REF].

Great tits were caught with mist nets from 22 to 31 November 2012 in a private forest in a rural area located 80 km south of Paris, at the Centre de Recherche en Ecologie . Twenty-three males and 19 females were caught and kept in outdoor aviaries (3 * 3 * 4 m), 4 adults in each of 9 aviaries and 2 adults in each of 3 aviaries. One female died before the start of the experiment but no deaths occurred during the experiment. Adults captured the same day were assumed to belong to the same winter flock and therefore were housed together in the same aviary independent of the sex-ratio. Indeed, outside the breeding season, passerines form roaming flocks [START_REF] Aplin | Social networks predict patch discovery in a wild population of songbirds[END_REF] and we preferred to maintain previously established groups in order to avoid fights in the aviaries. However, there were always members of several flocks mixed in each aviary captured same days. Birds were fed with mealworms -carotene, 0.2 ± 0.3 mg/kg of lutein and no zeaxanthin, [START_REF] Oonincx | An Investigation Into the Chemical Composition of Alternative Invertebrate Prey[END_REF] mixed with a premix of proteins, vitamins, minerals, amino acids and trace elements (NutriBird A19 Versele Laga), sunflower seeds (containing 1 mg/kg carotenoids, [START_REF] Peters | Condition-dependence of multiple carotenoid-based plumage traits: an experimental study[END_REF], and crushed peanuts mixed with a premix of fat components, vitamins, minerals, amino acids and trace elements (Orlux Uni Pâtée Premium Versele Laga, containing -carotene as indicated by the manufacturer). Water and food were given ad libitum. Water was changed every day and food was changed every three or four days. Birds were held in captivity between one and two weeks before the onset of the experiment to allow acclimatization. Each outdoor aviary contained many freshly cut branches and nests boxes (one for each bird).

On the day of capture, individuals were individually identified with a numbered aluminium ring (National Museum of Natural History), tarsus length was measured and eight breast feathers were taken to measure plumage reflectance (see § Plumage Reflectance).

2-Experimental design MANUSCRIT 1

We used a crossed design in which dietary carotenoid availability and physical activity were manipulated. A solution of carotenoids was added to the drinking water to a final concentration of 50 µg/g: 0.2 g of FloraGlo (5% lutein and 0.86% zeaxanthin, DSM France) in 200 mL of water. During the acclimatization phase, we observed that great tits drank about 5 g of water per day. In addition, over the course of the experiment, we observed that less than 20 g of water was drunk per day in each aviary (i.e. by 2 to 4 birds altogether). With a concentration of 50 µg/g carotenoids in the drinking water, birds therefore ingested a total of 50 to 500 µg carotenoids per day. In the same species, carotenoid concentration in plasma is recorded at about 30 µg/mL for a body mass of around 18g (Hõrak et al., 2004;Isaksson et al., 2007a) thus total quantity of circulating carotenoids could be estimated as 54 µg considering blood to represent 10% of body mass (Sturkie, 1986). In addition, it is known from supplementation experiments in poultry that the efficiency of carotenoid absorption in birds is about 20% of the used dose [START_REF] Surai | Carotenoids in avian nutrition and embryonic development. 1. Absorption, availability and levels in plasma and egg yolk[END_REF][START_REF] Surai | Natural Antioxidants in Avian Nutrition and Reproduction[END_REF]. Daily carotenoid intake should therefore have stayed within a physiological and natural range. Water was changed every day in all experimental groups at the same time of the day (11:00). There was no difference in water consumption between birds given carotenoid-supplemented water and control water (personal observation).

In order to induce a physical handicap, flight ability was decreased by plucking the fifth, seventh and ninth primary feathers from both wings [START_REF] Chai | Hummingbird hovering energetics during moult of primary flight feathers[END_REF]Verspoor et al., 2007).

Plucking these feathers has been shown to decrease body condition ( [START_REF] Sanz | Differential response by males and females to manipulation of partner contribution in the great tit (Parus major)[END_REF][START_REF] Patterson | Glucocorticoids, individual quality and reproductive investment in a passerine bird[END_REF] but see also [START_REF] Love | Manipulating developmental stress reveals sexspecific effects of egg size on offspring phenotype[END_REF]). Plucking feathers induces their regrowth, whereas clipping does not so our treatment manipulated flight ability, and consequently physical activity, and at the same time increased energy demand for feather growth (Sturkie, 1986;[START_REF] Chai | Hummingbird hovering energetics during moult of primary flight feathers[END_REF].

One week prior to the experiment, the adults were divided into three experimental groups: (i) carotenoid supplementation and physical handicap (C+/H+), (ii) no dietary supplementation with physical handicap (C-/H+) and (iii) control with no supplementation and no handicap (C-/H-). The experimental design could not be fully crossed and the treatment C+/H-was missing due to the lower than expected number of birds captured. Three aviaries with 4 adults (except one aviary with 3 adults in C-/H+) and one aviary with 2 adults per treatment were used. There were 8 males and 6 females in C+/H+, 8 males and 5 females in C-/H+ and 7 males and 7 females in C-/H-. Given that groups were composed before the start of the experiment, some aviaries had a biased sex-ratio due to the respected flock structure.

However, sex-ratio was similar in each treatment. MANUSCRIT 1

On the first experimental day, all birds were recaptured with mist nets (4 bags 210/1 16 mm mesh, Bonardi, Italy) in the aviaries. Blood samples were taken (See Laboratory analyses), body mass was measured and for experimental groups with handicap, the three wing feathers were plucked from each wing.

We ran the experiment for 21 days to ensure a sufficient feather growth without reducing the time left in the season for prospecting reproductive habitats (Isaksson and Andersson, 2008).

At the end of the experiment, a blood sample, a breast feather sample and morphometric measures (tarsus length and body mass) were taken. Variation in feather regrowth, measured using pictures of the right wing taken at the end of the experiment, was not explained by values of morphology (tarsus length: F 1,19 = 2.06, p = 0.17 and initial body mass: F 1,19 = 0.06, p = 0.94), sex (F 1,19 = 0.23, p = 0.64) or carotenoid supplementation (F 1,19 = 0.52, p = 0.45).

Great tits were released at the place where they had been caught.

3-Laboratory analyses

Blood samples of up to 250 µL were taken at the beginning and the end of the experiment from the brachial vein in heparinized capillary tubes for haematocrit, and a 1 or 2 µL drop of blood was smeared on a microscope slide for blood cell counts. Blood samples were placed at 4°C and left upright to measure sedimentation after 8h, i.e. the ratio of red blood cells to total blood volume, as an index of immune system activation (Glick, 2000;Campbell and Ellis, 2007) the number of leucocytes increases due to stress and infection (Glick, 2000;Campbell and Ellis, 2007), leading to elevated levels of immunoglobulins and fibrinogen in the blood (Sturkie, 1986;[START_REF] Svensson | Molt and migratory condition in Blue Tits: A serological study[END_REF]. Blood samples were centrifuged for 8 min at 1 000 rpm, and plasma was extracted and kept at -20°C until later laboratory analyses of antioxidant capacity.

Smears were air-dried, fixed in absolute methanol and stained with Giemsa (Sigma Giemsa modified 0.4% p/v) (Biard et al., 2010), then examined under oil immersion at 1 000x magnification to count the proportion of different types of leucocytes in a total of 50 leucocytes per smear in random order by the same person (MV). Only a total of 50 leucocytes could be counted (and not 100 as in other studies (Ots et al., 1998;[START_REF] Biard | An analysis of pre-and post-hatching maternal effects mediated by carotenoids in the blue tit[END_REF]Peters et al., 2007;Verboven et al., 2009) due to a lack of leucocytes (mean ± SE leucocytes per examination of 10 000 erythrocytes = 26 ± 17 as compared to other studies in great tits: 63 ± 3 (Peters et al., 2007) and 50 ± 23 (Ots et al., 1998)). Leucocyte counts can vary among populations [START_REF] Kilgas | Hematological parameters in broodrearing great tits in relation to habitat, multiple breeding and sex[END_REF]Davis et al., 2008), and the stress of captivity or seasonality MANUSCRIT 1 can cause low leucocyte counts (Hõrak et al., 1998;Davis et al., 2008;Bokony et al., 2012), but this is an unlikely explanation for these observation because we have found similar leucocyte counts in this same population during the reproductive period. Repeatability scores of our counts were highly significant (H/Ly ratio F 91,92 = 8.28, p < 0.01, intraclass correlation coefficient = 0.78 (Lessells and Boag, 1987)). High H/Ly ratio is often interpreted as a sign of physiological stress (Ots et al., 1998;Lobato et al., 2005;Campbell and Ellis, 2007), and for example, sustained flight has been shown to increase H/Ly [START_REF] Matson | Intense flight and endotoxin injection elicit similar effects on leukocyte distributions but dissimilar effects on plasma-based immunological indices in pigeons[END_REF].

The level of oxidative stress was measured through plasma total antioxidant capacity and ROM concentration test (d-ROMs test / Diacron, Grosseto, Italy) measures the concentration of hydroperoxides, which derive from the oxidation of several molecular substrates [START_REF] Miller | Oxidative Stress, Antioxidants, and Animal Function[END_REF][START_REF] Beckman | The Free Radical Theory of Aging Matures[END_REF]. Plasma (4 µL) was diluted 1:100 with acetate buffer (pH 4.8), mixed with 2 µL of N,N-diethyl-paraphenilenediamine and incubated for 75 min at 37°C. The acidic pH favours the release of iron and copper ions from plasma proteins. These metals catalyse the cleavage of ROOH leading to the generation of alkoxyl and alkyperoxyl radicals, two highly reactive and histolesive pro-oxidants. When these compounds react with N,N-diethyl-paraphenilenediamine they produce a complex whose intensity of pink is directly proportional to their concentration. The absorbance was read at 505 nm. The concentration of reactive oxygen metabolites (ROMs) was calculated according to a calibration curve obtained by measuring the absorbance of a standard solution and expressed as µmol of H 2 O 2 equivalents. The concentration of ROMs in two replicated subsamples was highly repeatable (F 82,83 = 1044.8, p < 0.01, intraclass correlation coefficient = 0.99). MANUSCRIT 1

Carotenoids and vitamins A and E were extracted and identified by high performance liquid chromatography as described elsewhere [START_REF] Ewen | Influences of carotenoid supplementation on the integrated antioxidant system of a free living endangered passerine, the hihi (Notiomystis cincta)[END_REF]. In brief,

4-Plumage colouration

Four replicate reflectance spectra were obtained from two sets of four feathers from each bird arranged in random order [START_REF] Quesada | Comparing plumage colour measurements obtained directly from live birds and from collected feathers: the case of the great tit Parus major[END_REF], using a USB 2000 + spectrometer with a DH 2000 deuterium-halogen light source (Ocean Optics, IDIL, Lannion, France) (Biard et al., 2005). We used a bifurcated fibre-optic probe (Ocean Optics QR200-7-SR-BX Premium 200µm) on which a cylindrical black plastic sheath was mounted to exclude ambient light and standardize measuring distance. Reflectance was calculated relative to a white standard (Ocean Optics spectralon WS-1-SL) with SpectraSuite software (Ocean Optics). Every three individuals, a dark and a white standards scan were obtained. Samples were taken randomly with respect to individual, aviary, treatments and time of sampling.

Three different parameters describing the variation in spectral shape were calculated for each spectrum. For great tit the visible spectrum ranges from 300 thought about 700 nm (Endler, MANUSCRIT 1 1990;Hart et al., 2000). As we were interested in colour produced by carotenoids, we absorbance, referred to as carotenoid chroma, as the difference in reflectance between maximum reflectance at the plateau of the spectrum and minimum reflectance at the wavelengths of maximum carotenoid absorption, divided by maximum reflectance ((R 700 -R 450 )/R 700 ) (Peters et al., 2007). As lutein is the main carotenoid present in great tit breast feathers (Örnborg, 2002;Isaksson and Andersson, 2007;Isaksson et al., 2007a), we measured lutein absorbance, calculated from mean reflectance in the 445-455 nm interval as -log 10 (mean reflectance) (Hõrak et al., 2001). Carotenoid chroma and lutein absorbance increase when carotenoid (lutein, zeaxanthin, beta-carotene, violaxanthin and neoaxanthin) or lutein content in feathers increases (Hill and McGraw, 2006), thereby giving yellower feathers (Hõrak et al., 2001;Örnborg, 2002;Peters et al., 2007). Feather structure, which causes variation in UV reflectance, is also a reliable cue of individual quality (Griffith et al., 2003;Limbourg et al., 2004;Limbourg et al., 2013). We thus calculated as the proportion of UV light reflected (300-400 nm reflectance divided by total reflectance (300-700nm)).

Repeatability of parameters within individuals and within sets of feathers within individuals, calculated as the in within sets of feathers (4 times): p<0.01, F 173,523 > 7.62, r Pearson > 0.55; within individuals (8 times): p<0.01, F 86,610 > 10.65, r > 0.65). For each individual we calculated carotenoid chroma, lutein absorbance and UV brightness, which were not inter-correlated (p > 0.10, -0.27 < r Pearson < 0.06), at the start and at the end of experiment.

5-Statistical analyses

All statistical analyses were performed in R v. Carotenoid supplementation increased plasma total carotenoid concentration at the end of the experiment. Significant differences between groups are indicated by different letters. Figure 3: Mean ± SE of carotenoid chroma as a function of carotenoid supplementation groups (black: control and grey: supplemented group) and time. Breast feathers of birds in the supplemented group were less yellow at the end of the experiment than they were at the start.

Significant differences between groups are indicated by different letters.

Introduction

Predicting the responses of organisms to environmental changes is an important challenge to ecologists and evolutionary biologists. In recent decades, global change has had a dramatic impact on the life history traits of organisms and ultimately on population dynamics (Stocker et al. 2013, Charmantier andGienapp 2014). Urban centres are the primary source of greenhouse-gas emissions and thus are implicated in global climate change (Stocker et al. 2013). The best-documented example of anthropogenic microclimate modification is the urban heat island effect: cities tend to have higher temperatures than rural areas especially at night (Escourrou 1990). Environmental temperature influences organisms in many ways, and in particular, temperature has a strong effect on phenology (Caro et al. 2013). Indeed, endotherms use environmental temperatures as a clue for fine-tuning the timing of reproduction or moult (Caro et al. 2013). In European blue tits (Cyanistes caeruleus) and great tits (Parus major), laying (Charmantier et al. 2008, Chmielewski et al. 2013, Kluen et al. 2011, Mainwaring et al. 2012, Naef-Daenzer et al. 2012, Nord and Nilsson 2011, Schaper et al. 2012, Visser et al. 2009) and hatching (Charmantier et al. 2008, Chmielewski et al. 2013) dates are negatively correlated with temperature. Moreover, low temperature during the laying phase is associated with delayed hatching of blue tits broods (Kluen et al. 2011, Nord andNilsson 2011).

Mothers can influence developmental temperature of their offspring by thermoregulation in viviparous species such as ewes [START_REF] Laburn | Effects on fetal and maternal body temperatures of exposure of pregnant ewes to heat, cold, and exercise[END_REF] or by modulating incubation behaviour in birds [START_REF] Webb | Thermal tolerance of avian embryos: a review[END_REF]). Incubation behaviour is thus crucial in the reproduction of egg-laying 4 MANUSCRIT 2 species. Maintaining temperatures within a narrow range is essential for embryonic development and exposure to temperatures outside optimal ones can be lethal to embryos or can reduce their success [START_REF] Gluckman | Early life events and their consequences for later disease: A life history and evolutionary perspective[END_REF]. Any variation in incubation temperature in the nest induces a change in nestling metabolism and also modifies incubation duration [START_REF] Webb | Thermal tolerance of avian embryos: a review[END_REF]. Finally incubation conditions may affect reproductive performance as observed in blue and great tits over a hundred-years period, during which fledging success was negatively influenced by average January to April environmental temperatures (Naef-Daenzer et al. 2012). Similarly, in blue tits, experimentally lowered incubation temperatures during eight or nine days decreased hatchling success, nestling tarsus length and increased nestling resting metabolic rate (Nord and Nilsson 2011). In contrast, the influence of temperature variation between hatching and fledging seems more complex, since it has been shown in the spotless starling (Sturnus unicolor) to depend on laying date; with a positive effect of increasing temperatures on wing and bill size in first clutches and a negative effect on nestling morphology in second clutches [START_REF] Salaberria | Effects of temperature and nest heat exposure on nestling growth, dehydration and survival in a Mediterranean hole-nesting passerine[END_REF]. Reproductive success increased when incubation temperatures were stable and high, but incubation or rearing temperatures above an optimum increased nestling dehydration and decreased fledging success [START_REF] Salaberria | Effects of temperature and nest heat exposure on nestling growth, dehydration and survival in a Mediterranean hole-nesting passerine[END_REF]). More variable and greater incubation temperature resulted in tree swallow (Tachycineta bicolor) nestlings depositing more corticosterone in their feathers [START_REF] Fairhurst | Nestling corticosterone response to microclimate in an altricial bird[END_REF], which suggests that temperature variation induced a stress response in nestlings [START_REF] Romero | Physiological stress in ecology: lessons from biomedical research[END_REF]). These negative effects of high or fluctuating temperature on the developing embryos can be mitigated through the behaviour of parents, especially by mothers in species where incubation is mainly provided by females.

During incubation, female birds may buffer egg temperature variation [START_REF] Boulton | How avian incubation behaviour influences egg surface temperatures: relationships with egg position, development and clutch size[END_REF] and start incubation before clutch completion to increase hatching asynchrony in response to climate change [START_REF] Lord | Incubation prior to clutch completion accelerates embryonic development and so hatch date for eggs laid earlier in a clutch in the great tit Parus major[END_REF]. Environmental temperature is one of the factors MANUSCRIT 2 modulating the allocation trade-off of energy between heating eggs and self-maintenance in [START_REF] Kluijver | Daily routines of the Great Tit, Parus m. major L[END_REF], Gosler 1993). Insufficient feeding by the male can induce the female to leave the nest to find food, causing egg temperature to drop. Then, when the female returns, more energy is needed to reheat the clutch than in cases where male feeding is sufficient [START_REF] Boulton | How avian incubation behaviour influences egg surface temperatures: relationships with egg position, development and clutch size[END_REF][START_REF] Ardia | Temperature and life history: experimental heating leads female tree swallows to modulate egg temperature and incubation behaviour[END_REF], Bryan and Bryant 1999[START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF][START_REF] Reid | Resource allocation between reproductive phases: the importance of thermal conditions in determining the cost of incubation[END_REF]. In the great tit, egg temperature when the female returns to the nest depends on clutch size, environmental temperature, duration of absence and initial egg temperature when the female left the nest [START_REF] Boulton | How avian incubation behaviour influences egg surface temperatures: relationships with egg position, development and clutch size[END_REF]. Moreover, nest composition influences incubation temperature. Nests with relatively little lining material are poorly insulated, as has been shown for several species (great and blue tit (Mainwaring et al. 2012), spiny-cheeked honeyeater (Acanthagenys rufogularis) and yellow-throated miner (Manorina flavigula) [START_REF] Heenan | The effect of wind on the rate of heat loss from avian cup-shaped nests[END_REF]). The mass of the cup lining material in great tit and blue tit nests decreased with increasing environmental temperature (Mainwaring et al. 2012, Deeming et al. 2012). Thus, parents may modify the thermal properties of the nest in response to environmental temperature variation. Heating and maintaining the eggs at a constant temperature represent an important energetic cost for females and depends on spatial and temporal factors. Indeed, when nests were experimentally heated, tree swallow females maintained higher body condition and fed nestlings at a higher rates (Perez et al. 2008).

