Sabrina Tartu
email: tartu.sabrina@gmail.com

Frédéric Angelier

John C Wingfield

Paco Bustamante

Pierre Labadie

Hélène Budzinski

Henri Weimerskirch

Jan Ove Bustnes

Olivier Chastel

Corticosterone, prolactin and egg neglect behavior in relation to mercury and legacy POPs in a long-lived Antarctic bird

Keywords: Snow petrel, Mercury, Persistent organic pollutants, Age, Reproduction

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INTRODUCTION

The parental phase is energy-demanding (Drent and Daan 1980) and individuals adopt different life-history strategies in order to cope with environmental stressors such as food shortage, predation or poor weather. In extreme environments, such as Polar Regions, animals often experience harsh and unpredictable environmental conditions, and as a result long-lived organisms such as seabirds may refrain from breeding or desert their brood (e.g [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]; Goutte et al. 2011a). At the physiological level, the release of glucocorticoid hormones (cortisol, corticosterone: CORT) during stressful events triggers physiological and behavioral adjustments that shift energy investment away from reproduction and redirects it towards selfpreservation and hence survival (Angelier and Wingfield, 2013;[START_REF] Ricklefs | The physiology/life-history nexus[END_REF][START_REF] Wingfield | Reproduction and Resistance to Stress: When and How[END_REF]. Stress hormones have therefore a strong connection to fitness traits such as breeding success, individual quality and survival (Angelier et al. 2009a[START_REF] Angelier | Hormonal correlates of individual quality in a long-lived bird: a test of the "corticosterone-fitness hypothesis[END_REF][START_REF] Bonier | Do baseline glucocorticoids predict fitness?[END_REF][START_REF] Bókony | Stress Response and the Value of Reproduction: Are Birds Prudent Parents?[END_REF][START_REF] Breuner | In search of relationships between the acute adrenocortical response and fitness[END_REF][START_REF] Goutte | Long-term survival effect of corticosterone manipulation in Black-legged kittiwakes[END_REF]Goutte et al. , 2011b;;[START_REF] Kitaysky | Dynamics of food availability, body condition and physiological stress response in breeding Black-legged Kittiwakes[END_REF]. Additionally, the hormone prolactin (PRL) can also mediate this lifehistory trade-off between reproduction and survival in free-living birds (reviewed in Angelier and Chastel 2009). The release of this anterior pituitary hormone stimulates and facilitates parental behaviour such as egg incubation and brood provisioning [START_REF] Buntin | Parental Care: Evolution, Mechanisms, And Adaptive Significance: Parental Care: Evolution, Mechanisms, And Adaptive Significance[END_REF]. In response to acute stress, circulating PRL levels has been shown to decrease in several bird species (Angelier et al. 2013;[START_REF] Chastel | Modulation of prolactin but not corticosterone responses to stress in relation to parental effort in a long-lived bird[END_REF], and this could ultimately trigger nest desertion if PRL levels remain low during a prolonged period [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]2009b;Angelier and Chastel 2009;[START_REF] Heidinger | Mellowing with age: older parents are less responsive to a stressor in a long-lived seabird[END_REF]. Importantly, this decrease in PRL levels varies between individuals and life-history stages, suggesting that birds can attenuate their PRL response to acute stress to ensure that reproduction is not inhibited when the fitness value of the current reproductive event is high (the 'brood value hypothesis'; [START_REF] Bókony | Stress Response and the Value of Reproduction: Are Birds Prudent Parents?[END_REF][START_REF] Lendvai | Reproduction and modulation of the stress response: an experimental test in the house sparrow[END_REF]). Thus, both CORT and PRL are very likely to mediate parental effort and parental investment in birds [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF](Angelier et al. , 2009b(Angelier et al. , 2013;;[START_REF] Chastel | Modulation of prolactin but not corticosterone responses to stress in relation to parental effort in a long-lived bird[END_REF][START_REF] Criscuolo | Corticosterone alone does not trigger a short term behavioural shift in incubating female common eiders Somateria mollissima, but does modify long term reproductive success[END_REF][START_REF] Groscolas | Spontaneous egg or chick abandonment in energy-depleted king penguins: A role for corticosterone and prolactin?[END_REF][START_REF] Koch | Prolactin-induced parental hyperphagia in ring doves: are glucocorticoids involved?[END_REF]) and any disruption of these major endocrine cascades may alter the ability of an individual to adjust reproductive decisions to environmental conditions [START_REF] Jenssen | Endocrine-Disrupting Chemicals and Climate Change: A Worst-Case Combination for Arctic Marine Mammals and Seabirds?[END_REF][START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]).

In addition to extreme environmental conditions, climate change and anthropogenic disturbances [START_REF] Clarke | Polar marine ecosystems: major threats and future change[END_REF][START_REF] Moline | High Latitude Changes in Ice Dynamics and Their Impact on Polar Marine Ecosystems[END_REF][START_REF] Smetacek | Polar ocean ecosystems in a changing world[END_REF], polar species are subjected to environmental pollution. Indeed, despite their remote location, polar areas are the fall-out region of contaminants which undergo long range transport such as persistent organic pollutants (POPs) and heavy metals (e.g. mercury: Hg). Indeed, because of climate characteristics, contaminants accumulate in the polar environment, where they may be bio-accumulated and for some compounds bio-magnified [START_REF] Bargagli | Environmental contamination in Antarctic ecosystems[END_REF][START_REF] Gordeev | Pollution of the Arctic[END_REF][START_REF] Risebrough | Bioaccumulation factors of chlorinated hydrocarbons between mussels and seawater[END_REF][START_REF] Wania | Peer Reviewed: Tracking the Distribution of Persistent Organic Pollutants[END_REF][START_REF] Wania | Assessing the Potential of Persistent Organic Chemicals for Long-Range Transport and Accumulation in Polar Regions[END_REF]. Moreover, long-lived organisms are thought to be highly sensitive to contaminants [START_REF] Rowe | The Calamity of So Long Life": Life Histories, Contaminants, and Potential Emerging Threats to Long-lived Vertebrates[END_REF], but there are surprisingly few data on the effect of age on contaminant levels, and it is not clear if seabirds accumulate POPs with increasing age (Bustnes et al., 2003a).

Marine apex predators, such as seabirds, are particularly exposed [START_REF] Gabrielsen | Levels and effects of persistent organic pollutants in arctic animals[END_REF][START_REF] Rowe | The Calamity of So Long Life": Life Histories, Contaminants, and Potential Emerging Threats to Long-lived Vertebrates[END_REF][START_REF] Van Den Brink | Directed transport of volatile organochlorine pollutants to polar regions: the effect on the contamination pattern of Antarctic seabirds[END_REF]) and several studies have reported breeding impairments in highly polluted seabirds [START_REF] Bustnes | Patterns of incubation and nest-site attentiveness in relation to organochlorine (PCB) contamination in glaucous gulls[END_REF](Bustnes et al. , 2003b[START_REF] Bustnes | Reproductive performance and organochlorine pollutants in an Antarctic marine top predator: The south polar skua[END_REF][START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF][START_REF] Verboven | Nest temperature and parental behaviour of Arctic-breeding glaucous gulls exposed to persistent organic pollutants[END_REF]. Such breeding impairments could originate from the ability of contaminants to act as endocrine disruptors and thus, to alter the functioning of endocrine axes [START_REF] Guillette | Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrine-disrupting contaminants[END_REF][START_REF] Ottinger | Reproductive consequences of EDCs in birds: What do laboratory effects mean in field species?[END_REF][START_REF] Ottinger | Assessing effects of environmental chemicals on neuroendocrine systems: potential mechanisms and functional outcomes[END_REF][START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF][START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF][START_REF] Tartu | The stress of being contaminated? Adrenocortical function and reproduction in relation to persistent organic pollutants in female black legged kittiwakes[END_REF][START_REF] Tyler | Endocrine Disruption in Wildlife: A Critical Review of the Evidence[END_REF]. Experimental studies have documented some effects of chemicals on glucocorticoids [START_REF] Love | Effects of Dietary PCB Exposure on Adrenocortical Function in Captive American Kestrels (Falco sparverius)[END_REF][START_REF] Odermatt | Glucocorticoid and mineralocorticoid action: Why should we consider influences by environmental chemicals?[END_REF], but the effects of contaminants on stress hormones in free-living organisms such as seabirds have rarely been studied [START_REF] Bergman | State of the science of endocrine disrupting chemicals 2012: an assessment of the state of the science of endocrine disruptors prepared by a group of experts for the United Nations Environment Programme and World Health Organization[END_REF][START_REF] Nordstad | Relationships between POPs and baseline corticosterone levels in black-legged kittiwakes (Rissa tridactyla) across their breeding cycle[END_REF][START_REF] Tartu | The stress of being contaminated? Adrenocortical function and reproduction in relation to persistent organic pollutants in female black legged kittiwakes[END_REF][START_REF] Verboven | Adrenocortical function of Arctic-breeding glaucous gulls in relation to persistent organic pollutants[END_REF]. It is therefore difficult to draw a general pattern of the relationships between contaminants and stress hormones.

Regarding the effects of contaminants on PRL, the knowledge is even poorer and only one study [START_REF] Verreault | Changes in prolactin in a highly organohalogen contaminated Arctic top predator seabird, the glaucous gull[END_REF]) has addressed relationships between PRL secretion and POPs. In glaucous gulls (Larus hyperboreus) baseline PRL levels and the rate of decrease in PRL levels tended to vary negatively with organohalogen contaminants in males only [START_REF] Verreault | Changes in prolactin in a highly organohalogen contaminated Arctic top predator seabird, the glaucous gull[END_REF]. Furthermore, numerous compounds are potential environmental contaminants (e.g. heavy metals, POPs), which may have different effects on hormones of the hypothalamopituitary-adrenal (HPA) axis, such as CORT but also PRL secretion. There is thus a need to determine whether different environmental contaminants can disrupt these hormones in freeliving organisms. The aim of this study was to investigate the potential roles of environmental contaminants such as Hg and some legacy POPs (i.e. polychlorinated biphenyls: PCBs; organochlorine pesticides: OCPs; and polybrominated diphenyl ethers: PBDEs) on two major endocrine mechanisms: stress hormones from the HPA axis: CORT, and a key pituitary 'parental hormone': PRL. The snow petrel (Pagodroma nivea) a contaminated Antarctic seabird [START_REF] Xie | Microanalysis of metals in barbs of a snow petrel (Pagodroma Nivea) from the Antarctica using synchrotron radiation X-ray fluorescence[END_REF][START_REF] Corsolini | Chlorinated biphenyls and pesticides in migrating and resident seabirds from East and West Antarctica[END_REF]Goutte et al. 2013, Tartu et al. unpublished data) provides an ideal species to address these questions. In this long-lived species (until ~50 years old, [START_REF] Chastel | High annual variability in reproductive success and survival of an Antarctic seabird, the snow petrel Pagodroma nivea[END_REF], CORT and PRL responses to acute stress are modulated in relation to parental investment and incubation commitment [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]Goutte et al. 2011c). For example, low stress-induced PRL levels are associated with a high probability of egg neglect, a frequently observed behaviour in snow petrels [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]). Further, thanks to an exceptional long-term banding survey (1964-present;[START_REF] Barbraud | Contrasting effects of the extent of sea-ice on the breeding performance of an Antarctic top predator, the Snow Petrel Pagodroma nivea[END_REF][START_REF] Chastel | High annual variability in reproductive success and survival of an Antarctic seabird, the snow petrel Pagodroma nivea[END_REF], many snow petrels are of known age, making it possible to address the effect of age on contaminant burden.

