Ce ´lia Bordier
email: celia.bordier@gmail.com.

Claire Saraux

Vincent A Viblanc

` Ne Gachot-Neveu

Magali Beaugey

Yvon Le Maho

Ce ´line Le Bohec

Inter-Annual Variability of Fledgling Sex Ratio in King Penguins

As the number of breeding pairs depends on the adult sex ratio in a monogamous species with biparental care, investigating sex-ratio variability in natural populations is essential to understand population dynamics. Using 10 years of data (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009) in a seasonally monogamous seabird, the king penguin (Aptenodytes patagonicus), we investigated the annual sex ratio at fledging, and the potential environmental causes for its variation. Over more than 4000 birds, the annual sex ratio at fledging was highly variable (ranging from 44.4% to 58.3% of males), and on average slightly biased towards males (51.6%). Yearly variation in sex-ratio bias was neither related to density within the colony, nor to global or local oceanographic conditions known to affect both the productivity and accessibility of penguin foraging areas. However, rising sea surface temperature coincided with an increase in fledging sex-ratio variability. Fledging sex ratio was also correlated with difference in body condition between male and female fledglings. When more males were produced in a given year, their body condition was higher (and reciprocally), suggesting that parents might adopt a sex-biased allocation strategy depending on yearly environmental conditions and/or that the effect of environmental parameters on chick condition and survival may be sex-dependent. The initial bias in sex ratio observed at the juvenile stage tended to return to 1:1 equilibrium upon first breeding attempts, as would be expected from Fisher's classic theory of offspring sex-ratio variation.

Introduction

In animal populations, determinants of adult sex ratio (ASR, i.e. the proportion of male to female adult individuals [START_REF] Mayr | The Sex Ratio in Wild Birds[END_REF]) are likely to occur before adulthood, due to variations in parental sexual allocation of resources (before and after birth, reviewed in [START_REF] Frank | Sex Allocation Theory for Birds and Mammals[END_REF]), and to fluctuations in sex-specific mortality of these offspring before recruitment into the breeding population. According to Fisher [START_REF] Fisher | The Genetical Theory Of Natural Selection[END_REF], parental investment into male and female offspring should be equal at the termination of parental care, as the production of any zygote requires one mother and father. Thus, the reproductive value of males and females should be equal [START_REF] Fisher | The Genetical Theory Of Natural Selection[END_REF] and any tendency to deviate from a 1:1 sex allocation should return to equilibrium by frequency-dependent selection [START_REF] Allen | Frequency dependent selection: homage to E. B. Poulton[END_REF][START_REF] Bull | How fundamental are Fisherian sex ratios?[END_REF]. Nonetheless, Hamilton pointed out that biased sex ratios were likely to occur in natural conditions [START_REF] Hamilton | Extraordinary Sex Ratios[END_REF], if the costs of producing male and female offspring were different. In such cases, a bias towards the less costly sex should be expected [START_REF] Fisher | The Genetical Theory Of Natural Selection[END_REF][START_REF] Hamilton | Extraordinary Sex Ratios[END_REF][START_REF] Trivers | Natural selection of parental ability to vary the sex ratio of offspring[END_REF][START_REF] Charnov | The Theory of Sex Allocation[END_REF]. In sexually dimorphic species for instance, the higher costs of producing the larger (e.g. male) sex should favour a production bias towards the smaller (e.g. female) one. Additionally, Trivers & Willard [START_REF] Trivers | Natural selection of parental ability to vary the sex ratio of offspring[END_REF] hypothesised that mothers in good condition may afford to invest more into the sex with greatest variance in reproductive success, when mothers in poor condition gain more by investing into the sex less affected by poor maternal rearing conditions [START_REF] Trivers | Natural selection of parental ability to vary the sex ratio of offspring[END_REF][START_REF] Nager | Experimental demonstration that offspring sex ratio varies with maternal condition[END_REF]. This seems to be the case in the red deer (Cervus elaphus) in which high population densities during female pregnancy lead to adverse nutritional stress causing mothers to produce fewer male than female offspring [START_REF] Kruuk | Population density affects sex ratio variation in red deer[END_REF]. Alternately, sex-ratio bias may also stem from the fitness benefits/costs associated with inter-sexual differences in competition/cooperation, favouring selection for the sex in which competition between parents and offspring is lowest and/or cooperation highest [START_REF] Hamilton | Extraordinary Sex Ratios[END_REF]. In particular, Clark's resource competition model ( [START_REF] Clark | Sex Ratio and Local Resource Competition in a Prosimian Primate[END_REF]; generalisation of Hamilton's model) highlights an additional cost of producing offspring for mothers, in that they must later compete for food/mate/ reproductive site resources. Consequently, mothers might favour the more dispersive sex. For instance, during years of El Nin ˜o events, Northern elephant seal (Mirounga angustirostris) females experience poor foraging conditions and high nutritional stress [START_REF] Crocker | Impact of El Nin ˜o on the foraging behavior of female northern elephant seals[END_REF]. Interestingly, offspring production is biased towards males in those years and this male-biased sex ratio has been suggested as an adaptive strategy to decrease later resource competition between mothers and daughters on similar food patches [START_REF] Lee | North Pacific Climate Mediates Offspring Sex Ratio in Northern Elephant Seals[END_REF].

Because it has direct consequences on breeder proportions [START_REF] Keyfitz | Population: facts and methods of demography[END_REF][START_REF] Caswell | Matrix population models[END_REF], understanding the dynamics of ASR and the reasons for its fluctuations is an important perspective in animal ecology (e.g. [START_REF] Trivers | Parental investment and sexual selection[END_REF], see [START_REF] Sheldon | Recent studies of avian sex ratios[END_REF] for a review in birds). In this study, we used a decade-long dataset to investigate sex-ratio variability in a wild seabird population of king penguins (Aptenodytes patagonicus). In these seasonally monogamous seabirds, bi-parental care is necessary for successful reproduction, so that a biased ASR should produce adults of the supernumerary sex that are unable to find mates and reproduce, resulting in a reduced number of breeders at the population scale with potential consequences on population dynamics. Surprisingly however, male-biased ASRs have been suggested in king penguin populations [START_REF] Bried | Why Do Aptenodytes Penguins Have High Divorce Rates?[END_REF][START_REF] Pincemy | Experiments on colour ornaments and mate choice in king penguins[END_REF][START_REF] Pincemy | Homosexual Mating Displays in Penguins: Homosexual Mating Displays in Penguins[END_REF][START_REF] Olsson | Survival in king penguins Aptenodytes patagonicus: temporal and sex-specific effects of environmental variability[END_REF] and so has male-biased mate competition [START_REF] Keddar | Male-biased mate competition in king penguin trio parades[END_REF]. Yet, the reason for such a bias, and life history stage at which it arises, remains unclear. Therefore, using over more than 4000 king penguins of 'La Baie du Marin' colony (Crozet Archipelago; Southern Ocean), we considered fledgling sex-ratio variability and its potential links with environmental parameters. The breeding biology of king penguins imposes strong constraints on parental investment. The absence of a nest [START_REF] Stonehouse | The King Penguin Aptenodytes patagonica of South Georgia. I. Breeding behaviourand development[END_REF] compels breeding birds to navigate through thousands of conspecifics to locate both partners and chicks [START_REF] Nesterova | Do penguins dare to walk at night? Visual cues influence king penguin colony arrivals and departures[END_REF]. In addition, chick development from hatching to fledging occurs over an exceptionally long period (over 11 months [START_REF] Stonehouse | The King Penguin Aptenodytes patagonica of South Georgia. I. Breeding behaviourand development[END_REF][START_REF] Barrat | Quelques aspects de la biologie et de l'e ´cologie du manchot royal (Aptenodytes patagonicus) des ı ˆles Crozet[END_REF][START_REF] Weimerskirch | The breeding biology and population dynamics of king penguins Aptenodytes patagonica on the Crozet Islands[END_REF]), encompassing the austral winter when chick growth is limited and mortality is high [START_REF] Weimerskirch | The breeding biology and population dynamics of king penguins Aptenodytes patagonica on the Crozet Islands[END_REF][START_REF] Stier | Starting with a handicap: phenotypic differences between early-and late-born king penguin chicks and their survival correlates[END_REF][START_REF] Olsson | Seasonal Effects of Timing and Reproduction in the King Penguin: A Unique Breeding Cycle[END_REF], chicks gather in cre `ches to limit predation pressure [START_REF] Bohec | The adaptive significance of cre `ches in the king penguin[END_REF] and increase thermoregulation [START_REF] Eichhorn | Heterothermy in growing king penguins[END_REF]. This long breeding cycle (over a year from courtship to fledging) also results in non-synchronised laying, early and late offspring being reared in highly heterogeneous conditions (i.e. differences in food resources [START_REF] Gauthier-Clerc | Seabird reproduction in an unpredictable environment: how king penguins provide their young chicks with food[END_REF] and bird colony density [START_REF] Viblanc | Energetic adjustments in freely breedingfasting king penguins: does colony density matter?[END_REF]). In particular, early and late chicks are known to differ in their phenotype at hatching [START_REF] Stier | Starting with a handicap: phenotypic differences between early-and late-born king penguin chicks and their survival correlates[END_REF] and to exhibit strong differential mortality during the winter [START_REF] Weimerskirch | The breeding biology and population dynamics of king penguins Aptenodytes patagonica on the Crozet Islands[END_REF][START_REF] Stier | Starting with a handicap: phenotypic differences between early-and late-born king penguin chicks and their survival correlates[END_REF][START_REF] Olsson | Seasonal Effects of Timing and Reproduction in the King Penguin: A Unique Breeding Cycle[END_REF]. Finally, as penguins are state-dependent breeders [START_REF] Gauthier-Clerc | State-dependent decisions in long-term fasting king penguins, Aptenodytes patagonicus, during courtship and incubation[END_REF], environmental conditions before the onset of breeding might affect parental body condition [START_REF] Mcnamara | State-dependent life histories[END_REF] and foraging efficiency, with potential consequences on chick growth and survival [START_REF] Gauthier-Clerc | State-dependent decisions in long-term fasting king penguins, Aptenodytes patagonicus, during courtship and incubation[END_REF][START_REF] Mcnamara | State-dependent life histories[END_REF]. Given the above features, king penguins provide an interesting model to investigate how sex ratio may be modulated by environmental factors and whether biased sex ratio may arise over early life history in this species, either due to differential parental allocation or sex-specific mortality. For instance, sex ratio might change between fledging and adulthood, as male and female juveniles may differ in their return rates to their natal colony [START_REF] Saraux | Effects of Individual Pre-Fledging Traits and Environmental Conditions on Return Patterns in Juvenile King Penguins[END_REF].