Moreover, in the water pipit (Anthus spinoletta), total energy expenditure for thermoregulation and basal metabolism for females were greater in colder areas and in first broods as compared to second broods (Rauter and Reyer 2000). In the zebra finch (Taeniopygia guttata), energetic cost increased with decreasing environmental temperature and increasing clutch size in a climate-controlled room (Nord et al. 2010). In the great tit, when minimum environmental temperature decreased overnight, the energy expenditure of 6 MANUSCRIT 2

females increased, and heating the nest during the night resulted in an increase in the total time spent incubating the following day (Bryan and Bryant 1999).

Other physiological processes can also be affected by environmental temperature through its effect on thermoregulation and metabolism. For example, when energetic metabolism increases, oxidative stress consequently increases (Isaksson et al. 2011, Finkel andHolbrook 2000). Oxidative stress is caused when oxidants (i.e. mainly reactive oxygen metabolites, ROM), produced by cellular activity, exposure to radiations or pollutants (Finkel & Holbrook 2000), outweigh antioxidants, potentially leading to cellular and molecular damage (Sies 1997). Oxidative stress is currently seen as a potential mediator of life history trade-offs, as allocation to immediate protection and/or repair may reduce energy available for other functions like reproduction while lack of allocation to protection or repair may lead to increased oxidative stress and decreased life expectancy [START_REF] Monaghan | Oxidative stress as a mediator of life history trade-offs: mechanisms, measurements and interpretation[END_REF]).

Furthermore, an increase in reproductive investment tends to increase oxidative stress that may in turn reduce fecundity or fertility, and the availability of dietary antioxidants may therefore constrain reproduction (Metcalfe and Alonso-Alvarez 2010). Organisms having finite energetic resources, a high investment in reproduction (in terms of clutch size but also parental behaviour) may also reduce the amount of energy available for other functions (Griffith and Buchanan 2010, Lindström 1999, Marshall and Uller 2007) including the immune system (Glick 2000). When the immune system is challenged, sedimentation rate and the relative number of leucocyte in the blood increase (Glick 2000, Ots et al. 1998). In addition, investment in reproduction may also induce a physiological stress [START_REF] Banbura | Spatial and Temporal Variation in Heterophil-to-Lymphocyte Ratios of Nestling Passerine Birds: Comparison of Blue Tits and Great Tits[END_REF], Hõrak et al. 1998, Lobato et al. 2005). When physiological stress increases heterophil concentration increases and lymphocyte concentration decreases (Ots et al. 1998, Davis et al. 2008), leading to an increase in the heterophil to lymphocyte ratio (H/Ly, (Glick 2000, Ots et al. 1998, Davis et al. 2008[START_REF] Clark | Atlas of Clinical Avian Hematology[END_REF])).

MANUSCRIT 2

Variation of environmental temperature thus influences bird reproduction in many ways, however, in the context of global temperature increase (Stocker et al. 2013), studies are still lacking to address the effects of temperature increase both on parental behaviour and health and on reproductive success. We address this question by experimentally increasing nest temperature at night by a few degrees during the incubation period. We chose the great tit as a model species, as it lives at various latitudes (Del Hoyo et al. 2007) and is thus able to adapt to a wide range of temperature variation. We measured the consequences of increased nest temperature on (i) parental behaviour (incubation and nestling feeding), (ii) parental health (estimated by body condition, immune status, physiological and oxidative stress) and (iii) parameters of reproductive success (hatching success, nestling body mass gain with age, nestling body condition, immune status, physiological and oxidative stress at fledging, and fledging success). We expected fewer days of incubation in heated nests compared to control ones, and that females of heated nests would be in better body condition than control females.

However, the effects of heating treatment on the antioxidant status and especially on oxidative damage were not straightforward to predict, because both positive (Alonso-Alvarez et al.

2004

) and negative [START_REF] Isaksson | Opposing effects on glutathione and reactive oxygen metabolites of sex, habitat, and spring date, but no effect of increased breeding density in great tits (Parus major)[END_REF]) relationships between reproductive investment and oxidative damages have been reported. Only females incubate the eggs in this species, so the treatment should only influence females. Finally, higher hatching and fledging success and better body condition were expected for nestlings in heated nests in comparison to control nests, because higher and potentially more stable incubation temperature should have a positive effect on early embryonic development.

Materiel and methods (2007) and [START_REF] Pazzuconi | Uova e nidi degli uccelli d'Italia[END_REF]). Out of a total of 62 nest boxes with a nest containing eggs, 29

were determined as occupied by a great tit pair, of which two started incubation before the experiment and three stopped laying at 4, 6 and 7 eggs. Twenty-four remaining nests were raised by 1 cm with a deck (a flower pot plastic saucer placed upside down) before incubation to allow inserting the heating system under the nest cup (see below).

Nests were visited every day to check whether incubation has begun, as it determined the starting day of the experimental treatment (between 29 of April and 8 of May). Twelve nests were randomly attributed to the experimentally heated nests group (H) and twelve to the control group (C) to be left unheated. Three nests were subsequently abandoned before incubation (all in the control group, with 8 or 9 eggs), which left us with a total of 21 nests for the experiment. During incubation all nests were visited every evening between 19:00 and 21:00 in random order. A hand warming pad (duration 7+ hours, Grabber Inc., USA) was inserted between the nest-, and control nests were handled in the same manner but no warming pad was added. Visits were stopped Adults were trapped in the nest while feeding nestlings aged of at least 6 days to limit the risk of clutch desertion (mean nestling age at capture; females: 6.05 ± 0.66, males: 7.92 ± 0.75; t = -1.87, df = 26.88, p = 0.07), and were sexed according to the size of the breast stripe, confirmed by the presence (female) or absence (male) of a brood patch. Adults were individually identified by a numbered aluminium ring (National Museum of Natural History).

Tarsus length and body mass were measured to the nearest 0.1 mm with a calliper, and to the nearest 0.25 g with a Pesola spring balance, respectively. A blood sample was taken from the brachial vein (see below).

All nestlings from each brood were weighed every two days in the afternoon (14:00-17:00) from day 1 to day 13 with a Pesola spring balance to the nearest 0.1 g. Nestlings were however not individually identified until day 14. Two broods were missed for the first weighing. One brood was missed at 11 days old, so mass measured the next morning at 10 am was used in its place (excluding these values from the analyses did not change the results presented here). Hatching date and success (nestling number divided by clutch size) and fledging date and success (fledgling number divided by hatchling number) were noted.

Parental feeding frequency was recorded when nestlings were 8 or 9 days old. As great tits feed significantly less often in the afternoon [START_REF] Gibb | Feeding rates of Great Tits[END_REF], we chose to record parental feeding in the morning and to weigh nestlings in the afternoon, so that any difference in parental behaviour could not be due to the perturbation induced at the nest by manipulating nestlings [START_REF] Gibb | Feeding rates of Great Tits[END_REF]. Each nest was observed for 60 minutes during the late morning (10:00-12:00) by one observer hidden by a canvas blind placed at least 30 m from the nest to minimize disturbance. The total number of entries and exits of the breeding pair was recorded and used in subsequent analyses.

A total of 136 nestlings from 15 nests (9 heated nest: 75 nestlings and 6 control nests: 41 nestlings) were individually identified with a numbered aluminium ring when 14-16 days old, tarsus length and body mass were measured, and a blood sample was taken. One nest was deserted before, and five nests after adult capture (3 nests in H group and 2 nests in C group, MANUSCRIT 2 respectively). Desertion rate post adult capture did not differ between heated and control groups (Chi ² = 0.28, df = 1, p = 0.60).

Laboratory analyses

Adult and nestling blood samples of up to 150 µL were taken from the brachial vein in heparinized capillary tubes for haematocrit, and a 1 or 2 µL drop of blood was smeared on a microscope slide for blood cell counts. Smears were air-dried, fixed in absolute methanol and stained with Giemsa (Sigma Giemsa modified 0.4% p/v) (Biard et al. 2010). Adult smears were examined under oil immersion at 1,000x magnification and the different types of leucocytes in a total of 50 leucocytes per smear were counted in random order by the same person (MV). Only a total of 50 leucocytes could be counted (as in Vaugoyeau et al. In Press but not 100 as in other studies (Ots et al. 1998[START_REF] Banbura | Spatial and Temporal Variation in Heterophil-to-Lymphocyte Ratios of Nestling Passerine Birds: Comparison of Blue Tits and Great Tits[END_REF][START_REF] Cirule | A rapid effect of handling on counts of white blood cells in a wintering passerine bird: a more practical measure of stress?[END_REF], Hõrak et al. 1998) due to a lack of leucocytes (mean ± SE leucocytes per examination of 10,000 erythrocytes = 26 ± 17 as compared to other studies in great tits: 90 ± 30 [START_REF] Cirule | A rapid effect of handling on counts of white blood cells in a wintering passerine bird: a more practical measure of stress?[END_REF] and 50 ± 23 (Ots et al. 1998)). Repeatability scores of our counts were highly significant (H/Ly ratio F 53,54 = 63.93, p < 0.01, intraclass correlation coefficient = 0.97 (Lessells and Boag 1987)). Blood samples were placed at 4°C and left upright to measure sedimentation rate after 8h as the ratio of red blood cells on total blood volume (Glick 2000, Campbell and Ellis 2007), finally they were centrifuged for 8 min at 1,000 rpm. Haematocrit and leucocyte buffy coat were measured as the amount of red blood cells and leucocytes relative to total blood volume in the capillary tubes (Ots et al. 1998, Hõrak et al. 1998). One nest from the heated group contained only two nestlings and these had poor body condition and extreme values for haematocrit and leucocyte buffy coat. Both were therefore excluded from the analyses involving these variables. High value of blood sedimentation rate and leucocyte buffy coat reflect an immune activation (Glick 2000, Ots et al. 1998). Low values of MANUSCRIT 2 haematocrit and high values of H/Ly (calculated as the proportion of heterophils on lymphocytes) are often interpreted as a sign of physiological stress (Glick 2000, Ots et al. 1998, Davis et al. 2008[START_REF] Clark | Atlas of Clinical Avian Hematology[END_REF].

The level of oxidative stress was measured through plasma total antioxidant capacity and ROM concentration. The total antioxidant capacity test (OXY-Adsorbent test, Diacron, Italy)

assesses the antioxidant capacity of plasma including both exogenous and endogenous antioxidants by measuring resistance to a massive oxidant attack of hypochlorous acid (HOCl). Plasma (3 µL) was diluted 1:100 with distilled water. A 200 µL aliquot of a HOCl solution was incubated with 5 µL of the diluted plasma for 10 min at 37°C, to which was added 5 µL of N,N-diethyl-paraphenilenediamine. This chromogen is oxidized by the residual HOCl and transformed into a pink derivative, so the intensity of pink is inversely related to the antioxidant capacity. The absorbance was read at 505 nm (Microplate reader model 680, Bio-Rad) and values were calculated according to a calibration curve run in each essay. Total antioxidant capacity was expressed as µmol of HOCl/mL neutralized (Costantini et al. 2011).

A low value indicates a low antioxidant capacity. Samples were measured twice and were found highly repeatable (F 152,153 = 8.27, p < 0.01, intraclass correlation coefficient = 0.78).

Mean values were used in this study. ROM concentration (d-ROMs test, Diacron, Grosseto, Italy) was measured by the concentration of hydroperoxides, which derive from the oxidation of several molecular substrates (Hõrak et al. 2004[START_REF] Hill | Condition-dependent traits as signals of the functionality of vital cellular processes[END_REF], Hill and Johnson 2012). The plasma (4 µL) was diluted 1:100 with acetate buffer (pH 4.8), mixed with 2 µL of N,Ndiethyl-paraphenilenediamine and then incubated for 75 min at 37°C. Low pH favours the release of iron and copper ions from plasma proteins. These metals catalyse the cleavage of ROOH, leading to the generation of the two highly reactive and histolesive pro-oxidants (alkoxyl and alkyperoxyl radicals). When these compounds react with N,N-diethylparaphenilenediamine they produce a pink colour whose intensity is directly proportional to Likelihood ratio tests were manually calculated for the random effect and are reported in Table 3. In order to test the effects of body condition and heating treatment on oxidative stress (total antioxidant capacity and ROM concentration), immune status (blood sedimentation rate and leucocyte buffy coat) and on stress response (haematocrit) in nestlings, models included tarsus length and body mass as fixed effects with two-way interaction with heating treatment and hatching date. When an interaction with treatment was significant and retained in the final model, data were subsequently analysed separately for heated and control nests. Residuals of all final linear models did not deviate from a normal distribution. Mean values are presented ± SE.

Results

Nest temperature and parental behaviour

Mean night and day temperatures (Fig 1), minimal and maximal temperatures (supplementary section) during incubation were significantly higher in heated than in control nests (Table 1).

Moreover, in heated nests, temperature was similar between night and day (t = 0, df = 21.80, p = 1), but lower during the night than during the day in control nests (t = 2.35, df = 14.62, p = MANUSCRIT 2 0.03). Minimal (supplementary section) and mean relative humidity were lower in heated than in control nests (Table 1) and similar during the day than during the night in both types of nests (heated nests: t = 0.75, df = 21.99, p = 0.46 and control nests: t = 0.65, df = 15.97, p = 0.53).

Duration of incubation was similar between heated and control nests (Table 1). Parental care, measured by feeding behaviour, was significantly influenced by heating treatment (Table 1):

in control nests, adults visited their nestlings significantly more often than adults from heated nests. However, there was no significant difference in brood size on the observation day (F 1,11 = 1.04, p = 0.33) or in the date of observation (F 1,11 = 1.28, p = 0.28) between heated and control nests, but number of visits was higher during rainy than dry days (9.4 2.5 and 8.4

2.5 respectively, F 1,11 = 5.33, p = 0.04).

Parental health

Neither body mass of females nor body condition of males differed according to heating treatment (females: t = -0.34, df = 7.17, p = 0.74; males: t = 1.20, df = 10.48, p = 0.26).

Blood sedimentation rates of females and males did not vary with heating treatment or date, and in females only, it significantly increased with body mass (Table 2A). Haematocrit, leucocyte buffy coat and H/Ly in both sexes did not vary with heating treatment, body condition or date (Table 2A and2B).

Only male total antioxidant capacity differed with heating treatment and date of capture, males from heated nests having higher antioxidant capacity than males from control nests (Table 2C). Female total antioxidant capacity and ROM concentration for both sexes did not vary with heating treatment or body condition (Table 2C).

Reproductive success MANUSCRIT 2

Hatching success was similar (Chi 2 = 1.49, df = 1, p = 0.22) between heated (88 of 97 eggs in 12 nests hatched, i.e. 90.7 %) and control nests (63 of 65 eggs in 9 nests hatched, i.e. 96.9 %).

Nestlings fledged at a similar age (t = -0.79, df = 9.06, p = 0.45) in heated (18.43 ± 0.48 3A). Nestling haematocrit and blood sedimentation rate increased with body mass but did not differ with heating treatment (Table 3B and3C). In a similar way, leucocyte buffy coat did not vary with any explanatory variable (Table 3B).

Finally, in nestlings, ROM concentration did not vary with nest heating treatment, body size or mass, while total antioxidant capacity significantly decreased with tarsus length, but was not affected either by heating treatment, body mass or hatching date (Table 3D).

MANUSCRIT 2

Discussion

In this study we experimentally increased mean nest temperature, especially during the night, throughout the incubation period to investigate the heat island effect on reproductive success, oxidative and physiological stress. Temperature currently increases globally and in particular during the night in cities (Stocker et al. 2013, Escourrou 1990). In birds, parental nest construction behaviour (Mainwaring et al. 2012, Deeming et al. 2012) and date of laying (Charmantier et al. 2008, Chmielewski et al. 2013, Kluen et al. 2011, Mainwaring et al. 2012, Naef-Daenzer et al. 2012, Nord and Nilsson 2011, Schaper et al. 2012, Visser et al. 2009) are influenced by temperature increase whereas great tit females were shown to increase the time spent incubating the following day when night temperature was experimentally increased (Bryan and Bryant 1999). We expected that heating treatment would decrease total incubation period and increase reproductive success and parental health. Although nest temperature was significantly increased by heating treatment, this did not significantly influence incubation duration, parental health except total antioxidant capacity of males, hatching and fledging success. In contrast, body mass of hatchlings was affected by heating treatment. Indeed, below 4 days old, nestlings from heated nests were significantly heavier but fledged at similar mass than nestlings from control nests.

Despite numerous experimental studies on the influence of environmental temperature on reproducing birds, the large diversity both in the type of heating treatment (at fixed temperature: climatic room with or without females (Nord et al. 2010[START_REF] Olson | Decoupling morphological development from growth in periodically cooled zebra finch embryos[END_REF] or in natura by Pelletier effect [START_REF] Ardia | Temperature and life history: experimental heating leads female tree swallows to modulate egg temperature and incubation behaviour[END_REF][START_REF] Magrath | Contrasting effects of reduced incubation cost on clutch attendance by male and female European starlings[END_REF], Perez et al. 2008[START_REF] Reid | Resource allocation between reproductive phases: the importance of thermal conditions in determining the cost of incubation[END_REF], or with constant heating effect: night-light (Bryan and Bryant 1999), hand warming pad [START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF])), and in the duration of the experiment (during few nights (Bryan and Bryant 1999[START_REF] Ardia | Temperature and life history: experimental heating leads female tree swallows to modulate egg temperature and incubation behaviour[END_REF][START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF], Magrath et al. 2005, Nord et

MANUSCRIT 2 2010, Perez et al. 2008) or over the whole incubation period [START_REF] Olson | Decoupling morphological development from growth in periodically cooled zebra finch embryos[END_REF][START_REF] Reid | Resource allocation between reproductive phases: the importance of thermal conditions in determining the cost of incubation[END_REF]), makes the comparison among studies difficult. As expected, we observed that nest temperatures in heated nest boxes were warmer than in control nest boxes in agreement with previous studies (Nord et al. 2010[START_REF] Ardia | Temperature and life history: experimental heating leads female tree swallows to modulate egg temperature and incubation behaviour[END_REF][START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF]). Temperature fluctuated more in control than in heated nest boxes. The nest heating treatment was successful in increasing nest temperature, consistent with similar experimental studies, that reported an increase in mean egg incubation temperature (Nord et al. 2010[START_REF] Ardia | Temperature and life history: experimental heating leads female tree swallows to modulate egg temperature and incubation behaviour[END_REF][START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF]). These previous studies however did not distinguish night from day temperatures. We additionally show that heating treatment resulted in daily temperatures fluctuating less in heated than in control nest boxes. In addition, our experimental increase in night temperature seems to mimic well the urban heat island effect, where temperature differences between urban and rural areas are negligible during daytime but increase rapidly after sunset, with higher night temperatures in cities than in rural neighbouring areas (Pickett et al. 2011). The urban heat island was first described observations, with night temperatures being 2.1°C warmer in the city than in the countryside [START_REF] Landsberg | 5 The Urban Heat Island[END_REF]. Our analysis of weather data from the national forecast agency indicated that between March and May 2012 to 2014, minimum monthly temperature was on average 4°C warmer in Paris than at the rural area. Our experiment increased only mean night temperature by 4°C, as measured in two heated nests that were deserted by parents, therefore resulting in similar temperature range as in urban areas in comparison to rural ones. In heated nest boxes where females incubated, temperature increased by 5.1°C during the day and by 7.2°C at night. This result suggests that the increase in temperature in heated nest boxes is not only due to the thermal effect of heaters but could also be due to the behaviour of females or thermic nest capacity. This, however, would deserve to be further investigated due to the 20 MANUSCRIT 2 small sample size available in this study to compare nests with and without incubating females. Perez et al. (2008) found that tree swallow females from nests heated during incubation visited nestlings more often than females from control nests. They hypothesized that normal incubation conditions were suboptimal and that heating treatment during incubation period allowed females to affect the trade-off between self-maintenance and parental investment in favour of the later (Perez et al. 2008). On the contrary, we found that adults brought food to their nestlings less often in heated than in control nests. Parental feeding behaviour was recorded in our study in the late morning and it is possible that adults from heated and control nests did not have the same feeding pattern over the course of the day. However, a lower feeding rate of parents in the heated group is consistent with the fact that their nestlings were significantly heavier at hatching, but finally fledged with a similar body mass than that of controls.