In that context, we investigated if POPs and/or Hg concentrations were related to 1) age, 2) CORT and/or PRL secretion and 3) parenting through egg-neglect behaviour. We predicted that POPs and/or Hg: 1) would increase with increasing age as a result of bio-accumulation;

2) would increase CORT and decrease PRL secretion; 3) would be higher in individuals that neglected their egg.

MATERIALS AND METHODS

Ethics statement

Animals were cared for in accordance with the guidelines of the ethics committee of the Institut Polaire Français Paul Emile Victor (IPEV) that specifically approved this study (Program no. 109,H. Weimerskirch).

Study site, blood sampling and body-condition

Snow petrels are Antarctic seabirds with a delayed sexual maturity (~10 years of age), a low fecundity (one egg per clutch and a maximum of one clutch per year) and a long lifespan (~50 years old) [START_REF] Chastel | High annual variability in reproductive success and survival of an Antarctic seabird, the snow petrel Pagodroma nivea[END_REF]. Adult males and females were handled during the 2010 late incubation period (8-21 January). A total of 49 birds (27 males and 22 females) were caught in 49 different nests and age was known for 47 of them (9-46 years old). Birds were captured by hand and were then bled according to the standardized capture/restraint stress protocol described by [START_REF] Wingfield | Modulation of the adrenocortical response to stress in birds[END_REF]. Immediately after capture (i.e. within 3 min), an initial blood sample (300 µl) was collected from the alar vein with a 1-mL heparinized syringe and a 25gauge needle. These initial blood samples were considered to reflect baseline levels of CORT and PRL [START_REF] Chastel | Modulation of prolactin but not corticosterone responses to stress in relation to parental effort in a long-lived bird[END_REF][START_REF] Romero | Collecting baseline corticosterone samples in the field: is under 3 min good enough?[END_REF]thereafter called 'baseline' sample).

After collection of the initial blood samples birds were placed into cloth bags, and a subsequent sample (300 µl) was collected 30 min after capture (thereafter called 'stressinduced' sample). During handling of the adult birds, their eggs were covered with cotton and kept warm. After these blood samples, each bird was put back in its nest. Snow petrels are tame and usually resume parental duties as soon as returned to their nest (e.g. [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]. After this acute stress protocol, petrels were left undisturbed at their nest for 20 min and were then captured again and blood sampled within 3 min of recapture (thereafter called 'post-stress' sample) to monitor how quickly hormone levels may return to baseline after a stressor. This blood sample was taken before CORT and PRL concentrations returned to normal, allowing us to effectively monitor the stress recovery. All birds were weighed to the nearest 2 g using a spring balance and their skull length (head + bill) was measured to the nearest 0.5 mm. Body condition index (thereafter 'body condition') was calculated as the residuals between body mass and skull length (regression: F 1,47 = 20.28, p < 0.001, R²=0.35).

After capture and blood sample, each nest was monitored twice a day until the manipulated petrel was relieved by its mate. We were therefore able to know whether a bird neglected its egg during the incubation bout following capture/restraint stress protocol (thereafter called 'egg neglect behaviour'). Leaving eggs unattended temporarily is common in Procellariiform birds [START_REF] Boersma | Egg neglect in the Procellariiformes: reproductive adaptations in the Fork-tailed Storm-Petrel[END_REF][START_REF] Chaurand | Incubation routine, body mass regulation and egg neglect in the blue petrel Halobaena caerulea[END_REF]. Distant foraging and unpredictable weather increase the probability to delay an individual's returning to relieve its incubating partner [START_REF] Boersma | Egg neglect in the Procellariiformes: reproductive adaptations in the Fork-tailed Storm-Petrel[END_REF]. Eggs left unattended for a long period are less likely to hatch successfully [START_REF] Boersma | Egg neglect in the Procellariiformes: reproductive adaptations in the Fork-tailed Storm-Petrel[END_REF]Wheelwright, 1979, Angelier et al. 2007). In snow petrels were egg-neglect is often observed (Angelier et al;[START_REF] Gabrielsen | Levels and effects of persistent organic pollutants in arctic animals[END_REF], both parents incubate the single egg four bouts lasting ca. 4 to 8 days while the partner is feeding at sea (Ryan and Atkins 1989). In two sampled birds, the egg was predated during the incubation bout following the capture/restraint stress protocol. Egg-neglect data were available for 47 birds.

Molecular sexing and hormone assay

Blood samples were centrifuged, and plasma was decanted and stored at -20°C until assayed.

After centrifugation, red cells were kept frozen for molecular sexing as well as for Hg determination. The sex was determined by polymerase chain reaction amplification of part of two highly conserved genes (CHD) present on the sex chromosomes at UMR 7372 -CNRS-Université de La Rochelle, as detailed in [START_REF] Weimerskirch | Population sex ratio variation in a monogamous long-lived bird, the wandering albatross[END_REF]. Plasma concentrations of CORT were determined first by radioimmunoassay at UMR 7372 -CNRS-Université de La Rochelle, as previously described [START_REF] Lormée | Sex-specific patterns in baseline corticosterone and body condition changes in breeding Red-footed Boobies Sula sula[END_REF]. Plasma concentrations of PRL were determined with the remaining plasma by a heterologous radioimmunoassay at UMR 7372 -CNRS-Université de La Rochelle, as detailed in [START_REF] Cherel | Seasonal and fasting-related changes in circulating gonadal steroids and prolactin in king penguins, Aptenodytes patagonicus[END_REF]. The PRL assay has previously been validated in snow petrels [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]). All samples were run in one assay for both hormones. To measure intra-assay variation, we included 4 different referents 10 times in the CORT and PRL assays. From this, the intra-assay variation was 6.7% for total CORT and 7.8% for PRL. CORT and PRL concentrations were measured in baseline, stressinduced and post-stress samples.

Organic pollutants determination in plasma

POPs were measured in plasma samples collected from 15 females and 21 males only, since in 13 birds the remaining plasma volumes were too low. The targeted compounds included 7 indicator PCBs (CB-28, -52, -101, -118, -138, -153 and -180), 11 OCPs (HCB, Gamma HCH, Heptachlore, cis-chlordane, trans-nonachlor, 2,4' DDE, 4,4' DDE, 4,4' DDD, 2,4' DDT, 4,4' DDT and mirex) and two PBDE (BDE-47 and BDE-99). Certified solutions containing all analytes in isooctane at 2 ng•µL -1 each were obtained from LGC Standards (Molsheim, France). To a plasma sample of 100 µL, internal standards (1 ng each) were added gravimetrically: CB-30, -103, -155 and -198 were used to quantify PCBs, p,p'-DDT-d8 was used to quantify OCPs and F-BDE-47 was used to quantify BDE-47 and BDE-99; standards were provided by either Dr Ehrenstorfer GmbH, Cambridge Isotope Laboratory (via Cluzeau Info Labo, Sainte-Foy-La-Grande, France) or Chiron (via BCP Instruments, Irigny, France).

POPs were extracted with 1 mL of pentane:dichloromethane (90:10; v/v) ; after centrifugation (2000 rpm, 2min at 4°C), the organic layer was collected and the operation was repeated.

Both extracts were combined and purified on an acid silica gel column (40% H 2 SO 4 ). After extract loading, analytes were eluted with 3 x 5 mL of pentane/dichloromethane (90/10; v/v).

Extracts were then concentrated using a RapidVap vacuum evaporation system from Labconco (Kansas City, MO, USA) to a volume of 1 mL and further concentrated under a gentle stream of nitrogen (40°C) after addition of 100 µL of isooctane as solvent keeper.

Octachloronaphtalene (1 ng) was finally added to determine the recovery rate for each internal standard, for each sample (68-108%). Final extracts were analysed by gas chromatography coupled with electron capture detection (GC-ECD) as described elsewhere [START_REF] Tapie | PBDE and PCB contamination of eels from the Gironde estuary: From glass eels to silver eels[END_REF].

Quality control consisted in the analysis of standard solutions (NIST SRM 2261 and SRM 2262) and of procedural blanks (clean and empty glass tubes treated like a sample, one blank for 8 samples). Recoveries for standard solutions ranged from 89 to 104 % with standard deviations lower than 13 % (n=4). Chicken plasma samples (Sigma-Aldrich, S t Quentin Fallavier, France) spiked with all analytes (3 ng•g -1 each) were analysed; the recovery rates were in the range 77-103 % with coefficients of variation lower than 17 % (n=5), except for CB-52 (22%) and mirex (29%). POP concentrations were blank corrected and the detection limit (LoD) was set at two times the mean blank value; for analytes that were not detected in blanks, LoD was determined as the concentration with a signal to noise ratio of 3 in spiked chicken plasma samples. Overall, LoDs ranged from 0.03 to 0.34 ng•g -1 wet weight (ww).

Additionally, plasma total lipids were measured on an aliquot of 10 µL by the sulfo-phosphovanillin (SPV) method for colorimetric determination [START_REF] Frings | Improved Determination of Total Serum Lipids by the Sulfo-Phospho-Vanillin Reaction[END_REF].

Hg determination in blood cells

Total Hg was measured as described in details in [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North Eastern Atlantic waters: Influence of geographical origin and feeding ecology[END_REF]. Briefly, from freeze-dried and powdered red blood cells (hereafter called 'blood') in an Advanced Hg Analyzer spectrophotometer (Altec AMA 254). At least two aliquots ranging from 5 to 10 mg were analyzed for each individual and quality assessment was measured by repeated analyses of certified reference material TORT-2 (lobster hepatopancreas, NRCC; certified value 0.27±0.06 µg•g -1 ; with recoveries of 98 to 102%) and blanks, empty sample container, run every 20 samples. Hg concentrations are expressed in µg•g -1 dry weight (dw).

Statistical analyses

All analyses were performed using R 2.13.1 (http://r-project.org/). We first tested intercorrelations between the different families of POPs detected by using linear models (LM).