Materials and Methods

Ethics

Most animals in this study were handled only once (during their first moult) for marking with a subcutaneous transponder tag and measurement of morphological features. A smaller number of individuals had also been previously handled as chicks (in their first month) for marking with a small external plastic pin (Fishtag, Floytag) and blood-sampling. All procedures employed during the field work were approved by the Ethical Committee of the French Polar Institute (Institut Paul Emile Victor -IPEV) and conducted in accordance with its guidelines, also complying with French laws including those relating to conservation and animal welfare. Authorizations to enter the breeding site (permits n ˚2005-191 Handled animals were removed from the colony in order to minimize the disturbance to neighbouring birds and carried a few meters away for manipulation. They were hooded to reduce their stress and manipulations lasted between 5 and 10 minutes. The transponder tags weigh 0.8 g and have no known adverse effects. They were shown to have no effect on survival of king penguins [START_REF] Froget | Is penguin banding harmless?[END_REF] or breeding success, recruitment or survival of great tits (Parus major) [START_REF] Nicolaus | Effect of PIT tags on the survival and recruitment of Great Tits[END_REF]. Furthermore, concerns about infections should be minimal, as transponder tags are kept sealed sterile in iodine capsules (Betadine) and removed from the capsules only by the process of injecting them into the bird. Ve ´te ´dine soap and alcoholic antiseptic solutions were used to disinfect the skin and the injecting needle before each insertion. Flesh wounds did not appear infected thereafter (personal observations on a sample of recaptured birds). Blood was sampled in small quantities, taking into account the age of the chick (1 mL for fledglings and 100 mL for younger chicks).

Long-term monitoring and sex-ratio determination

Our study was conducted in the king penguin colony of 'La Baie du Marin', on Possession Island, Crozet Archipelago (46 ˚259S, 51 ˚459E). From 2000 to 2009, we marked 3787 king penguin chicks that we later monitored using an automatic system (ANTAVIA), based on radio-frequency identification (RFID) of individuals born in a subpart of the colony [START_REF] Gendner | A new application for transponders in studying penguins[END_REF]. Around the peak of fledging, chicks in advanced moult were captured on the edge of the colony and implanted with passive transponder tags under the skin of their leg, without any other external mark. Upon capture, the chick's head was covered with a hood to keep it calm. A 1-mL blood sample was collected from its marginal flipper and used for sex determination based on genomic DNA (see below). Because those chicks were initially captured right before fledging, we did not know whether they were issued from an early (laying prior to Jan. 1 st ) or late reproduction [START_REF] Stonehouse | The King Penguin Aptenodytes patagonica of South Georgia. I. Breeding behaviourand development[END_REF][START_REF] Weimerskirch | The breeding biology and population dynamics of king penguins Aptenodytes patagonica on the Crozet Islands[END_REF]. Thus, we captured and marked 200 early and 200 late chicks per year from 2007 to 2009 (,3-4 week old). Those chicks were temporarily tagged with a small external plastic pin (Fishtag, Floytag). Of those, 225 chicks (198 early chicks and 27 late chicks) survived to fledging and were re-captured, transponder-tagged and bloodsampled at that time. Finally, as part of a separate study, 20 early and 20 late chicks were captured in 2009, and 173 early and 197 late chicks were captured in 2010, right after hatching (,1 week old). Those were marked with a fishtag, blood sampled (,100 mL), and used to investigate the changes in sex ratio between hatching and fledging.

Chick sex was determined from DNA extracted from blood samples following the protocol described by Sambrook et al. [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF]. Sex determination relied on polymerase chain reaction (PCR) amplification of two parts of the sex chromosome CHD1 gene, which in birds differs in size between the Z and W chromosomes [START_REF] Griffiths | Sex determination in birds using two CHD genes[END_REF][START_REF] Ellegren | Sex ratio adjustment in relation to paternal attractiveness in a wild bird population[END_REF]. Females were characterized by displaying both a Wspecific fragment and a Z-specific fragment, while males showed only the shorter Z-fragment. All individuals were tested with primers F1/R1 [START_REF] Kahn | Chromosome-Specific Intron Size Differences in the Avian CHD Gene Provide an Efficient Method for Sex Identification in Birds[END_REF], and in each year, ca. 10% of them were controlled at random using another pair of primers P2/P8 [START_REF] Griffiths | A DNA test to sex most birds[END_REF] to confirm sex determinations. Sex ratio was calculated as the proportion of males in the population (given as a %).

Chick morphological measurements and body condition

Standardised measurements of flipper length, beak length (¡1 mm) and body mass were collected on all but 3 chicks upon capture right before fledging (N54101). Flipper and beak lengths are highly repeatable measurements [START_REF] Fahlman | Accounting for body condition improves allometric estimates of resting metabolic rates in fasting king penguins, Aptenodytes patagonicus[END_REF] and provide good descriptors of structural size. However, because those measurements are correlated (Spearman's rank correlation; p,0.001, rho50.32, N54101), we established a Structural Size Index (SSI) using principal component analysis as previously described in [START_REF] Saraux | Effects of Individual Pre-Fledging Traits and Environmental Conditions on Return Patterns in Juvenile King Penguins[END_REF]. The first principal component of the analyses explained 79% of the variation in bill and flipper length and was retained as the SSI (SSIf ledging~PC1~0:26|Beakz0:97|Flipper) .

Because body mass is highly variable in king penguins and perhaps associated with differences in nutritional status as well as structural size, OLS-regression residuals of body mass on structural size are likely to provide a better reflection of the actual energy stores of the animal [START_REF] Schulte-Hostedde | Restitution of mass-size residuals: validating body condition indices[END_REF]. Thus, we calculated body condition as the residuals of an OLS-regression of chicks' body mass on SSI [START_REF] Saraux | Effects of Individual Pre-Fledging Traits and Environmental Conditions on Return Patterns in Juvenile King Penguins[END_REF][START_REF] Dobson | Comparison of color and body condition between early and late breeding king penguins[END_REF].

To determine if the sexual size dimorphism occurred at hatching or fledging, we also calculated body condition from an OLS regression of body mass on structural size at hatching over 173 early and 197 late-hatched king penguin chicks in 2010 (SSIhatching~PC1~0:28|Beakz0:96|Flipper) .

Life-history determination of transponder-tagged birds

Once implanted with a transponder tag, chicks are automatically identified throughout their lifetime in the ANTAVIA sub-colony by RFID antennas. Those antennas are buried underground at unique transit pathways in and out of the sub-colony and allow continuous monitoring of bird movements and presence within the colony [START_REF] Gendner | A new application for transponders in studying penguins[END_REF]. Such data have yielded detailed information on individuals' lives after fledging, including data on first return rates to their natal colony [START_REF] Saraux | Effects of Individual Pre-Fledging Traits and Environmental Conditions on Return Patterns in Juvenile King Penguins[END_REF], reproduction [START_REF] Descamps | The annual breeding cycle of unbanded king penguins Aptenodytes patagonicus on Possession Island (Crozet)[END_REF][START_REF] Bohec | Population dynamics in a long-lived seabird: I. Impact of breeding activity on survival and breeding probability in unbanded king penguins[END_REF][START_REF] Saraux | Chick-provisioning strategies used by king penguins to adapt to a multiseasonal breeding cycle[END_REF], age-specific survival and breeding performances (Le Bohec et al., unpublished data). Thus, monitoring individuals tagged from fledging allows assessment of changes in sex ratio throughout different life-history stages. This information was used to investigate changes in sex ratio between fledging, first return to the colony, and first reproductive attempt. For this analysis, only the first 4 cohorts of tagged chicks were used, as most of the birds from latter cohorts had not yet started to breed.