Since it is demonstrated that heating nests decreased energetic expenditure for females in the water pipit (Rauter and Reyer 2000), zebra finch (Nord et al. 2010) and pectoral sandpiper (Calidris melanotos) [START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF]), we expected that adults from heated nests would be in better condition than those from control nests. In the water pipit, female mass loss during the night decreased when environmental temperature increased (Rauter and Reyer 2000) and during incubation tree swallow females from heated nests were heavier than control females (Perez et al. 2008). However, in the spotless starling, females from heated nests were lighter than females from control nests in the first two days after hatching [START_REF] Magrath | Contrasting effects of reduced incubation cost on clutch attendance by male and female European starlings[END_REF]). In the present study, we found no difference between female or male body condition from heated and control nests, as also found by Bryan and Bryant (1999) and [START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF]. In the same way, contrary to expectations, heating treatment did not influence adult immune status, physiological stress, or an aspect of oxidative stress levels, ROM MANUSCRIT 2 concentration. The lack of a detectable effect of heating the nest during incubation could be due to the delay between the end of the treatment and capture, which may have allowed individuals to compensate for the costs of incubation. In previous studies, adults were captured during incubation (Rauter and Reyer 2000, Nord et al. 2010[START_REF] Cresswell | The energetic costs of egg heating constrain incubation attendance but do not determine daily energy expenditure in the pectoral sandpiper[END_REF], Perez et al. 2008) or in the first two days after hatching [START_REF] Magrath | Contrasting effects of reduced incubation cost on clutch attendance by male and female European starlings[END_REF]. In our case, we did not capture adults earlier, since the weather during this particular spring was very cold and rainy [START_REF] Eden | [END_REF], which made great tits in our population more sensitive to perturbation at the nest and more prone to desertion (pers. obs., and see Methods). Our results suggest that energy expenditure was too moderate to impact adult health until chick rearing, or that it did not influence their health or body condition at all.

Finally this result may not be so surprising if we considers the wide latitudinal distribution and hence temperature range tolerance of great tits (Del Hoyo et al. 2007).

The failure of an egg to hatch may result from infertility, genetic defects, improper chemical exchange rates between the egg and the environment, or failures in parental incubation [START_REF] Webb | Thermal tolerance of avian embryos: a review[END_REF]. Under the hypothesis that females in heated nests would spend less energy to heat the nest and clutch than females in control nests, we expected that this saved energy would be reallocated to parental investment through feeding behaviour for example, which could therefore increase fledging success. However, we did not find any difference in reproductive success, as also found in [START_REF] Reid | Resource allocation between reproductive phases: the importance of thermal conditions in determining the cost of incubation[END_REF] and [START_REF] Magrath | Contrasting effects of reduced incubation cost on clutch attendance by male and female European starlings[END_REF]. In the spotless starling, fledging success was higher and nestlings fledged at an earlier age in heated than control nests [START_REF] Reid | Resource allocation between reproductive phases: the importance of thermal conditions in determining the cost of incubation[END_REF], but it was not the case in our study. Fledging success was positively related to nestling mass at 3 days old, which was significantly greater in heated as compared to control nests, but this difference was not strong enough to lead to a detectable effect of heating treatment on fledging success. Thus, it seems that a higher and more stable incubation temperature did not significantly influence reproductive success contrary to expected MANUSCRIT 2 [START_REF] Deeming | Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles[END_REF] or that females could not reallocate energy to health or to reduce physiological and oxidative stress.

In our study, nestlings from heated nests were heavier than nestlings from control nests at 1-4 days old, consistent with previous studies in tree swallows (Perez et al. 2008) and spotless starlings [START_REF] Reid | Resource allocation between reproductive phases: the importance of thermal conditions in determining the cost of incubation[END_REF]. In zebra finches, heavier hatchlings for the same size in heated nests probably resulted from incubation temperature being maintained closer to the optimum, and growth of soft tissues such as muscles and organs being quicker than that of other tissues like bone [START_REF] Olson | Decoupling morphological development from growth in periodically cooled zebra finch embryos[END_REF]. Moreover, embryos of zebra finches exposed to periodic cooling were smaller at 12 days of incubation than embryos under optimal incubation conditions (37.4

°C) although mean incubation temperature was kept high (35.4 °C) [START_REF] Olson | Periodic cooling of bird eggs reduces embryonic growth efficiency[END_REF] showing the importance of constant and high temperature incubation. Four days after hatching however, the initial difference in nestling mass between treatments disappeared (Figure 2) as reported in tree swallows (Perez et al. 2008) and in spotless starlings [START_REF] Reid | Resource allocation between reproductive phases: the importance of thermal conditions in determining the cost of incubation[END_REF].

Slower nestling mass gain pattern may be due to a decrease in parental investment, if the parental feeding rate in heated nests was consistently lower as compared to controls over the course of chick rearing as when recorded 8 or 9 days after hatching. However, nestlings from heated nest fledged at a similar body mass as controls, and other indicators of juvenile quality were not affected by the heating treatment. We did not find any difference in morphology (tarsus length and body mass before fledging), immune status (blood sedimentation rate and leucocyte buffy coat), physiological stress (haematocrit) or oxidative stress (total antioxidant capacity and ROM concentration) between nestlings from heated and from control nests. We expected that a more stable incubation temperature would increase nestling health. However, heating treatment did not significantly influence the various aspects of nestling phenotype that were measured at fledging but we cannot rule out that heating might have a long-term influence in adult life [START_REF] Deeming | Egg Incubation: Its Effects on Embryonic Development in Birds and Reptiles[END_REF]. Indeed, nestling metabolic rate and then MANUSCRIT 2 that of future adult has been shown to be increased by low developmental temperature (Nord andNilsson 2011, Webb 1987) and tropical bids had lower basal metabolic rate than birds living in temperate areas [START_REF] Wiersma | Tropical birds have a slow pace of life[END_REF]. So living in hot environment seemed related with a low metabolic rate [START_REF] Mckechnie | Sources and significance of variation in basal, summit and maximal metabolic rates in birds[END_REF] and could be necessary for urban birds. As studies that link pre-hatching environment, post-fledging phenotype and fitness are absent, influence of environmental temperature on future adult metabolic rate is speculative.

Global warming is not limited to urban areas as mean and extreme temperature increase in all ecosystems (Stocker et al. 2013), so our results give some insights on the potential future consequences of high temperature on early developmental conditions and on the trade-off between self-maintenance and parental investment during reproduction in passerine populations. Finally, if high temperature has indeed a negative influence on parental investment in nestling feeding, it could be important to limit heat island effect in city by increasing green roofs and walls (Oberndorfer et al. 2007, Susca et al. 2011). Indeed, green roofs and walls decreased albedo effect and improve air and water purity (Oberndorfer et al. 2007, Susca et al. 2011).

MANUSCRIT 2 Table 3: Models investigating variation in (A) morphology as a function of hatching date and 749 heating treatment and their interaction, and in (B) immune status, (C) physiological stress and 750 (D) oxidative stress as a function of tarsus length, body mass and the interaction between 751 hatching date and heating treatment, in great tit nestlings aged 14-16 days, i.e. at Treatment effects on minimal and maximal of temperature and of percent of humidity during incubation period were tested using Welch t-tests. Heated nests had higher minimal and maximal temperature (Figure 1) and lower minimal percent of humidity (Table 1). Therefore, maximal humidity was similar between heated and control nests (Table 1).

Introduction

Animal colour patterns represent a compromise between thermoregulation, crypsis to avoid detection by predators and conspicuousness for intraspecific communication [1,2]. The development of coloured signals in animals involves a variety of structural elements and pigments. In most species of birds, pigment-based colouration mainly results from carotenoids, producing bright red, orange, and yellow colouration, or melanins, producing black, brown, red or grey colours [2]. Vertebrates cannot produce carotenoids, they must acquire them from food [2] whereas they synthesize melanin pigments from amino acids [3].

Carotenoid and melanin-based colouration are signals of body condition, immune status and reproductive investment [2]. Life in urban habitats may influence plumage colouration through availability and quality of food resources and this can perturb ecological processes such as partner choice or reproductive investment [1,2]. However, relatively little is known about differences in carotenoid or melanin-based plumage colours between urban and rural populations of birds or the relationship between signal and physiology status in the relatively new urban environment. Some studies indeed indicate that carotenoids are less available in urban than rural areas [START_REF] Isaksson | The chemical pathway of carotenoids: from plants to birds[END_REF][START_REF] Isaksson | Carotenoid diet and nestling provisioning in urban and rural great tits Parus major[END_REF] whereas heavy metals, which can serve as catalysts of melanin synthesis [3], are more available [START_REF] Pickett | Urban ecological systems: Scientific foundations and a decade of progress[END_REF]. Urban birds could thus be expected to display more intense melanin-based but less intense carotenoid-based colouration than rural animals. Male and female great tits (Parus major) living near heavy metal pollution sources had larger black breast stripe than those from unpolluted sites [START_REF] Dauwe | Melanin-and carotenoid-dependent signals of great tits (Parus major) relate differently to metal pollution[END_REF] possibly because heavy metals catalyse melanin synthesis [3]. In the same species, adults living near heavy metal pollution sources were less yellow than rural ones [START_REF] Dauwe | Melanin-and carotenoid-dependent signals of great tits (Parus major) relate differently to metal pollution[END_REF], as were adults living in the city [START_REF] Hõrak | Carotenoid-based plumage coloration reflects hemoparasite infection and local survival in breeding great tits[END_REF][START_REF] Isaksson | Plasma Glutathione and Carotenoid Coloration as Potential Biomarkers of Environmental Stress in Great Tits[END_REF], though these had similar plasma carotenoid concentration to rural adults [START_REF] Isaksson | Sexual, seasonal, and environmental variation in plasma carotenoids in great tits, Parus major[END_REF]. Several mechanisms may explain why carotenoid-based signals are duller in cities. Carotenoids have multiple physiological functions in addition to their role in pigmentation, including regulating and stimulating MANUSCRIT 3 immune function [START_REF] Biard | Carotenoid-based bill colour is an integrative signal of multiple parasite infection in blackbird[END_REF][START_REF] Romero-Diaz | Independent sources of condition dependency and multiple pathways determine a composite trait: lessons from carotenoid-based plumage colouration[END_REF] and scavenging damaging oxygen species [START_REF] Isaksson | The chemical pathway of carotenoids: from plants to birds[END_REF][START_REF] Surai | Distribution of carotenoids from the yolk to the tissues of the chick embryo[END_REF]. In urban areas, therefore, available carotenoids maybe be required to neutralize free radicals from heavy metal pollution [START_REF] Koivula | Metal-related oxidative stress in birds[END_REF] or for the immune system because of high disease risk in urban habitats [START_REF] Bradley | Urbanization and the ecology of wildlife diseases[END_REF]. A negative relationship between carotenoid-based colouration and parasite load has often been documented in animals in natural habitats [START_REF] Biard | Carotenoid-based bill colour is an integrative signal of multiple parasite infection in blackbird[END_REF][START_REF] Romero-Diaz | Independent sources of condition dependency and multiple pathways determine a composite trait: lessons from carotenoid-based plumage colouration[END_REF][START_REF] Kilgas | Variation in assemblages of feather bacteria in relation to plumage color in female great tits[END_REF] but not yet in urban ones [START_REF] Hõrak | Carotenoid-based plumage coloration reflects hemoparasite infection and local survival in breeding great tits[END_REF], although the prevalence of several parasites is higher in cities [START_REF] Bradley | Urbanization and the ecology of wildlife diseases[END_REF]. When dietary carotenoids are scare as is the case in cities and carotenoids are required for protection against the effects of pollution [START_REF] Eeva | Environmental pollution affects the plumage color of great tit nestlings through carotenoid availability[END_REF] little may be available for other functions such as immune response or colouration.

Urbanization is increasing globally [START_REF] Pickett | Urban ecological systems: Scientific foundations and a decade of progress[END_REF] but to date few studies have investigated the potential links between carotenoid or melanin based colouration with immune and stress status in urban habitats. Furthermore if link between coloration and adult fitness is altered in the urban habitat, partner selection as well as population dynamics might be impacted. Here, we investigated how carotenoid-based and melanin-based colouration in breeding great tits varied with body condition and immune response measured by higher sedimentation and leucocytes rates and lower haematocrit rate [START_REF] Glick | Avian Physiology -Chapter 26 -Immunophysiology[END_REF][START_REF] Ots | Haematological health state indices of reproducing Great Tits: Methology and sources of natural variation[END_REF], in two populations of great tits; one urban and one rural.

The great tit is a small Eurasian passerine inhabiting a large variety of habitats, from pristine forests to city parks. This bird has yellow carotenoid-based and black melanin-based plumage.

As urban environments suffer from heavy metal pollution [START_REF] Pickett | Urban ecological systems: Scientific foundations and a decade of progress[END_REF] we expected that black breast stripe would be larger because melanin synthesis is stimulated by heavy metals.

Moreover, we expected that in the urban population, yellow feathers would be less brightly coloured due to lower carotenoid availability [START_REF] Isaksson | The chemical pathway of carotenoids: from plants to birds[END_REF][START_REF] Isaksson | Carotenoid diet and nestling provisioning in urban and rural great tits Parus major[END_REF] and more pollution in the urban population as compared to the rural one [START_REF] Pickett | Urban ecological systems: Scientific foundations and a decade of progress[END_REF]. Finally, we expected these differences in coloured signals to MANUSCRIT 3 be related with environmental characteristics such as aerial pollution (NO 2 ). Moreover, we tested how melanin-based and carotenoid-based colouration varied with body condition, sex, age and immune status to determine whatever colouration was a reliable signal of individual quality in the two habitats.

Materials and methods

Data on breeding great tits were collected from 2012 to 2014 in two forest sites and six parks and one cemetery in Paris. Nitrogen dioxide (NO 2 ) is emitted by motor traffic and is highest close to roads [START_REF] Bignal | The ecological effects of diffuse air pollution English Nature Research Report[END_REF]. Data on aerial NO 2 were collected by AirParif (http://www.airparif.asso.fr) in five stations, (one in Fontainebleau, four in Paris). We used one measurement every five days to compare stations and years (see supplementary material).

We associated each urban park with the station nearest to it (< 3 km) and both rural habitats CEREEP). Since feathers are grown between July and August [START_REF] Isaksson | Sexual, seasonal, and environmental variation in plasma carotenoids in great tits, Parus major[END_REF] we used mean aerial NO 2 concentration during July and August of year before capture.

Nest boxes (Schwegler wood-concrete nest boxes 2M, Valliance, Saint Pierre La Palud, France) were evenly distributed within each site, and regularly inspected and adults were trapped on the nest while feeding nestlings aged at least 6 days to limit the risk of clutch desertion, and were sexed according to the presence (female) or absence (male) of a brood patch (Table 1). Over the three studied breeding seasons, of 234 rural tits, 24 were captured twice and 6 were captured each year. Of the 67 urban tits, 7 were captured twice and 3 were captured each year. Data from first clutches only are used here. Great tits were aged according to their wing feathers moult pattern (yearling versus older birds [START_REF] Svensson | Identification guide to European passerines[END_REF]). Eight yellow breast feathers were plucked and stored in individual zip-lock bags in the dark until further analyses.

MANUSCRIT 3

Tarsus length and body mass were measured to the nearest 0.1 mm with a calliper, and to the nearest 0.25 g with a Pesola spring balance, respectively. To estimate immune status, we collected blood samples from the brachial vein in heparinized capillary tubes. Blood samples, were placed at 4°C and left upright to measure sedimentation after 8h. Thereafter, We measured sedimentation after 8h, then centrifuged each sample for 8 min at 1 000 rpm.

Haematocrit and leucocyte buffy coat were measured as the relative amount of red blood cells and leucocytes in total blood volume in the capillary tubes. Immune challenge leads to higher sedimentation rate, and leucocyte proportion and lower haematocrit [START_REF] Glick | Avian Physiology -Chapter 26 -Immunophysiology[END_REF][START_REF] Ots | Haematological health state indices of reproducing Great Tits: Methology and sources of natural variation[END_REF].

Three pictures were taken to measure breast stripe size according to a method described in the literature [START_REF] Figuerola | Measurement of plumage badges: an evaluation of methods used in the Great Tit Parus major[END_REF][START_REF] Quesada | The role of melanin-and carotenoid-based plumage coloration in nest defence in the Great Tit[END_REF] using ImageJ software v1.43u. Birds were positioned inside a gutter carved in a polystyrene platform equipped with a millimetre grid to standardize subsequent measures.

The breast stripe size was measured from a 3 cm wide area beginning 2 cm below upper border of tie and including the entire width, and calculated as the area covered (cm²).

Repeatability of breast stripe area measured twice from the same picture (intraclass [START_REF] Lessells | Unrepeatable repeatabilities -a common mistake[END_REF], p < 0.01, F Four replicate reflectance spectra were obtained from two sets of four feathers arranged in random order using a USB 2000 + spectrometer with a DH 2000 deuterium-halogen light source (Ocean Optics, IDIL, Lannion, France) [START_REF] Biard | Effects of carotenoid availability during laying on reproduction in the blue tit[END_REF]. We used a bifurcated fibre-optic probe (Ocean Optics QR200-7-SR-BX Premium 200µm) on which a cylindrical black plastic sheath was mounted to exclude ambient light and to standardize measuring distance. Reflectance was calculated relative to a white standard (Ocean Optics spectralon WS-1-SL) with SpectraSuite MANUSCRIT 3 software (Ocean Optics). After every three individuals, a dark and a white standard scan were obtained. Samples were taken randomly with respect to individual, habitat type and time of sampling. Two different parameters describing the variation in spectral shape were calculated for each spectrum. For great tit, the visible spectrum ranges from about 300 through about 700 nm [START_REF] Endler | On the measurement and classification of colour in studies of animal colour patterns[END_REF][START_REF] Hart | Visual pigments, oil droplets, ocular media and cone photoreceptor distribution in two species of passerine bird: the blue tit (Parus caeruleus L.) and the blackbird (Turdus merula L.)[END_REF]. As we were interested in colour produced by carotenoids, we calculated the relative reflectance at the s as the difference in reflectance between maximum reflectance at the plateau of the spectrum and minimum reflectance at the wavelengths of maximum carotenoid absorption, divided by maximum reflectance ((R700-R450)/ R700) [START_REF] Peters | The condition-dependent development of carotenoid-based and structural plumage in nestling blue tits: males and females differ[END_REF]. When feathers contain more carotenoids, carotenoid chroma increases [START_REF] Hõrak | Carotenoid-based plumage coloration reflects hemoparasite infection and local survival in breeding great tits[END_REF][START_REF] Peters | The condition-dependent development of carotenoid-based and structural plumage in nestling blue tits: males and females differ[END_REF][START_REF] Örnborg | Ultraviolet coloration and colour communication in blue cits Parus caeruleus[END_REF] with the feathers appearing more intensely yellow.