Second, we used generalized linear model (GLM) with normal errors and an identity link function to test whether ƩPOPs or Hg were influenced by sex, body-condition and age (dependent variable: ƩPOPs and Hg, independent factor and variables: sex, body-condition and age). Third, we tested whether CORT and PRL kinetics differed between male and females by using repeated measures GLM with the time of sampling (baseline, stress-induced and post-stress levels) as the repeated measures; (dependent variable: CORT and PRL concentrations, independent factors: sex, time and their interaction). Fourth, we tested whether CORT concentration (baseline, stress-induced and post-stress) was related to ƩPOPs and Hg (dependent variable: CORT, independent factor and variables: ƩPOPs, sex, age, Hg, body-condition and their interaction with sex). For PRL we analysed males and females separately (dependent variable: PRL, independent variables: ƩPOPs, age, Hg, body-condition) because in incubating snow petrels, females bear higher PRL concentrations than males [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]. Finally, we tested if the probability of neglecting the egg was related to ƩPOPs and Hg in males and females separately (dependent variable: egg neglect; independent variables: ƩPOPs, age, Hg, body-condition). To test the relationships between contaminants and egg-neglect behaviour (yes or no) we used GLM with binomial error and logit link.

Dependent continuous variables were previously tested for normality with a Shapiro-Wilk test and were log-transformed when necessary. Selected models were then checked for assumptions, that is, constancy of variance and residual normality. We performed all our model selection starting from the most general model that included all the variables/factors of interest and their interactions and we removed step by step the non-significant interactions, variables or factors. For POPs statistical analyses, concentrations below LoD were assigned LoD value, and only compounds detected in at least 70% of the individuals were included into the sum of POPs [START_REF] Noël | Persistent organic pollutants (POPs) in killer whales (Orcinus orca) from the Crozet Archipelago, southern Indian Ocean[END_REF].

RESULTS

Contaminants: concentrations and compounds

Out of the 20 POP targeted compounds, 15 could be detected but the concentrations of four OCPs (cis-chlordane, trans-nonachlor, heptachlor and 2,4'-DDE) and one PBDE (BDE-99)

were systematically below LoD. The most abundant compounds were the PCBs, with the PCBs 101 and 118 reaching the highest concentrations, closely followed by CB-138 and CB-153 (Table 1). Of the OCPs, HCB had the highest concentrations followed by 4,4'-DDE (Table 1). Only four PCBs (-CB101, -118, -138 and -153) and four OCPs (HCB, gamma HCH, 4,4' DDE and 2,4' DDT) were detected in at least 70% of the individuals and were thus included into the analyses. ƩPCBs and ƩOCPs were positively correlated (PCBs vs.

OCPs: LM, F 1,34 = 18.1, p<0.001, R² = 0.35). Thus the global pollutant burden was described as a sum of POPs (hereafter 'ƩPOPs'). In incubating snow petrels, blood Hg averaged 1.91 ± 0.75 µg•g -1 dw, specifically in males 1.94 ± 0.77 µg•g -1 dw (range: 0.89 -4.01) and in females 1.87 ± 0.73 µg•g -1 dw (range: 0.74 -3.70).

Relationship between contaminants, sex, body-condition and age

During the incubation period, ƩPOPs was not statistically different between male and female snow petrels (GLM, F 1,34 = 3.21, p = 0.082). Hg concentrations were not related to sex neither (GLM, F 1,47 = 0.12, p = 0.734). ƩPOPs and Hg concentration, respectively, were unrelated to the body-condition index (ƩPOPs: F 1,34 = 1.05, p = 0.313, interaction with sex: F 1,33 = 2.18, p = 0.149; Hg: F 1,46 = 3.30, p = 0.076, interaction with sex: F 1,45 = 0.14, p = 0.714). ƩPOPs were not related to age (F 1,34 = 0.88, p = 0.355, interaction with sex: F 1,33 = 0.61, p = 0.441), and neither were Hg concentrations (F 1,45 = 2.05, p = 0.159 interaction with sex: F 1,44 = 0.16, p = 0.693). Finally, ƩPOPs and Hg were not related (F 1,34 = 2.09, p = 0.157).

CORT and PRL kinetics: response and recovery to acute stress protocol

CORT concentrations significantly increased over 30 min from 4.7 ± 3.4 to 37.8 ± 8.6 ng•ml -1 and then declined 20 min post-stress to 23.6 ± 12.6 ng•ml -1 (GLMM, time as factor, F 2,94 = 269.98, p <0.001), without sex difference (sex: F 1,47 = 0.28, p = 0.600; time × sex interaction: F 2,94 = 0.08, p = 0.926). PRL concentrations significantly decreased over time (GLMM, time as factor, F 2,94 = 144.26, p <0.001) and females had higher PRL concentrations than males (sex: F 1,47 = 152.72, p <0.001; time × sex interaction: F 2,94 = 3.79, p = 0.026). In females, PRL decreased from 239.5 ± 36.3 to 169.4 ± 32.1 ng•ml -1 after 30 min and until 165.4 ± 30.8 ng•ml -1 20 min post-stress. In males PRL concentrations decreased from 139.5 ± 30.7 to 90.9 ± 24.7 over 30 min and they reached 83.4 ± 24.0 ng•ml -1 20 min post-stress.

Relationships between contaminants and CORT concentrations

CORT absolute concentrations (baseline, stress-induced and post-stress) were not related to sex, neither to age, Hg, body-condition and their interaction with sex (Table 2). ƩPOPs were not related to baseline CORT (Fig. 1.A, Table 2), but increasing concentration of ƩPOPs was positively related to increasing concentration of stress-induced and post-stress CORT (Fig.

1.B-C, Table 2).

Relationships between contaminants, PRL concentrations and egg-neglect

In females we did not find any relationship between PRL (baseline, stress-induced and poststress) and ƩPOPs, age, Hg, or body-condition (p>0.08 for all tests). In males baseline and post-stress PRL concentrations were not related to ƩPOPs, age, Hg, or body-condition (p>0.07 for all tests), but increasing blood Hg concentration was related to decreasing stressinduced PRL concentration: i.e. after 30 min restraint the most contaminated males were less likely to maintain high concentrations of PRL (GLM, F 1,25 =5.6, p=0.0263; Fig. 2). Eleven females and eight males were observed neglecting their egg, out of 21 and 26, respectively. Blood Hg concentration was higher in males that were more likely to neglect their egg (GLM, χ²=, p = 0.019, Fig. 3) a relationship not found in females (GLM, χ²=0.1, p = 0.796, Fig. 3).

Finally, egg neglect behaviour was not related to ƩPOPs, age or body-condition in any sex (p>0.4 for all tests).

DISCUSSION

The present study is the first to report plasma POP concentrations in the long-lived, Antarctic snow petrel. Firstly, there were no relationships between plasma ƩPOPs or blood Hg and age, suggesting that long-lived seabird are able to eliminate much of their contaminant burden.

Secondly, ƩPOPs and Hg seem related to different hormonal pathways involved in reproductive decisions; ƩPOPs may disrupt the HPA axis whereas Hg was related to PRL secretion in males and consequently to egg-neglect behaviour.

Contaminants and age

Although snow petrels are very long-lived and thus exposed to contaminants over many years, no evidence was found that contamination was age-related, neither for ƩPOPs nor Hg. POPs and Hg measured in blood (plasma and red blood cells, respectively) can be correlated to levels found in storage organs and also adipose tissues, in birds but also chelonians and humans [START_REF] Henriksen | Validation of the use of blood samples to assess tissue concentrations of organochlorines in glaucous gulls, Larus hyperboreus[END_REF][START_REF] Henny | Nineteenth Century Mercury: Hazard to Wading Birds and Cormorants of the Carson River, Nevada[END_REF];[START_REF] Pauwels | Comparison of Persistent Organic Pollutant Residues in Serum and Adipose Tissue in a Female Population in Belgium, 1996-1998[END_REF][START_REF] Wayland | Concentrations of Cadmium, Mercury and Selenium in Blood, Liver and Kidney of Common Eider Ducks from the Canadian Arctic[END_REF][START_REF] Keller | Organochlorine contaminants in sea turtles: Correlations between whole blood and fat[END_REF][START_REF] Van De Merwe | Using blood samples to estimate persistent organic pollutants and metals in green sea turtles (< i> Chelonia mydas)[END_REF]Szumiło et al. 2013;Fromant et al. unpublished data). Thus, blood contaminant concentration may be a good proxy of contaminant burden in other organs.

The relationship between Hg and age in seabirds is often contradictory, for example liver Hg was found to decrease, increase or be unrelated to age [START_REF] Furness | Pollutant levels in the great skua Catharacta skua[END_REF][START_REF] Hutton | Accumulation of heavy metals and selenium in three seabird species from the United Kingdom[END_REF]Thompson et al. 1991). For blood, the relationship between Hg contamination and age is also not clear: no relationship was found between age and Hg contamination [START_REF] Gonzáles-Solís | Metals and selenium as bioindicators of geographic and trophic segregation in giant petrels Macronectes spp[END_REF][START_REF] Tavares | Influence of age, sex and breeding status on mercury accumulation patterns in the wandering albatross Diomedea exulans[END_REF]) but in pre-breeding snow petrels and incubating cape petrels (Daption capense), a negative relationship was found between blood Hg and age (Tartu et al., unpublished data). This relationship was, however more likely the result of an age-related change in feeding ecology. With regard to POPs, it seems that in seabirds, concentrations in different tissues and blood increase until a steady-state is reached, often before the age of breeding [START_REF] Donaldson | Organochlorine and Heavy Metal Residues in Breast Muscle of Known-Age Thick-Milled Murres (Uria lomvia) from the Canadian Arctic[END_REF][START_REF] Newton | Organochlorine contamination and age in sparrowhawks[END_REF][START_REF] Van Den Brink | Fluctuating concentrations of organochlorine pollutants during a breeding season in two Antarctic seabirds: Adelie penguin and southern fulmar[END_REF], and for breeding birds, most studies have not observed any age-related POP accumulation [START_REF] Bustnes | Age and accumulation of persistent organochlorines: A study of arctic-breeding glaucous gulls (Larus hyperboreus)[END_REF][START_REF] Newton | Organochlorine contamination and age in sparrowhawks[END_REF]. In this study, all snow petrels were breeders, and presumably they had already reached their steady-state levels.

