Amandine Gamble
email: amandine.gamble@gmail.com

Romain Garnier

Audrey Jaeger

Hubert Gantelet

Eric Thibault

Pablo Tortosa

Vincent Bourret

Jean-Baptiste Thiebot

Karine Delord

Henri Weimerskirch

• Audrey Jaeger

Jérémy Tornos

Christophe Barbraud

Thierry Boulinier

Exposure of breeding albatrosses to the agent of avian cholera: dynamics of antibody levels and ecological implications

Keywords: Capture-mark-recapture, Disease ecology, Immuno-ecology, Maternal antibodies, Seabird, Serological dynamics, Survival

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The threat infectious diseases represent for biodiversity has recently been acknowledged and the ability to predict the effect of a pathogen on host populations is an important challenge for conservation [START_REF] Smith | The role of infectious diseases in biological conservation[END_REF][START_REF] Wobeser | Conservation, biodiversity and infectious disease: scientific evidence and policy implications[END_REF]). However, it requires understanding the transmission dynamics, which can directly depend on the capacity of hosts to survive infection and to mount an immune response [START_REF] Keeling | Modeling infectious diseases in humans and animals[END_REF]). An often neglected but critical compartment of the host population is thus the proportion of individuals surviving infection. These individuals may, for instance, constitute a reservoir compartment [START_REF] Viana | Assembling evidence for identifying reservoirs of infection[END_REF] or limit transmission through a herd immunity effect [START_REF] Iverson | Injecting epidemiology into population viability analysis: avian cholera transmission dynamics at an Arctic seabird colony[END_REF]. For instance, breeding females can mount a protective immune response and passively transmit it to their offspring [START_REF] Boulinier | Maternal transfer of antibodies: raising immuno-ecology issues[END_REF]. Protection could thus potentially be transferred from surviving individuals to potentially highly susceptible ones. Immunological, and particularly serological, data can be especially useful to track past exposure and quantify epidemiological dynamics (e.g., Pepin et al. 2017;[START_REF] Borremans | Estimating time of infection using prior serological and individual information can greatly improve incidence estimation of human and wildlife infections[END_REF] while informing on host responses, notably when collected in longitudinal settings (i.e., repeated sampling of individuals; [START_REF] Cizauskas | Frequent and seasonally variable sublethal anthrax infections are accompanied by short-lived immunity in an endemic system[END_REF][START_REF] Buzdugan | Inference of the infection status of individuals using longitudinal testing data from cryptic populations: towards a probabilistic approach to diagnosis[END_REF]. Such data can thus be used to estimate the proportion of individuals that survived the previous exposure to infection.

Bacterial agents known for their pathogenicity in domesticated animals are commonly encountered in the wild, but variations in susceptibility among host populations and compartments of host populations are largely underestimated, especially when several strains circulate [START_REF] Benskin | Bacterial pathogens in wild birds: a review of the frequency and effects of infection[END_REF]. For instance, avian cholera, caused by Pasteurella multocida (Pm), has been repeatedly implicated in mass mortality events of adult waterfowl and seabirds in the northern hemisphere (e.g., [START_REF] Descamps | Avian cholera, a threat to the viability of an Arctic seabird colony?[END_REF][START_REF] Österblom | Adult survival and avian cholera in common guillemots Uria aalge in the Baltic Sea[END_REF][START_REF] Wille | A pelagic outbreak of avian cholera in North American gulls: scavenging as a primary mechanism for transmission?[END_REF], while it seems to principally affect nestlings in the seabird community of Amsterdam Island (37°49ʹS, 77°33ʹE, southern Indian Ocean) where recurrent epizooties have been recorded since the mid-1980s [START_REF] Weimerskirch | Diseases threaten Southern Ocean albatrosses[END_REF][START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF][START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]). In addition, some evidence suggest that exposure of waterfowl to Pm results in a short-lived immune response [START_REF] Samuel | Comparison of methods to detect Pasteurella multocida in carrier waterfowl[END_REF](Samuel et al. , 2005a, b), b), contradicting the suggested, but not fully explored, herd immunity effect leading to avian cholera epizootics fadeout in a population of common eiders (Somateria mollissima; [START_REF] Iverson | Injecting epidemiology into population viability analysis: avian cholera transmission dynamics at an Arctic seabird colony[END_REF]. Hence, despite avian cholera being an important pathogen for both domestic [START_REF] Christensen | Fowl cholera[END_REF] and wild birds, basic knowledge on host responses to these bacteria is insufficient to be able to predict its impact on wild populations.