Feather structure has been shown to be a reliable cue of individual quality and to induce variations in UV colour which is visible to birds [2]. We thus measured as the proportion of reflected light in the 300-400 nm interval of the total brightness (300-700nm 2).

Great tit males had larger breast stripes than females (3.28 ± 0.06 cm² and 1.76 ± 0.06 cm², respectively), and breast stripe size differed among years (Fig 2 C).

Finally, carotenoid and melanin based signals were not related to immune status (Table 2).

Discussion

We investigated the influence of urbanisation on the expression of two different visual signals, carotenoid-based and melanin-based plumage colouration in the great tit. As heavy metals can catalyse melanin production [3] and are in high concentration in cities [START_REF] Pickett | Urban ecological systems: Scientific foundations and a decade of progress[END_REF], we expected that urban individuals, and especially those from more polluted sites, would have larger black breast stripes than rural ones. Moreover, as carotenoids are less available in urban than in rural habitats and as carotenoid participate in scavenging free radicals induced by pollution [START_REF] Isaksson | The chemical pathway of carotenoids: from plants to birds[END_REF][START_REF] Surai | Distribution of carotenoids from the yolk to the tissues of the chick embryo[END_REF], we expected that urban great tits would be less yellow, especially those from more polluted sites, experiencing higher oxidative stress that consumes carotenoids, than rural ones, . We found that habitat (urban versus rural) influenced carotenoid chroma but not black breast stripe size. Surprisingly, carotenoid chroma was higher in urban than rural area, though it decreased with increasing NO 2 pollution.

Variation in UV brightness varied significantly only among years. Two reasons could explain this lack of influence of habitat or pollution on UV brightness, (i) feather structure was not influenced by habitat or pollution UV and (ii) variation was too low in our populations to be measured. Carotenoid-based coloured signal varied with habitat, but contrary to previous studies [START_REF] Hõrak | Carotenoid-based plumage coloration reflects hemoparasite infection and local survival in breeding great tits[END_REF][START_REF] Isaksson | Plasma Glutathione and Carotenoid Coloration as Potential Biomarkers of Environmental Stress in Great Tits[END_REF], urban birds had higher overall mean carotenoid chroma than rural ones but MANUSCRIT 3 carotenoid chroma deceased with increasing NO 2 concentrations. Moreover, in urban birds, carotenoid chroma increased with decreasing NO 2 concentrations (F 1,38 = 3.78, p = 0.05).

Higher carotenoid chroma of urban birds than rural ones could be due higher diversity or availability of carotenoids in the urban habitat. Diet, feather or yolk analyses in urban area could elucidate the nature and proportion of carotenoids consumed by birds in urban areas.

The negative correlation between carotenoid chroma and NO 2 concentrations could be explained if carotenoids are diverted into detoxifying NO 2 instead of to feather colour in urban settings [START_REF] Koivula | Metal-related oxidative stress in birds[END_REF]. Unlike in other studies [START_REF] Hõrak | Carotenoid-based plumage coloration reflects hemoparasite infection and local survival in breeding great tits[END_REF][START_REF] Kilgas | Variation in assemblages of feather bacteria in relation to plumage color in female great tits[END_REF], we found not covariation between carotenoid-based colouration and immune system characters or body condition as if carotenoids were not limiting or were not used in response to immune system activation.

Previous studies have found larger breast stripes near a heavy metal source (factory) [START_REF] Dauwe | Melanin-and carotenoid-dependent signals of great tits (Parus major) relate differently to metal pollution[END_REF] but smaller breast stripes of Spanish urban than forest great tits [START_REF] Senar | Selection based on the size of the black tie of the great tit may be reversed in urban habitats[END_REF]. Here, we found that melanin-based colouration varied only by sex with pollution or habitat having no detectable influence. The lack of influence of habitat or NO 2 concentration in our study could be due to the absence of difference in heavy metal concentrations between rural and urban areas or due to the lack plasticity or sensitivity in studied populations. Variation in melanin-based colouration among years suggests phenotypic plasticity for the trait. Breast stripe size increased over the three study years, which could relate to meteorological characteristics (temperature, pluviometry) or other chemical pollutant concentrations.

Our study revealed no effect of habitat or pollution on melanin-based colouration contrary than expected though additional future research should measure chemical pollution along food chain in cities to see whether a larger pollution gradient or larger samples would reveal a link.

Similar phenotypes between populations did not mean that natural or sexual selections based on the black breast stripe were similar between habitats [START_REF] Senar | Selection based on the size of the black tie of the great tit may be reversed in urban habitats[END_REF]. In contrast carotenoid colouration was more intense in urban areas, confirming that urbanization can influence the MANUSCRIT 3 nature and availability of carotenoids, though we do not know if the supply or the allocation is altered. The modification in colour could modify partner choice in sexual selection. If sexual selection will differ between habitats, it could be conduct to split up population and transform urban area as ecological trap.

MANUSCRIT 3 2). Carotenoid chroma was lower in rural area than in urban area.

Introduction

The eggshell separates a developing embryo from the external environment. It allows water and gas exchange while being a barrier for parasites and bacteria. It also provides calcium during embryo development, permitting skeletal growth and influencing embryo survival (Nys 2010;Solomon 1997). In birds, eggshell thickness is related to calcium availability and egg mass [START_REF] Ar | The avian egg: mass and strength[END_REF]Solomon 1997). Eggshell structure is determined by calcium addition in crystal morphology (Rodriguez-Navarro and Garcia-Ruiz 2000) and is produced in the uterus during the night preceding laying (Sturkie 1986). Eggshell production is costly for females, since it represents about 10% of the female calcium pool (Sturkie 1986). Calcium is provided by food, especially snails (Graveland 1996), with environments poor in calcium having fewer and smaller snails (Jubb et al. 2006), as is observed in urban habitats [START_REF] Lososova | Diversity of Central European urban biota: effects of human-made habitat types on plants and land snails[END_REF]. Thus in urban areas, the lack of snails could limit bird reproductive success. In calcium-poor environments, birds produce thinner eggshells and hatching success decreases (Solomon 1997). In the blue tit (Cyanistes caeruleus), higher calcium availability increases hatching success but not female or nestling body condition [START_REF] García-Navas | Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus[END_REF]. In great tits (Parus major), higher calcium availability is related to greater eggshell mass (Gosler et al. 2005).

In birds, eggshells may be pigmented and variation in colour pattern reflects eggshell structure and quality (Gosler et al. 2005;Nys 2010;Solomon 1997). Brown eggshell pigmentation is produced by protoporphyrin which is synthetized in the bloodstream during the anabolism of haemoglobin (Baird et al. 1975) and is transferred to the egg from the shell gland via epithelial cells during mineralization (Rodriguez-Navarro and Garcia-Ruiz 2000).

The functional significance of variation in egg shell pigmentation is still poorly understood, but several studies point to a relationship between eggshell pigmentation and female or male condition, egg quality (Holveck et al. 2012;Martínez-de la Puente et al. 2007;[START_REF] Martínez-Padilla | Does egg colouration reflect male condition in birds?[END_REF]) and even nest or nestling success [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF]. In great tits, clutches with abnormally pigmented eggs (totally white eggshell) are deserted more often and have lower hatching success than clutches with spotted eggs (Graveland 1996). In blue tits, eggs with larger and less evenly-distributed spots have higher antibody concentrations, suggesting that both the quantity and distribution of brown pigments reflect the transfer of maternal immune compounds to egg yolks (Holveck et al. 2012). In blue tits, females that lay more spotted eggs were in poorer body condition and have higher cellular concentration of stress proteins and marginally lower total immunoglobulin levels in the blood (Martínez-de la [START_REF] Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF]). However, blue tit male provision nestlings more and nestling success is greater when spottiness is more widely distributed [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF], and in European kestrels (Falco tinnunculus) mates of females that lay more pigmented eggs are in better condition, estimated as mass corrected by wing length [START_REF] Martínez-Padilla | Does egg colouration reflect male condition in birds?[END_REF].

Eggshell colour and pattern is influenced by genetics [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF] and environment. In calcium-poor environments, eggshell of both blue and great tits have more brown pigmentation (Gosler et al. 2005;Graveland 1996), and brown pigmentation of Eurasian sparrowhawk (Accipiter nisus) eggs increases with increasing pesticide contamination, possibly because pesticides increased anabolism of haemoglobin (Jagannath et al. 2008). In great tits (Gosler et al. 2005;Graveland 1996), Eurasian sparrowhawk (Jagannath et al. 2008) and northern lapwing (Vanellus vanellus) [START_REF] Bulla | Eggshell spotting does not predict male incubation but marks thinner areas of a shorebird's shells[END_REF]) eggshells with more brown pigmentation are thinner. Moreover, the brown spots themselves are thinner than nonpigmented areas of the shell in northern lapwing eggs [START_REF] Bulla | Eggshell spotting does not predict male incubation but marks thinner areas of a shorebird's shells[END_REF] but not in great tits (Hargitai et al. 2013). It has been hypothesised that protoporphyrin, in great and blue tits, can replace calcium when this is rare [START_REF] García-Navas | Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus[END_REF]Gosler et al. 2011;Gosler et al. 2005;Hargitai et al. 2013;Holveck et al. 2012;Magi et al. 2012; Martínez-de la Puente et al.

2007).

Given the complex relationships between the different characteristics of the eggshell and reproductive success in birds, we investigate the influence of habitat type on egg morphology and eggshell characteristics (thickness and pigmentation) and attempt to identify the associated consequences on reproductive success. We selected urban and rural populations of blue and great tits, small passerine species with maculated eggs breeding along urban-rural habitat gradients (Del Hoyo et al. 2007). We expected eggs with thinner and more pigmented eggshell, and lower reproductive success in urban area than in rural ones because snails are rare [START_REF] Lososova | Diversity of Central European urban biota: effects of human-made habitat types on plants and land snails[END_REF]).

Materials and Methods

Egg measurement

Following recent studies (Brulez et al. 2014b;Holveck et al. 2012), we analysed eggshell pigmentation using digital image analyses instead of visual scoring [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF]Gosler et al. 2005). Since spots on great and blue tits eggshells are not evenly distributed [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF], Gosler et al. 2005), four high-resolution photos (6 016 × 4 016 pixels) were taken to obtain a comprehensive view of the egg. Three pictures were taken by rolling the egg on its long axis (without overlapping), and one picture was taken of the crown, all against a black background that allowed clear differentiation between egg and background and under standardized conditions [START_REF] Mallard | An automated image analysis system to measure and count organisms in laboratory microcosms[END_REF]) using a digital camera controlled by a computer (Nikon D300 and CameraControlPro). The black background was framed with a white background to maintain the same overall balance between black and white. Constant strong homogeneous lighting was provided by four LED bulbs (Pikaline, 16W, 650 lumens).

Pictures were taken while eggs were still frozen, then eggs were returned to 80°C within 3 minutes.

Using ImageJ software v1.43u, black and white backgrounds were deleted by adjusting were extrapolated by fitting an From these values, egg shape (ratio width/length; the smaller the ratio, the rounder the egg shape) and volume (V = 0.51 length width) were estimated according to [START_REF] Hoyt | Practical methods of estimating volume and fresh weight of bird eggs[END_REF]. Using the tool (with Y for luminance and U and V as components of chroma) were adjusted for each picture to separate the spotted surface from the white background (Holveck et al. 2012). The total area of spotted surface, the number of spots, and maximum, minimum and mean size of spots were then measured. The estimated proportion of total area of spotted surface / eggshell surface) was used thereafter. All egg and eggshell pigmentation parameters were highly repeatable on the same picture (pictures of different eggs analysed twice, blue tits: n = 18, F 17,18 > 6.42, p < 0.01, intraclass correlation coefficient > 0.73 (Lessells and Boag 1987), great tits: n = 12, F 11,12 > 4.47, p < 0.01, intraclass correlation coefficient > 0.63).

Since egg and eggshell measurements estimated on the three pictures on the long axis from a subsample of 53 blue tits and 61 great tits eggs were found highly repeatable (blue tits: F 53,108 > 3, p < 0.01, intraclass correlation coefficient > 0.60, great tits: F 61,124 > 3, p < 0.01, intraclass correlation coefficient > 0.50), we selected one of the pictures that contained no freezing-induced crack, at random, for the rest of the eggs collected.

Eggshells were later removed from partially defrosted eggs, and washed with demineralised water. Using a Mitutoyo micrometer (reference 7313), the thickness of the shell including membrane at the crown was measured to the nearest 0.01mm. Since thickness has been found to be smaller on dark spots than on the white matrix in northern lapwing (Bulla et In tables 3 and 4, significant p-values from the final models are presented in bold and initial values are also given in italics for variables that were not retained in the final model. Mean ± SE are presented.

Results

1. Scoring principal components of egg morphology and eggshell pigmentation

For both species and both characters (egg morphology and pigmentation), PCA revealed two principle axes with eigenvalues greater than one.

In both species eggs were larger and heavier for larger values on EggPC1 (Table 1A), so this axis represents egg size (Solomon 1997). Eggshells were thicker with higher relative mass for larger values on EggPC2, so this axis represents eggshell thickness (Table 1A).

In both species eggshell was more pigmented with larger spots on the sides and crown and a larger proportion of the eggshell surface pigmented for larger values of PigPC1, (Table 1B), so this first axis represents the degree of pigmentation and size of spots. When PigPC2 increased, eggshells had more spots on sides and crown for larger values of PigPC2 (Table 1B), so this second axis represents of spots number independently of the extend of these spots for sides and crown.

EggPC2 was negatively correlated with PigPC2 for blue tits and positively correlated with PigPC1 for great tits (Table 2), so in blue tits thicker shells had fewer spots whereas in great tits thicker shells had larger spots with more of the surface pigmented. 3).

In great tits, EggPC1 scores differed among years but not with female body condition or habitat (Fig 1A, Table 3). EggPC2 scores were similar in urban and rural habitats (Fig 1B ), differed among years and were positively related to clutch size (Table 3).

In both species, PigPC1 (spot size and degree of pigmentation) did not vary with habitat or female body condition and only differed among year (Fig 1C andTable 3).

In blue tits, PigPC2 (spot number) did not differ between habitats ( 3). PigPC2 was not significantly influenced by female body condition.

Reproductive success

In blue tits, hatching and fledging successes were lower in the urban than the rural area (Fig 4).

In great tits, hatching success was higher and fledging success lower in Paris than in rural area (Fig 2,Table 4). Hatching success in great tits was greater for low scores of EggPC1 i.e. for smaller eggs and for high values of PigPC1 i.e. larger spots and more pigmentation (Table 4).

Fledging success was greater for fledglings hatched from eggs with high PigPC1 scores i.e.

with larger spots and more pigmentation, with low values of EggPC2 i.e. thinner eggshell, with low scores of PigPC2 i.e. fewer spots and laid by females with poorer body condition (Table 4). Hatching and fledging successes of great tits differed among years.

Discussion

Although there is no longer any doubt that egg characteristics profoundly affect reproductive success in birds [START_REF] Ar | The avian egg: mass and strength[END_REF]Nys 2010;Solomon 1997), few studies have analysed the effect in the context of urbanization. The urban environment differs from rural habitats in calcium availability, the main element necessary for producing eggshells. This study therefore attempts to shed new light on the possible influence of an urban environment on egg morphology, eggshell quality and their consequences in terms of reproductive success.

Studying urban and rural populations of blue and great tits, which lay maculated eggs, we found that eggs of urban blue tits were smaller and lighter than those of rural blue tits, and that eggs of urban great tits had fewer spots and a higher hatching success than those of rural great tits. In contrast, blue tit s hatching success was lower in urban habitats. Finally, fledging success of both species was also lower in urban habitats. Moreover, in accordance with previous studies (Griffith and Buchanan 2010;Solomon 1997), we found that egg mass and volume were positively related to female body condition in blue tits. In contrast, we found no relation between eggshell thickness and female body condition for either species.

The hypothesis that bird eggshells should be thinner in urban than rural areas based on the observed lower snail abundance and diversity [START_REF] Lososova | Diversity of Central European urban biota: effects of human-made habitat types on plants and land snails[END_REF] was not verified in our study. For both species eggshell thickness (EggPC2) was similar in urban and rural areas.

This unexpected result could be due to the high capacity of blue and great tits to use anthropogenic calcium sources such as grit or eggshell of other species in calcium poor environments. Indeed, a recent study on urban and rural soils in the United-States showed that calcium concentration increased in cities due to chemical pollution (calcium chloride used as de-icing agent) and cement (Trammell et al. 2011).We could test this by exanimating nest 13 MANUSCRIT 4 material, droppings and pellets as in Graveland (1996). Alternatively snails may be similarly abundant in Paris as in our rural study site, which needs to be verified in the field.

In this study, we observed that egg size (EggPC1) was lower in urban than in rural habitats in blue tits and a similar tendency was observed in great tits. Low egg volume and mass in urban habitats can ensue from different characteristics of the urban environment such as a low quality of food. Indeed, Isaksson (2009) showed that urban leaves and caterpillars contained fewer carotenoids than rural ones, which could decreased female body condition. Moreover, heavy metal pollution is higher in cities than in neighbouring forests (Fritsch et al. 2012;Roux and Marra 2007), and can contaminate birds [START_REF] Costa | Assessing heavy metal pollution using Great Tits (Parus major): feathers and excrements from nestlings and adults[END_REF]Roux and Marra 2007) and decrease immune response and body condition [START_REF] Snoeijs | Heavy metal exposure affects the humoral immune response in a free-living small songbird, the great tit (Parus major)[END_REF]). Despite the negative influence urban factors may have on female body condition, only one study has reported lower female body conditon in urban areas compared to rural areas [START_REF] Chávez-Zichinelli | How stressed are birds in an urbanizing landscape? Relationships between the physiology of birds and three levels of habitat alteration[END_REF], whereas other studies have found no difference (Bokony et al. 2012;Isaksson et al. 2008). If egg size is a good predictor of nestling mass and survival (Solomon 1997;[START_REF] Williams | Intraspecific variation in egg size and egg composition in birds: effect on offspring fitness[END_REF], we may predict that the smaller eggs in urban areas should be accompanied by low hatching and fledging success. Surprisingly, we found better hatching success for small eggs in great tits and better fledging success for hatchlings from eggs with thinner eggshells in both species. A further analysis of egg components is needed to understand why great tits had higher hatching success in Paris and for smaller eggs whereas blue tits had a lower hatching success in Paris with no effect of egg size.

Brown pigmentation on eggshells has been suggested to compensate for a lack of calcium [START_REF] García-Navas | Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus[END_REF]Gosler et al. 2011;Gosler et al. 2005;Hargitai et al. 2013;Holveck et al. 2012;Magi et al. 2012;Martínez-de la Puente et al. 2007), with eggshells being more pigmented in calcium-poor areas (Solomon 1997). The proposed underlying mechanism was that protoporphyrin, synthetised during the anabolism of haemoglobin (Baird et al. 1975), is Martínez-de la [START_REF] Puente | Are eggshell spottiness and colour indicators of health and condition in blue tits Cyanistes caeruleus?[END_REF][START_REF] Martínez-Padilla | Does egg colouration reflect male condition in birds?[END_REF]). However we found no relationship between eggshell pigmentation and female body condition, so there is no evidence for a mechanism that could ensure the honesty of such a signal. Nonetheless, eggshell pigmentation could reflect investment into antioxidants or immune components placed in the egg yolk (Holveck et al. 2012;Martínez-de la Puente et al. 2007;[START_REF] Martínez-Padilla | Does egg colouration reflect male condition in birds?[END_REF]), which we did not measure in this study. If this were the case, eggshell pigmentation could still serve as a reliable signal that males should use to moderate their paternal investment.