Environmental descriptors

Climate and oceanographic conditions

To investigate how sex ratio may be influenced by climatic conditions, we used indicators of processes acting both at local and global scales [START_REF] Stenseth | Ecological Effects of Climate Fluctuations[END_REF][START_REF] Stenseth | Weather packages: finding the right scale and composition of climate in ecology[END_REF]. A large-scale climatic index, the Southern Oscillation Index (SOI; calculated on a monthly basis as the fluctuation in the air pressure difference between Tahiti and Darwin) was obtained from the Australian Bureau of Meteorology. Prolonged periods of negative SOI values are usually associated with warm ocean temperatures in our study area, typical of El Nin ˜o events [START_REF] Deser | El Nin ˜o events and their relation to the Southern Oscillation: 1925-1986[END_REF]. We used Sea Surface Temperature (SST, in ˚C), as a local proxy of prey abundance and distribution, a good indicator for marine predators such as king penguins [START_REF] Saraux | Effects of Individual Pre-Fledging Traits and Environmental Conditions on Return Patterns in Juvenile King Penguins[END_REF][START_REF] Bohec | King penguin population threatened by Southern Ocean warming[END_REF][START_REF] Saraux | Reliability of flipperbanded penguins as indicators of climate change[END_REF][START_REF] Pe ´ron | Projected poleward shift of king penguins' (Aptenodytes patagonicus) foraging range at the Crozet Islands, southern Indian Ocean[END_REF]. Indeed, by affecting primary production, SST has strong consequences on prey abundance [START_REF] Gregg | Ocean primary production and climate: Global decadal changes[END_REF]. Monthly SST values were obtained from the National Ocean and Atmospheric Administration of the USA and averaged for the feeding areas (46-60 ˚S, 46-56 ˚E) of king penguins during the summer (Polar Front, PF) and the winter (Marginal Ice Zone, MIZ) [START_REF] Charrassin | Utilisation of the oceanic habitat by king penguins over the annual cycle[END_REF]. We previously found that breeding success in our study colony was mostly affected by the SOI and SST conditions of the same year [START_REF] Bohec | King penguin population threatened by Southern Ocean warming[END_REF]. However, we tested for SOI and SST effects over four different periods: 6-and 12mo time-lag prior to laying (prior to the mean annual laying date of early breeders, May-Oct. or Nov.-Oct.), 6-mo time-lag after laying (after the mean annual laying date of early breeders, Nov.-Apr.), and over the entire breeding cycle (from laying to fledging, Nov.-Oct.). The different time-periods yielded similar results, so that results are given only over one period: the summer season, i.e. the 6-mo period following the onset of reproduction (the mean annual laying date of early breeders). Finally, we recorded the mean annual laying date of early breeders from 2004 to 2009 as a proxy of environmental conditions before breeding onset, and calculated the mean annual breeding success of the colony from 2000 to 2009 (Le Bohec et al., unpublished data) as a proxy of the annual environmental conditions endured during reproduction. Early years and/or years of high breeding success could be viewed as more favourable years of high resource availability.

Colonial breeding conditions

Because colony density of king penguins varies to a large extent over the course of a breeding season and may affect the physiological status of breeding parents [START_REF] Viblanc | Energetic adjustments in freely breedingfasting king penguins: does colony density matter?[END_REF], we considered whether colony density might affect chick sex ratio. Each year, starting in 2004, we calculated yearly colony density indices in the ANTAVIA subcolony by counting breeding individuals on pictures taken in December (peak density of the breeding season). We further calculated a second annual density index as the proportion of transponder-tagged birds monitored as breeding in a given year among tagged birds alive and of breeding age.

Statistics

All statistics were run using R v. 2.13.0 [START_REF]R: A language and environment for statistical computing[END_REF]. Because chicks were randomly sampled in the absence of their parents, we could not know whether sampled chicks may come from the same breeding pairs from year to year. This prevented us from looking for pseudo-replication in our data and using Generalized Linear Mixed Models (GLMMs) that would take adult identity into account. Yet, it should be noted that no more than 500-600 chicks were marked per year in a part of the colony retaining ca. 8,000 breeding pairs, so that the probability of pseudoreplication should be low. Therefore, Generalized Linear Models (GLMs; maximum likelihood approach) fitted to a binomial distribution were used to evaluate sex ratio with environmental factors or laying period. As all independent variables (e.g. laying period) were not available for all individuals, we could not run a single general model and use an information theoretical approach to select variables. Instead, we had to compute separate models for different independent variables. Still, in the case of climatic variables, we ran a single global model with SST and SOI as independent variables. The most appropriate models were selected using Akaike's Information Criterion (AIC). Adjusted R 2 values are indicated along with p-values. Data were checked for normality and for between-group homoscedasticity and between-year comparisons were performed using Wilcoxon rank-sum tests. Sex-ratio comparisons across life-history stages were performed using a binomial proportion test ('prop.test' package in R). Variables were considered significant for P,0.05. Bonferroni corrections were considered for multiple testing in three occasions, i.e. comparisons of sex ratio between lifehistory stages (Pv 0:05 3 ) comparisons of body condition between males and females for each year (Pv 0:05 10 ), and finally pairwise comparisons of sex ratio between years (Pv 0:05 45 ). In the first 2 cases, results were similar regardless of whether the Bonferroni adjustment was applied or not, indicating their robustness. In the last case (pairwise comparison of annual sex ratios), differences appeared. As Bonferroni adjustments are often criticized for being too conservative preventing from detecting significant differences, we decided to present the results without this adjustment on Figure 1. The number of birds is given as N.

Results

Sex ratio at fledging and inter-annual variability

Over the 10-year study period, fledging sex ratio was slightly biased towards males (1941 females vs. 2071 males), implying that an overall greater proportion of male offspring successfully fledged from 2000 to 2009, i.e. 51.62% of male (GLM; z52.05, P50.04, N54012; upper-right panel, Fig. 1). However, fledging sex ratio varied significantly between years (Pairwise tests given in Fig. 1) and significant male-bias was observed in only 2 of the study years (GLM; z52.90, P50.004, N5383 and z52.84, P50.004, N5295 for 2001 and 2002, respectively; Fig. 1). Because the data were limited to 10 points, we could not perform breakpoint analyses, but from a visual inspection (Fig. 1) it seems that the proportion of fledged males increased from 2000 to 2002 (44.37% to 58.31%, z52.39, P50.02, N5820), before decreasing continuously over 2002-2007, from 58.31% to 47.45% (z523.39, P,0.001, N52392). Finally, sex ratio at fledging was stable over the last 3 years (2007-2009) of the study (z50.70, P50.48, N51546).

Sex ratio and breeding timing: Early vs. late-hatched chicks

In 2010, the proportion of hatched males tended to be greater for early (57.95%) than for late (48.66%) chicks (GLM; z521.90, P50.06, N5419; Fig. 2). Also in 2010, we found that chick sex ratio did not vary between hatching and fledging (which included the austral winter period), both in early (-5.90%; Proportion test; P50.47, N5195/73) and late (+1.34%; P51, N5224/6) chicks (see Fig. 2).

From 2007 to 2010, we determined fledgling sex ratio for chicks hatched either early or late in the season (Fig. 2). Although the overall proportion of males appeared higher in late chicks (62.50% vs. 52.35% for early chicks), this difference was not statistically significant over the years (GLM; z50.88, P50.38, N5317). Similarly, we observed no significant difference in early-vs. late-chick sex ratio when years were considered separately (GLM 2007 ; z50. [START_REF] Stonehouse | The King Penguin Aptenodytes patagonica of South Georgia. I. Breeding behaviourand development[END_REF]

Sex ratio and life-history stages

Sex ratio at chick-fledging (55.22%), when juveniles first return to the colony (54.62%), or at first breeding attempt (53.27%) did not vary among cohorts hatched between 2000 and 2003. Sex ratio did not change significantly between fledging and first returns (Proportion test; P50.83, N51112/866), between first returns and first breeding attempts (P50.65, N5866/597), or between fledging and first breeding attempts (P50.47, N51112/597).

Fledging sex ratio and environmental factors

Climatic parameters

Over the 10-year study (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009) and 4012 individuals, the most appropriate models, selected by AIC, did not show any effect of SOI or SST on fledgling sex ratio. Indeed, the null model was retained regardless of the tested period (before breeding, summer after laying, and entire breeding cycle).

However, it is interesting to note that the period of decreasing male proportion at fledging (2002-2007, except in 2006) coincided with negative values of SOI during the period of incubation and chick-brooding. Also, variability in fledging sex ratio tended to increase with SST (Fig. 3), i.e. warmer years produced very unbalanced sex ratios (whether in favour of males or females) compared to colder years during which the sex ratio was close to 1:1.

From 2004 to 2009, the mean annual laying date of early breeders did not affect annual sex ratio at fledging (GLM; z520.02, P50.99). Similarly, the mean annual breeding success of the colony over the 10-year study (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009) was not related to fledgling sex ratio (GLM; z51.07, P50.28).

Colonial environment

From 2004 to 2009, sex ratio at fledging was not related to colony density (global colony-counts of individual penguins; GLM; z50.76, P50.44). Colony density as estimated only by counting tagged birds reproducing in the colony from 2007 to 2009, yielded similar results (GLM; z50.94, P50.35).

Fledging sex ratio and chicks' structural size and body condition

According to the structural size index, no dimorphism was observed at hatching neither for the 2010 early-hatched king penguin chicks (Wilcoxon; W54378, P50.86, N5173), nor for the 2010 late-hatched chicks (Wilcoxon; W54378, P50.24, N5197).

At fledging, however, chicks exhibited sexual size dimorphism, males being larger than females according to the structural size index (3.36¡0.26 vs. 23.59¡0.25, Wilcoxon; W51372092, P,0.001, N54101). However, when looking at body condition at fledging, the difference between males and females was not constantly in favour of males (Fig. 4A). Males had a higher body condition than females only in 2004 (Wilcoxon; W521025, P50.003, N5449).