On Amsterdam Island, avian cholera outbreaks recurrently affect seabirds, providing a unique opportunity to gain insights on the responses of long-lived hosts to such a pathogenic agent in the wild. Nestling die-offs appear to particularly affect the locally abundant but globally endangered Indian yellow-nosed albatross (Thalassarche carteri; [START_REF] Weimerskirch | Diseases threaten Southern Ocean albatrosses[END_REF][START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. In contrast, impact of the pathogen on adult albatrosses appears limited, with no associated effect on the population [START_REF] Rolland | Assessing the impact of fisheries, climate and disease on the dynamics of the Indian yellow-nosed albatross[END_REF]) despite a few infected individuals found dead [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. A potential disease-induced mortality of adults could have a strong demographic effect in such a long-lived species. However, it is not clear if adult birds are even exposed to the infectious agent. Notably, the pathogen also threatens the endangered endemic Amsterdam albatross (Diomedea amsterdamensis; [START_REF] Rivalan | Combined impacts of longline fisheries and climate on the persistence of the Amsterdam Albatross Diomedia amsterdamensis[END_REF]. Indeed, the entire population of this endemic species breeds in relative close proximity to the affected yellow-nosed albatross population, and predating and scavenging brown skuas (Stercorarius antarcticus) may act as epidemiological bridges between the two populations [START_REF] Boulinier | Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?[END_REF].

In long-lived species such as albatrosses, antibody responses could be expected to persist longer than in waterfowl, because Procellariiformes are slow-living animals, characterized notably by a long lifespan and a long chickrearing period [START_REF] Lee | Linking immune defenses and life history at the levels of the individual and the species[END_REF][START_REF] Garnier | Evolution of the temporal persistence of immune protection[END_REF][START_REF] Ramos | Long antibody persistence and transgenerational transfer of immunity in a long-lived vertebrate[END_REF]. The maintenance of high individual levels of specific antibodies over several breeding seasons could strengthen herd immunity, but also allow the long-term transfer of potentially protective antibodies from breeding females to offspring via the egg yolk [START_REF] Gasparini | Related concentrations of specific immunoglobulins against the Lyme disease agent Borrelia burgdorferi sensu lato in eggs, young and adults of the kittiwake (Rissa tridactyla)[END_REF][START_REF] Garnier | Maternal antibody persistence: a neglected life-history trait with implications from albatross conservation to comparative immunology[END_REF]. Vaccination against Pm is widely considered the most effective way to protect poultry flocks [START_REF] Ahmad | Development of immunization trials against Pasteurella multocida[END_REF]) and autogenous vaccines are routinely used to reduce the risk of emergence of Pm and the mortality it causes [START_REF] Christensen | Fowl cholera[END_REF]. On Amsterdam Island, vaccination has proven efficient to protect yellownosed albatross nestlings after direct vaccination [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF], while non-vaccinated nestlings appear to die without mounting an immune response. If vaccination of adults led to the maintenance of high antibody levels over several breeding seasons, it could open important conservation perspectives by considering the vaccination of breeding females to protect offspring via maternal antibody transfer [START_REF] Garnier | Maternal antibody persistence: a neglected life-history trait with implications from albatross conservation to comparative immunology[END_REF].

In this context, the goal of this study was to improve our understanding of the role of birds that have survived exposure to Pm. This was done by assessing (1) whether adult yellow-nosed albatrosses show serological evidence of exposure to Pm, and if yes, (2) whether they are able to survive subsequent exposure, and (3) whether they can mount and maintain an immune response leading to the transfer of antibodies from breeding females to their offspring. Based on the previous observations suggesting no impact of the epizootics on adult survival in the recent years [START_REF] Rolland | Assessing the impact of fisheries, climate and disease on the dynamics of the Indian yellow-nosed albatross[END_REF], we explored predictions from two scenarios. In a first scenario, only a small proportion of adults is exposed to the bacteria, for instance, if outbreaks happen in the colony when attendance by adults is low (late in the breeding cycle). In that case, we would expect a low-to-null seroprevalence in non-vaccinated adults, independently of any potential disease-induced mortality. In a second scenario, a significant proportion of adults is exposed, survive exposure because diseaseinduced mortality is low, and mount a detectable immune response. In that case, we would expect a moderate-tohigh seroprevalence in non-vaccinated adults. Both scenarios would lead to a high annual survival of adults, with no effect of vaccination on survival, but different seroprevalences. In addition, if high antibody levels are maintained several years following vaccination, maternally inherited antibodies could be detected in young nestlings. To explore these scenarios, we assessed the proportion of adults mounting a humoral immune response to Pm exposure, the persistence of antibody levels over several years, adult annual survival, and the transfer of these antibodies to nestlings in both naturally exposed and vaccinated adult yellow-nosed albatrosses breeding on Amsterdam Island. To do so, we set up a capture-recapture program including the monitoring of both non-vaccinated and vaccinated breeders in a colony recurrently affected by avian cholera outbreaks. In addition to the recording of individual resighting histories, breeding adults and their offspring were screened for anti-Pm antibodies for 4 consecutive years using two different immunoassays. Considering the threat avian cholera can represent for wild bird populations [START_REF] Descamps | Avian cholera, a threat to the viability of an Arctic seabird colony?[END_REF][START_REF] Phillips | The conservation status and priorities for albatrosses and large petrels[END_REF][START_REF] Uhart | Review of diseases (pathogen isolation, direct recovery and antibodies) in albatrosses and large petrels worldwide[END_REF], the results of the present study may have important implications from basic eco-immunology of host-parasite interactions [START_REF] Anderson | Infectious diseases of humans: dynamics and control[END_REF] to applied conservation.