Indeed, we have indirect evidence that eggshell pigmentation indicates the quality of maternal investment into egg components because eggshell pigmentation correlated with reproductive success in our study. Hatching and fledging successes of great tits increased with increasing to degree of pigmentation and spot size, while fledging success of both species decreased with increasing spot number. A negative relationship between spot number and fledging success could be due to contamination by pollutants transmitted by the mother to the egg (Jagannath et al. 2008;Tsipoura et al. 2008) or to a lack of calcium in the eggshell or differences in maternal investment of antioxidants (Holveck et al. 2012;Martínez-de la Puente et al. 2007;[START_REF] Martínez-Padilla | Does egg colouration reflect male condition in birds?[END_REF] or other critical resources. Indeed, eggshell provides calcium needed for the embryo development and influences survival (Nys 2010;Solomon 1997). If the eggshell pigmentation is due to a lack of calcium, as hypothesized in previous studies [START_REF] García-Navas | Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus[END_REF]Gosler et al. 2011;Gosler et al. 2005;Hargitai et al. 2013;Holveck et al. 2012;Magi et al. 2012;Martínez-de la Puente et al. 2007), we would expect the degree of pigmentation to be negatively related to fledging succes, which was not the case in this study.

Moreover, great tits feldging success increased with increasing eggshell pigmentation. Our study thus gives one more clue that eggshell pigmentation is not necessarily due to low calcium availability or that pigmentation is not only due to protoporphyrin replacing calcium, as suggested by a recent study reporting that the link between pigmentation and protoporphyrine quantity was not direct (Brulez et al. 2014a). In our study, pigmentation of eggshell was related to egg and eggshell quality and fledging succes in both species, so pigmentation predicts reproductive success. We did not however identify here the underlying mechanisms inducing eggshell pigmentation. More studies would be needed to elucidate these mechanisms and especially to why and how pigmentation in both species varied among years.

With our white and yolk current analysed, we would be confirmed that eggshell pigmentation is correlated with quantity of essential nutriment and ressources in egg. (degree of pigmentation and spot size) and D) PigPC2 (spot number) for blue tit and great tit eggs according to habitat (dark grey: urban area, light grey: rural area). Stars indicate significant differences between habitats (*: p=0.05; ** p<0.05, Table 3). 4). Hatching success (%)

Blue tit Great tit

Fledging success (%)

  et chez les amphibiens (Sun and Narins 2005; Cunnington and Fahrig 2010)).

Figure 1 :

 1 Figure 1 : La dynamique des populations est influencée par les différentes composantes des

Figure 3 :

 3 Figure 3 : Les effets maternels agissent sur le phénotype du jeune indépendamment du génotype transmis

  la réponse des espèces aux caractéristiques environnementales des villes uées par les changements globaux et les réponses possibles des populations. En effet, comme les villes concentrent les principales caractéristiques des changements globaux (température, luminosité, pollution chimique et fond sonore plus fort), elles peuvent donner des clés pour comprendre les réponses des espèces aux changements présents et futurs. Enfin, selon ements globaux sur les écosystèmes. En effet, pour eux, la modification des habitudes et des infrastructures dans les villes peut encore influencer le futur et est même essentielle pour que les changements globaux à venir soient moins importants que ceux e (Revi et al. 2014).

  faible qualité ou quantité de nourriture disponible peut réduire (Yom-Tov and Hilborn 1981; Isaksson and Andersson 2007). Donc, et le succès de reproduction devraient varier en fonction des changements globaux mais nous ignorons si les caractéristiques environnementales vont avoir la même influence en milieu urbain. Les populations réagissent de manière variable mais une diminution de la taille et du nombre s nourrissage sont quand-même attendus. Le but de cette thèse est de comprendre le rôle des effets maternels sur le phénotype des oiseaux urbain à travers la description et la mise en relation des variations environnementales, phénologiques et phénotypiques, entre et au sein de populations aviaires, à différents niveaux Pour répondre à ces questions, trois populations (Figure 5) de deux petits passereaux présents le long du gradient urbain-rural et faciles à manipuler, ont été sélectionnés : les mésanges bleues (Cyanistes caeruleus) et les mésanges charbonnières.

Figure 4

 4 Figure 4 des petits passereaux. Les flèches vertes présentent les réponses hypothétiques des populations aux changements globaux et les bleues les effets parentaux prédits en réponse aux changements globaux.

Figure 6 :Figure 7 :

 67 Figure 6 : Aires de distribution A) de la mésange bleu et B) de la mésange charbonnière issus de BirdLife International (2014) (http://www.birdlife.org, 03/09/2014)

Figure 8 :

 8 Figure 8 : Suivi corrélatif réalisé dans les trois populations et sur les deux espèces de mésanges

3 .

 3 , sont moins jaunes. En période de reproduction le suivi corrélatif réalisé (Manuscrit 3) montrer que la coloration due à la structure de la plume ou due aux pigments est influencée par le statut immunitaire, le stress physiologique ou la condition corporelle des mésanges charbonnière. à travers le suivi corrélatif de savoir comment la disponibilité en caroténoïdes joue sur le stress oxydatif hors de la période de reproduction, une expérience a été réalisée afin de mieux comprendre les liens n physique et la disponibilité en caroténoïdes. On trouve ainsi qu après la mue en milieu rural, les individus les plus grands ont les plumes du torse plus jaunes (Manuscrit 1, Is oxidative status influenced by dietary carotenoids and physical activity after moult in the great tit (Parus major)?). Par contre, comme pendant la saison de reproduction, trouvé entre et une activation du système immunitaire (N = 41, t = -0.10, p = 0.92), le stress physiologique (N = 41, t = -1.27, p = 0.21), ou oxydatif (N = 41, t < -1.93, p > 0.07). es due à une quantité suffisante disponible . Dans les populations étudiées, aucun lien la mue la sélection de partenaires se base en partie sur la couleur donc il est possible que dans les populations étudiées, le choix du partenaire soit différent des études précédentes. Homogamie et succès de reproduction Comme décrit destination du partenaire. Les individus partenaires leur ressemblant (homogamie positive) ou dissemblables (homogamie négative) (Danchin et al. 2005). Précédemment nous avons vu que la coloration des mésanges charbonnières basée sur les caroténoïdes était différente entre la zone urbaine et la zone rurale possiblement due à une variabilité des caroténoïdes disponibles. est plus forte chez les mésanges charbonnières parisiennes que chez les rurales. Comme il y a pas de différence mesurée entre mâles et femelles au sein des habitats (Manuscrit 3), nous pouvons nous demander et le succès de reproduction vont être influencés par la coloration des plumes jaunes du torse. En effet, malgré le manque de corrélation entre la coloration basée sur les caroténoïdes peut y avoir une sélection des partenaires basé sur la couleur. Comme la coloration jaune des adultes peut-être corrélée à leurs investissements dans la reproduction, les liens entre coloration du couple et ses ont été analysés. Les parties Matériel & Méthodes ainsi que Résultats sont détaillées dans la Box 1. Il y a effectivement une homogamie positive chez les mésanges charbonnières intensité du jaune des plumes du torse comme montré précédemment

Figure 2 :

 2 Figure 2 : Moyenne ± SE de la date (A et B) et la taille (C et D) des premières pontes de mésanges bleues (barres à pois, A et C) et des mésanges charbonnières (barres pleines, B et D (zone urbaine en noire et zone rurale en gris). La taille et la date de ponte varient en fonction des années et plus particulièrement la date de ponte est largement retardée en 2013 pour les deux espèces.

Figure 9 :

 9 Figure 9 : A) Spectre des huit mesures sur un même individu. Calcul sur un spectre de B)caroténoïdes chroma ((R700-R450)/R700) et de C) la brillance dans les UV (Aire sous la courbe de 300-400 nm / Aire sous la courbe de 300-700 nm)

Figure 10 :

 10 Figure 10 : Bilan des différences entre les populations de mésanges bleues (A) et charbonnières (B)

Figure 11 :ManuscritsManuscrit 1 : 2 :Manuscrit 3 : 4 : 1

 1112341 Figure 11 : Bilan des résultats obtenus lors de la thèse grâce au suivi corrélatif (flèche simples pleines) et aux expérimentations en milieu rural (flèches simples en pointillé) uniquement chez les mésanges charbonnières (flèches simples grises) ou chez les deux espèces (flèches simples noires).

  ROM concentration. Total antioxidant capacity test (OXY-Adsorbent test / Diacron, Grosseto, Italy) assesses the antioxidant capacity of plasma including both exogenous and endogenous antioxidants by measuring the ability to resist a massive oxidant attack of hypochlorous acid (HOCl). Plasma (3 µL) was diluted 1:100 with distilled water. A 200 µL aliquot of a HOCl solution was incubated with 5 µL of the diluted plasma for 10 min at 37°C. Then 5 µL of N,N-diethyl-paraphenilenediamine, which is oxidized by the residual HOCl and transformed into a pink derivative, was added. The absorbance was read at 505 nm and values were calculated according to a calibration curve run alongside each essay. Total antioxidant capacity was expressed as µmol of HOCl/mL neutralized(Costantini et al., 2011). A low value indicates a low antioxidant capacity. Samples were measured twice and were found highly repeatable (F 161,162 = 45.1, p < 0.01, intraclass correlation coefficient = 0.96). Mean values were used in this study.

  20 µL of plasma was homogenized with 40 µL of ethanol and then twice extracted by centrifugation with 40 µL of hexane. After that, hexane extracts were pooled and evaporated at 60-65°C under a flow of nitrogen and the residue was dissolved in 100 µL of a 50:50 mix of dichloromethane and methanol. Total carotenoid concentration of each sample was quantified by injecting 25µL of purified extract into a Spherisorb type S5NH2 5 µm C18 reverse-phase column (250 × 4•6 mm, Phase Separation Ltd., Clwyd, UK) with a mobile phase of methanol distilled water /min (using Thermo Spectra System P1000 isocratic pumps), and detected with a UV/Vis absorbance detector at 445 nm (using Thermo SpectraSystem P100 isocratic pumps). The HPLC was calibrated using lutein standards (DSM France) (Relative Standard Deviation (RSD%) = 0.60 at 1 ppm, 0.71 at 2 ppm at 0.28 at 4 ppm,[START_REF] Rachieru | Validation of a method to determine vitamine E (alpha tocopherol) from feed inegredientes by HPLC using reverse phase chromatography[END_REF] ---tocopherol) concentrations were detected with a Spherisorb type ODS2 3-µm C18 reverse-phase column (150 x 4.6 mm Phase Separation, Clwyd, UK), by injecting 25µL of extract with a mobile phase of methanol-distilled water (97:3) at a flow rate of 1.05 mL/min using fluorescence detection for 295 nm wavelength excitation and 330 nm wavelength emission for vitamin E ---tocopherol were identified by comparison with the retention time of tocopherol standards (Sigma, Poole, UK), which were also used to calibrate the HPLC (retinol: RSD% = 1.49 at 1 ppm, 0.76 at 2 ppm et 0.28 at 4 ppm and alpha-tocopherol: RSD% = 1.88 at 0.5 ppm, 1.79 at 1 ppm et 0.37 at 2 ppm).

Figure 1 :

 1 Figure 1: Mean ± SE of total plasma carotenoid concentration (µg/mL) as a function of carotenoid supplementation groups (black: control and grey: supplemented group) and time.

Figure 2 :

 2 Figure 2: Mean ± SE of ROM concentration (µmol of H 2 O 2 /mL) as a function of carotenoid supplementation groups (black: control and grey: supplemented group) and time. ROM concentration marginally increased at the end of the experiment for carotenoid supplemented birds.

Figure 4 : 1 Figure 1

 411 Figure 4: Mean ± SE of UV brightness as a function of handicap treatment (white: control, light grey: handicapped group) and time. UV brightness of breast feathers decreased during the experiment in handicapped birds whereas it did not change for non-handicapped birds.Significant differences between groups are indicated by different letters.

  Figure 2 865

MANUSCRIT 2 when

 2 the first egg had hatched. During the experiment, a data logger (Hygrochron Temperature/Humidity Logger iButton DS1923, Maxim IC, USA) was placed in each nest cup under the eggs before incubation and set to record at 5 min intervals.After all nests included in the experiment had started to incubate and before the first hatching, the warming effect of the warming pads in the absence of the female was measured in two close nest-boxes out of the three containing a nest and eggs and found deserted before incubation and moreover before treatment (heated or control) attribution (see above) (n = 2, clutch sizes = 4 and 7 eggs, nest heights = 65 and 80 mm, respectively for heated and control nests). Temperature was measured over three days and four nights by a data logger placed in the same conditions as in the experimental nests. To limit pseudo-replication and inflation of degrees of freedom, we used mean nocturnal and diurnal temperature per day. In absence of the female, we found an average increase of 4°C in heated nest-box during the night (between 8:00 pm and 8:00 am to cover night time session of birds and heating time of warming pad, t = -2.96, df = 5.73, p = 0.03, Welch test) and no difference during the day (between 8:00 am and 8:00 pm, t = -1.49, df = 5.73, p = 0.25). These temperatures could be compared with daily temperatures in Paris (urban area) and at the CEREEP (rural area) calculated from monthly data provided by the national forecast agency (Météo France) between March and May 2012 to 2014. Minimum monthly temperature was indeed higher in urban area than in the rural area (2.7 ± 1.2°C and -2.7 ± 1.0°C, respectively; t = -3.35, df = 15.5, p < 0.01), maximum(23.3 ± 1.2°C and 23.8 ± 1.0°C, respectively) and mean temperatures were similar (11.7 ± 1.0°C and 10.0 ± 1.0°C, respectively; t = 0.32, df = 16, p = 0.76 and t = -1.15, df = 16.0, p = 0.27 respectively).

  daysold) and control nests(18 ± 0.26 days-old). Fledging success was similar (Chi 2 = 0.02, df = 1, p = 0.88) between heated (55 of 88 nestlings fledged, i.e. 62.5 %) and control nests (41 of 63 nestlings fledged, i.e. 65 %). In a logistic regression model, fledging success did not vary with heating treatment (F 1,20 = 1.23, p = 0.27, estimate (H) = -0.43 ± 0.39) and was positively related to mean nestling mass at 3 days old (F 1,20 = 8.90, p < 0.01, estimate ± SE = 0.85 ± 0.29).Nestling mass varied over time in different ways between heated and control nests (age x heating treatment interaction; F 19,902 = 17.31, p < 0.01,Fig 2). Indeed, at 1 day old, nestlings from heated nests were significantly heavier (2.25 ± 0.07 g) than nestlings from control nests (1.75 ± 0.06 g, F 16,102 = -5.63, p = 0.02, Fig 2, results were qualitatively the same at 3 days old when body mass was available for nestlings of all nests). However, from 4 days until fledging, nestlings from heated and control nests had similar mass (Fig 2). At 14-16 days old, i.e. at the last capture before fledging, nestling tarsus length did not vary with heating treatment or hatching date, whereas body mass significantly increased with tarsus length and did not significantly differ with heating treatment (Fig 2) nor vary with hatching date (Table

  Figure 2

  ,15 = 265.30) or once from each of three pictures of each individual (intraclass , p < 0.01, F 146, 435 = 39.83, , p < 0.01, F 63, 186 = 137.25, intraclass correlation , p < 0.01, F 83, 246 = 45.68 for 2012, 2013 and 2014 respectively) were high and and the mean of the three measurements was used thereafter.

Figure 1 :

 1 Figure 1: Variation in A) carotenoid chroma (ratio), B) UV brightness (ratio) and C) breast

Figure 2 :Figure 1 : 4

 214 Figure 2: Variation in A) carotenoid chroma (ratio), B) UV brightness (ratio) and C) breast

2 .

 2 Habitat and maternal influences on egg morphology and eggshell pigmentation In blue tits, birds from Paris had significantly smaller scores for EggPC1 (egg size) than did rural birds (Fig 1A), the EggPC1 scores differed among years and increased with increasing female body condition, whereas EggPC2 (eggshell thickness) showed no significant variation among habitats (Fig 1B), years or with female body condition (Table

  Fig 1D) whereas in great tits, PigPC2 scores were lower in Paris than in the rural area, suggesting that eggs of Parisian birds had fewer spots (Fig 1D, Table

Figure 1 :

 1 Figure 1: Mean ± SE of A) EggPC1 (egg size), B) EggPC2 (eggshell thickness), C) PigPC1

Figure 2 :

 2 Figure 2: Hatching success (number of eggs hatched relative to clutch size) and fledging

  Figure 1

Box 1 : Suivi corrélatif de la coloration basée sur les caroténoïdes des mésanges charbonnières

  Le but de cette étude est on état de santé au moment de la reproduction es populations et va influencer leurs succès de reproduction. Pour répondre à ces questions les données utilisées sont celles présentées dans le manuscrit 3. Des modèles linéaires mixtes ont été réalisés sur les variables couleurs (fonction lme package nlme & fonction Anova vraisemblance ainsi que sa probabilité ont été calculées manuellement pour chaque effet aléatoire. » ont été réalisés (fonction glm package base & fonction Anova package car, type 3). Le succès s deux paramètres de couleurs de la femelle puis du mâle en tant que facteurs présentés sous forme de tableaux. Les p-values des variables significatives sont mises en gras et les valeurs initiales des variables non gardées dans les modèles finaux sont en italique.

	Tableau de relation Habitat Année Site femelles les plus jaunes. Tableau de relation des couleurs de la femelle expliqué par celles du mâle, Réflectance dans les UV Caroténoïdes chroma F p Effet F p Effet 2.20 1,76 0.14 Urbain = Rural 1.20 1,76 0.27 Urbain = Rural 53.51 4,78 < 0.01 2010 = 2011 = 2012 > 2013 = 2014 52.74 4,77 < 0.01 2011 < 2010 = 2012 = 2013 < 2014 1.35 1,76 0.25 n.s. 7.31 1,77 < 0.01 + 0.01 1,76 0.96 n.s. 0.01 1,76 0.95 n.s. < 0.01 LRT: 133.46 0.04 LRT: 3.07 < 0.01 LRT: 49.03 < 0.01 LRT: 35.92 Réflectance dans les UV Caroténoïdes chroma F p Effet F p Effet Habitat 0.12 1,74 0.73 Urbain =Rural 3.82 1,75 0.05 Urbain > Rural Année 58.76 4,76 < 0.01 2010 = 2011 > 2012 = 2013 = 2014 36.95 4,75 < 0.01 2010 = 2011 = 2012 = 2013 < 2014 1.39 1,74 0.24 n.s. 5.93 1,75 0.01 + 0.09 1,74 0.77 n.s. 0.01 1,74 0.98 n.s. Identité < 0.01 LRT: 380.12 < 0.01 LRT: 9.07 Site < 0.01 LRT: 47.17 < 0.01 LRT: 40.70 Les femelles sont mâles les plus jaunes. production, des modèles Mâles F p Effet F p Effet Date de ponte (DP) 0.16 1,232 0.69 n.s. 0.13 1,183 0.72 n.s. Habitat 0.21 1,232 0.65 Urbain = Rural 1.51 1,183 0.22 Urbain = Rural Année 4.04 4,232 0.40 2010 = 2011 = 2012 = 2013 = 2014 7.74 4,183 0.10 2010 = 2011 = 2012 = 2013= 2014 Caroténoïdes chroma 0.68 1,232 0.41 n.s. 9.33 1,183 < 0.01 + Réflectance dans les UV 0.01 1,232 0.95 n.s. 12.23 1,183 < 0.01 + DP × Habitat 0.29 1,232 0.59 n.s. 1.35 1,183 0.25 n.s. Femelles Mâles F p Effet F p Effet Date de ponte (DP) 11.8 1,232 < 0.01 + 0.71 1, 302 0.34 n.s. Habitat 87 1,232 < 0.01 Urbain < Rural 0.03 1, 183 0.86 Urbain = Rural Année 131.7 4,232 < 0.01 2012 < 2010 = 2011 = 2013 < 2014 221.95 4,302 < 0.01 2012 = 2013 < 2010 = 2011 < 2014 Caroténoïdes chroma 7.03 1,232 < 0.01 + 1.66 1,183 0.20 n.s. Réflectance dans les UV 19.2 1,232 < 0.01 + 0.22 1,183 0.64 n.s. binomiaux « logit Femelles DP × Habitat 2.14 1,232 0.14 n.s. 1.10 1,183 0.29 n.s.

ogie et le succès de reproduction : L pport du suivi corrélatif

		(Box 2 et Manuscrit 4 : Characteristics of egg and eggshell
	influence reproductive success in urban and rural populations of blue tits (Cyanistes (co En particulier, le
	caeruleus) and great tits (Parus major)). Pour aller au-delà du simple impact du milieu, une milieu urbain est connu pour avoir une biodiversité et espèces natives plus
	faibles par rapport aux écosystèmes naturels	(Manuscrit 2, How does
	night temperature during incubation influence reproduction in the great tit (Parus major)?) et
	a permis de montrer une influence de la hausse des températures nocturnes sur le
	comportement des parents et la condition corporelle des jeunes	.
	1.
	analysés (Box 2).
	taille de ponte est plus faible que celle en milieu rural. Pour les mésanges charbonnières
	a pas de différence entre les deux habitats (urbain versus rural). Donc contrairement à ce qui
	était attendu les populations de mésanges bleues et charbonnières
	et du fond sonore (Sun and entre elles malgré la hausse de température (Manuscrit 2). De plus, la taille
	Narins 2005; Barber et al. 2010; Cunnington and Fahrig 2010; Francis et al. 2011) peu diminuée. Il peut y avoir deux explications à ce résultat, 1) soit les mésanges ne peuvent
	une diminution de la qualité	s, 2) soit
	et/ou de la quantité de nourriture disponible investissement des femelles malgré les différences environnementales. A priori la
	biodiversité) (Isaksson and Andersson 2007; Isaksson 2009), peuvent influencer négativement première explication peut être . En effet, la date de
	la qualité d	des jeunes en milieu urbain.	un ou plusieurs
	stimuli environnementaux. La deuxième explication a été
		(Box 2).