The yearly difference in body condition between males and females was correlated with sex ratio at fledging (r50.67, P50.04; Fig. 4B). Higher male proportions were associated with a higher male body condition when compared to females.

Discussion

Over a 10-year period, fledgling sex ratio in our king penguin population was slightly male-biased. This finding is in line with previous data on male-biased ASR both in the present [START_REF] Bried | Why Do Aptenodytes Penguins Have High Divorce Rates?[END_REF][START_REF] Bohec | Population dynamics in a long-lived seabird: I. Impact of breeding activity on survival and breeding probability in unbanded king penguins[END_REF] and other king penguin colonies (on Kerguelen [START_REF] Pincemy | Experiments on colour ornaments and mate choice in king penguins[END_REF][START_REF] Pincemy | Homosexual Mating Displays in Penguins: Homosexual Mating Displays in Penguins[END_REF] and South Georgia [START_REF] Olsson | Survival in king penguins Aptenodytes patagonicus: temporal and sex-specific effects of environmental variability[END_REF] archipelagos). Interestingly, biased ASR in birds appears to mainly result from adult sex-specific mortality, rather than biased offspring sex ratios. Indeed, in a review of 140 estimates of offspring sex ratio, only few (17%) differed significantly from 1:1 (9.3% female biased and 7.8% male biased) [START_REF] Donald | Adult sex ratios in wild bird populations[END_REF]. Yet, despite slightly higher annual survival on average for males vs. females (94.2% vs. 90.7%), this difference is not significant ( [START_REF] Bohec | Population dynamics in a long-lived seabird: I. Impact of breeding activity on survival and breeding probability in unbanded king penguins[END_REF] and unpublished results over 1999-2010). Our result on biased sex ratio at fledging is thus relatively unusual for birds. Yet, it should be noted that despite a high sample size, the bias was only barely significant, indicating that fledgling sex ratio was mainly maintained close to the Fisher equilibrium of 1:1. In agreement with previous studies in adults [START_REF] Barrat | Quelques aspects de la biologie et de l'e ´cologie du manchot royal (Aptenodytes patagonicus) des ı ˆles Crozet[END_REF][START_REF] Fahlman | Accounting for body condition improves allometric estimates of resting metabolic rates in fasting king penguins, Aptenodytes patagonicus[END_REF], we found that king penguin fledglings also exhibited slight sexual dimorphism in favour of males, probably a result of differential growth, as no sexual dimorphism was observed at hatching for king penguin chicks (though size at hatching was available only for one cohort). Higher costs of raising males might therefore be expected. Given the equalization of fitness of male and female offspring at the termination of parental care, theory would then predict an even or slightly female-biased sex ratio at fledging [START_REF] Fisher | The Genetical Theory Of Natural Selection[END_REF]. Of course, for this to be true, costs of raising male and female offspring actually need to differ and remain to be accurately measured (e.g. in terms of energy allocation). Nonetheless, our results might thus appear in this regard to contradict Fisher's theory because a significant male-bias occurred [START_REF] Fisher | The Genetical Theory Of Natural Selection[END_REF]. However, this is perhaps not surprising given that life histories of birds clearly violate several assumptions of the standard sex allocation models (e.g. extended biparental care, overlapping generations, see [START_REF] Komdeur | Adaptive sex allocation in birds: the complexities of linking theory and practice[END_REF] for a review).

In contrast to the overall limited sex-ratio bias, we observed high interannual variability in fledging sex ratio ranging from 44% to 58% of males. Such fluctuation at fledging may result from yearly fluctuations in primary sex ratio and/or from differential chick survival between hatching and fledging. Unfortunately, we only had one year of data on hatching sex ratio, so that our Panel A: male and female body conditions are given as solid line, open circles and dotted line, full circles, respectively. Letters in superscript indicate differences between years in the average (male and female) body condition. Average values not sharing a common superscript are different for P,0.05. Panel B: the grey area corresponds to the years in which male body condition is higher than female body condition, while the white area presents the opposite. Note that the horizontal solid line indicates the balanced sex ratio at fledging. Every point situated above corresponds to a higher proportion of males, while below corresponds to a higher proportion of females. doi:10.1371/journal.pone.0114052.g004 findings have to be considered preliminary. Nonetheless, they suggest that sex ratio could be biased as soon as hatching, but also that sex-biased chick mortality may occur between hatching and fledging. Finally, we found that biased sex ratios at fledging were associated with biased body conditions in male and female fledglings. When sex ratio was male-biased, males presented a higher body condition than females, and reciprocally. Because fledgling body condition affects post-fledging return rates and may be viewed as an index of chick quality [START_REF] Saraux | Effects of Individual Pre-Fledging Traits and Environmental Conditions on Return Patterns in Juvenile King Penguins[END_REF], this may suggest that parents adopt sex-specific production strategies depending on yearly environmental conditions. For instance, larger males may be more vulnerable than females to food shortage [START_REF] Nager | Parental condition, brood sex ratio and differential young survival: an experimental study in gulls (Larus fuscus)[END_REF] in specific years, making it a better option to produce females under harsh environmental conditions [START_REF] Trivers | Natural selection of parental ability to vary the sex ratio of offspring[END_REF]. Also, parents in poor condition (e.g. nutritional stress or lack of experience) may favour the less costly sex either to avoid impairing future fitness or because reproduction is likely to fail, i.e. the 'cost of reproduction hypothesis' [START_REF] Williams | Natural selection, the cost of reproduction and a refinment of Lack's principle[END_REF][START_REF] Cockburn | Sex ratios in birds and mammals: can the hypotheses be disentangled? Sex Ratios: concepts and research methods[END_REF][START_REF] Nager | Experimental demonstration that offspring sex ratio varies with maternal condition[END_REF][START_REF] Velando | Experimental manipulation of maternal effort produces differential effects in sons and daughters: implications for adaptive sex ratios in the blue-footed booby[END_REF]. Alternatively, females may survive better than males in poor years, even under equal investment, because of lesser energy requirements during the growth period.

In our study, the proportion of hatching males tended to be higher early in the season (58 vs. 49% late in the season), although our small sample size of late fledglings prevents us from definite conclusions. Such seasonal variability in sex ratio has been previously reported in the painted turtle (Chrysemys picta) [START_REF] Bowden | Environmental sex determination in a reptile varies seasonally and with yolk hormones[END_REF], and may result from poor foraging and nutritional conditions, because of mismatch with peak resource productivity (e.g. [START_REF] Moreno | The effects of hatching date and parental quality on chick growth and cre `ching age in the chinstrap penguin Pygoscelis antarctica: a field experiment[END_REF][START_REF] Durant | Climate and the match or mismatch between predator requirements and resource availability[END_REF]). In king penguins, late breeding birds spend longer foraging trips at sea during chick-rearing and build up larger fuel reserves than early birds, reflecting the well-known trade-off between adult maintenance and offspring care/survival in long-lived birds [START_REF] Gauthier-Clerc | Seabird reproduction in an unpredictable environment: how king penguins provide their young chicks with food[END_REF][START_REF] Williams | Natural selection, the cost of reproduction and a refinment of Lack's principle[END_REF]. If male chicks indeed require larger amounts of food than females, a change in parental provisioning late in the season may affect male mortality, thus explaining the observed female bias in sex ratio.

Fledging sex ratio was relatively insensitive to environmental descriptors measured in this study. Yearly population (colony) density did not affect fledging sex ratio. Although effects of population density on sex ratio have been reported in mammals [START_REF] Kruuk | Population density affects sex ratio variation in red deer[END_REF], those effects were mediated by maternal condition affected by high competition on nutritional resources [START_REF] Kruuk | Population density affects sex ratio variation in red deer[END_REF]. However, intra-specific competition may be less of a problem for king penguins, for which foraging success appears to be more influenced by local oceanic characteristics of food patches than by density-dependent processes (see [START_REF] Pu ¨tz | Foraging strategy of king penguins (Aptenodytes patagonicus) during summer at the Crozet Islands[END_REF] for a discussion). Similarly, global climatic conditions (SOI) did not appear to have an effect on fledging sex ratio. However, whether this is a result of sex ratio truly being insensitive to global oceanographic conditions or because only relatively mild La Nin ˜a/El-Nin ˜o events occurred over the 10-year monitoring period (low amplitude in annual SOI variation, see [START_REF] Vargas | Modelling the effect of El Nin ˜o on the persistence of small populations: The Gala ´pagos penguin as a case study[END_REF]) remains to be examined. Finally, no effect of yearly local environmental conditions (SST) on fledging sex ratio was highlighted in this study. Although we did record a significant bias in fledging sex-ratio variability with increasing SST (cold years were associated with sex ratios close to 1:1, whereas warm years resulted in highly biased sex ratios), this bias was not consistently in favour of either sex, and our large sample sizes suggest that yearly changes in sex ratio were likely just related to stochasticity in environmental parameters (including SST) affecting chick mortality.

Finally, if understanding which parental/environmental variables influence offspring sex ratio is fundamental to the breeding and evolutionary biology of king penguins, one should bear in mind that it is actually the ASR that is a key parameter to population dynamics. In our study colony, although the sex-ratio bias was 2 percentage points lower at recruitment (i.e. first breeding attempt) than at fledging (suggesting a return towards equilibrium at recruitment), this decrease was not statistically significant.