Materials and methods

Study population

Fieldwork was carried out on Amsterdam Island, in Entrecasteaux cliffs, where approximately 20,000 pairs of yellow-nosed albatrosses nest between September and March each year [START_REF] Rolland | Assessing the impact of fisheries, climate and disease on the dynamics of the Indian yellow-nosed albatross[END_REF]Electronic Supplemental Material [ESM] 1, Fig. S1.1). Yellow-nosed albatrosses lay one egg per year in the early September and eggs hatch in late November-mid December [START_REF] Jouventin | Biologie et frequence de reproduction chez l'albatros à bec jaune (Diomedea chlororhynchos)[END_REF]. Nest attendance by adults is high until January, but drops thereafter. Nestlings are thus on their own on their nest until fledging in April, except for punctual feeding visits from their parents. No albatross is present in Entrecasteaux cliffs between April and August. The study was focused on a naturally delimited subcolony of approximately 250 breeding pairs of yellow-nosed albatrosses.

Vaccine development

Following a process designed for the poultry industry, an autogenous vaccine was produced from a Pm strain isolated from the carcass of a sooty albatross nestling found dead in Entrecasteaux cliffs in 2011-2012 (strain reference D2C;[START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. This strain was characterized as serotype Heddleston 1, Namioka 7 [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF]. The vaccine was constituted from killed bacteria of this strain mixed with a mineral oil adjuvant to obtain a water-in-oil emulsion (Ceva Biovac, France). This vaccine has been available since 2013. The formulation was modified in 2015 to induce a higher and more durable response. More details on the vaccine production and its use in albatross nestlings can be found in [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF].

Field sampling and vaccination design

During the late incubation and the early chick-rearing periods (November-December) in 2013, 134 breeding adults from 70 nests were captured, blood-sampled (0.5-1 mL from the metatarsal veins), and marked with metal and alphanumeric Darvic ® plastic leg bands allowing individual identification without recapture. Each captured adult individual received a subcutaneous injection of 0.5 mL in the back of the neck of either the vaccine (67 "vaccinated" individuals) or a placebo (0.9% sodium chloride solution; 67 "NaCl-injected" individuals). When observed during the following weeks, birds from either group were recaptured and blood-sampled to assess their response to injection (37 NaCl-injected and 34 vaccinated individuals). These birds then also received a second injection of either NaCl or the vaccine. A short-time booster effect was detected by MAT 1 year but not 2 years after vaccination (ESM 1, Fig. S3.1) and no effect on raw return rate 1 year after vaccination was recorded. Birds injected twice in 2013 were grouped with birds injected once in the following analyses to be conservative with regards to the inter-annual persistence of antibody levels following vaccination. When both partners from a nest were captured, they received the same treatment. Birds from both treatment groups were spatially mixed and distributed throughout the study subcolony.

All banded individual observed within the study subcolony during the three following breeding seasons (2014-2015, 2015-2016, and 2016-2017) were recaptured and blood-sampled to monitor the inter-annual variations of specific antibody level. Most of the captures occurred during the early chick-rearing period and no individual was sampled more than three times within a season.

In 2015, after obtaining the first results of the immunoassays quantifying the anti-Pm antibody response and interannual antibody persistence (see "Results"), it was decided to attempt to boost the anti-Pm antibody levels of the birds that had been vaccinated 2 years before. Thirteen individuals were injected in December 2015 with a new formula of the vaccine, while keeping 13 others as control (injecting them with NaCl). The use of the new formula of the vaccine, although preventing our ability to disentangle a potential booster effect from an effect of the new vaccine formulation, was motivated by the aim to maximize the potential to obtain the persisting levels of antibodies.