Les données du suivi corrélatif de populations ont été utilisées pour mesurer la différence de phénologie et de succès de reproductions chez les mésanges bleues et les mésanges

Box 2 : Suivi corrélatif de la reproduction des mésanges bleues et charbonnières

				t aussi que le taux de
	recrutement permette aux populations de se maintenir. Les données de recrutement ne sont des tests de ² ont été réalisés avec la fonction chisq.test du
	package stats sous R sur la première ponte.		er des tests
	de ², les sites de zone suburbaine (ZSU) et de la zone urbaine (ZU) ont été regroupés par contre les sites de la
	zone rurale ont été étudiés de manière séparée (Site 1R et 2R) car ils ont une occupation très différente des jeunes au moment du baguage a été analysé car la survie du jeune au premier hiver ont des effectifs suffisants pour être analysés seuls.
			(Naef-Daenzer et al. 2001). La condition s dans le tableau 1. globale des deux sites
	de la zone rurale varie avec les années (Site 1 : ² 4 = 42.46, p < 0.01, Site 2 : ² 4 = 56.37, p < 0.01) contrairement au
		-urbaines ( ² 2 = 3.82, p = 0.15) et urbaine ( ² 1 = 1.16, p = 0.28). Quand les
	espèces sont séparées, le même pattern est retrouvé pour les mésanges charbonnières (Site 1R : ² 4 = 63.30, p < mésanges bleues urbaines ne sont pas en moins bonne condition corporelle que leurs voisines 0.01, Site 2R : ² 4 = 27.09, p < 0.01, ZSU : ² 1 = 0.02, p = 0.97, ZU : ² 2 = 1.56, p = 0.46) et pour les mésanges
	rurales contrairement aux jeunes mésanges charbonnières urbaines de qui sont plus grosses : ² 4 = 7.85, p = 0.10, Site 2R :
	² 4 = 19.75, p < 0.01, ZSU : ² 1 = 0.74, p = 0.39, ZU : ² 2 = 2.00, p = 0.37).
	Quelles que			1R est toujours moins
	utilisé que la zone urbaine ( ² 3 > 13, p < 0. 01).
	des jeunes, contrairement aux études précédents (Isaksson and Andersson 2007; Brahmia et
	Tableau 1 :	ccupation des nichoirs selon les espèces (B : mésanges bleues, C : mésanges
	al. 2013) peut-être due à une qualité et une quantité de nourriture disponible similaire entre les charbonnières et ND : espèces non déterminées), les années et les zones
	2010 sites ou à une compensation de la part des parents travers les effets parentaux mais aussi à un 2011 2012 2013 2014 Site 1R 3B + 1C + 1ND 9B + 8C + 0ND 11B + 24C + 2ND 6B + 2C + 0ND 11B + 3C + 1ND
	Site 2R 43B + 23C + 0ND effectif limité. ZSU	26B + 43C + 0ND	32B + 49C + 3ND	20B + 26C + 1ND 26B + 31C + 1ND 0B + 10C + 0ND 17B + 17C + 0ND
	ZU		19B + 25C + 5ND	15B + 16C + 3ND 10B + 22C + 2ND
	charbonnière à Paris par rapport à la zone rurale peut-être expliquée par les plus hautes la variation de phénologie a été analysée.
	températures en ville. Afin de		; Nys
	Afin de quantifier la variation entre les sites et les années de la date et de la taille de la première ponte des 2010) effets parentaux et les succès de reproduction des mésanges charbonnières, une expérience de modèles linéaires mixtes ont été utilisés (fonction lme du package nlme & fonction Anova du package car, type
		ont été mis en facteur fixe	et
	chauffage des nids a été réalisée. site ont été		4) permet de
	conclure que la pigmentation globale chez les mésanges bleues et le nombre de tâches chez
	les mésanges charbonnières sont corrélés à la qualité de la coquille. De plus, nous avons
	montré que le nombre de tâches est inférieur sur les coquilles parisiennes des mésanges
	charbonnières et comme pour la mésange bleue le nombre de tâches est négativement lié au
	cont
	plus faibles pour les deux espèces par rapport aux populations rurales. Le nombre de poussin

mis en facteur aléatoire. Les sorties de modèles sont présentées dans le tableau 2. Le log de la vraisemblance ainsi que sa probabilité ont été calculées manuellement pour chaque effet aléatoire. Chez les

	Taille de ponte		Mésanges bleues		Mésanges charbonnières
		F	p	Effet	F	p	Effet
	Année	12.80 4,143 0.01	2011 = 2012 = 2013 =	24.51 4,157 < 0.01 2012 = 2013 < 2010 =
				2014 < 2010			2011 = 2014
	Habitat	7.75 1,143	< 0.01 Urbain < Rural	0.16 1,157	0.69	Urbain = Rural
	Nichoir		0.50	LRT : -1.86		0.50	LRT : -2.10
	Site		0.03	LRT : 3.30		0.50	LRT : -0.90
	mésanges charbonnières ni la date, ni la taille de ponte ne sont influencées		(Figure 2).
	Tableau 2						du site. Les p-value
	des variables significatives sont en gras.
	Date de ponte		Mésanges bleues		Mésanges charbonnières
		F	p	Effet	F	p	Effet
	Année	296.52 4,143 < 0.01 2014 < 2010 = 2011 =	303.27 4,157 < 0.01 2014 < 2010 = 2011 =
				2012 < 2013			2012 < 2013
	Habitat	0.01 1,143	0.97	Urbain = Rural	0.06 1,157	0.81	Urbain = Rural
	Nichoir		0.50	LRT : 0.01		0.50	LRT : -4.29
	Site		0.50	LRT : -10.75		0.50	LRT : -32.33

D

  Le but de cette partie est de voir si la morphologie des poussins est différente entre les populations.

	Comme le tarse et la masse sont corrélés pour les deux espèces (mésange bleue : N = 1493, t = 48.17, p < 0.01,
	r pearson = 0.78, mésange charbonnière : N = 1503, t = 40.65, p < 0.01, r pearson = 0.72)
	corporelle des jeunes indépendamment de la taille, des modèles linéaires mixtes (fonction lme package nlme &
	fonction Anova package car, type 3, méthode REML) ont exploré la variation des résidus de la masse régressé
	sur la longueur du tarse en fonction de	et du site comme facteurs fixes et le fait que le jeune se soit
	envolé ou pas ainsi que l						Le log de la
	vraisemblance ainsi que sa probabilité ont été calculés manuellement pour chaque effet aléatoire.
	Tableau
			Mésanges bleues			Mésanges charbonnières
		F	p	Effet		F	p	Effet
	Année	68.61 4,1378	< 0.01 2010 < 2011 = 2012	21.67 4,1383	< 0.01 2012 < 2010 = 2011
				< 2013 < 2014				= 2013 = 2014
	Habitat	2.99 1,1378	0.08	Urbain > Rural		5.73 1,1383	0.02	Urbain > Rural
	Envol	9.77 1,1378	< 0.01 non envolé < envolé 41.00 1,1384	< 0.01 non envolé < envolé
	Nichoir		0.5	LRT : -237.88			0.5	LRT : -198.10
	Site		< 0.01 LRT : 26.30			< 0.01 LRT : 21.38

III. Discussion et perspectives

	Cette étude se base sur les données récoltées pendant un suivi de populations de trois ans pour
	la zone urbaine, cinq pour la zone rurale
	répond à plusieurs contraintes logistiques, entre autres, le site le plus urbain devait-être
	composé de parcs et jardins de Paris. Paris en tant que capitale de la France devrait pouvoir se
	; Pickett et comparer aux autres capitales européennes et en effet, elle est comparable en nombre
	al. 2011),	s à	s nocturnes allait diminuer le coût de plus densément
	2) a peuplée au monde loin devant les autres capitales européennes, ce qui en fait une ville
	montré que	trouvés dans cette étude à
		s, néanmoins, les effets mis en valeurs ici peuvent donner des pistes sur le
	parents mais influençait sur le poids des devenir des populations aviaires en zone urbaine.	la fréquence de nourrissage des
	adultes.
	entre les populations. En particulier chez les mésanges charbonnières, la coloration basée sur
	les caroténoïdes varie entre les populations et avec la pollution au contraire de la coloration
	basée sur la mélanine. Afin de mieux comprendre comment des oiseaux urbains peuvent-être
	composi plus jaunes il faut revenir à la méthode de mesure et comparer les résul -
	et si il y a une base génétique et/ réalisée. La réflectance des plumes a été mesurée quatre fois deux lots de quatre donc chaque
	paramètres environnementaux sur la reproduction des oiseaux en ville permettrait de lancer individu a huit spectres (Figure
	des actions concrètes dans le but de favoriser la biodiversité aviaire en zone urbaine.

  ).

			par contre selon
	-mue une activité physique diminue la brillance dans les UV et une
	supplémentation en lutéine et zéaxanthine diminue le jaune des plumes. Cela explique en
	partie pourquoi les mésanges rurales sont moins jaunes. En effet, la proportion de zéaxanthine
	par rapport aux autres caroténoïdes semble être		en milieu urbain
	(Isaksson 2009). Cette hypothèse va bientôt pouvoir être vérifiée	HPLC de la
	composition en caroténoïdes des vitellus congelés		utilisés dans le
	manuscrit 4.
	Même si la variation
			le
	positive mesurée. La corrélation entre la couleur des adultes et leurs succès de reproduction
	couleur des plumes des jeunes permettra d	plus d éléments sur la variation de la

Table 1 :

 1 2.15.0 (R Development CoreTeam 2012). We used linear mixed models with backward eliminati criterion to select the best predictive model. Final models were maximum-likelihood (REML) linear mixed models, run using the function lme in the package nlme and the function Anova in the package car, with type III sum of squares). Total carotenoid, retinol and total vitamin E ROM concentration, total carotenoids, vitamin A and vitamin E), on stress response (blood sedimentation and ratio H/Ly) and on plumage colour (lutein absorbance, carotenoid chroma and UV brightness), all models included as fixed effects: tarsus length and body mass, and three-way interactions among carotenoid supplementation or physical handicap with sex and time of experiment (start or end of the experiment). When final models included significant interactions, difference of least squares means were tested between levels of treatments or sex with adjusted values of p, using the Tukey-Kramer method in SAS v. 9.3 (SAS Institute Inc., Cary, NC, USA).Means are presented ± SE. At the start of the experiment, plasma composition (antioxidant capacity, ROM concentration, vitamin A, E and total carotenoid concentrations), immune system activation (blood sedimentation), stress response (H/Ly), colour and morphological values were not significantly different among groups or aviaries (F 1,11 < 2 and p > 0.05).Aberrant values were deleted for ROM concentration (2 values higher than 10 µmol of H 2 O 2 equivalents). One male was not weighed at the end of experiment. Total carotenoid concentration was missing for 2 individuals at the start and 2 others at the end of the experiment due to insufficient plasma. Retinol and vitamin E values were missing for one female at the end of experiment for the same reason. MANUSCRIT 1 Marie Vaugoyeau: designed experiment, captured, manipulated and fed birds in aviaries, did antioxidant system and blood smears analyses, did statistical analyses and wrote article Beatriz Decencière and Samuel Perret: captured and fed birds in aviaries Filiz Karadas: ran carotenoids and vitamins analyses Sandrine Meylan and Clotilde Biard designed experiment, captured, manipulated birds and helped for statistical analyses and wrote article. MANUSCRIT 1 Mean ± SE for all measured parameters for males and females at the start and at the 829 end of experiment for each treatment group 830

	plasma concentrations and H/Ly ratio were log-transformed. No residuals of final models
	deviated from a normal distribution.
	All statistical models included aviaries and individual identity as random factors, which were

not significant for any model (aviaries: Z 1,12 < 1.06 and p > 0.14 and individual identity: Z 1,41 < 1.28 and p > 0.20). According to predictions 1 and 2, in order to test the effects of physical activity and dietary carotenoid availability on the antioxidant system (total antioxidant MANUSCRIT 1 capacity,

Table 2 :

 2 Summary of the effects of carotenoid supplementation and physical handicap treatments on plasma composition, antioxidant system, immune and physiological stress, and feather colour in adult great tit ("0" indicates no significant effect, "-" a significant negative effect, "(-)" a slightly negative effect and "+" a significant positive effect)

	Carotenoid supplementation	Physical
		handicap

Table S1 :

 S1 Results from a linear mixed model with log-transformed total carotenoid, and vitamin E and retinol concentrations (µg/mL) as 881 dependent variables. For information, initial values of statistical tests are also presented for variables that were not retained in the final model (in 882

	sex × handicap	2.20 1,25	0.14	-0.51 ± 0.35 (M×H+)	0.26 1,25 0.61	-0.56 ± 1.24 (M×H+)	0.02 1,25	0.90 0.09 ± 0.67 (M×H+)
	italics). Significant p-values in the final models are in bold. time × sex × 883 884 carotenoid suppl 0.41 1,28 0.52 0.28 ± 0.44 (End×M×C+)	0.85 1,28 0.36	-1.56 ± 1.71 (End×M×C+)	1.28 1,32	0.26	1.08 ± 0.96 (End×M×C+)
	time × sex × handicap	Total carotenoid concentration p Estimate ± SE 0.01 1,28 F df 0.96 -0.02 ± 0.47 (End×M×H+)	Vitamin E concentration p Estimate ± SE 0.02 1,28 0.90 F df 0.22 ± 1.74 (End×M×H+)	F df 2.99 1,32	Retinol concentration p Estimate ± SE -1.68 ± 0.97 0.08 (End×M×H+)
	intercept 885	1.01 1,33	0.31	0.16 ± 0.16	65.23 1,36 <0.01	-1.65 ± 0.20	571.61 1,40 <0.01	-2.30 ± 0.10
	tarsus length	2.70 1,25	0.10	0.19 ± 0.12	2.91 1,25 0.10	0.84 ± 0.42	1.98 1,25	0.16	0.29 ± 0.20
	body mass	1.00 1,28	0.32	0.08 ± 0.08	0.09 1,28 0.76	0.07 ± 0.28	0.49 1,32	0.49	-0.10 ± 0.14
	time	2.98 1,33	0.08	0.28 ± 0.17 (End)	0.29 1,28 0.60	-0.50 ± 0.92 (End)	0.50 1,32	0.48	0.35 ± 0.49 (End)
	sex	1.06 1,25	0.30	-0.27 ± 0.26 (M)	0.72 1,25 0.39	-0.73± 0.90 (M)	0.06 1,25	0.80	-0.12 ± 0.48 (M)
	carotenoid suppl	0.39 1,9	0.53	-0.14 ± 0.22 (C+)	1.26 1,9	0.26	-1.13 ± 1.01 (C+)	0.03 1,9	0.86	0.09 ± 0.53 (C+)
	handicap	0.02 1,9	0.89	-0.03 ± 0.22 (H+)	0.30 1,9	0.58	0.53 ± 0.98 (H+)	0.31 1,9	0.58	-0.28 ± 0.51 (H+)
	time × sex	0.12 1,28	0.73	0.12 ± 0.34 (End×M)	0.04 1,28 0.83	0.28 ± 1.27 (End×M)	0.46 1,32	0.50	0.46 ± 0.68 (End×M)
	time × carotenoid suppl	14.61 1,33 <0.01	0.83 ± 0.22 (End×C+)	0.52 1,28 0.47	0.97 ± 1.35 (End×C+)	1.02 1,32	0.31	-0.75 ± 0.74 (End×C+)
	sex × carotenoid suppl	0.37 1,25	0.54	0.22 ± 0.36 (M×C+)	1.08 1,25 0.30 1.25 ± 1.27 (M×C+)	0.01 1,25	0.99	-0.01 ± 0.69 (M×C+)
	time × handicap	4.67 1,33	0.03	-0.49 ± 0.23 (End×H+)	0.23 1,28 0.63	0.63 ± 1.30 (End×H+)	0.58 1,32	0.45	0.55 ± 0.73 (End×H+)
										35
	MANUSCRIT 1 MANUSCRIT 1

Table S2 :

 S2 Results from a linear mixed model with total antioxidant capacity (µmol of HOCl/mL) and ROM concentration (µmol of H 2 O 2 equivalents) as dependent variables. For information, initial statistical tests are also presented for variables that were not retained in the final model (in italics). Significant p-values in the final models are in bold.