To conclude, our results over a 10-year period hint to potential effects of oceanographic conditions on fledging sex ratio. With a predicted increase in warm climate episodes (IPCC 2013, [START_REF]Climate change[END_REF]), investigating king penguin sex ratio over later life-history stages appears the next step to assess its importance in modulating population trajectories and population persistence over time [START_REF] Veran | Demographic origins of skewed operational and adult sex ratios: perturbation analyses of two-sex models[END_REF]. From results to date, it appears that an expectation of considerable fluctuations in fledging sex ratio might accompany further ocean warming.

  issued on the 21 st of November 2005, 2006-67 issued on the 6 th of November 2006, 2007-149 on the 24 th of October 2007, 2008-98 issued on the 5 th of September 2008, 2009-57 issued on the 26 th of August 2009, 2010-79 issued on the 3 rd of September 2010) and handle birds (permits n ˚99/346/AUT issued on the 30th of November 1999, 00/240/AUT issued on the 5 th of September 2000, 01/315/AUT issued on the 4 th of July 2001, 01/322/AUT issued on the 16 th of August 2001, 2003-113 and 2003-114 issued on the 7 th of October 2003, 2004-182 and 2004-183 issued on the 14 th of December 2004, 2005-203 issued on the 1 st of December 2005, 2006-73 issued on the 6 th of November 2006, 2007-144 issued on the 24 th of October 2007, 2008-71 issued on the 5 th of September 2008, 2009-59 issued on the 29 th of August 2009, 2010-67 issued on the 3 rd of September 2010) were delivered first by the French 'Ministe `re de l'Ame ´nagement du Territoire et de l'Environnement' and then by the 'Terres Australes et Antarctiques Franc ¸aises' (TAAF).

Figure 1 .

 1 Figure1. Sex ratio of fledging king penguins over a 10-year period. Sex ratio is presented as male proportion (%). Dotted lines represent the 95% confidence interval. The grey area of the figure (fledging males ,50%) corresponds to female-biased sex ratios, while the white area corresponds to malebiased sex ratios. Points not sharing a common superscript are different for P,0.05. Sample sizes are given in brackets. The insert shows the average sex ratio over the 10-year study (*significant difference for P,0.05). doi:10.1371/journal.pone.0114052.g001

  , P50.81, N529; GLM 2008 ; z51.08, P50.28, N5124; GLM 2009 ; z50.49, P50.62, N585; GLM 2010 ; z520.10, P50.92, N579).

Figure 2 .

 2 Figure 2. Sex ratio of king penguin chicks produced either early or late during the 2007-2010 breeding seasons. Sex ratio is presented as male proportion (%). White and grey bars correspond to early and late chicks, respectively. In 2010, chick sex was determined both at hatching and fledging. Sample sizes are specified in the bars. doi:10.1371/journal.pone.0114052.g002

Figure 3 .

 3 Figure 3. Changes in sex ratio of fledging king penguins according to the Sea Surface Temperature (SST) over a 10-year period. Sex ratio is presented as male proportion (%). SST ( ˚C) was averaged over the summer season, i.e. the 6-mo period following the onset of reproduction (mean annual laying date of early breeders) (see methods). doi:10.1371/journal.pone.0114052.g003

Figure 4 .

 4 Figure 4. Yearly changes in male and female fledging body condition (BC; panel A) and yearly sex ratio at fledging according to fledging BC difference between males and females (panel B) in king penguins. Panel A: male and female body conditions are given as solid line, open circles and dotted line, full circles, respectively. Letters in superscript indicate differences between years in the average (male and female) body condition. Average values not sharing a common superscript are different for P,0.05. Panel B: the grey area corresponds to the years in which male body condition is higher than female body condition, while the white area presents the opposite. Note that the horizontal solid line indicates the balanced sex ratio at fledging. Every point situated above corresponds to a higher proportion of males, while below corresponds to a higher proportion of females.

PLOS ONE | DOI:10.1371/journal.pone.0114052 December 10, 2014

Acknowledgments

We are deeply grateful to F.S Dobson for his insights on sex-ratio theory and major improvements on the manuscript and to S. Descamps for helpful comments in revising the paper. We are also grateful to P. Ulrich and E. Boras as well as all the students (M. Bonzi, N. Muller, M. Schaming, L. Tischmacher) that contributed to sexing individuals in the lab. We also thank M. Ballesteros, D. Beaune, C. Bricaud, N. Chatelain, G. Conan, S. Descamps, J. Dutel, M. Gauthier-Clerc, C. Gilbert, S. Gravier, E. Grosbellet, A. Hergott, L. Kernaleguen, T. Lebard, J. Legrand, M. Le Vaillant, S. Mangin, V. Mosch, O. Prudhomme, S. Que ´me ´neur, M. Ripoche, A. Simon, E. Taquet, N. Vetter, and C. Villemin, for their help with field work.

This work was supported by the Institut Polaire Franc ¸ais -Paul-Emile Victor, the Fondation Bettencourt-Schueller, the Fondation des Treilles and the Centre National de la Recherche Scientifique (Programme Zone Atelier de Recherches sur l'Environnement Antarctique et Subantarctique), grants from the Marie Curie Intra European Fellowship (FP7-PEOPLE-IEF-2008, European Commission; project No 235962) (to C.L.B.) and V.A.V. was supported by a postdoctoral fellowship from the AXA Research Fund during the time of writing. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors confirm that all data underlying the findings are fully available without restriction. Data have been deposited to Figshare (http://dx.doi.org/10.6084/m9.figshare.1228056).

Author Contributions


Ahmed Fakhry

Françoise Pillier

Catherine Debiemme-Chouvy
email: catherine.debiemme-chouvy@upmc.fr

Templateless electrogeneration of polypyrrole nanostructures: Impact of the anionic composition and pH of the monomer solution

Keywords: Polypyrrole, templateless nanostructure synthesis, electrochemical route, nanowires, nanofibers, interfacial pH

Different superhydrophilic polypyrrole nanostructures can be electrosynthezised in the presence of anions of weak acid (monohydrogenophosphate) and non-acidic anions (perchlorate) without the need for templates. Actually the type of nanostructures formed depends both on the concentration of anions at the electrode and on the interfacial pH.

Depending on the anion composition of the pyrrole aqueous solution the film electrogenerated under a given applied potential is either a very thin membrane (10-20 nm) consisting of overoxidized polypyrrole or a tridimensional film with oriented nanowire array or a network of more or less interconnected nanofibers. The formation of such nanostructures is explained by a side reaction which is water oxidation. Since this reaction is pH-dependent, the pH of the pyrrole solution is one of the key parameter for the synthesis of such nanostructures. The reaction mechanism is discussed and compared to those proposed in the literature for nanofiber network electrosynthesis. Actually in the monomer solution, the role of the anions of weak acid is twofold. On the one hand they allow to limit the decrease of the interfacial pH during pyrrole oxidation and on the other hand to decrease the interfacial anion concentration, so that water oxidation takes place with formation of hydroxyl radicals and dioxygen nanobubbles.

Introduction

Among the conducting organic polymers, polypyrrole (PPy) is one of the most widely used due to its high electronic conductivity, biocompatibility and good stability in air and aqueous media. These properties allow its use for various applications such as (bio)chemical sensors, 1- 3 biomedical devices, [START_REF] Sirivisoot | Nanocomposite Materials[END_REF][START_REF] Shoa | [END_REF] batteries, 6,7,8 fuel cells, 9,10 electrochemical supercapacitors, [START_REF] Zhitomirsky | Ariyanayagamkumarappa and I[END_REF]12,13 corrosion protection 14,15 and so on. 16,17 For all of these applications the structure of the polymer is important, and it is notably interesting to have nanostructures with very high specific surface. These materials can be synthesized in the presence or in the absence of templates. 18,19,20 Polypyrrole can be prepared by using either a chemical or an electrochemical route. To control over the location of the deposit and the polymer thickness, the electropolymerization can be considered as the main method. It is an attractive technique that allows the one-step formation of a polymer film onto the electrode surface. 21,22 Whatever the method used, the oxidation of pyrrole (Py) leads to the formation of polypyrrole under its oxidized form, form doped with anions (A -), according to the following reaction:

- - ) n ( n - e 2) - n ) ((2 H 1) - 2(n ] A n , [(Py) A n Py n            (1)
where  stands for the doping level of the polymer, it is generally between 0.25 and 0.33.