Nestlings of injected successful breeders of the 2014-2015, 2015-2016, and 2016-2017 cohorts were blood-sampled at approximately 5, 10, 30, 60, and 90 days of age to study the transfer and decay of maternal antibodies. In the absence of sexual dimorphism, adult birds were sexed using a molecular technique [START_REF] Fridolfsson | A simple and universal method for molecular sexing of non-ratite birds[END_REF] for comparison of mother's and nestling's antibody levels at hatching. The age of nestlings for which hatching date was not known was estimated using their tarsus length (ESM 1, Fig. S1.2).

Captured individuals were handled on site for only a few minutes. Biosecurity measures were followed to avoid spreading infectious agents between individuals (ESM 1). Within hours of collection, blood samples were centrifuged and plasma extracted. Plasma was kept at 4 °C in the field, then at -20 °C in the laboratory until analyzed. Sample sizes of the different treatment groups are summarized in ESM 1, Table S1.1.

Immunological assays

Detection of antibodies in plasma samples was used as an indication of exposure to Pm antigens (natural or vaccination). Until now, a single strain of Pm has been repeatedly isolated from dead birds, rather supporting a single introduction event of the bacterium on the island [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]; seropositivity against Pm is thus likely to inform on exposure to this strain, although the circulation of other, potentially less pathogenic, strains cannot be excluded. Given the classical caveats of the use of serology in wildlife [START_REF] Gilbert | Deciphering serology to understand the ecology of infectious diseases in wildlife[END_REF]; e.g., test sensitivity and specificity bellow unity, individual heterogeneity in response), anti-Pm antibody levels in plasma samples were measured using two complementary immunoassays detecting different immunoglobulins to maximize the probability to detect past exposure events. First, specific agglutinating antibodies (mostly IgM and IgY) were measured using a microagglutination test (MAT) developed from the Pm strain D2C isolated on Amsterdam Island (SEROPAST ® , Ceva Biovac, France; see [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF]. Individuals were considered seropositive if agglutination was observed at the tenfold or more dilution. Results are expressed as titers (log2[last positive dilution/10] + 1). Second, a commercial indirect enzymelinked immunosorbent assay (ELISA) detecting chicken IgY against the Heddleston serotypes 1, 3, and 4 (ID Screen ® Pasteurella multocida Chicken and Turkey Indirect, IDvet, France) was used. Results are expressed as optical densities (ODs). The use of anti-chicken IgY to detect yellow-nosed albatross IgY has previously been validated using an experimental design and the positivity threshold was adapted using a method relying on fitting a mixture of normal distributions to the values of ODs (see [START_REF] Garnier | Interpreting ELISA analyses from wild animal samples: some recurrent issues and solutions[END_REF]). The two assays, thus, only partly overlap in their targeted immunoglobulins: they detect different isotypes, directed against different epitopes, which may or may not be agglutinating. In addition, the MAT was manufactured using the Pm strain isolated from the field and does not rely on an intermediate conjugate; as such it is expected to be more sensitive and more specific than the ELISA to detect exposure of albatrosses to Pm on Amsterdam Island (ESM 1). Overall, 697 samples collected from 134 adults and 83 nestlings were analyzed (ESM 1, Table S2.1).

Immunological data analyses

Annual variations in seroprevalences in "non-vaccinated individuals" (individuals before vaccination and NaClinjected individuals) were assessed based on the data collected during the late incubation and early chick-rearing period using logistic regressions with serological status (seronegative or seropositive) as the response variable and breeding season as an explanatory variable. As many individuals were sampled several times and a large proportion of individuals were partners, we used generalized linear mixed models in the 'lme4 ' R package [START_REF] Bates | Fitting linear mixedeffects models using lme4[END_REF], with the individual and the nest as random effects; likelihood ratio test (LRT) χ 2 are reported. The Wilcoxon rank-sum test with a Bonferroni correction was used for comparisons of antibody levels between groups of different treatments and between periods. One individual was removed from all the analyses because of consistent surprisingly high ODs (ESM 1, Fig. S2.3). For the figures, the seroprevalences were calculated as the proportion of individuals tested seropositive at least once among all tested individuals during a given period. Sample sizes are reported in the corresponding figures. All statistical analyses were conducted in R 3.3.3 (R Development Core Team 2017) and codes are available in ESM 2.