			Antioxidant capacity		ROM concentration
		F df	p	Estimate ± SE	F df	p	Estimate ± SE
	intercept	1062.16 1,39 <0.01 258.07 ± 7.92	16.55 1,39 <0.01 1.47 ± 0.36
	tarsus length	0.29 1,25	0.59 -3.63 ± 6.49	0.53 1,25 0.47 -0.02 ± 0.19
	body mass	0.01 1,33	0.94 -0.09 ± 4.44	0.12 1,28 0.73 -0.01 ± 0.12
	time	31.21 1,39	<0.01 -46.90 ± 8.40 (End)	0.31 1,39 0.58 -0.27 ± 0.48 (End)
	sex	17.72 1,28	<0.01 -24.19 ± 5.75 (M)	0.19 1,25 0.67 -0.86 ± 0.40 (M)
	carotenoid suppl	0.60 1,9	0.44 -12.78 ± 16.17 (C+)	0.01 1,10 0.92 -0.06 ± 0.62 (C+)
	handicap	1.35 1,10	0.25 -1.47 ± 9.10 (H+)	0.61 1,9	0.44 0.18 ± 0.49 (H+)
	time × sex	0.10 1,33	0.76 -5.54 ± 18.35 (End×M)	0.30 1,33 0.59 1.06 ± 0.55 (End ×M)
	time × carotenoid suppl	0.32 1,33	0.57 11.32 ± 20.00 (End×C+)	3.80 1,39 0.05 1.61 ± 0.82 (End×C+)
	sex × carotenoid suppl	0.59 1,25	0.44 15.32 ± 19.96 (M×C+)	1.60 1,27 0.21 1.05 ± 0.54 (M×C+)
	time × handicap	4.97 1,39	0.03 23.06 ± 10.35 (End×H+)	2.08 1,28 0.15 -0.30 ± 0.57 (End×H+)
	sex × handicap	2.93 1,25	0.10 -32.25 ± 19.59 (M×H+)	0.98 1,25 0.32 -0.03 ± 0.54 (M×H+)
	time × sex × carotenoid suppl 0.03 1,33	0.86 -4.51 ± 25.90 (End×M×C+)	0.81 1,35 0.37 -2.33 ± 0.75 (End×M× C+)
	time × sex × handicap	0.80 1,33	0.37 23.19 ± 26.23 (End×M×H+)	1.96 1,28 0.16 0.66 ± 0.77 (End×M×H+)
	MANUSCRIT 1

Table S3 :

 S3 Results from a linear mixed model with blood sedimentation rate and H/Ly ratio as dependent variables. For information, statistical tests are also presented for variables that were not retained in the final model (in italics). Significant p-values in the final models are in bold.

			Blood sedimentation rate			H/Ly ratio
		F df	p	Estimate ± SE	F df	p	Estimate ± SE
	intercept	7.90 1,38 <0.01 0.48 ± 0.17	57.17 1,36 <0.01 -2.18 ± 0.32
	tarsus length	0.26 1,25 0.61	-0.01 ± 0.01	0.54 1,25 0.46 -0.20 ± 0.30
	body mass	3.71 1,38 0.05	0.02 ± 0.01	0.42 1,30 0.51 0.11 ± 0.18
	time	20.20 1,38 <0.01 -0.06 ± 0.01 (End)	0.13 1,36 0.72 -0.22 ± 0.22 (End)
	sex	11.77 1,27 <0.01 -0.10 ± 0.03 (M)	1.41 1,27 0.23 0.13 ± 0.37 (M)
	carotenoid suppl	2.11 1,9	0.15	0.06 ± 0.04 (C+)	2.80 1,10 0.09 0.88 ± 0.52 (C+)
	handicap	0.90 1,10 0.34	-0.02 ± 0.02 (H+)	2.41 1,9	0.12 -1.06 ± 0.68 (H+)
	time × sex	0.08 1,33 0.78	-0.01 ± 0.05 (End×M)	0.02 1,36 0.89 0.58 ± 0.31 (End ×M)
	time × carotenoid suppl	3.05 1,33 0.09	-0.09 ± 0.05 (End×C+)	0.15 1,30 0.70 -0.24 ± 0.61 (End×C+)
	sex × carotenoid suppl	2.37 1,25 0.12	-0.08 ± 0.05 (M×C+)	5.36 1,27 0.02 -1.32 ± 0.57 (M×C+)
	time × handicap	0.89 1,33 0.34	0.05 ± 0.05 (End×H+)	0.38 1,30 0.54 -0.36 ± 0.60 (End×H+)
	sex × handicap	8.83 1,27 < 0.01 -0.10 ± 0.03 (M×H+)	1.47 1,25 0.23 0.87 ± 0.76 (M×H+)
	time × sex × carotenoid suppl 2.40 1,33 0.12	0.11 ± 0.07 (End×M×C+)	0.06 1,30 0.81 0.19 ± 0.81 (End×M× C+)
	time × sex × handicap	0.25 1,33 0.61	-0.03 ± 0.07 (End×M×H+)	0.87 1,30 0.35 0.75 ± 0.82 (End×M×H+)
	MANUSCRIT 1

Table S4 :

 S4 Results from a linear mixed model with lutein absorbance, carotenoid chroma (ratio) and UV brightness (ratio) as dependent variables.For information, initial statistical tests are also presented for variables that were not retained in the final model (in italics). Significant p-values in the final models are in bold.

		Abstract			(M×H+)					(M×H+)
	time × sex × carotenoid suppl Urban areas are characterised by heat islands, with higher temperatures during the night, as 1.57 1,33 0.21 -32.53 ± 26.40 (End×M×C+) 0.62 1,33 0.43 0.03 ± 0.04 (End×M×C+)	1.50 1,33	0.22	(End×M×C+) -0.01 ± 0.001
	Lutein absorbance p Estimate ± SE 0.83 5.40 ± 26.68 (End×M×H+) compared to neighbouring rural areas. Temperature affects many life stages of organisms, Carotenoid chroma F df F df p Estimate ± SE time × sex × handicap 0.05 1,33 1.03 1,33 0.31 0.04 ± 0.04 (End×M×H+) especially early development. In egg-laying species, incubation represents an energetic cost	F df 0.01 1,33	UV brightness Estimate ± SE 0.001 ± 0.008 0.92 p (End×M×H+)
	intercept for parents and parental behaviour during incubation may therefore influence offspring 533.84 1,39 0.01 188.51 ± 8.16 4.64 1,37 0.03 -0.52 ± 0.24	5444.45 1,39 <0.01	0.23 ± 0.003
	tarsus length body mass development. Incubation behaviour is indeed accompanied by a change in feeding behaviour 0.06 1,25 0.81 -1.56 ± 5.88 7.72 1,28 <0.01 0.03 ± 0.01 0.29 1,33 0.59 1.68 ± 4.10 1.99 1,37 0.16 0.01 ± 0.01	1.00 1,25 2.75 1,33	0.32 0.10	-0.002 ± 0.002 -0.002 ± 0.001
	time	1.49 1,39 leading to a trade-off between incubation temperature and adult health. Currently, few studies 0.22 -13.62 ± 11.15 (End) 0.12 1,37 0.73 0.004 ± 0.01 (End)	0.02 1,39	0.90	-0.01 ± 0.01 (End)
	sex	0.01 1,28 have investigated the impact of environmental temperature during incubation on the 0.93 0.89 ± 10.61 (M) 0.63 1,25 0.43 0.02 ± 0.03 (M)	8.60 1,27	< 0.01 -0.01 ± 0.003 (M)
	carotenoid suppl handicap reproductive behaviour of female birds. We addressed this question by experimentally 0.68 1,9 0.41 13.07 ± 15.22 (C+) 0.24 1,10 0.63 -0.008 ± 0.02 (C+) 3.46 1,9 0.06 -28.15 ± 14.76 (H+) 0.01 1,9 0.94 -0.002 ± 0.03 (H+)	2.28 1,9 5.94 1,10	0.13 0.01	-0.01 ± 0.004 (C+) -0.01 ± 0.004 (H+)
	time × sex increasing the nest temperature at night by a few degrees (4°C) during the incubation period, 4.67 1,39 0.03 32.18 ± 14.89 (End×M) 4.47 1,33 0.04 -0.06 ± 0.03 (End×M)	1.01 1,33	0.32	(End×M) 0.005 ± 0.005
	time × carotenoid suppl	1.33 1,33	0.25	23.00 ± 20.38 (End×C+)	4.36 1,37 0.04	-0.04 ± 0.02 (End×C+)	0.46 1,33	0.50	0.004 ± 0.006 (End×C+)
	sex × carotenoid suppl	0.16 1,25	0.69	-7.95 ± 19.23 (M×C+)	0.01 1,25 0.94	0.004 ± 0.04 (M×C+)	0.01 1,25	0.91	0.01 ± 0.01 (M×C+)
	time × handicap	2.69 1,33	0.10	32.21 ± 15.79 (End×H+)	0.94 1,33 0.33	-0.03 ± 0.03 (End×H+)	6.50 1,39	0.01	-0.01 ± 0.003 (End×H+)
	sex × handicap	0.73 1,25	0.39	15.35 ± 18.84	0.05 1,25 0.82 0.01 ± 0.04 (M×H+)	6.80 1,27	<0.01	0.004 ± 0.003
		MANUSCRIT 1 temperature during incubation decreased nestling feeding behaviour but did not affect parental
		health in terms of body condition, immune status, physiological and oxidative stress. Third,
		nestling mass at hatching was greater but nestling mass gain was slower in heated nests than
		in control nests, resulting in similar fledging mass. The present study demonstrates that
		increased environmental temperature during incubation influenced nestling development in
		the great tit and especially hatchlings mass. Temperature increase had no effect on nestlings
		or adults					or future
		reproduction, especially could decrease basal metabolic rate.
		MANUSCRIT 2

using the great tit (Parus major) as a model species. We measured the consequences of a daily mean increase in nest temperature during incubation on (i) parental behaviour (incubation and nestling feeding), (ii) parental health (estimated by body condition, immune status, physiological and oxidative stress) and (iii) reproductive success (nestling body condition, growth, i.e. mass gain, hatching and fledging success and nestling immune status, physiological and oxidative stress). This study yields three major results. First, we found that heating the nest did not modify the duration of incubation. Second, increasing nest

  Data were collected in 2013 in a population of great tits breeding in a private forest in a rural area near Paris (80 km), at the Centre de Recherche en Écologie Expérimentale et Prédictive 120 nest boxes (Schwegler woodconcrete nest boxes 2M, Valliance, Saint Pierre La Palud, France) evenly distributed across a homogenous deciduous woodland, and used by blue tits as well as great tits. Nest boxes were regularly inspected to determine laying date, and nest height was measured with a ruler to the next mm on the day the first egg was laid. To identify the species before incubation, one egg was weighed when clutch size was 6 or 7 eggs. When its mass was greater than 1.30 g, it was considered to have been laid by a great tit (mean ± S.E. egg mass of blue tits = 1.15 ± 0.02 g and of great tit = 1.60 ± 0.03 g; n=133 and n=145 eggs, respectively, data collected in the same population during the reproductive seasons 2010 to 2012, see alsoDel Hoyo et al.

1. Data collection and experimental protocol

MANUSCRIT 2

  Absorbance was read at 505 nm (Microplate reader model 680, Bio-Rad).The concentration of ROMs was calculated according to a calibration curve obtained by measuring the absorbance of a standard solution and was expressed as µmol of H 2 O 2 equivalents. The concentration of ROMs was highly repeatable (12 samples analysed twice, 2 was no significant difference in the nestling age at which males and females were captured according to heating treatment (males: H: 7.5 ± 0.8 days old and C: 8.6 ± 1.6 days old; t = 0.62, df = 5.93, p = 0.56, females: H: 5.6 ± 0.9 days old and C: 6.6 ± 1.1 days old; t = 0.73, df = 14.92, p = 0.48). However, although nests were randomly attributed to treatments, adult tarsus length happened to differ significantly by heating treatment in interaction with sex (F 1,7 = 14.43, p < 0.01); the tallest females being found in the heated group (H: 22.66 ± 0.14 mm, C: 22.12 ± 0.16 mm, t = -2.58, df = 12.87, p = 0.02) and the tallest males in the control group (H: 22.43 ± 0.18 mm, C: 23.16 ± 0.14 mm, t = 3.25, df = 10.93, p < 0.01). Body mass was significantly and positively correlated with tarsus length in males (r Pearson = 0.59, t 11 = 2.45, p = 0.03) but not in females (r Pearson = 0.13, t 13 = 0.48, p = 0.64). As an index of body condition, body mass was used for females, and residuals of the regression of body mass on tarsus length

	nest into account (Likelihood ratio test = -229.92, p = 0.5), including two-ways interactions with heating treatment and age. Linear mixed models with heating treatment and nest identity as a random effect were used to test for differences between treatments at each nestling age (Fig 2, likelihood ratio test < -50, p = 0.5). their concentration. F 11,12 = 30.77, p < 0.01, intraclass correlation coefficient = 0.94). Variation in nestling body mass, tarsus length, blood sedimentation rate, haematocrit,
	leucocyte buffy coat, total antioxidant capacity and ROM concentration (log-transformed)
	3. Statistical analyses before fledging (i.e. at 14-16 days old) were analysed using restricted maximum-likelihood
	All statistical analyses were performed in R v. 2.15.0 (R Development CoreTeam 2012). Due linear mixed models, with nest identity as a random effect, and backward elimination of
	to small sample size, treatment effects were tested using Welch t-tests for hatching date,
	duration of incubation period, incubation temperature (nocturnal and diurnal mean, minimal
	and maximal during incubation period (supplementary section) per nest), percent of humidity
	Linear models (function lm, package base & function Anova, package car, type III) with
	backward elimination of	Information Criterion (AIC) were used to select
	the best predictive model for adult blood sedimentation rate, leucocyte buffy coat,
	haematocrit, H/Ly, total antioxidant capacity and ROM concentration. In order to test the
	models (function lm, package base & function Anova, package car, type III) with nest identity effects of adult body condition and heating treatment on oxidative stress (total antioxidant
	as a random effect (likelihood ratio test = -1.56, p = 0.5) and backward elimination of factors capacity and ROM concentration), immune status (blood sedimentation rate and leucocyte
	were used to select the best predictive model. buffy coat) and stress response (haematocrit and H/Ly), models included body mass for
	Neither laying date, nest height, egg mass, incubation date, nor clutch size differed between females and residuals of body mass on tarsus length for males, two-ways interaction with date
	experimental groups (t-test, N control = 9, N Heated = 12, all t < 0.7, and p > 0.16). To be able to of capture and heating treatment. The effect of heating treatment on nestling mass with
		13
	MANUSCRIT 2 MANUSCRIT 2

(nocturnal and diurnal mean, minimal and maximal during incubation period (supplementary section) per nest), and Chi² tests for hatchling and fledging success.

(function glm, package base & function Anova, package car, type III) including the interaction between heating treatment and mean nestling mass at 3-days old (first day for which all nestling mass were available) was used to test the influence of hatchling mass and treatment on fledging success. As parental feeding behaviour can be modulated by treatment but also by brood size, date of observation and slightly by rain

[START_REF] Gibb | Feeding rates of Great Tits[END_REF]

we used linear compare physiological measures of adults between control and heated nests it was important to verify that all were caught during the same window of the nestling period. Indeed, there MANUSCRIT for males (F 1,11 = 5.89, p = 0.03, estimate = 1.25 ± 0.51). Statistical analyses were run separately for males and females.

Welch t-tests were used to test for the effect of heating treatment on adult body condition. increasing age was tested using a restricted maximum-likelihood linear mixed model with nest identity as a random effect to take the non-independence of nestlings sharing the same factors by AIC (package nlme, function lme & function Anova, package car, type III).

Table 1 :

 1 Comparison of nest temperature and humidity, incubation duration, clutch size and parental behaviour in control (C) and heated (H) nests. Significant p-values are in bold.

		Mean ± SE	Heating effect
		C nests	H nests	df	t	p
	Mean day temperature (°C)	20.7 ± 0.5	25.9 ± 1.8	12.85	-2.74	0.02
	Mean night temperature (°C)	18.6 ± 0.7	25.9 ± 2.0	13.81	-3.44	< 0.01
	Mean day humidity (%)	63.1 ± 2.2	49.9 ± 3.7	17.21	3.05	< 0.01
	Mean night humidity (%)	61.1 ± 2.3	45.9 ± 3.8	17.35	3.40	< 0.01
	Incubation duration (days)	11.7 ± 0.4	12.0 ± 0.3	13.91	-0.64	0.53
	Clutch size	8.22 ± 0.4	9.2 ± 0.5	16.85	-1.48	0.16
	Feeding behaviour (visits/hour) 14.0 ± 2.9	5.5 ± 1.8	1,13	8.93	0.01

Table 2 :

 2 Models investigating variation in adult great tits immune status (A) physiological 741 stress (B) and oxidative stress (C) as a function of heating treatment, body mass (females) or 742 body condition (males, see methods) and date of capture. The interaction between date and 743 heating treatment was tested but was never significant (females: F 1,9 < 3.30, p > 0.10, males: 744 F 1,6 < 0.40, p > 0.50). For information, initial statistical tests from the full starting models are 745 also presented for variables that were not retained in the final model (in italic).

	Significant p-

  the last 752 capture before fledging. For information, initial statistical tests from the full starting models 753 are also presented for variables that were not retained in the final model (in italic). Significant 754 p-values in the final models are in bold. Likelihood Ratio Tests (LRT in table) were manually 755 calculated for random effect of individuals and nest identities. Heating treatment did not 756 Temperature recorded in the nests over the course of the experiment, i.e. during incubation. Mean ± SE temperature are given for each 12h period (data for day temperatures are placed at each graduation, and those for nights between graduations). Grey values represent control nests and black values represent heated nests, numbers indicate the number of nests in which temperature was recorded each night. The late temperature increase in heated nest is due to last nests still to hatch, which had high temperatures at the end of

	Nest identity Date of hatching Figure legends Figure 1		0.41 1,55	0.5 0.52	LRT: -17.50	0.003 ± 0.004
	affect nestling phenotype. B) Immune status 757 Blood sedimentation rate Tarsus length Body mass Date of hatching Heating treatment Date of hatching × Heating treatment Heating treatment Date of hatching × Heating treatment Individual identity Nest identity D) Oxidative stress Tarsus length 8 8 Total antioxidant capacity Figure 1: incubation. 11 11 11 12 8	3.54 1,57 5.94 1,57 0.28 1,9 1.25 1,9 1.29 1,9 2.13 1,8 2.11 1,8 8.02 1,57 < 0.01 0.06 0.01 0.59 0.26 0.26 0.14 0.15 0.5 0.5 8 9 9 8 12 12 12 12 12 LRT: -2.48 e -14 (H) 3.02 ± 2.39 -0.03 ± 0.02 0.010 ± 0.005 -0.01 ± 0.01 (DH:H) -0.02 ± 0.02 (H) 2.44 ± 1.68 (DH:H) -0.02 ± 0.01 LRT: -1.90 -20.10 ± 7.41 8 7
	Individual identity Body mass	7	0.33 1,56	0.5 0.57	Nestlings LRT: -5.68 e -14	-1.51 ± 2.81
	F 0.59 1,9 df Figure 2: Body mass of nestlings (mean ± SE) over the growth period in the nest as a function P Estimate ± SE Nest identity 0.5 LRT: -28.56 Date of hatching 0.44 2.54 ± 3.02
	A) Morphology Heating treatment of nestling age (day 0 = hatching) and heating treatment (grey: control nests, black: heated 0.45 1,9 0.50 (H) 463.75 ± 721.06
	Tarsus length Leucocytes buffy coat Date of hatching × Heating treatment nests). Stars indicate significant differences between treatments at each nestling age step (*: p 0.45 1,9 0.50 (DH:H) -3.43 ± 5.33
	Date of hatching Tarsus length Individual identity = 0.05; ** p < 0.05, linear mixed models). 0.23 1,9 0.04 1,55	0.63 0.93 0.5	(H) -0.02 ± 0.03 -0.001 ± 0.001 LRT: -7.77 e -10
	Heating treatment Body mass Nest identity		0.01 1,9 0.04 1,55	0.96 0.85 0.5	LRT: -20.17	0.57 ± 8.33 0.001 ± 0.001
	Date of hatching × Heating treatment Date of hatching	0.01 1,9 0.94 1,8	0.98 0.33	(DH:H) -0.01 ± 0.06 -0.001 ± 0.001
	Individual identity Heating treatment ROM concentration		0.06 1,8	0.5 0.80	LRT: -1.71 e -11 (H) -0.04 ± 0.15
	Nest identity Date of hatching × Heating treatment Tarsus length	0.07 1,8 0.40 1,56	0.5 0.80 0.53	LRT: -0.35 (DH:H) 0.001 ± 0.001 -0.13 ± 0.21
	Individual identity Body mass		0.71 1,56	0.5 0.40	LRT: -2.57 e -9 0.06 ± 0.07
	Body mass before fledging Nest identity Date of hatching	0.26 1,9	< 0.01 0.61		LRT: 70.89 0.03 ± 0.05
	Tarsus length Heating treatment		68.34 1,74 < 0.01 0.13 1,9 0.72	1.31 ± 0.16 (H) 6.08 ± 13.00
	Date of hatching C) Physiological stress Date of hatching × Heating treatment	0.01 1,9 0.14 1,9	0.92 0.71		0.05 ± 0.09 -0.05 ± 0.10
	Heating treatment Haematocrit Individual identity		2.86 1,11	0.09 0.5	(H) -0.86 ± 0.40 LRT: -5.12 e -13
	Date of hatching × Heating treatment Tarsus length Nest identity	3.00 1,9 0.01 1,55	0.08 0.98 0.05	(DH:H) 0.27 ± 0.17 -0.01 ± 0.01 LRT: 2.69
	Individual identity Body mass 758		5.54 1,56	0.5 0.02	LRT: 0	0.006 ± 0.003
	MANUSCRIT 2 MANUSCRIT 2 MANUSCRIT 2 MANUSCRIT 2

Table 1 :

 1 Comparison of nest temperature and humidity in control (C) and heated (H) nests.

	Significant p-values are in bold.
		Mean ± SE	Heating effect
		C nests	H nests	df	t	p
	Minimal temperature (°C)	15.3 ± 0.7	19.5 ± 1.4	16.29	-2.70	0.02
	Maximal temperature (°C)	23.4 ± 0.5	30.5 ± 1.7	12.90	-3.87	< 0.01
	Minimal humidity (%)	53 ± 3	34 ± 4	18.97	3.79	< 0.01
	Maximal humidity (%)	73 ± 1	70 ± 3	15.07	0.85	0.41
	Figure 1: Minimal (diamond) and maximal (square) remperature recorded in the nests over the
	course of the experiment, i.e. during incubation. Mean ± SE temperature are given for each
	12h period (data for day temperatures are placed at each graduation, and those for nights
	between graduations). Grey values represent control nests and black values represent heated
	nests. The late temperature increase in heated nest is due to last nests still to hatch, which had
	high temperatures at the end of incubation.