From reaction (1) it should be noted on the one hand that the interfacial pH decreases since protons are released and on the other hand that the presence of anions is essential. Li has shown that the Py polymerization rate is directly proportional to the anion concentration of the monomer solution (for [A -] < 1 M). 23 For electrochemical PPy synthesis, various parameters such as the applied potential or the anodic current, 24 the concentration of monomer, 25 the nature of the supporting electrolyte, 26 and the nature of the working electrode 27 have an influence on the growth kinetics and the final structure of the polymer. 28,29 Polypyrrole nanostructures have been obtained using pyrrole solution containing either monohydrogenophosphate 28 or mixing of monohydrogenophosphate and dihydrogenophosphate (phosphate buffer solution (PBS)) [30][31][32][33][34][35] or carbonate. 36,37 For example, it has been shown that in the presence of only monohydrogenophosphate or carbonate anions, ultra thin overoxidized PPy films are formed. 38,39,40 In the presence of monohydrogenophosphate and low concentration of perchlorate ions oriented PPy nanostructures have been obtained. 28 As far as the mechanism of formation of overoxidized PPy film is concerned, it has been reported that when only anions of weak acids (named weak-acid anions), such as monohydrogenophosphate, acetate, carbonate, monohydrogenocarbonate or tartrate, which confer a relatively high pH to the Py solution, are present in a pyrrole aqueous solution the electrooxidation of the monomers leads to a pinhole-free very thin (~ 10-20 nm) nonconductive overoxidized polypyrrole (OPPy) film, under potentiostatic or potentiodynamic conditions. 38 Actually this overoxidation is indirectly related to the release of protons at the electrode/solution interface during Py oxidation (Reaction 1). Indeed, the protons which are released during Py polymerization are captured by the weak-acid anions. Thus if the current is high, i.e. if a lot of protons are produced, at a moment very few or no anions are available at the electrode/solution interface so that the Py oxidation cannot occur anymore. If a high potential is applied at the electrode, water oxidation takes place leading to the generation of hydroxyl radicals which react with the already deposited PPy film resulting in its overoxidation:

H 2 O → OH • + H + + e - (2)
PPy + x However, when non-acidic anions such as perchlorate are added to a pyrrole solution containing a high concentration of K 2 HPO 4 salt, polypyrrole nanowires (NWs) which are surrounded by OPPy are obtained. 28 Under potentiostatic conditions, the length of the NWs is a function of the electrode polarization duration. In this case the polymeric film is conductive and superhydrophilic. Herein, we report for the first time on the influence of the perchlorate concentration of a pyrrole aqueous solution which contains a high concentration of monohydrogenophosphate ions (0.2 M) on the electropolymerization process and its effects on the morphology of the films. Additionally, the influence of the pH of the pyrrole solution is also studied. The reaction mechanism which leads to the synthesis of nanostructure without the use of template or of chemical additives is also discussed and compared to those reported in the literature for electrosynthesis of polypyrrole nanofibers.

Experimental section

Pyrrole solution was distilled before use. Py solution, K 2 HPO 4 , LiClO 4 , K 3 Fe(CN) 6 and K 4 Fe(CN) 6 salts were purchased from Aldrich. All the solutions were prepared with bidistilled water.

The electrochemical experiments were performed in a classical three-electrode electrochemical cell. A platinum wire was used as counter electrode and a saturated calomel electrode (SCE) as the reference one. The working electrode was either Pt (S = 0.07cm²) or

Au/glass or a-CNx substrates for SEM observations. The pyrrole polymerization was conducted under potentiostatic conditions. The potentiostat used was an Autolab PGSTAT30 (Ecochemie).

After the PPy film electrogeneration, its superhydrophilic character was checked by performing the drop test. This consists on putting a small water droplet on the polypyrrole film and to check whether the drop spreads over the entire surface of the film or not. This test allows one to detect the presence of polypyrrole nanostructures as confirmed by scanning electron microspoy (SEM) observations. The film morphology was examined under a field emission gun scanning electron microscope (FEG-SEM), Ultra55 Zeiss, operating at 3 kV.

The chemical composition of the polymers was determined by X-ray photoelectron spectroscopy (XPS). The XPS measurements were performed on a VG ESCALAB 250i-XL spectrometer using monochromatic Al K radiation as the X-ray source. The survey spectra were recorded with a pass energy (PE) of 100 eV, in the constant analyzer energy mode. For the detection of trace of elements (doping anions of the PPy film), the PE was fixed at 50 eV and the number of scans was 49. The detection angle was 90° with respect to the plane of the sample.

Results and discussion

Using a pyrrole solution containing 0.2 M K 2 HPO 4 , pH around 8.9, the electropolymerization of the monomer was performed under potentiostatic conditions, at 0.78 V/SCE. In order to investigate the influence of the presence of non-acidic anions on the electrochemical process, several experiments were conducted using various concentrations of LiClO 4 , from 10 -4 M to 5.10 -2 M. Figure SI-1 shows the chronoamperograms recorded during these experiments.

From this figure, it is obvious that the anodic current (Py polymerization rate) depends on the perchlorate concentration even though the initial concentration of monohydrogenophosphate ions is high.

For perchlorate concentrations lower than 8.10 -4 M, the anodic current which is high at the very beginning of the polarization of the electrode decreases rapidly up to a very low and nearly constant value (curves A and B in Figure SI-1). The shape of these chronoamperometric curves is similar to the one obtained in the absence of perchlorate in the medium, also the anodic charge (Q=It) is similar, it is about 0.015 C cm -2 (after 600 s of polarization). 28 40 The films electrogenerated under these conditions are overoxidized (nonconductive) films. For LiClO 4 concentrations higher than 5.10 -4 M, the shape of the chronoamperometric curves is different from the previous ones. First, the anodic current decreases rapidly too but after it increases more or less slowly indicating that the films are at least partially conductive.

Moreover these films are superhydrophilic according to the water droplet test indicating that they are nanostructured.

All these findings are summarized in Figure 1 that shows the variation of the anodic charge according to the perchlorate concentration of the pyrrole solution (0.15 M Py + 0.2 M K 2 HPO 4 ). Even if the perchlorate concentration is low in comparison with the initial monohydrogenophosphate one its influence on the anodic charge is important. Indeed, for given experimental conditions, the higher is the perchlorate ion concentration the higher is the anodic charge. After 600 seconds of polarization at 0.78 V/SCE, for a perchlorate concentration of 0.1 mM the anodic charge is 0.9 mC whereas for a concentration of 20 mM the charge is more than a hundred time higher, about 100 mC. However, it is noteworthy that when the PPy films are electrogenerated in the same experimental conditions but in the absence of HPO 4 2-i.e. just in the presence of 0.15 M pyrrole and 0.02 M LiClO 4 , the anodic charge is substantially higher, equals to 800 mC (after 600 s at 0.78V/SCE), and the shape of the chronoamperometric curve is different (not shown). The electrode is coated with a very thick black film which is not superhydrophilic. Therefore one can conclude that the film obtained under this condition (without K 2 HPO 4 ) is not nanostructured.

(A) (B) (C) (D)

Notice that the same experiments were done applying two different potentials, 0.75V/SCE and 0.8V/SCE. The results are similar except that the anodic currents are higher for the higher applied potential (not shown). The shape of the current-time curves remains identical.

The SEM images reported in Fig. 2 allow one to compare the different morphologies of PPy films obtained under the same experimental conditions except the perchlorate concentration of the pyrrole solution.

Actually, for very low concentration of LiClO 4 (below 8.10 -4 M), ultra thin non-conductive overoxidized polypyrrole films are obtained (Fig. 2A). For LiClO 4 concentrations higher than 8.10 -4 M, the films are conductive and superhydrophilic as previously described. For intermediate perchlorate concentrations, between 8.10 -4 M and 2.10 -3 M, well oriented nanowires (NWs) are obtained (Fig. 2B) whereas for the highest concentrations (> 2.10 -3 M)

the PPy nanostructures are much longer and are more or less interconnected leading to networks (Figs. 2C and2D). These SEM images show that with increasing perchlorate ion concentration, the density and length of the polypyrrole nanostructures increase which is in good agreement with the increase of the anodic charge (Fig. 1). It is clear that with increasing the perchlorate concentration, the nanostructures change from oriented polypyrrole nanowires to polypyrrole networks consisting of very long nanofibers more or less interconnected. The length of the PPy nanostructures can be monitored just by varying the electrode polarization duration. Indeed, for perchlorate concentrations higher than 5 10 -4 M, the anodic charge increases with the polarization time (see Fig. SI-2) indicating that the polymer is continuously electrogenerated. It has been shown that in the presence of 1 mM perchlorate the NW growth rate is constant with time about 0.8 nm s -1 (at 0.75 V/SCE). 28 The nature of the polymeric films was confirmed by the cyclic voltametry performed in the presence of a rapid monoelectronic redox system (Fe(CN) 6 4-/3-in 0.5M KCl aqueous solution).

The current-potential responses are reported in Figure 3. For the films obtained with perchlorate concentrations lower than 8.10 -4 M, the voltammograms are flat, no currents are detected (see Fig. 3A), the oxidation and the reduction of the redox probe do not occur. This confirms that the electrode is coated with a compact insulator film i.e. with an overoxidized PPy film. Indeed, as previously mentioned, same result has been obtained for films electrogenerated in pyrrole solution containing only HPO 4 2-anions. 38,40 On the contrary, for LiClO 4 concentration higher than 8.10 -4 M (Fig. 3B), anodic and cathodic currents are detected and their values increase with the perchlorate concentration used for the electrogeneration of the PPy deposit on the Pt electrode (see Fig. 4). For comparison, the curve recorded at a bare Pt electrode is shown too. ) and the ratio between them if exists. Figure 5 shows the XPS survey spectra obtained for deposits electrogenerated in the presence of 0.1 mM (Fig. 5A) and 5 mM (Fig. 5B) of the perchlorate ion. For the lower concentration no Cl (208 eV) or P (104 eV) contribution are detected in the survey spectrum (Fig. 5-A). The signals recorded in the energy range of the P2p and Cl2p do not show any features (49 scans, pass energy (PE) 50 eV). This finding confirms that the films are overoxidized i.e. they are dedoped, they do not contain any anions.

Moreover, the shape of the background of the survey spectrum confirms that the film is nonconductive (overoxidized). It should be noted that the gold substrate is detected (Au4f: 82 eV) this implies that the OPPy film is very thin about 10 nm, in good agreement with the SEM image shown in Fig. 2A.