Capture-recapture analysis

To explore a potential effect of vaccination on the annual survival of breeding adults, resightings of banded individuals were recorded between the breeding season 2013-2014 and 2016-2017 on the study site and analyzed in a capture-recapture framework [START_REF] Lebreton | Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF]). To increase the power of the analysis, 109 non-injected individuals newly marked in December 2014 or December 2015 as part of a parallel study [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF] were included. Marked individuals were thus divided in three treatment groups: NaCl-injected, vaccinated, and non-injected. The capture histories of marked individuals were built as series of 0 s (not observed during the breeding season; i.e., dead or alive but not seen) and 1 s (observed at least once during the breeding season; i.e., alive and seen). Each pair received a different code and a random-effect capture-recapture model [START_REF] Choquet | Estimating demographic parameters from capture-recapture data with dependence among individuals within clusters[END_REF]) was used, allowing to account for the potential dependence of fates (survival and resighting) between the members of pairs. The use of such approaches allows to account for potentially heterogeneous resighting probabilities and thus limits the risk of biased estimations of survival probabilities. First, we used U-CARE to assess the goodness-of-fit of the data set to a Cormack-Jolly-Seber (CJS) model (Choquet et al. 2009a). Then, individual resighting histories were analyzed by running a CJS model in E-SURGE (Choquet et al. 2009b). Our initial model included a fixed vaccination effect, a fixed breeding season effect plus their interaction and a pair random effect on resighting probability, and a fixed vaccination effect and a pair random effect on survival probability (Table 1). Survival probability was constrained to be constant between seasons to allow parameter identifiability [START_REF] Little | Identifiability and redundancy: theoretical considerations[END_REF]). The among pair variation in survival or resighting (θ) for the ith individual of pair j was modeled as follows:

where β 0 is the mean survival or resighting probability on the logit scale and the b j 's are independent and identically distributed as an univariate normal distribution N(0,σ 2 ) where σ 2 is the variance among pairs [START_REF] Choquet | Estimating demographic parameters from capture-recapture data with dependence among individuals within clusters[END_REF]. Parameters were estimated by maximization of the likelihood [START_REF] Lebreton | Modeling survival and testing biological hypotheses using marked animals: a unified approach with case studies[END_REF]) and models including different fixed effects were then compared using AIC corrected for small sample size (AICc; [START_REF] Burnham | Model selection and multimodel inference: a practical information-theoretic approach[END_REF]. Random effects of pair were tested using LRTs at the 5% significant level following [START_REF] Choquet | Estimating demographic parameters from capture-recapture data with dependence among individuals within clusters[END_REF]. In total,

log it( (ij)) = 0 + b j ,
243 individuals from 130 nests were included in the analysis, corresponding to 670 resighting events.

Results

Immune response after natural exposure

Evidence of natural exposure of adults to Pm was found. Seroprevalence according to MAT in non-vaccinated adults was low during the 2013-2014 breeding season (2%, 3/133; Fig. 1) and significantly higher during the three following seasons (LRT χ 2 = 14.21, p < 0.01; ESM 1, Tables S3.1-2), reaching 33% (8/24) in 2016-2017. After 2014-2015, 7/23 (observed/potential) seroconversion events were observed (ESM 1, Fig. S3.2). Seroprevalence according to ELISA was low and stable during the 4 years of the study, varying between 4 and 7% (LRT χ 2 = 2.04, p = 1.00; ESM 1, S3). As expected, the MAT was thus more sensitive compared to the ELISA for detecting natural exposure.

Most NaCl-injected individuals that seroconverted were resighted the following years (Fig. 2c; ESM 1, Fig. S3.2). Regarding the persistence of specific antibody levels following natural exposure, after 2014-2015, 6/10 seroreversion events were observed based on the MAT results (Fig. 2c; ESM 1, Fig. S3.2). Specific antibody response after natural exposure may thus be short-lived at the inter-annual scale. Overall, these results suggest some exposure to Pm in 2014-2015 and the following years.

Immune response after vaccination

The initial vaccine injection (received in 2013-2014) induced a strong short-lived immune response. Most individuals responded to vaccination during the weeks following injection (MAT: 85%, 34/40; ELISA: 61%, 22/36; ESM 1, Fig. S3.3). Notably, all the individuals recaptured 3 weeks or more after vaccination were MAT-positive (21/21). Positive individuals after vaccination showed higher antibody levels in the weeks following injection than naturally exposed individuals (i.e., positive non-vaccinated individuals) during the whole study (MAT: W = 1249, p < 0.01; ELISA: W = 168, p = 0.04; Fig. 2a,b). Thus, vaccination induced a strong and immediate-specific immune response detected by both MAT and ELISA. One year after vaccination (i.e., 2014-2015), 76% (38/50) of the recaptured vaccinated individuals had specific antibodies levels detectable by MAT, with a higher proportion among those that had received two injections of the vaccine (17/20 for the birds that received two injections in 2013 versus 14/27 for the birds that received one). At the same time, seroconversion was observed in 30% (17/56) of the recaptured non-vaccinated individuals (Fig. S3.2). Assuming similar exposure to the bacterium of vaccinated and non-vaccinated individuals and 100% seroconversion of vaccinated individuals, vaccinated individuals had thus a 0.66 probability to remain MAT-positive 1 year after vaccination (see detailed calculation in ESM 1). In contrast, seroprevalence and antibody levels measured by ELISA 1 year after vaccination were not higher in vaccinated individuals than in NaCl-injected individuals (Fig. 2a,b; ESM 1, Fig. S3.3), independently of the numbers of injections received (ESM 1, Fig. S3.1), suggesting no persistence of ELISA-detected antibody levels over a year after the initial vaccination. More precisely, the modeled vaccine response would suggest a peak OD ± SD of 0.29 ± 0.08 reached 26 ± 9 days after vaccination and a return to seronegativity by 61 ± 23 days after vaccination (ESM 1, Fig. S3.5).