MANUSCRIT 3

  = -1.22, df = 60.98) and leucocytes (p = 0.91, t = -0.12, df = 46.30) rates did not differ between urban and rural habitat. Means are presented ± SE. No residuals of final models deviated from normal distribution. All statistical models included site (forests and the different urban parks) and individual identity as random factors, for which Likelihood Ratio Tests were manually calculated (Table2).Breast stripe size did not differ between habitats or with pollution(Fig 1C and 2C, Table

	Carotenoid chroma, UV brightness and breast stripe size were examined with linear mixed yearling birds. UV brightness did not differ between habitats or with pollution (Fig 1B and
	models and backward e 2B), but only by year (Fig 2B).	nformation Criterion (AIC) to select
	the best predictive model (package nlme, function lme, method REML & package car,
	function Anova, type III). We tested the effect of NO 2 concentration, year and three way
	interaction with sex, age and habitat as fixed factors. Moreover, as coloured signals may vary
	with individual parameters [8, 16] we included age, blood sedimentation, haematocrit,
	leucocytes buffy coat, residuals of the regression of body mass on tarsus length (F 1,296 =
	229.15, p < 0.01, estimate = 0.98 ± 0.06). Welch t-test were used to test for the effect of
	habitat on immune status. Sedimentation (p = 0.59, t = -0.54, df = 79.42), haematocrit (p =
	). Repeatability of parameters among sets of feathers within individuals and among individuals (between sets of feathers: p < 0.01, F 1125,3396 > 37,; 0.23, t Results
	between individuals:	p < 0.01, F 562,2244 > 32), and
	individual means were subsequently used for each parameter. The two colour variables were NO2 concentration was similar among years (27.8 ± 1.42.µg/m³, 27.2 ± 2.4 µg/m³ and 32.3 ±
	significantly negatively correlated (r Pearson = -0.60, t = -12.70, df = 290, p < 0.01). 3.0 µg/m³ respectively, F 2,142 = 1.22, p = 0.30) but differed among habitats (F 1,142 = 94.72, p <
	0.01), the rural station showing far lower values (4.86 ± 0.39 µg/m³) than urban stations
	All statistical analyses were carried out with R software v. 2.15.0 (R Development CoreTeam
	(35.20 ± 1.52 µg/m³). Interaction year and habitat was not significant (F 2,140 = 0.25, p = 0.78).
	2012). To test the hypothesis of higher pollution in urban area, we compared NO 2
	concentration between urban and rural habitats and among years using linear models (package Breast feathers were brighter in the urban habitat than in the rural one (Fig 1A), and in old
	base, function lm & package car, function Anova, Type III Sum of Squares). Immune status adults than in yearlings (Table 2), decreased with NO 2 pollution (Fig 2A) and differed among
	years (Fig 2A). Urban and old birds therefore had brighter yellow plumage than rural and
	MANUSCRIT 3 MANUSCRIT 3

and colouration did not differ between the two rural sites (compared with Welch t-test; N=36 from

Fontainebleau and 198 from CEREEP, p > 0.11, t < 1.70, df > 35.6)

.

  = 180, great tits : 8.9 ± 0.1 eggs, n = 205; data used in this article). Collected eggs were frozen at -20°C immediately after weighing in the field lab, and then transferred to -80°C until further analyses in the laboratory. Nests were followed to record hatching success (number of hatchlings relative to clutch size minus the removed egg) and fledging success (number of fledged nestlings relative to the number of hatchlings). Adults were trapped at the nest while feeding nestlings aged at least 6 days to limit the risk of clutch desertion, and were sexed by the presence (female) or absence (male) of a brood patch. Adults were individually identified by a numbered aluminium ring ( Paris). Tarsus length and body mass were measured to the nearest 0.1 mm with a calliper, and to the nearest 0.25 g with a Pesola spring balance, respectively and used hereafter to estimate female body condition. As size and pigmentation of eggs are expected to vary with female body condition

	1. Data collection
	Data were collected from 2010 to 2014 in a population of great and blue tits breeding in a
	private forest in a rural area near Paris (80 km), at the Centre de Recherche en Écologie
	(48°52'N, 2°21'E). The rural study area contained 120 nestboxes (Schwegler wood-concrete
	nest boxes 2M, Valliance, Saint Pierre La Palud, France, floor area: 113 cm²) evenly
	distributed throughout a homogenous deciduous woodland, and used by both species. The
	urban study area contained 60 nestboxes (40 Schwegler 2M and 20 handmade nestboxes in
	wooden panel, floor area: 105 cm²) installed in deciduous areas of 6 urban parks and 1
	cemetery. Nestboxes were regularly inspected to determine laying dates. Only first clutches
	were used and one egg, chosen at random, was removed and weighed before incubation had
	begun, i.e. when clutch size was 6 or 7 eggs in the rural and 5 or 6 eggs in the urban area

(mean ± SE completed clutch size and number of clutches in the urban area: blue tits : 9.3 ± 0.

3 eggs, n = 27; great tits : 8.2 ± 0.2 eggs, n = 29; in the rural area: blue tits : 10.6 ± 0. 2 eggs, n

[START_REF] De Coster | Intraclutch variation in avian eggshell pigmentation: the anaemia hypothesis[END_REF] Holveck et al. 2012; Martínez-de la Puente et al. 2007

) we analysed relation between female body condition and egg and eggshell size and pigmentation.

Table 2 .

 2 al. higher than 0.60 are in bold in Table 1. Potential relationships between principal components of the two PCA and shown in Second, we investigated how egg morphology and pigmentation varied with habitat, year, breeding parameters (laying date and clutch size) and female body condition estimated as the residuals of the regression of body mass on tarsus length (blue tits: F 1,176 = 17.08, p < 0.01, estimate = 0.20 ± 0.05, great tits: F 1,173 = 56.54, p < 0.01, estimate = 0.72 ± 0.10). Linear mixed models included habitat, year, female body condition and egg morphology (EggPC1 and 2) as fixed explanatory variables (package nlme, function lme, method REML & package car, function Anova, type III sum of squares) and female identity as a random factor to correct the non-independence of some eggs. Indeed some eggs were produced by the same female in different years (blue tits: 1 female laid during 4 seasons, 3 during 3 seasons and 23 twice, great tits: 1 female laid during 5 seasons, 3 during 4 seasons, 9 during 3 seasons and 18 twice). Likelihood Ratio Tests were manually calculated for the random effect and given in

Table 3 .

 3 Backward elimination of was used to select the best predictive model. Third, we investigated which parameters (type of habitat, year, laying date, clutch size, eggshell colouration (PigPC1 and 2) or egg morphology (EggPC1 and 2), female body condition) were the best predictors for hatching and fledging success. To that purpose, s were used (package base, & package car, function Anova, type III).

Table 4 )

 4 2, . Hatching success was greater for eggs with low values of PigPC2 i.e. with fewer spots (Table4). Fledging success was greater for eggs from later laid clutches, with higher values of EggPC1 i.e. larger eggs, lower values of EggPC2 i.e. thinner shells, low scores of PigPC2 i.e. fewer spots and by females with poorer body condition. Fledging success differed among years (Table

Table 1 :

 1 Principal component analyses of egg characteristics in blue tits (n = 206) and great tits (n = 234): A). Eigenvalues and percentage of variance explained by the first (EggPC1) and second (EggPC2) principal components, and their correlation coefficients with egg morphology variables, and B) Eigenvalues and percentage of variance explained by the first (PigPC1) and second (PigPC2) principal components, and their correlation coefficients with variables describing eggshell pigmentation on side (S) and crown (C) pictures. Significant loadings are indicated in bold.

	Blue tit	Great tit

Table 2 (

 2 EggPC1 and EggPC2) and principal components of eggshell pigmentation (PigPC1 and PigPC2) for blue tit and great tit

			PigPC1			PigPC2
		t	Df	p	r Pearson	t	Df	p	r Pearson
	Blue tit
	EggPC1 -0.76 176 0.45 -0.06 -0.25 176 0.81 -0.02
	EggPC2	1.17 176 0.24 0.09 -2.43 176 0.02 -0.18
	Great tit
	EggPC1	0.87 173 0.38 0.07	1.64 173 0.10 0.12
	EggPC2	2.08 173 0.04 0.16	1.20 173 0.23 0.09

Table 3 :

 3 Results from linear models investigating variation in egg size (EggPC1), eggshell 506 thickness (EggPC2), degree of eggshell pigmentation and spots size (PigPC1) and spot 507 number (PigPC2) as a function of year, type of habitat, clutch size, laying date and female 508 body condition in blue tit and great tit eggs. Initial values of variables that were not retained 509 in final models are presented in italic, and significant p-values in the final models are in bold. 510Likelihood Ratio Tests (LRT) were manually calculated for the random effect of female 511

	512	identity.
				Blue tit				Great tit
		F	Df	p	Estimate ± SE	F	Df	p	Estimate ± SE

Comme les changements globaux augmente la température et la luminosité et avance la date de débourrage (Tableau 1), la date de reproduction devrait-être avancée ce qui a été mesuré chez les oiseaux[START_REF] Møller | Effects of Climate Change on Birds[END_REF] Charmantier and Gienapp 2014). Malgré le constat Ainsi, les dates de ponte des mésanges charbonnières anglaises ont avancé au fil des ans en on de température(Charmantier et al. 2008) alors que celles des mésanges charbonnières des Pays-Bas ont variées au cours des années sans montrer de pattern[START_REF] Visser | Warmer springs lead to mistimed reproduction in great tits (Parus major)[END_REF][START_REF] Visser | Adaptive phenological mismatches of birds and their food in a warming world[END_REF]. Il y a donc une réponse différente entre les populations qui peut-être dû à des différences génétiques ou à de la plasticité phénotypique et/ou à des soins parentaux différents. Figure 2 : Influence des changements globaux et synchronisation entre ressource et besoin.[START_REF] Visser | Shifts in caterpillar biomass phenology due to climate change and its impact on the breeding biology of an insectivorous bird[END_REF]

MANUSCRIT 2

Remerciements

Acknowledgements

This study was conducted in full compliance with French laws and regulation, including recommendations for ethical treatment of experimental animals in France. We thank DSM for kindly providing FloroGlo and lutein standards. We also would like to thank Anaïs Bompard, Ewen Georgelin, Lisa Jacquin, Julie Legoupi and Sylvain Londe, who helped constructing aviaries. We thank Julie Legoupi and Claudy Haussy who helped analysing antioxidant capacity and ROM concentration of plasma. We thank Jacqui Shykoff for discussions we had about this article and her suggestions. Finally, we would like to thank two anonymous reviewers for their constructive and detailed comments.

Acknowledgements

This study was conducted in full compliance with French laws and regulation, including recommendations for ethical treatment of experimental animals in France. We acknowledge funding from Emergence-UPMC-2011 research program. We thank Benjamin Burguet for help in the field. We also thank Julie Legoupi who helped analysing total antioxidant capacity and ROM concentration in the lab. We thank Jacqui Shykoff for discussions we had about this article and her corrections.

Acknowledgements

This study was conducted in full compliance with French laws and regulation, including recommendations for ethical treatment of experimental animals in France. We thank Cannelle Tassin de Montaigu for help in the lab with measuring breast feather colour, and the numerous students involved in fieldwork each year for their help. We thank Jacqui Shykoff for discussions we had about this article and her suggestions. We acknowledge funding from Emergence-UPMC-2011 research program.

Acknowledgements

We acknowledge funding from Emergence-UPMC-2011 research program. We thank Clément Dubois and Manon Nivière for help in the lab and picture analyses. We thank the CRBPO for providing ringing facilities and authorizing the ringing program on these bird populations. We thank Jacqui Shykoff for discussions we had about this article and her suggestions.

This study was conducted in full compliance with French laws and regulations, including recommendations for ethical treatment of experimental animals in France.

Funding

We acknowledge funding from Emergence-UPMC-2011 research program

Competing interest

Authors declare no competing interests.

Author contribution

Verboven, N., Verreault, J., Letcher, R. J., Gabrielsen, G. W. and Evans, N. P. (2009). Nest temperature and parental behaviour of Arctic-breeding glaucous gulls exposed to persistent organic pollutants. Anim. behav. 77, 411-418. Verspoor, J. J., Love, O. P., Rowland, E., Chin, E. H. and Williams, T. D. (2007). Sexspecific development of avian flight performance under experimentally altered rearing conditions. Behav. Ecol. 18, 967-973. Woodall, A. A., Britton, G. and Jackson, M. J. (1996). Dietary supplementation with -tocopherol levels and susceptibility of tissues to oxidative stress. Br. J Nutr. 76, 307-317.

Is oxidative status influenced by dietary carotenoids and physical activity after moult in the great tit (Parus major)?

Marie Vaugoyeau 1 , Beatriz Decencière 2 , Samuel Perret 2 , Filiz Karadas 3 , Sandrine Meylan

Aerial pollution

Aerial NO 2 concentration was measured every day by AirParif. To compare stations, we need data that could be considered independent. We tested for independence by calculating correlations between concentration at D day and at D + 1, D + 2 and so forth for each station and year (Figure 1). For all station and year combinations data taken at five-day intervals

were not correlated and were therefore used to compare stations.

2012) and to be thicker at the crown in great tits (Gosler et al. 2005), the same observer (M.V.) measured shell thickness three times at different points of white areas at the crown.

Thickness was highly repeatable (blue tits: F 228,458 = 21.63, p < 0.01, intraclass correlation coefficient = 0.88, great tits: F 254,510 = 35.08, p < 0.01, intraclass correlation coefficient = 0.92), and the mean of the three measurements was used thereafter. Eggshells were then dried at 80°C overnight and weighed (to the next relative eggshell mass (eggshell mass / fresh egg mass 100) was used thereafter.

The repeatability of egg measures within clutches was tested using clutches found deserted before incubation, and was found to be significant in all cases (blue tits: N = 7 clutches and 36 eggs, all F 6,23 > 3, p < 0.02, intraclass correlation coefficient > 0.52, great tits: N = 8 clutches and 55 eggs, all F 7,40 > 2.5, p < 0.04, intraclass correlation coefficient > 0.40). One egg was thus considered representative of its clutch, and in the case of deserted clutches, one egg was chosen at random and its measurements used in further analyses.

Statistical analyses

All statistical analyses were performed in R v. 2.15.0 (R Development CoreTeam 2012).

First, the relationships among egg morphology variables and the relationships among eggshell pigmentation pattern variables were investigated with PCA analysis using the package FactoMineR with function PCA. We retained only those axes with eigenvalues at least equal to one [START_REF] Kaiser | The application of electronic-computers to factor analysis[END_REF]. The principal components EggPC1 and EggPC2 were determined from the correlation matrix of thickness and relative mass of eggshell, egg shape and volume and egg fresh weight, while the principal components PigPC1 and PigPC2 were determined from the correlation matrix of the number of spots, maximum, minimum and mean size of spots and proportion of spotted surface for side and crown pictures. Factors with contributions transferred by the shell gland to the egg (Rodriguez-Navarro and Garcia-Ruiz 2000) and would act as a shock-absorber between calcite crystals of the eggshell [START_REF] Burhans | Songbird abundance and parasitism differ between urban and rural shrublands[END_REF]. If calcium was less available in urban areas, we expected that urban eggs would be more pigmented than rural ones. Contrary to this expectation, we found little variation in eggshell pigmentation between urban and rural areas except in the great tits, whose spot number (PigPC2) was lower in Paris than in the rural area. One explanation for our findings could be, as mentioned above, that calcium is not limiting in Paris (Trammell et al. 2011). Alternatively, eggshell pattern may be highly heritable [START_REF] Gosler | Inheritance and variation in eggshell patterning in the great tit Parus major[END_REF] with no genetic differentiation for the trait between our urban and rural populations [START_REF] García-Navas | Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus[END_REF]). However, we did find a relationship between eggshell thickness and number of spots for both species, but in opposite directions. In blue tits thinner-shelled eggs had more spots, and in great tits thinner-shelled eggs were less pigmented with smaller spots. Thus pigmentation can be a signal for other aspects of eggshell quality, but in a species-specific way.

These findings are thus consistent for correlation between protoporphyrin based pigmentation and eggshell thickness [START_REF] García-Navas | Experimental evidence for the role of calcium in eggshell pigmentation pattern and breeding performance in Blue Tits Cyanistes caeruleus[END_REF]Gosler et al. 2011;Gosler et al. 2005;Hargitai et al. 2013;Holveck et al. 2012;Magi et al. 2012;Martínez-de la Puente et al. 2007).

Indeed, in blue tits, the degree of pigmentation and size of spots increased when eggshell thickness decreased. Moreover, in great tits, the number of spots (PigPC2) increased when eggshell thickness increased. So, it seemed that eggshell pigmentation was reliable information of eggshell thickness in both species, which could influence male partner investment as measured in [START_REF] Sanz | Eggshell pigmentation pattern in relation to breeding performance of blue tits Cyanistes caeruleus[END_REF].

Another nonexclusive hypothesis advanced to explain the variation of eggshell pigmentation is that eggshell pigmentation signals egg or female quality and can be used by males to moderate their investment to nest and brood care [START_REF] Aviles | Sexual selection based on egg colour: physiological models and egg discrimination experiments in a cavity-nesting bird[END_REF]Holveck et al. 2012;