Besides, seabirds can biotransform PCBs and eliminate POPs through their preen gland [START_REF] Borgå | Bioaccumulation of PCBs in Arctic seabirds: influence of dietary exposure and congener biotransformation[END_REF][START_REF] Henriksen | Levels and congener pattern of polychlorinated biphenyls in kittiwakes (Rissa tridactyla), in relation to mobilization of body-lipids associated with reproduction[END_REF][START_REF] Solheim | The Preen Gland -an Organ for Excretion of Persistent Organic Pollutants in Black-legged Kittiwake (Rissa tridactyla). Master thesis[END_REF], in the same line Hg can be excreted through feather growth [START_REF] Bearhop | Dynamics of mercury in blood and feathers of great skuas[END_REF]. These mechanisms could partially explain the lack of association between blood contaminants and increasing age. Also we have to remain cautious as we hypothesized that in snow petrels, as in other bird species, blood contaminants would represent levels in internal tissues [START_REF] Henny | Nineteenth Century Mercury: Hazard to Wading Birds and Cormorants of the Carson River, Nevada[END_REF]Szumiło et al. 2013;[START_REF] Wayland | Concentrations of Cadmium, Mercury and Selenium in Blood, Liver and Kidney of Common Eider Ducks from the Canadian Arctic[END_REF][START_REF] Henriksen | Validation of the use of blood samples to assess tissue concentrations of organochlorines in glaucous gulls, Larus hyperboreus[END_REF]Fromant et al. unpublished data). However, we have no evidence for this relationship in snow petrels. Additionally, following food intake or lipid mobilization, contaminant levels in blood may fluctuate more than those in adipose tissues or liver and this could mask a hypothetical contaminant/age relationship.

POPs: Concentrations and relationship with the CORT stress response

Very few studies have examined blood concentrations of POPs in adult Antarctic seabirds, and among the few studies, comparisons are made difficult due to different analytical methods. Indeed, POPs are often described as a "sum of compounds" and the compounds taken into the sum vary among studies. However, some OCPs are often reported individually since their detrimental effects have been well identified. This is the case of the HCB a relatively volatile compound [START_REF] Calamari | Role of plant biomass in the global environmental partitioning of chlorinated hydrocarbons[END_REF] principally used in fungicide formulations [START_REF] Barber | Hexachlorobenzene in the global environment: Emissions, levels, distribution, trends and processes[END_REF]. Higher concentrations of this compound are commonly found in species restricted to the Antarctic region than those in temperate regions (e.g. [START_REF] Van Den Brink | Directed transport of volatile organochlorine pollutants to polar regions: the effect on the contamination pattern of Antarctic seabirds[END_REF].

HCB was the OCP with the highest concentrations in incubating snow petrels, but much lower than the concentrations found in other high-Antarctic species such as south polar skuas (Catharacta maccormicki) from Svarthamaren (71° 53' S, 05° 10' E) in Dronning Maud Land (Antarctica): i.e. HCB concentrations were 10-fold lower, and mirex concentrations 100-fold lower [START_REF] Bustnes | Organochlorines in Antarctic and Arctic Avian Top Predators: A Comparison between the South Polar Skua and Two Species of Northern Hemisphere Gulls[END_REF][START_REF] Bustnes | Reproductive performance and organochlorine pollutants in an Antarctic marine top predator: The south polar skua[END_REF]. These results could be the consequence of a different trophic level, toxicokinetic factors (e.g. metabolism, clearance rate), compound-specific physicochemical properties (K ow , half-life) or depend on the bio-availability of contaminants in the breeding area [START_REF] Walker | Principles of Ecotoxicology, Fourth Edition[END_REF]).

The Arctic is more contaminated by POPs than Antarctica [START_REF] Bustnes | Organochlorines in Antarctic and Arctic Avian Top Predators: A Comparison between the South Polar Skua and Two Species of Northern Hemisphere Gulls[END_REF][START_REF] Choi | Passive Air Sampling of Polychlorinated Biphenyls and Organochlorine Pesticides at the Korean Arctic and Antarctic Research Stations: Implications for Long-Range Transport and Local Pollution[END_REF], HCB concentrations in snow petrels were slightly lower than those measured in plasma of incubating black-legged kittiwakes (Rissa tridactyla) from Svalbard: 1.85 ± 1.41 ng•g -1 ww versus 2.5 ± 0.44 ng•g -1 ww respectively (Tartu et al. unpublished data). In comparison, incubating glaucous gulls (Larus hyperboreus) had HCB concentrations in plasma much higher than snow petrels [START_REF] Verreault | New organochlorine contaminants and metabolites in plasma and eggs of glaucous gulls (larus hyperboreus) from the norwegian arctic[END_REF]: on average ~400 ng•g -1 lipid weight whereas in snow petrels concentrations given in lipid weight average ~200 ng•g -1 . In both blacklegged kittiwakes and glaucous gulls there is evidence of CORT disruption by POPs [START_REF] Nordstad | Relationships between POPs and baseline corticosterone levels in black-legged kittiwakes (Rissa tridactyla) across their breeding cycle[END_REF][START_REF] Verboven | Adrenocortical function of Arctic-breeding glaucous gulls in relation to persistent organic pollutants[END_REF]. Indeed, in both species increasing POP concentrations were related to higher baseline CORT concentrations and for male glaucous gulls, higher POP concentrations were related to decreasing stress-induced CORT concentrations. In the present study, increasing ƩPOPs were not related to baseline CORT, but to stress-induced and post-stress CORT concentrations. Hence, the most polluted birds released more CORT when subjected to a handling stress protocol, and those concentrations remained high 20 minutes post-stress. These results are in accordance with the recent finding that POPs, and especially PCBs are associated with a higher adrenocortical response to an acute stress in pre-laying female black-legged kittiwakes [START_REF] Tartu | The stress of being contaminated? Adrenocortical function and reproduction in relation to persistent organic pollutants in female black legged kittiwakes[END_REF]. However, although post-stress CORT concentrations were admittedly higher in the most contaminated snow petrels they did not decrease more slowly than in less polluted birds, indicating that negative feedback from CORT on the hypothalamus and the pituitary was functional. One possible explanation for the over-release of CORT could be related to an increase of the number of adreno-corticotrophic-hormone (ACTH) receptors (ACTH-R) on the adrenals.

ACTH is one of the few polypeptide hormones having a positive trophic effect on its own receptors [START_REF] Beuschlein | ACTH-receptor expression, regulation and role in adrenocortial tumor formation[END_REF][START_REF] Penhoat | Corticotropin positively regulates its own receptors and cAMP response in cultured bovine adrenal cells[END_REF]). Thus, an increase of ACTH-R in the most POP contaminated snow petrels may be the consequence of an excess of ACTH input to adrenals. This suggests that ƩPOPs may alter the functioning of the pituitary by stimulating ACTH release and/or that ƩPOPs may mimic ACTH and bind to ACTH-R, which in that case would mobilize more ACTH-R from the adrenals of the most contaminated individuals. However this study is correlational, we cannot confirm without experimental support that the observed relationship is not the consequence of other intrinsic or extrinsic factors. Yet, an exacerbated secretion of CORT in response to a stressful often mirrors poor fitness related traits as lowered parental investment (Angelier et al. 2009a;[START_REF] Bókony | Stress Response and the Value of Reproduction: Are Birds Prudent Parents?[END_REF]Goutte et al. 2011b;[START_REF] Lendvai | Reproduction and modulation of the stress response: an experimental test in the house sparrow[END_REF] or an impacted survival [START_REF] Blas | Stress response during development predicts fitness in a wild, long lived vertebrate[END_REF][START_REF] Goutte | Long-term survival effect of corticosterone manipulation in Black-legged kittiwakes[END_REF][START_REF] Romero | Using the reactive scope model to understand why stress physiology predicts survival during starvation in Galápagos marine iguanas[END_REF]). Nevertheless we did not find any relationship between POPs and parenting in terms of PRL concentration contrary to the study of [START_REF] Verreault | Changes in prolactin in a highly organohalogen contaminated Arctic top predator seabird, the glaucous gull[END_REF] or egg-neglect behaviour.

Hg: concentrations and relationships with stress-induced PRL and egg neglecting

Hg concentrations in incubating snow-petrels were within the range of those measured in the blood of south polar skuas breeding in Adélie land (2.15 ± 0.17 µg•g -1 dw, [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF]. In comparison with an Arctic breeding seabird, we also found comparable concentrations in incubating black-legged kittiwakes, (average 1.6 ± 0.5 µg•g -1 dw, Tartu et al. unpublished data). Contrary to other studies on free-ranging birds [START_REF] Franceschini | The corticosterone stress response and mercury contamination in free-living tree swallows, Tachycineta bicolor[END_REF][START_REF] Herring | Mercury Exposure May Suppress Baseline Corticosterone Levels in Juvenile Birds[END_REF][START_REF] Wada | Suppressed Adrenocortical Responses and Thyroid Hormone Levels in Birds near a Mercury-Contaminated River[END_REF]), we did not find any relationship between Hg and CORT secretion. Hg is well-known for its negative effects on breeding (reviewed in [START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF]). However, to the best of our knowledge, no studies have described relationships between Hg and PRL in free-living organisms. In humans, urinary Hg concentration was negatively correlated to plasma PRL [START_REF] De Burbure | Prolactin Changes as a Consequence of Chemical Exposure: de Burbure and Bernard Respond[END_REF][START_REF] Lucchini | Neurotoxic effect of exposure to low doses of mercury[END_REF][START_REF] Lucchini | Application of a Latent Variable Model for a Multicenter Study on Early Effects Due to Mercury Exposure[END_REF]. In the present study, we found a similar relationship in incubating male snow petrels: increasing Hg concentrations were related to decreasing stress-induced PRL concentrations.

PRL is an anterior pituitary hormone, and a previous study on polar seabirds has described relationships between Hg and another anterior pituitary hormone: luteinizing hormone (LH, Tartu et al. 2013, Tartu et al. unpublished data). Hg seemed to disrupt LH secretion via a lack of Gonadotropin-Releasing-Hormone (GnRH) input from the hypothalamus [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. GnRH release is controlled by an area of the hypothalamus called zona incerta [START_REF] Ben-Jonathan | Dopamine as a Prolactin (PRL) Inhibitor[END_REF]. Interestingly, this area also participates in the secretion of dopamine, a neuro-transmitter which is the principal antagonist of PRL (reviewed in [START_REF] Ben-Jonathan | Dopamine as a Prolactin (PRL) Inhibitor[END_REF]. Moreover, it has been well established that organic and inorganic Hg can stimulate the spontaneous release of dopamine in laboratory rodents [START_REF] Faro | Effects of Methyl Mercury on the in Vivo Release of Dopamine and Its Acidic Metabolites DOPAC and HVA from Striatum of Rats[END_REF][START_REF] Faro | Intrastriatal Administration of Methylmercury Increases In Vivo Dopamine Release[END_REF][START_REF] Faro | Comparative effects of organic and inorganic mercury on in vivo dopamine release in freely moving rats[END_REF][START_REF] Minnema | Effects of methylmercury on neurotransmitter release from rat brain synaptosomes[END_REF]) but also in wild larvae of a fish (Fundulus heteroclitus, [START_REF] Zhou | Neurotransmitter levels in two populations of larval Fundulus heteroclitus after methylmercury exposure[END_REF] and in wild American minks Mustela vison, where Hg induced a decrease of dopaminergic receptors and ligand affinity interpreted as an adaptive mechanism to prevent the hyper-stimulation of the dopaminergic system [START_REF] Basu | Effects of mercury on neurochemical receptor-binding characteristics in wild mink[END_REF]. Additionally, when subjected to a stress, dopamine concentrations in blood increase (e.g. [START_REF] Finlay | The Effects of Stress on Central Dopaminergic Neurons: Possible Clinical Implications[END_REF]. Stress-induced dopamine synthesis in male snow petrels may thus be enhanced by Hg contamination, and result in a decrease of stress-induced PRL concentrations but not baseline or post-stress PRL concentrations. The fact that the most polluted birds quickly decrease their PRL concentrations when exposed to stress may highly affect their parental investment: they would be more likely to neglect their egg than less polluted birds.