The new vaccine injection received 2 years after the initial vaccination induced a longer immune response. All individuals that received that injection in 2015-2016 mounted an immune response detectable by MAT andELISA 1 year later (i.e., 2016-2017;Figs. 2c, S3.4). These individuals had higher specific antibody levels than 1 year after the initial vaccination (MAT: paired W = 36, p = 0.05, n = 10; ELISA: paired W = 55, p < 0.01, n = 10), and than other individuals that had received two injections in 2013-2014 1 year after vaccination (MAT: W = 147, p < 0.01, n = 27; ELISA: W = 140, p < 0.01, n = 24). NaCl-injected and individuals vaccinated in 2013 but not in 2015-2016 (independently from the number of injections received in 2013) had lower levels of specific antibodies in 2016-2017 (Fig. 2a, b) (Fig. 2a, b). This suggests that the injection of the newly formulated vaccine received 2 years after the initial vaccination induced a stronger and more persistent immune response than the first vaccination.

Maternal transfer of antibodies

Vaccination of breeding females led to the transfer of maternal antibodies to their offspring. Specific antibodies were detected by MAT in the first blood sample of a high proportion (> 0.50) of nestlings of parents vaccinated the previous year (i.e., in the 2014-2015 cohort after vaccination of parents in 2013 and the 2016-2017 cohort after parents had received a new injection in 2015, Fig. 3a). Notably, within the 2014-2015 cohort, 8/9 nestlings of females that had received two vaccine injections the previous year (versus 2/10 for females that had received one) and, within the 2016-2017 cohort, 4/4 nestlings of females that received a new vaccine injection the previous year were detected as A low proportion of the other nestlings were detected seropositive at their first blood sample (from zero for nestlings of non-vaccinated adults in 2014-2015 and 2015-2016 to 2/4 for nestlings of vaccinated adults 3 years after vaccination). In seropositive nestlings, antibody titers were low at hatching, ranging from 0 to 4, and decayed after hatching, with most nestlings becoming seronegative by 21 day; only one maintained a detectable antibody level for at least 38 days (Fig. 3b). Of all nestlings, specific antibodies were detected in only two of the 2016-2017 nestlings using ELISA (OD = 0.54 and 0.81, aged of 6 and 14 days, respectively). These individuals were associated with females vaccinated in 2013 and 2015 who had high ODs in December 2016 (OD = 0.47 and 1.32, respectively).

Capture-recapture analysis

Capture-recapture modeling suggested that adult survival was high and independent of vaccination against Pm. In more detail, the goodness-of-fit test suggested no lack of fit of the data to a CJS model (χ 2 = 7.14, p = 0.21). Excluding a pair random effect on resighting probability (Table 1, model 2) markedly increased the AICc value of the initial model. For resighting, the assumption of an absence of variance among the pairs (σ = 0) was rejected (σ ± SE = 5.939 ± 2.743, p < 0.001), indicating high heterogeneity among pairs in resighting probability. Removing the effects of treatment or season on resighting (models 3 and 4 versus 1) greatly increased the deviance, but resulted in large model uncertainty based on AICc values. Including a pair random effect on survival only slightly improved the deviance of the model compared with the treatment survival model (model 1 versus 6). For survival, the assumption σ = 0 could not be rejected (σ ± SE = 0.794 ± 1.098, p = 0.137). Therefore, survival probabilities of individuals associated in pairs were independent. Removing the vaccination effect on survival (model 7) markedly decreased the AICc and a vaccination effect on survival was 5.7 less likely than a constant survival, suggesting that adult survival was independent of vaccination against Pm. Model averaged resighting probabilities were estimated between 0.806 and 0.913 for all years and treatments, and survival probability [CI 95%] was estimated at 0.924 [0.824; 0.965].