For the highest perchlorate concentration used to electrogenerate the PPy film, the Cl2p contribution is clearly visible in the survey spectrum (Fig. 5-B). In this spectrum no P is detected. However the P2p range exhibits a slight contribution. Actually the integration of Cl2p and P2p peaks (both recorded with a PE of 50 eV) indicates that the concentration of each element in the PPy nanostructures is about 96 % of Cl and 4 % of P. Therefore under our experimental conditions, logically, the polymer is doped with the anions without acid-base properties i.e. which are still ionized because they do not capture the protons released during pyrrole oxidation (reaction 1) on the contrary to weak-acid anions.

The nature of the anions and the release of protons during the oxidation of pyrrole (Reaction 1) are important and decisive for the elaboration of polypyrrole nanostructures. Indeed we assume that the nanostructuration of the films is due to the oxidation of some water molecules which leads to the formation of hydroxyl radicals (Reaction 2). These radicals can react with the already deposited film to overoxidize it or with themselves, leading to H 2 O 2 that is further

oxidized to O 2 : 2 OH • → H 2 O 2 (4)
H 2 O 2 → O 2 + 2H + + 2e - (5)
In the presence of anions having no acid-base property (perchlorate ions in the present case)

when their concentration in the electrolytic media is sufficient, the increase of the anodic current over time implies the presence of conductive areas on the formed film. Considering all our experimental results, we have suggested that these areas are present due to the formation of O 2 nanobubbles which protect the polypyrrole film from the hydroxyl radicals avoiding its overoxidation. This allows the electrochemical growth of the polypyrrole nanowires. This mechanism is summarized in the scheme presented in Figure 6. Obviously, when the rate of formation of hydroxyl radicals is very slow, the reaction of PPy overoxidation is the predominant one with respect to reaction (4) which is a second-order reaction with respect to the hydroxyl radical. It should be emphasized that the standard potential (E 0 ) of water oxidation reaction is a function of the pH of the medium: E 0 = 0.99 -0.06 pH V/SCE, at 25°C. Thus, for an applied potential of 0.78 V/SCE, from a kinetic point of view, water oxidation can occur if the interfacial pH is higher than 3.5, at 25°C. In the presence of 0.2 M monohydrogenophosphate anions and 1 mM perchlorate anions, for pyrrole solutions of pH lower than 4, after polarization for 600 seconds at 0.78 V/SCE, the resulting anodic charge is high. The PPy films prepared under these experimental conditions have a classical cauliflower-like structure (see Fig. 7A). On the contrary, for Py solutions having very high pH (>9), the resulting anodic charge is extremely low and OPPy films are formed (Fig. 7A). Besides, in order to confirm the influence of the nature of the anions similar experiments have been conducted in the presence of only perchlorate anions. In this case the influence of the pH is low, the anodic charge is persistently high (diamond scatters, Fig. 7B) and the films are not superhydrophilic i.e. they are not nanostructured. In the presence of only phosphates as anions in the Py solution (square scatters, Fig. 7B), and when the pH is lower than 4 (pHs at which water oxidation does not occur), conductive PPy films which are not superhydrophilic are electrogenerated. All these findings are in very good agreement with the mechanism proposed in Fig. 6.

B

Taking into account this mechanism, under a high applied potential, another way to limit the Py electrooxidation so that water oxidation takes place is to use a low pyrrole concentration.

Then, in the case of a high concentration of non-acidic anion (perchlorate) and low concentration of pyrrole, PPy nanostructures should be obtained. Preliminary results shown in PPy nanofibers have also been electrogenerated at 0.75V/SCE for 200 s in a phosphate buffer solution (pH = 6.9) containing gelatin (from 0.002 to 0.02 wt%), 0.07 M LiClO 4 and 0.15 M pyrrole at 60°C. 30 The authors suggested that the gelatin molecules act as a soft-template.

Using gelatin we have obtained the same results. However similar structures have been also obtained without addition of gelatin in the Py solution (0% gelatin: Q=36.8 mC ; 0.002% gelatin: Q=23.6 mC ; 0.02% gelatin: Q=18.9 mC) (see current-time curves and SEM images in Fig. SI-3). Therefore, we think that the mechanism proposed herein (Figure 6) is applicable to explain the nanostructure formation with or without gelatin. One can notice from Fig. SI-3 that in the presence of gelatin the Py polymerization rate is lower. This is likely due to an increase of the Py solution viscosity which leads to a decrease of the diffusion rate of the species from the bulk of the solution to the electrode.

under potentiostatic conditions (0.8 V/SCE). It was proposed that the presence of hydrogen bonding between phosphate anions and pyrrole oligomers is essential to produce nanostructures. Systematic investigations have indicated that a relatively high concentration of PBS, high pH and low oxidation potential favor the formation of PPy nanofibers.

Therefore, the formation mechanism we propose in this study (Fig. 6) perfectly allows one to explain the electrosynthesis of PPy nanostructures and can be an interesting compromise to the various mechanisms proposed in the literature.

Conclusion

Different polypyrrole nanostructures were obtained by the electropolymerization of pyrrole in the presence of both monohydrogenophosphate and perchlorate ions which are weak-acid anions and non-acidic anions, respectively. The influence of different parameters were studied especially that of the concentration of perchlorate ions. The results clearly show that this parameter plays an important role in the formation and in the development of superhydrophilic polypyrrole nanostructures. The proposed reaction mechanism is different from those described in the literature. It permits notably to explain the formation of polypyrrole well-oriented nanowires. The effect of the pH of the pyrrole solution was also highlighted. It appears that this parameter is indeed a key point for obtaining polypyrrole nanostructures since the mechanism is based on the formation of hydroxyl radicals and nanobubbles of dioxygen due to water oxidation.

1 )Figure 1 :

 11 Figure 1: Anodic charge vs. perchlorate concentration of the Py solution. Charge passes though a Pt electrode polarized at 0.78V/SCE for 600 s. Electrolyte: LiClO 4 + 0.2 M K 2 HPO 4 + 0.15 M Py. OPPy: overoxidized PPy. SH PPy: superhydrophilic PPy electrodeposit.

Figure 2 :

 2 Figure 2: SEM images from PPy films electrogenerated in (A) 10 -4 M LiClO 4 + 0.2 M K 2 HPO 4 + 0.15 M Py ; (B) 10 -3 M LiClO 4 + 0.2 M K 2 HPO 4 + 0.15 M Py ; (C,D) 5.10 -2 M LiClO 4 + 0.2 M K 2 HPO 4 + 0.15 M Py at 0.78 V/SCE for 600 s (A, C, D) or 1000 s (B). (A, B, D) cross section,  = 65°. (C) Top view. Substrates: (A) a-CNx, (B, C, D) Au/mica. Scale bars: (A) 100 nm, (B) 200 nm, (C, D) 1 m.

Figure 3 :

 3 Figure 3: Cyclic voltamograms at a Pt electrode coated with a polypyrrole film obtained under potentiostatic conditions (0.78 V/SCE) in 0.15 M Py + 0.2 M K 2 HPO 4 + LiClO 4 aqueous solution. LiClO 4 concentrations are indicated in the graph (A: 10 -4 to 10 -3 M ; B: 2 10 -3 M to 2 10 -2 M). Electrolyte: 10 -2 M Fe(CN) 6 4-/3-+ 0.5 M KCl. Potential scan rate : 10 mV s -1 .

Figure 4 :

 4 Figure 4: Anodic current peak in 10 -2 M Fe(CN) 6 4-/3-+ 0.5 M KCl aqueous solution vs. the anodic charge due to (O)PPy film electrogeneration at 0.78V/SCE for 600 s in LiClO 4 (concentrations in mM are indicated in the graph) + 0.2 M K 2 HPO 4 + 0.15 M Py solution.Data are from Fig.3.

Figure 5 :

 5 Figure 5: XPS survey spectra obtained from films electrogenerated in 0.15 M Py + 0.2 M K 2 HPO 4 + 10 -4 M (A) or 5 10 -3 M (B) LiClO 4 , at 0.78 V/SCE for 600 seconds, on Au/mica substrates.

Figure 6 :

 6 Figure 6: Schematic model showing the process which takes place depending on the perchlorate concentration of a 0.15 M Py + 0.2 M K 2 HPO 4 solution on the electrode polarized at 0.78 V/SCE. : Py oxidation ; : water oxidation (OH  and/or O 2 production).

Figure 7 :

 7 Figure 7: Anodic charge vs. pH of the Py solution after 600 s of polarization at 0.78 V/SCE. Electrolyte: A: 0.15 M Py + 0.2 M K 2 HPO 4 + 10 -3 M LiClO 4 ± H 3 PO 4 or KOH to adjust the pH (initial pH = 8.9) ; B: () same as A ; () 0.15 M Py + 0.2 M LiClO 4 ; () 0.15 M Py + 0.2 M K 2 HPO 4 . Inset A: SEM images of the films obtained at pH 3 and 11 (Substrate: Au/mica). Scale bars: 1 m (pH 3), 200 nm (pH 11). SH PPy: superhydrophilic PPy film.

Figure 8 :

 8 Figure 8: A: Current density vs. time a Pt electrode (S = 0.07 cm 2 ) polarized at 0.75 V/SCE in 0.2 M LiClO 4 + Py aqueous solution. Py concentrations are indicated in the graph. Anodic charge: 1.3 mC (1 mM) ; 7 mC (1.5 mM) ; 11.5 mC (3 mM) ; 24.3 mC (5 mM). B: SEM image of a PPy film electrogenerated in 5 10 -3 M Py + 0.2 M LiClO 4 aqueous solution at 0.75 V/SCE for 300s. Scale bar: 200 nm.