This goes together with the fact that in males, where PRL concentrations were lower than in females, the most polluted individuals were more likely to neglect their egg. In females, PRL concentrations and egg-neglect behaviour were not related to Hg, maybe their PRL concentrations remained sufficiently high to prevent egg-neglect, a behaviour associated with poor hatching success and chick mortality [START_REF] Boersma | Egg neglect in the Procellariiformes: reproductive adaptations in the Fork-tailed Storm-Petrel[END_REF][START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF].

CONCLUSION

In conclusion, there were no relationships between age and POPs or Hg, which is in line with most other studies. However we report significant relationships between contaminants and hormones involved in reproductive decisions. Over time, the action of POPs and Hg may jeopardize the maintenance of long-lived species populations. Indeed in long-lived species, that are expected to maximize their own survival rather than that of their brood, an exacerbated stress response as a consequence of POPs contamination and a decrease of PRL for the most Hg polluted males, are additional threats that may encourage individuals to refrain from breeding or desert their brood. To confirm the reported relationships, this study would greatly benefit from further experimental support. Table 1: Concentrations of plasma persistent organic pollutants and blood Hg (µg•g -1 ) of incubating female and male snow petrels. For POPs, concentrations are given in wet-weight (ng•g -1 ww) in the first rows and lipid-weight (ng•g -1 lw) in the second rows.

Figure 1 :

 1 Figure 1: Relationships between plasma ƩPOP concentrations (ng•g -1 wet weight) and plasma CORT concentrations (ng•ml -1 ) in incubating snow-petrels (A) baseline, (B) stress-

Figure 2 :

 2 Figure 2: Relationship between total blood Hg (µg•g -1 dry weight) and stress-induced plasma PRL concentrations (ng•ml -1 ) in incubating snow-petrels. White circles denote females and

Figure 3 :

 3 Figure 3: Relationship between blood Hg (µg•g -1 dry weight) and egg-neglect behaviour in

Acknowledgements

The authors thank M. Kriloff, S. Lucchini, F. Amélineau, E. Cottin, and A.M. Thierry for their help in the field, C. Trouvé, C. Parenteau, S. Ruault, C. Churlaud, A. Carravieri, L.

Peluhet and P. van Delft for their assistance in hormone, Hg and POPs assays and three anonymous referees for their very useful comments on a previous version of the manuscript.

Role of the funding source

This project was funded by Agence Nationale de la Recherche (ANR PolarTop, O. Chastel), the French Polar Institute (IPEV, Program 109, H. Weimerskirch) and the 7th research program of the European Community FP7/2007-2013 (Marie-Curie Fellowship, no. 237034, F. Angelier). The funders played no part in the design, analysis, or reporting of the study.

Conflict of interest statement

The authors declare no conflict of interest.


Sabrina Tartu
email: tartu.sabrina@gmail.com

Paco Bustamante

Aurélie Goutte

Yves Cherel

Henri Weimerskirch

Jan Ove Bustnes

Olivier Chastel

Age-related mercury contamination and relationship with luteinizing hormone in a long-lived Antarctic bird

Keywords: Pagodroma nivea, mercury, age, Luteinizing hormone, Antarctica, stable isotopes, sex-related segregation

Seabirds, as long-lived top predators, accumulate contaminants such as mercury (Hg), an established endocrine disruptor. In long lived species hormonal secretion varies with age; therefore, Hg-induced endocrine disruption may be exacerbated in some age classes. Here we investigated relationships between blood total Hg and luteinizing hormone (LH, a key pituitary hormone for the onset of breeding), in pre-laying known-age (11-45 years old) snow petrels (Pagodroma nivea) from Adélie Land, Antarctica. We predicted that 1) blood Hg would increase with advancing age as a consequence of bio-accumulation; and that 2) increasing blood Hg would be related to decreased concentrations of LH in the most Hgcontaminated individuals. Hg concentrations were higher in females than in males (p<0.001), and contrary to our prediction, decreased with advancing age in males (p=0.009) and tended to do so in females (p=0.06). The analysis of stable isotopes (δ 13 C and δ 15 N) suggested that this unexpected pattern could originate from age and sex-related variations in trophic niche, and hence Hg exposure. Regarding LH, our prediction was only supported in young birds (≤ 23 years) where baseline LH was inversely correlated with Hg concentrations (p=0.04). Hg burden did not predict baseline LH or GnRH-induced LH in birds that were more than 23 years old. These results show that age and contaminants may interfere with major endocrine mechanisms and, together with other recent studies, support the view that Hg could be connected to LH secretion and could then impair the fitness of long-lived birds.

Introduction

Mercury (Hg) is a ubiquitous contaminant, present in aquatic biota with elevated concentrations in fish, some mammals and seabirds [START_REF] Scheuhammer | Effects of Environmental Methylmercury on the Health of Wild Birds, Mammals, and Fish[END_REF], [START_REF] Gabrielsen | Pollution in the Barents Sea. Ecosystem Barents Sea[END_REF]. Diet is the principal route of contamination: Hg bio-accumulates into individuals [START_REF] Koeman | Mercury and selenium in marine mammals and birds[END_REF], [START_REF] Honda | Age-related accumulation of heavy metals in bone of the striped dolphin, Stenella coeruleoalba[END_REF], [START_REF] Wagemann | Comparison of Heavy Metals in Two Groups of Ringed Seals (Phoca hispida) from the Canadian Arctic[END_REF], [START_REF] Dietz | Geographical differences of zinc, cadmium, mercury and selenium in polar bears (Ursus maritimus) from Greenland[END_REF] and bio-magnifies along the food web [START_REF] Bargagli | Biomagnification of mercury in an Antarctic marine coastal food web[END_REF], [START_REF] Blévin | Wide Range of Mercury Contamination in Chicks of Southern Ocean Seabirds[END_REF]. Seabirds are top predators which often bear elevated levels of Hg, and are thus considered good bio-monitors to describe Hg bio-accumulation and biomagnification in the marine environment [START_REF] Furness | Birds as monitors of pollutants[END_REF]. Seabirds are also long-lived organisms, and therefore would accumulate Hg in their tissues over a long life-span [START_REF] Rowe | The Calamity of So Long Life": Life Histories, Contaminants, and Potential Emerging Threats to Long-lived Vertebrates[END_REF]. Although it has been well established that seabird nestlings show lower Hg concentrations than adults [START_REF] Carravieri | Mercury exposure in a large subantarctic avian community[END_REF], very few studies have examined the influence of age on Hg contamination in adults and the patterns are not clear [START_REF] Furness | Pollutant levels in the great skua Catharacta skua[END_REF], [START_REF] Hutton | Accumulation of heavy metals and selenium in three seabird species from the United Kingdom[END_REF], [START_REF] Furness | Mercury levels in the plumage of red-billed gulls Larus novaehollandiae scopulinus of known sex and age[END_REF], [START_REF] Thompson | Mercury Accumulation in Great Skuas Catharacta skua of Known Age and Sex, and Its Effects Upon Breeding and Survival[END_REF], [START_REF] Tavares | Influence of age, sex and breeding status on mercury accumulation patterns in the wandering albatross Diomedea exulans[END_REF]. In this study, we report the results of a crosssectional study on known-age adult snow petrels Pagodroma nivea, a small (≈ 400 g) but very long-lived Antarctic seabird (≈50 years) [START_REF] Chastel | High annual variability in reproductive success and survival of an Antarctic seabird, the snow petrel Pagodroma nivea[END_REF]. We made use of an exceptional long-term banding study (1964-to present, [START_REF] Chastel | High annual variability in reproductive success and survival of an Antarctic seabird, the snow petrel Pagodroma nivea[END_REF]) to identify the age of each bird and measured blood total Hg concentrations. Blood Hg concentrations reflect dietary exposure over the last several weeks and also a complex equilibrium between ingestion, deposition and mobilization [START_REF] Rowe | The Calamity of So Long Life": Life Histories, Contaminants, and Potential Emerging Threats to Long-lived Vertebrates[END_REF], [START_REF] Bearhop | Dynamics of mercury in blood and feathers of great skuas[END_REF], [START_REF] Bearhop | Bioamplification of Mercury in Great Skua Catharacta skua Chicks: the Influence of Trophic Status as Determined by Stable Isotope Signatures of Blood and Feathers[END_REF]. In seabirds, blood Hg is strongly correlated to Hg concentrations in storage organs [START_REF] Gochfeld | Tissue distribution of mercury in normal and abnormal young Common Terns[END_REF], [START_REF] Wayland | Concentrations of Cadmium, Mercury and Selenium in Blood, Liver and Kidney of Common Eider Ducks from the Canadian Arctic[END_REF], [START_REF] Henny | Nineteenth Century Mercury: Hazard to Wading Birds and Cormorants of the Carson River, Nevada[END_REF]; blood Hg is thus considered as a good proxy of overall Hg burden. Given the ability of Hg to bio-accumulate we would expect blood Hg concentrations to be positively related to age in adult snow petrels. As mentioned previously, diet is the principal route of Hg contamination and to further interpret the possible Hg/age relationship, we used the isotopic niche as a proxy of the trophic niche, with δ 13 C and δ 15 N values indicating the birds' foraging habitats and trophic position, respectively [START_REF] Newsome | A niche for isotopic ecology[END_REF]. Indeed this possible Hg/age relationship could originate from an age-related change in trophic niche. Snow petrels forage in close association with ice-pack and during summer they mainly feed their chicks with fish (up to 95% of the preys [START_REF] Ridoux | The diets of five summer breeding seabirds in Adélie Land, Antarctica[END_REF]). To our knowledge no studies have described the trophic niche of snow petrels during the inter-breeding period, and there is no evidence of age and/or sex related trophic niche segregation.