Discussion

Despite critical implications for the understanding of disease dynamics and the implementation of potential management strategies, temporal patterns of exposure to bacteria and features of host humoral responses are often unknown in wild populations. Here, we provide novel information on these issues in a system involving Pm in a population of a long-lived and threatened species, by having repeatedly sampled breeding individuals over a 4-year period. We found evidence of adult exposure to the pathogen despite high annual survival, highlighting that adults are able to survive exposure. Indeed, the adult survival of Indian yellownosed albatrosses estimated in this study is close to the adult survival of Atlantic yellow-nosed albatrosses (Thalassarche chlororhynchos) estimated on Gough Island (mean ± SE 0.92 ± 0.01), and higher than the one estimated on Tristan Island (0.84 ± 0.02; [START_REF] Cuthbert | Demography and population trends of the Atlantic yellow-nosed albatross[END_REF], from where avian cholera has never been reported to our knowledge. Immune response after exposure or vaccination was not life-long but long enough to provide information on the recent epizootics dynamics, especially when using the MAT assay specifically developed with the locally isolated Pm strain, suggesting that serology can be a useful tool to track the patterns of Pm exposure in the wild. In addition, the detection of specific antibodies in nestlings 1 year after vaccination of breeding females provides insight into possible applications of these tools for conservation, although more work is needed to understand the mechanisms involved in antibody-level persistence.

Immune response after natural exposure

Our study shows that, despite high annual survival, a large proportion of adult yellow-nosed albatrosses from Amsterdam Island are exposed to the agent of avian cholera. This is consistent with results in geese, suggesting that adult birds can survived exposure to Pm (Samuel et al. 2005a, b), although it had not been quantified. It, however, contrasts with the other waterfowl and seabird populations in which avian cholera induced high adult mortalities [START_REF] Leotta | Outbreaks of avian cholera in Hope Bay, Antarctica[END_REF][START_REF] Descamps | Avian cholera, a threat to the viability of an Arctic seabird colony?[END_REF][START_REF] Österblom | Adult survival and avian cholera in common guillemots Uria aalge in the Baltic Sea[END_REF][START_REF] Wille | A pelagic outbreak of avian cholera in North American gulls: scavenging as a primary mechanism for transmission?[END_REF]). This heterogeneity is not surprising given what is generally known of the variability of Pm virulence depending on the strain and the host [START_REF] Christensen | Fowl cholera[END_REF]. On Amsterdam Island, the previous work suggests the circulation of a unique strain of Pm among seabirds [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF], although more work is needed to confirm the lack of Pm diversity, notably by considering potential non-pathogenic strains circulating among healthy animals that could induce cross-reacting immune responses. Individuals surviving infection to a pathogenic agent can play an important role in its maintenance [START_REF] Viana | Assembling evidence for identifying reservoirs of infection[END_REF]) and circulation at different spatial scales [START_REF] Boulinier | Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?[END_REF]) by potentially carrying it.

Observations of seroconverting and seroreverting individuals each season since 2014-2015 suggest that the high seroprevalences observed these years correspond to several outbreaks, and not just to the maintenance of antibodies after the 2014-2015 outbreak. Years of high seroprevalences (2014-2015, 2015-2016, and 2016-2017) correlate with years of low fledging success recorded in yellow-nosed albatrosses on Amsterdam Island [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF][START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF], indicating that seroprevalence may be used to track outbreaks. In addition, low seroprevalences were observed in yellow-nosed albatrosses in the first year of the study despite the fact that Pm outbreaks and low fledging success had been recorded in the previous years [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]. This suggests that antibody levels reached in response to exposure to Pm may not persist over successive breeding seasons. Similarly, levels of antibodies produced by ducks after experimental infection wanes within a few months [START_REF] Samuel | Comparison of methods to detect Pasteurella multocida in carrier waterfowl[END_REF]). Short-lasting immunity against Pm may, thus, impact the dynamics of epizootics but not be sufficient to lead to fadeouts. These results contradict what has been suggested, but not assessed, in eider ducks exposed to Pm in the Canadian Arctic [START_REF] Iverson | Injecting epidemiology into population viability analysis: avian cholera transmission dynamics at an Arctic seabird colony[END_REF].

The short-lasting humoral response observed also suggests that individuals might not maintain Pm infection over the years and that the contribution of chronically infected adults to the inter-annual maintenance of Pm may be limited, unless it occurs in seronegative birds. Carrying of the bacteria by seronegative birds has been reported in previous studies on other species [START_REF] Samuel | Comparison of methods to detect Pasteurella multocida in carrier waterfowl[END_REF]), but duration is not known [START_REF] Wobeser | Conservation, biodiversity and infectious disease: scientific evidence and policy implications[END_REF] for a review). The main affected species may also not be the sole reservoir. Studies on the roles of brown rats (Rattus norvegicus; Curtis 1983) and brown skuas, which may be year-round residents on the island and probably highly exposed to Pm through foraging, are, hence, needed. Brown skuas may notably play a key role as spreaders of Pm within the island [START_REF] Boulinier | Migration, prospecting, dispersal? What host movement matters for infectious agent circulation?[END_REF].