Figure 8

 8 Figure 8 indicate that it is the case.

  OH

Another example concerns the synthesis of PPy nanofibers which were electrosynthesized by the use of normal pulse voltammetry (a series of potential pulses was imposed from -0.05 to 0.75 V/SCE). 31 In this case, the pyrrole monomers were dissolved in 0.2 M PBS containing 0.1 M LiClO 4 . When the anodic potential was applied, the pyrrole monomers are polymerized and converted to PPy fiber at the electrode/solution interface and during the cathodic polarization, polymerization stops and PPy changes from oxidized state to the reduced state.

The authors suggested that when the potential varies from cathodic to anodic, PPy nuclei are formed on the electrode surface and then, as the polymerization proceeds, these nuclei continuously grow together. However it has been noted by the authors that using classical cyclic voltammetry method (0 to 0.75 V/SCE, 100 cycles, potential scan rate of 50 mV s -1 ) PPy nanostructures were also obtained. The same mechanism was suggested for obtaining PPy nanowires in a solution containing phosphate buffer and organic sulfonic acids such as ptoluenesulfonic acid. 34 PPy films having hollow structures have also been prepared under galvanostatic conditions (5 mA cm -2 ) in p-toluenesulfonate alkaline pyrrole solution (pH = 9). 41 Under the same experimental conditions but at pH 1.5, it has been observed that the PPy films have classical cauliflower-like morphology. The authors have proposed that the growth mechanism implies the formation of micelles which can act as a template with the assistance of hydroxyl ions. A similar mechanism has been proposed concerning the formation of PPy conical nanocontainers from pyrrole aqueous solution (pH = 6) containing 40 mM D-camphorsulfonic acid and 1 mM NaCl. 32 Finally similar superhydrophilic polypyrrole nanofibers have been obtained by Li et al. 35 using an aqueous solution containing 0.1 M Py, 0.07 M LiClO 4 and 0.2 M PBS (pH 6.8)


Yuming Fang

Junle Wang

Jing Li

Romuald Pépion

Patrick Le Callet

AN EYE TRACKING DATABASE FOR STEREOSCOPIC VIDEO

Keywords: stereoscopic video, visual attention, eye tracking database, human fixation

We present a large-scale eye tracking database for stereoscopic video. A set of participants were involved in this eye tracking experiment. The human fixation maps were created as the ground truth for stereoscopic video from the gaze data from participants. To the best of our knowledge, this is the first large-scale eye tracking database of visual attention modeling for stereoscopic video. The details of the processing operations and properties of the database are described in this paper.

INTRODUCTION

Visual attention is an important characteristic in the Human Visual System (HVS) for visual information processing. It is a cognitive process to selectively process the significant visual information while ignoring others in visual scenes. Generally, the visual information captured by human eyes is much more than that the HVS can process. During viewing the visual scene, the HVS cannot recognize the components and their relationship in the visual scene immediately. Selective attention would filter out redundant visual information to focus on the most important visual information to process. These important visual information are also termed as salient regions.

In the past decades, there have been many studies investigating visual attention mechanisms from various fields of biology, psychology, neuroscience, computer vision, etc. Many computational models of visual attention have been proposed for saliency detection in 2D images/video. To evaluate the performance of computational models of visual attention, many eye tracking databases have been built on 2D images/video in the public domain. With the rapid development of stereoscopic display, there is an increasing interest in various emerging applications for 3D multimedia in the research community. Currently, there are several saliency detection models proposed for 3D images/video [START_REF] Fang | Saliency detection for stereoscopic images[END_REF]. To evaluate the performance of the saliency detection models for stereoscopic images, some studies have conducted eye tracking experiments to obtain the ground truth.

At present, there are less studies focusing on building saliency detection models of stereoscopic video in the research community, compared with visual attention modeling for stereoscopic images. Different from visual attention modeling for 3D images, computational models of stereoscopic video have to consider the influence of the motion factor and this causes the visual attention modeling for stereoscopic video complicated. Another reason is that the lack of ground truth of eye tracking dataset for stereoscopic video have limited the studies of computational models of stereoscopic video. In this study, we investigate the human prediction on stereoscopic video based on eye tracking experiments. The study tries to provide some cues for visual attention modeling for stereoscopic video. Furthermore, the built eye tracking database provide a benchmark for performance evaluation of computational models of stereoscopic video for the research community.

THE EYE TRACKING DATABASE

Stereoscopic Video Sequences

The stereoscopic video dataset in this study includes 41 video sequences in total: 24 video sequences are from RMIT3DV database [START_REF] Cheng | RMIT3DV: Pre-Announcement of a Creative Commons Uncompressed HD 3D Video Database[END_REF]; 3 video sequences are from Elephant Dream project; 2 video sequences are from Okugai; 12 video sequences are from the IVC stereoscopic video database.

RMIT3DV Database

The database is composed of video sequences by considering diverse content with different factors such as level of 3D effect, aesthetic composition, variations in colour, environment, motion, texture,light, etc. [START_REF] Cheng | RMIT3DV: Pre-Announcement of a Creative Commons Uncompressed HD 3D Video Database[END_REF]. The stereoscopic video in this database were natively captured by a Panasonic AG-3D camera using Blackmagic Hyperdeck Shuttles and Intel SATA3 SSDs to record the video content. These video sequences are recorded with the resolution of 1920 × 1080 and the format is 10-bit 4:2:2 YUV at 25 fps without audio content. More details about this database can be referred to the original paper [START_REF] Cheng | RMIT3DV: Pre-Announcement of a Creative Commons Uncompressed HD 3D Video Database[END_REF].

A set of 12 stereoscopic video were captured by using a Panasonic AG-3DA1 3D camera [START_REF] Urvoy | Nama3ds1-cospad1: Subjective video quality assessment database on coding conditions introducing freely available high quality 3d stereoscopic sequences[END_REF]. Each video consists of two sequences of left and right views with full-HD resolution (1920 × 1080). This database includes both indoor and outdoor scenes taken in University of Nantes. Without using any depth range sensors during recording the stereoscopic video, the depth maps of the IVC stereoscopic video database are estimated by the optical flow approach on the stereo-pair video frames. The ground truth disparity maps are computed as the 'left-to-right' disparity map which represents the displacement of each pixel in the left view.

Procedures

The stereoscopic video sequences were displayed on a 26inch (552 × 323 mm) Panasonic BT-3DL2550 LCD screen with the resolution of 1920 × 1200 pixels and the refresh rate of 60Hz. Each screen pixel subtends 61.99 arcsec at a 93 cm viewing distance. The maximum luminance of the display is 180 cd/m 2 , which yields a maximum luminance of about 60 cd/m 2 when watched through glasses. The stereoscopic video sequences were viewed by subjects through a pair of passive polarized glasses. The environment luminance was adjusted for each subject with an appropriate size for the pupil during the eye tracking experiment. The eye movements were recorded by the SMI RED (4 firewire) remote eye-tracker working at 60Hz. During the eye tracking experiments, we asked the observers not to move and check them in live.

The stereoscopic video sequences were presented in a random order for the subjective test. During the experiment, subjects were required to do a free-viewing to the screen. The calibration test was performed at the beginning of the experiment, and repeated every eight video sequences. Subjects could ask for a break before each calibration. Each subject was required to have at least one break during the viewing test. All the experiments were conducted from 09:00 to 13:00 a.m. or 2:00 to 6:00 p.m. to avoid the feeling of fatigue as much as possible.

There were 40 subjects ranging from 19 to 44 years old involving the experiment. The average age of subjects is 26.02 years. All the subjects has either normal or corrected-tonormal visual acuity, verified by three pretests prior to the start of the experiment: the Snellen chart was used to check the visual acuity of subjects; the Ishihara color test was used to check color vision of subjects; the Randot stereo test was used to check 3D visual acuity of subjects. They were also naive to the purpose of the experiment and were compensated for their participation.

Fixation Density Map

After recording the gaze points by the eye tracker, we create the fixation density map as the ground truth for saliency es- timation for stereoscopic video. The gaze point maps from each eye were first created. The left gaze point maps were created by directly using the coordinates of the gaze positions of the left eye, while the right gaze maps were created by adding a displacement, horizontally and vertically, on the coordinates of each right-eye gaze point. We calculated the displacement of each gazed point from the 'right-to-left' disparity map computed by the same approach as the one used to crete the ground truth disparity maps of the IVC stereoscopic video database.

We summed these two gaze point maps as the final gaze point map and filtered it by a two-dimension Gaussian kernel based on the following two factors: (1) the decrease of visual acuity with increasing eccentricity from the fovea, and (2) the decrease in the accuracy of the eye tracker. The standard deviation of the Gaussian kernel is set as 2 degrees of the visual angle to guarantee the best visual acuity within the fovea. We provide one sample of the original video frame and its fixation density map in Fig. 1.

SUMMARY

This paper presents a large-scale eye tracking database for stereoscopic video sequences. The procedures and details of the database are illustrated. The database provides a benchmark for performance evaluation of computational models of visual attention for stereoscopic video. We will release this database to the research community in the future.

Fig. 1 .

 1 Fig. 1. First: the stereoscopic video frame; Second: the fixation density map.