Taking age into consideration is crucial when considering the consequences of Hg contamination. Indeed, several studies on vertebrates have reported breeding impairment from Hg including suppression of reproduction, reduced clutch size, poor breeding performances or unusual courtship behavior [START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF], [START_REF] Drevnick | Effects of Dietary Methylmercury on Reproductive Endocrinology of Fathead Minnows[END_REF], [START_REF] Frederick | Altered pairing behaviour and reproductive success in white ibises exposed to environmentally relevant concentrations of methylmercury[END_REF], [START_REF] Evers | Adverse effects from environmental mercury loads on breeding common loons[END_REF], [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. Indeed, in its methylated form, Hg is highly toxic and is known to disrupt the reproductive endocrine system [START_REF] Tan | The endocrine effects of mercury in humans and wildlife[END_REF]. For instance, in an Arctic seabird, the black-legged kittiwake Rissa tridactyla, a recent study has reported that Hg could interfere with the secretion of luteinizing hormone (LH) [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF] a major hormone for the onset of breeding [30]. In vertebrates, the onset of breeding involves the activation of the hypothalamic-pituitary-gonadal axis (hereafter HPG axis): increasing day length activates the expression of a neuro-hormone, the Gonadotropin Releasing Hormone (GnRH) which triggers the secretion of LH from the pituitary gland. LH, in concert with follicle-stimulating hormone (FSH), promotes gonadal maturation, sex steroid secretion and in turn, sexual behaviors [30].

In black-legged kittiwakes plasma baseline LH concentrations of non-breeding males were negatively correlated to blood Hg concentrations [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. By using the injection of exogenous GnRH, a common and powerful method to test the functionality of the pituitary through its ability to release LH [START_REF] Schoech | Delayed breeding in the cooperatively breeding Florida scrub-jay (Aphelocoma coerulescens): inhibition or the absence of stimulation?[END_REF], [START_REF] Goutte | Stress and the timing of breeding: Glucocorticoid-luteinizing hormones relationships in an arctic seabird[END_REF], [START_REF] Goutte | Why do some adult birds skip breeding? A hormonal investigation in a long-lived bird[END_REF], the authors suggested that Hg contamination may disrupt GnRH secretion from the hypothalamus in black-legged kittiwakes [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. The effect of Hg on LH secretion is supported by experimental investigations: Hg-fed male rats had decreased concentrations of LH and testosterone [START_REF] Ramalingam | Effect of mercuric chloride on circulating hormones in adult albino rats[END_REF]. LH secretion could thus be a potential target for Hg. In addition LH secretion is also closely related to age across vertebrates [START_REF] Saal | Reproductive senescence: Phenomena and mechanisms in mammals and selected vertebrate. The physiology of reproduction[END_REF] including long-lived seabirds like the snow petrel [START_REF] Goutte | Why do some adult birds skip breeding? A hormonal investigation in a long-lived bird[END_REF]. Regarding our study model, the snow petrel, we therefore predict a negative relationship between blood Hg and baseline LH in the most contaminated age class. To investigate the underlying mechanism of such possible relationship, we used a GnRH challenge to test the ability of the pituitary to release LH in relation to Hg. If GnRH-induced LH concentrations are negatively correlated to blood Hg, this would indicate that Hg affects the ability of the pituitary to properly release LH.

Material and methods

Ethics statement

Animals were cared for in accordance with the guidelines of the ethics committee of the Institut Polaire Français Paul Emile Victor (IPEV) that specifically approved this study (Program no. 109, H. Weimerskirch).

Capture, blood sampling and GnRH-challenge

The study was conducted on Adélie land (66°40' S, 140°01' E), Antarctica. Many snow petrels were of known age, because chicks have been banded each year since 1964 [START_REF] Chastel | High annual variability in reproductive success and survival of an Antarctic seabird, the snow petrel Pagodroma nivea[END_REF].

Twenty-nine males and 16 females, from 11 to 45 years old, were handled during the prelaying period (i.e. the courtship and mating period, a few days after arrival to the colony), from 11 to 23 November 2008. Only one bird was captured at each nest and the sex of the birds was previously determined by molecular sexing [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF]. Snow petrels were caught by hand and a first blood sample was collected from the alar vein with a syringe immediately after capture (mean ± S.D.: 3 min ± 5 s) to determine baseline plasma LH concentrations. Then, 40 birds were immediately injected with 0.1 ml of a GnRH solution in the second alar vein ([Gln8], Sigma Lot 121H04314). The GnRH was dissolved in a physiological solution to yield a dosage of 0.6 mg for 0.1 ml (about 1.5 mg kg -1 body mass in 1 ml of 0.9% saline solution, as validated in this species [START_REF] Goutte | Why do some adult birds skip breeding? A hormonal investigation in a long-lived bird[END_REF]). In snow petrels, a GnRH injection elicits a maximal increase of LH after 10 minutes, and this response documents the ability of the pituitary to release LH. In this species, measuring LH levels 30 minutes post-GnRH challenge can inform on an individual's capacity to maintain elevated LH concentrations [START_REF] Goutte | Why do some adult birds skip breeding? A hormonal investigation in a long-lived bird[END_REF]. Blood samples were therefore collected from alar veins at 10 and 30 min after the GnRH injection to measure plasma LH concentrations. LH changes from 0 to 10 min post-GnRH injection [(LH t=10 -LH t=0 )/LH t=0 ] and LH changes from 10 to 30 min post-GnRH injection [(LH t=30 -LH t=10 )/LH t=10 ] were used as indicators of different individuals' abilities to release LH. Blood samples were centrifuged and plasma (for LH assay) and red blood cells (for total Hg and stable isotopes assays) were stored at -20°C. Adults were weighted to the nearest 2 g using a spring balance and their skull length (head + bill) was measured to the nearest 0.5 mm using a caliper. In snow petrels, skull length appears to be a reliable measurement of the overall size of a bird [START_REF] Angelier | Age-specific reproductive success in a long-lived bird: do older parents resist stress better?[END_REF] and adult scaled mass index was then calculated [START_REF] Peig | New perspectives for estimating body condition from mass/length data: the scaled mass index as an alternative method[END_REF].

Hg analysis

Total Hg was measured as described in [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North Eastern Atlantic waters: Influence of geographical origin and feeding ecology[END_REF] from freeze-dried and powdered red blood cells (hereafter called blood) in an Advanced Hg Analyzer spectrophotometer (Altec AMA 254).

Prior and after freeze-drying, blood samples were weighed to determine the percentage of water in blood to facilitate comparisons with other studies. At least two aliquots ranging from 5 to 10 mg were analyzed for each individual and quality assessment was measured by repeated analyses of certified reference material TORT-2 (lobster hepatopancreas, NRCC; certified value 0.27±0.06 µg•g -1 ). Hg concentrations are expressed in µg•g -1 dry weight (dw).

Stable isotopes analysis

The isotopic niche of snow petrels was used as a proxy of their trophic niche during the prebreeding period, as blood cells is a metabolically active tissue that integrates periods of weeks before sampling [START_REF] Hobson | Turnover of 13 C in Cellular and Plasma Fractions of Blood: Implications for Nondestructive Sampling in Avian Dietary Studies[END_REF], [START_REF] Hobson | Assessing Avian Diets Using Stable Isotopes II: Factors Influencing Diet-Tissue Fractionation[END_REF] and [START_REF] Bearhop | Factors That Influence Assimilation Rates and Fractionation of Nitrogen and Carbon Stable Isotopes in Avian Blood and Feathers[END_REF]. The isotopic method was validated in the southern Indian Ocean, with δ 13 C values of seabirds indicating their foraging habitats [START_REF] Cherel | Geographical variation in carbon stable isotope signatures of marine predators: a tool to investigate their foraging areas in the Southern Ocean[END_REF], [START_REF] Jaeger | Seabird satellite tracking validates the use of latitudinal isoscapes to depict predators' foraging areas in the Southern Ocean[END_REF] and their δ 15 N values increasing with trophic level [START_REF] Cherel | Isotopic niches and trophic levels of myctophid fishes and their predators in the Southern Ocean[END_REF]. Before isotopic analysis, blood samples were freezedried and powdered. Sub-samples of homogenized blood powder were then weighed (~0.4 mg) with a microbalance and packed in tin containers. Relative abundances of C and N isotopes were determined with a continuous flow mass spectrometer (Thermo Scientific Delta V Advantage) coupled to an elemental analyzer (Thermo Scientific Flash EA 1112). Isotopic results are presented in the  notation relative to Vienna PeeDee Belemnite and atmospheric N 2 for δ 13 C and δ 15 N, respectively. Replicate measurements of internal laboratory standards (acetanilide) indicate measurement errors < 0.10 ‰ for both  13 C and  15 N values. The C:N mass ratios of the samples were calculated as the ratio between the mass percentages in carbon and nitrogen. The consistently low C:N values of blood cells verified that low lipid content did not necessitate lipid extraction [START_REF] Cherel | Isotopic Discrimination between Food and Blood and Feathers of Captive Penguins: Implications for Dietary Studies in the Wild[END_REF].

Hormone assays

Plasma LH concentrations were assayed by radioimmunoassay as validated for snow petrels [START_REF] Goutte | Why do some adult birds skip breeding? A hormonal investigation in a long-lived bird[END_REF]. The lowest detectable concentration of LH was 0.05 ng•ml -1 and the intra-assay coefficient of variation was 6.2% (three duplicates). In 4 birds plasma levels were too low to measure LH.

Statistics

All analyses were performed using R v. 2.13.1. We used GLM with normal error and identity link function to explain: (i) blood Hg concentration in relation to age, blood δ 13 C and δ 15 N, scaled mass index, and interactions with sex, (ii) blood δ 13 C and δ 15 N values in relation to age, age², scaled mass index and interactions with sex (linear and/or quadratic). To address the interaction between LH, Hg and age, the dataset was split into two groups using a recently developed Bayesian approach ('broken stick' modelling technique [START_REF] Thiebot | Gentlemen first? "Broken stick" modelling reveals sex-related homing decision date in migrating seabirds[END_REF], [START_REF] Authier | Breaking the sticks: a hierarchical change-point model for estimating ontogenetic shifts with stable isotope data[END_REF], based on beta regression analysis with 'betareg' package in R, [START_REF] Cribari-Neto | Beta Regression in R[END_REF]. This method will infer the 'age limit' where blood Hg concentration starts translates statistically as a change-point in the distribution of blood Hg concentration versus age. To estimate the age limit of the changepoint T i , in blood Hg concentrations we used a profile likelihood approach [START_REF] Authier | Breaking the sticks: a hierarchical change-point model for estimating ontogenetic shifts with stable isotope data[END_REF]: the likelihood was computed for each age. The value of T i that maximized the likelihood was thus evaluated, and an approximate confidence interval for T i was computed with a Likelihood Ratio Test with 1 df. This method bypasses the need to manually inspect the data for identification of a change-point, which could only be uncertain in our case owing to the high variance observed in individual blood Hg concentrations. Thanks to this method we were able to distinguish a 'break' in Hg distribution at 23 years old in our data set. Thus we considered two groups: birds that were until 23 years old (N=31) and birds that were more than [START_REF] Newsome | A niche for isotopic ecology[END_REF]

Results

Hg concentrations in relation to age, sex and trophic niche

In snow petrels dw blood was equivalent to 35.5 ± 0.3 % of ww blood and Hg concentration averaged 2.7 ± 1.1 (range: 1.0; 5.3) µg•g -1 dw During the pre-laying period, blood Hg concentrations were not related to sampling date (GLM, F 1,43 = 0.5, p=0.488) and were higher in females than in males (Table 1). Blood Hg concentration decreased with increasing age in males (GLM, F 1,27 = 7.9, p=0.009, Fig. 1) and this negative relationship was only close to statistical significance in females (GLM, F 1,14 = 4.0, p=0.06, Fig. 1). Females had significantly higher δ 13 C and lower δ 15 N values than males (Table 1). A significant U-shaped relationship between blood δ 13 C values and age was found, i.e. young and old birds had higher δ 13 C values than middle-aged ones (Fig. 2, Table 2). Blood δ 15 N values were not related to age (Table 2). Blood Hg concentrations increased with increasing blood δ 13 C in females while in males the similar pattern was found but only close to statistical significance (Fig. 3A,Table 3). Regarding the relationships between blood Hg and δ 15 N values, a positive relationship was found in females only (Fig. 3B, Table 3). Scaled mass index was not related to blood Hg concentration, δ 13 C or δ 15 N values and interactions with sex (p>0.099 for all tests).

Hg, age and plasma LH

Following GnRH injections, absolute LH concentrations (baseline: 8.4 ± 0.5 ng•ml -1 ) significantly increased (10 min: 11.5 ± 0.8 ng•ml -1 ), then decreased (30 min: 9.1± 0.8 ng•ml -1 ; generalized linear mixed model (GLMM), time as factor: F 2,61 = 12.57, p<0.001) these LH variations were not different between sexes or age classes (≤23 y.o. versus >23 y.o.) (p>0.08 for all tests). Absolute plasma LH concentrations (baseline, after 10 and after 30 minutes) were significantly higher in males compared to females (p<0.009 for all tests) and were not related to sampling date (p>0.123 for all tests) or to scaled mass index (p>0.07 for all tests).

In birds that were ≤ 23 y.o., plasma baseline LH significantly decreased with increasing Hg concentration in blood (Fig. 4A,Table 4) and LH changes from 0 to 10 minutes and from 10 to 30 minutes following the GnRH challenge were unrelated to blood Hg concentration (Fig. 4). In birds that were >23 y.o., plasma baseline LH concentration, LH changes from 0 to 10 minutes and from 10 to 30 minutes, were unrelated to blood Hg concentration (Fig. 4D-E-F, Table 4).

4B-C, Table

Discussion

Hg exposure, trophic niche and age

In pre-laying snow petrels, blood Hg concentrations were in the range of concentrations found in other species from the Southern ocean [START_REF] Tavares | Influence of age, sex and breeding status on mercury accumulation patterns in the wandering albatross Diomedea exulans[END_REF], [START_REF] Anderson | Influence of trophic position and foraging range on mercury levels within a seabird community[END_REF], [START_REF] González-Solís | Metals and selenium as bioindicators of geographic and trophic segregation in giant petrels Macronectes spp[END_REF] and equivalent to those reported at the same location (Adélie Land) in south polar skuas Catharacta maccormicki (2.2 ± 0.2 µg•g -1 dw [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF]). Blood Hg concentrations were higher in females than in males. In birds, Hg sex differences are often observed and are more likely to be related to trophic guild than other extrinsic or intrinsic factors [START_REF] Robinson | Sex Differences in Mercury Contamination of Birds: Testing Multiple Hypotheses with Meta-Analysis[END_REF]. In the present study we have an evidence of trophic guild difference between sexes; indeed stable isotopes analyses showed that foraging habitat (δ 13 C values) was positively related to blood δ 13 C values especially in females. According to the latitudinal  13 C gradient in oceanic waters of the Southern Ocean [START_REF] Cherel | Geographical variation in carbon stable isotope signatures of marine predators: a tool to investigate their foraging areas in the Southern Ocean[END_REF], [START_REF] Jaeger | Seabird satellite tracking validates the use of latitudinal isoscapes to depict predators' foraging areas in the Southern Ocean[END_REF], blood δ 13 C values suggest the use of a high Antarctic habitat [START_REF] Cherel | Isotopic niches of emperor and Adélie penguins in Adélie Land, Antarctica[END_REF]. More precisely, the δ 13 C values close to -23 ‰ depict warmer waters at more northern latitudes and the δ 13 C values close to -26 ‰ colder waters at more southern latitudes, but still within the Antarctic zone [START_REF] Cherel | Isotopic niches of emperor and Adélie penguins in Adélie Land, Antarctica[END_REF]. Hg distribution, and hence its bioavailability is known to be heterogeneous in the Southern Ocean [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. The positive relationship between  13 C values and Hg concentrations means that Hg might be more bioavailable for snow petrels at more northern latitudes (δ 13 C values close to -23 ‰).

Trophic position, as depicted by blood δ 15 N values, was positively related to blood Hg concentration in females only. The enrichment of Hg with increasing trophic level has been described in food-webs and species communities [START_REF] Blévin | Wide Range of Mercury Contamination in Chicks of Southern Ocean Seabirds[END_REF], [START_REF] Furness | Birds as monitors of pollutants[END_REF] but more rarely within a given species. For example, Hg enrichment with increasing δ 15 N values was found in chicks but not in adults of great skuas Catharacta skua [START_REF] Bearhop | Bioamplification of Mercury in Great Skua Catharacta skua Chicks: the Influence of Trophic Status as Determined by Stable Isotope Signatures of Blood and Feathers[END_REF]. In Adélie land, snow petrels feed their chicks mainly with fish [START_REF] Ridoux | The diets of five summer breeding seabirds in Adélie Land, Antarctica[END_REF], but the adults' diet is still unknown. When compared to other Antarctic seabirds and their prey [START_REF] Cherel | Isotopic niches of emperor and Adélie penguins in Adélie Land, Antarctica[END_REF], the large range of blood δ 15 N values suggests that self-feeding breeding adults fed from a krill-to a fish-based diet, depending on individuals. The higher blood δ 15 N values of males indicate they fed more on fish than females. However and unexpectedly despite their higher trophic level, males are less Hg contaminated than females, suggesting once more that females may feed in areas where Hg is more bioavailable. This is in agreement with the δ 13 C values-Hg concentration relationship. Alternatively, detoxification processes could be more efficient in males. Noticeably, their stable isotope values indicate that the most contaminated males fed on fish at more northern latitudes than the others, suggesting again a latitudinal effect on Hg availability [START_REF] Cossa | Mercury in the Southern Ocean[END_REF]. To summarize, females probably fed at more northern latitudes and at a lower trophic position compared to males. However, females were more Hg contaminated and Hg contamination was positively related to foraging habitat and trophic position, while in males no relationships were found. These results could be the consequence of sex-specific foraging preferences [START_REF] Lecomte | Patterns of aging in the long-lived wandering albatross[END_REF], [START_REF] Thiers | Foraging zones of the two sibling species of giant petrels in the Indian Ocean throughout the annual cycle: implication for their conservation[END_REF] or maybe sex-specific physiological constraints. Indeed, females are 20 % lighter than males consequently they could be more sensitive to the harsh Antarctic climate during winter and constraint to forage in more northern latitudes where Hg is more bioavailable. Contrary to our prediction blood Hg concentration decreased with increasing age in pre-laying snow petrels, especially in males. The relationship between Hg and age in seabirds is poorly documented and often contradictory. For example, in organs involved in storage and detoxification as the liver, Hg concentration may decrease, increase, with advancing age or may not be related to age [START_REF] Furness | Pollutant levels in the great skua Catharacta skua[END_REF], [START_REF] Hutton | Accumulation of heavy metals and selenium in three seabird species from the United Kingdom[END_REF] and [START_REF] Thompson | Mercury Accumulation in Great Skuas Catharacta skua of Known Age and Sex, and Its Effects Upon Breeding and Survival[END_REF]. In excretory tissues as feathers, Hg reaches a plateau until individuals start breeding, and then feather Hg concentration decreases [START_REF] Tavares | Influence of age, sex and breeding status on mercury accumulation patterns in the wandering albatross Diomedea exulans[END_REF].

Regarding blood Hg, two studies on Procellariiforms found no relationship between Hg and age [START_REF] Tavares | Influence of age, sex and breeding status on mercury accumulation patterns in the wandering albatross Diomedea exulans[END_REF], [START_REF] González-Solís | Metals and selenium as bioindicators of geographic and trophic segregation in giant petrels Macronectes spp[END_REF]. However in south polar skuas, breeding experience (which is tightly related to age) was negatively related to blood Hg [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF], as found in our study. This pattern could originate from an age-related change in trophic niche i.e. older individuals would forage in areas where Hg is less bioavailable and/or at a lower trophic level than younger ones. Indeed, trophic niche segregations by age, has been observed in some Procellariform species [START_REF] Lecomte | Patterns of aging in the long-lived wandering albatross[END_REF], [START_REF] Thiers | Foraging zones of the two sibling species of giant petrels in the Indian Ocean throughout the annual cycle: implication for their conservation[END_REF]. In our study, a quadratic relationship was found between foraging habitat (δ 13 C values) and age. This suggests that, compared to middle-aged birds, young and old individuals foraged at higher latitudes which may be more Hg contaminated. Isotopic signature of blood represents the isotopic niche during the last month preceding sampling [START_REF] Cherel | Isotopic niches of emperor and Adélie penguins in Adélie Land, Antarctica[END_REF]. As birds have been sampled in November (a few days after their arrival on the breeding area) those values may mirror the trophic niche used during late winter and suggest that young and old snow petrels do not use the same foraging areas than middle-aged birds at that time. And why older individuals bear lower Hg concentrations than younger birds could be the consequence of an increase in Hg excretion rate with increasing age or the selective disappearance of the most contaminated individuals over time. One may assume that Hg excretion opportunities increase with age; as older individuals had more opportunities to excrete Hg, through molting, and egg-laying for females, than younger birds. But there is no evidence for supporting such an assumption. Also, our study was cross-sectional, and since in its methylated form Hg is highly toxic, the possibility that most of the highly contaminated birds died selectively over time cannot be excluded. In Adult common loons (Gavia immer) blood Hg concentrations of 2-3 µg•g -1 (ww) are considered at moderate risk to effects of Hg contamination [START_REF] Champoux | Assessment of mercury exposure and potential effects on common loons (Gavia immer) in Québec[END_REF]. In snow petrels ww blood Hg concentrations would reach 1.6 µg•g -1 and 1.9 µg•g -1 , for the most contaminated male and female respectively; these concentrations are close to the threshold level for moderate risk in common loons and snow petrels could maybe be more sensitive to lower Hg concentrations. However, in free-living birds, Hg has so far never been reported as affecting survival probabilities, even in highly contaminated areas [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF], [START_REF] Mitro | Common Loon Survival Rates and Mercury in New England and Wisconsin[END_REF], [START_REF] Wayland | Survival rates and blood metal concentrations in two species of free-ranging North American sea ducks[END_REF], [START_REF] Hallinger | Mercury exposure and survival in free-living tree swallows (Tachycineta bicolor)[END_REF], [START_REF] Goutte | Demographic consequences of heavy metals and persistent organic pollutants in a vulnerable long-lived bird, the wandering albatross[END_REF].

LH secretion in relation to age and Hg

In the present study, plasma baseline LH decreased with increasing blood Hg in individuals until 23 y.o., which were those that bore the higher concentrations of blood Hg. This negative relationship between plasma baseline LH and blood Hg could either originate from a Hg-related dysfunction of the pituitary (i.e. an inability to produce enough LH) and /or of the hypothalamus (i.e. a lack of endogenous GnRH production). Since LH changes (0-10 min and 10-30 min) following an exogenous input of GnRH were not related to Hg, this means that Hg does not disrupt the capability of the pituitary to release LH in birds until 23 y.o.: their pituitary is thus functional. This rather suggests, as found in black-legged kittiwakes, of a problem originating from the hypothalamus, which may not release enough GnRH in response to high Hg exposure [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. However, contrary to black-legged kittiwakes [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF], Hg contamination did not result in an over-release of LH post-GnRH injection in snow petrels. In black-legged kittiwakes, the over-release of LH was interpreted as an increase of GnRH receptors on the pituitary in response to the low level of GnRH secreted by the hypothalamus [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. In snow petrels, the concentrations of blood Hg are slightly higher than in black-legged kittiwakes (2.7 ± 1.1 µg•g -1 against 2.0 ± 0.5 µg•g -1 , respectively) and LH secretion post-GnRH injection was less pronounced in snow petrels compared to kittiwakes (averagely 1.4-fold and 2.0-fold, respectively). It is always difficult to compare different species as hazardous effects of Hg could be highly species-specific [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF]: e.g. average blood Hg concentrations in brown skuas were much higher than in south polar skuas (8.2 ± 0.2 and 2.2 ± 0.2 µg•g -1 dw, respectively) however south polar skuas suffered from higher Hginduced breeding failure than their closely relative [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF]. Thus, it is possible that black-legged kittiwakes could be more sensitive to Hg than snow petrels and would react more strongly to slightly lower Hg concentrations. In birds that were more than 23 y.o., blood Hg concentrations were lower than in the ≤23 age class, and no relationship appeared between plasma LH and blood Hg concentrations. However sample size was low for individuals aged above 23, and such interpretation must be taken cautiously. The onset of breeding is importantly controlled by an increase of circulating LH in the plasma [START_REF] Follett | Luteinizing hormone in the plasma of white-crowned sparrows (Zonotrichia leucophrys gambelii) during artificial photostimulation[END_REF], [START_REF] Sharp | A Comparison of Variations in Plasma Luteinizing Hormone Concentrations in Male and Female Domestic Chickens (gallus Domesticus) from Hatch to Sexual Maturity[END_REF], if these concentrations are not high enough individuals will be less receptive to mating behaviors [START_REF] Bentley | Rapid inhibition of female sexual behavior by gonadotropin-inhibitory hormone (GnIH)[END_REF].

In snow petrels a large proportion of young individuals do not breed each year [START_REF] Goutte | Age and the timing of breeding in a long-lived bird: a role for stress hormones?[END_REF], this nonbreeding behavior could thus be related to Hg contamination.

Conclusion

This study shows the importance of considering age in eco-toxicological studies. We reported an unexpected negative relationship between age and blood Hg concentrations in adult snow petrels and the use of stable isotopes suggests that this pattern could originate from an age and sex-related variation in late wintering areas, and hence Hg bioavailability. It is now possible to map migration and winter distribution of relatively small birds such as snow petrels via miniature loggers (geolocation sensors, GLS, e.g. [START_REF] Frederiksen | Multicolony tracking reveals the winter distribution of a pelagic seabird on an ocean basin scale[END_REF]). These devices could thus open new research possibilities to confirm the influence of age on the wintering areas used by this Antarctic species and to document how migratory strategies and winter distribution can affect the uptake of contaminants. We also showed that the age of the birds and their contaminant burden could influence the secretion of a key pituitary hormone for the onset of breeding (LH). This result, together with recent findings on black-legged kittiwakes, supports the view that Hg is tightly connected to LH secretion and then can impair the decision to breed [START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF] and long-term reproductive success [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF] in polar seabirds. Anthropogenic Hg emissions are expected to rise in the coming decades [START_REF] Streets | Projections of Global Mercury Emissions in 2050[END_REF], probably resulting in increasing Hg exposure for Antarctic top predators. This, coupled with a wide range of environmental perturbations in Antarctic regions could exacerbate the demographic responses of top predators to Hg [START_REF] Goutte | Demographic responses to mercury exposure in two closely-related Antarctic top predators[END_REF].

  (N=14) hereafter '≤ 23 y.o.' and '> 23 y.o.', respectively), blood Hg concentrations were significantly higher in '≤ 23 y.o' birds than in the '> 23 y.o.' ones (GLM, F 1,43 =4.8, p=0.034). Finally (iii) absolute plasma baseline LH concentrations, LH changes from 0 to 10 minutes, and changes from 10 to 30 minutes were tested as functions of blood Hg concentration (linear) in relation to age classes ('≤ 23 y.o.' and '> 23 y.o.'). We performed all our model selection starting from the most parameterized model that included all the variables/factors and their interactions and we removed step by step the non-significant interactions, variables or factors. Selected models were checked for assumptions and values were log-transformed when necessary, we used GLM with normal error and identity link function to explain LH concentrations in relation with Hg and interaction with sex. Values are means ± SD.

Table 4 .

 4 Modelling the relationships between blood Hg concentration a) baseline plasma LH concentrations, b) LH change from 0 to 10 minutes and c) LH change from 10 to 30 minutes in pre-laying snow petrels ≤ 23 years old and > 23 years old.

Figure 1 :

 1 Figure 1: Blood Hg concentration in relation to age in pre-laying snow petrels. Hg

Figure 2 :

 2 Figure 2: Blood δ 13 C values in relation to age in pre-laying snow petrels. δ 13 C values

Figure 3 :

 3 Figure 3: Blood Hg concentration in relation with blood δ 13 C and δ 15 N values in pre-laying snow petrels. (A) Hg concentration increased with increasing δ 13 C values for females and this relationship was close to significance for males (P=0.06), while (B) Hg concentration increased with increasing δ 15 N values in females only (white circle). Solid line refers to linear regression for females and dashed line to linear regression

Figure 4 :

 4 Figure 4: Plasma LH concentrations in relation to blood Hg concentration. In pre-laying

Table 1 .

 1 Blood Hg concentrations, δ13 C and δ15 N values in pre-laying snow petrels in relation to sex. Values are means ± SD with ranges in parentheses.

		Males	Females	Sex differences
	Blood values			SS* Df	F	p-value
	Hg (µg•g -1 ww) 2.2 ± 0.9 (1.0 ; 4.5)	3.6 ± 1.0 (2.0 ; 5.3)	18.7 1,43 21.9 >0.001
	δ 13 C (‰)	-25 ± 0.3 (-25.6 ; -24.4) -24.5 ± 0.6 (-25.6 ; -23.6) 1.9 1,43 10.1 >0.001
	δ 15 N (‰)	9.3 ± 0.6 (8.5 ; 12.5)	10.1 ± 1.0 (8.2 ; 10.4)	6.5 1,43 8.3 0.006

*SS = Sum of squares

Table 2 .

 2 Modelling the effects of age (years), age², sex and interactions as a function of blood δ13 C and δ15 N values (‰) in pre-laying snow petrels.

	Dependent variable	Independent variable	SS*	Df	F	p-value
	a) δ 13 C	Age 2	1.0	1,32	5.0	0.033
		Age	0.9	1,32	4.5	0.041
		Sex	1.5	1,32	7.5	>0.001
		Age 2 × Age	0.8	1,32	4.1	0.052
		Age 2 × Sex	0.7	1,32	3.7	0.063
		Age × Sex	0.7	1,32	3.4	0.074
		Age 2 × Age × Sex	>0.1	1,32	>0.1	0.862
	b) δ 15 N	Age 2	>0.1	1,32	>0.1	0.809
		Age	>0.1	1,32	>0.1	0.876
		Sex	5.3	1,32	6.1	0.019
		Age 2 × Age	0.2	1,32	0.2	0.644
		Age 2 × Sex	0.6	1,32	0.7	0.421
		Age × Sex	0.7	1,32	0.8	0.369
		Age 2 × Age × Sex	>0.1	1,32	>0.1	0.927

*SS = Sum of squares

Table 3 .

 3 Modelling the relationships between blood δ 13 C, δ15 N values (‰) and blood Hg concentration (µg•g -1 dw) in male and female pre-laying snow petrels.

	Dependent variable	Sex	Independent variable	SS Df	F	p-value
	Hg	Males	δ 13 C	2.8 1,27 3.9 0.060
			δ 15 N	1.7 1,27 2.2 0.150
		Females δ 13 C	5.7 1,14 9.1 0.009
			δ 15 N	8.6 1,14 20.6 >0.001

*SS = Sum of squares

Acknowledgments

The authors thank M. Kriloff and E. Antoine for their help in the field, C. Trouvé, C. Parenteau, S. Dano and C. Churlaud for their assistance in hormone and Hg assays, C.

Barbraud for his help in statistical analyses, J.B. Thiébot and M. Authier for providing the R code for the 'broken sticks' method and C. Breuner for improving the English and the manuscript. We also thank two anonymous referees for their constructive comments.