Immune response after vaccination

Vaccination of adult yellow-nosed albatrosses induced an immune response, but detectable antibody levels dropped to below detection within a year in a large proportion of individuals. However, a new injection 2 years after the initial one using a new vaccine formulation induced high levels of antibodies that were maintained over the following year. Considering the frequency of the outbreaks (indicated by low fledging success; [START_REF] Jaeger | Avian cholera outbreaks threaten seabird species on Amsterdam Island[END_REF]) and the high seroprevalence in non-vaccinated individuals, it is likely that most of the individuals have been exposed prior to vaccination. The observed difference in antibody-level maintenance between the first and second vaccine injection is thus less likely to be due to a booster effect than to an effect of a difference in the vaccine formulation, although natural (re)exposure or vaccination are likely inducing different responses. Overall, our results open perspectives regarding the maintenance of anti-Pm antibody levels for several years after vaccination.

Maternal transfer of antibodies

The detection of specific antibodies in nestlings of vaccinated females suggests that, as observed in other seabird host-infectious agent systems (e.g., [START_REF] Gasparini | Related concentrations of specific immunoglobulins against the Lyme disease agent Borrelia burgdorferi sensu lato in eggs, young and adults of the kittiwake (Rissa tridactyla)[END_REF][START_REF] Garnier | Maternal antibody persistence: a neglected life-history trait with implications from albatross conservation to comparative immunology[END_REF], anti-Pm antibodies can be transferred from mother to offspring via the egg yolk. However, low antibody levels in mothers the years following the initial vaccination prevented the transmission of sufficient quantity of antibody for a potentially protective level to be maintained [START_REF] Grindstaff | Initial levels of maternally derived antibodies predict persistence time in offspring circulation[END_REF]. Indeed, avian cholera outbreaks are usually recorded several weeks after hatching [START_REF] Weimerskirch | Diseases threaten Southern Ocean albatrosses[END_REF][START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF]. Future work should, thus, focus on testing different vaccination designs in breeding adults (notably based on different vaccine formulations and injection schedules) to assess if it is possible to induce a strong long-lasting immune response. Such response could lead to the transfer of sufficient antibody levels to nestlings over several breeding seasons, as observed in another procellariform species [START_REF] Ramos | Long antibody persistence and transgenerational transfer of immunity in a long-lived vertebrate[END_REF], to ensure potentially protective levels at the time of the outbreaks.

Perspectives for disease surveillance and management

Vaccination of wild populations, despite numerous constraints and keeping in mind the importance of ethical issues, represents a powerful conservation tool [START_REF] Plumb | Vaccination in conservation medicine[END_REF]. Vaccination programs can be developed with different purposes, including elimination of the infectious agent(s) or direct protection of a target population. On Amsterdam Island, attention is strongly focused on the rare endemic Amsterdam albatross, but other species, potentially involved in Pm circulation or maintenance, also need to be considered. Avian cholera appears to kill thousands of nestlings of the locally abundant yellow-nosed albatross and their immunization, active by direct vaccination (previously proven in [START_REF] Bourret | Vaccination protects endangered albatross chicks against avian cholera[END_REF], or passive by maternally transferred antibodies, could thus be a potentially important management tool if maintained over series of years to protect several cohorts. Vaccination of parts of the yellow-nosed albatross local population to limit negative demographic effects and vaccination of brown skuas to limit the spread of the bacteria within the island or direct precautionary vaccination of Amsterdam albatrosses are possible options that could be considered. The use of vaccination for conservation in Procellariiformes would benefit from the longevity and faithfulness of individuals to their breeding site, which should help to strengthen potential local herd immunity effects. In any case, a priori modeling would be a useful step to determine which components of the community may be more pertinent to vaccinate and with which potentially optimal design [START_REF] Haydon | Low-coverage vaccination strategies for the conservation of endangered species[END_REF][START_REF] Garnier | Maternal antibody persistence: a neglected life-history trait with implications from albatross conservation to comparative immunology[END_REF]. The results of the current study thus clearly open perspectives, but empirical work is still required regarding the potential effects of vaccination on shedding and nestling protection via maternal antibody transfer.

In addition, our study revealed that serology could be useful to track the circulation of a supposedly highly lethal disease agent in the wild. This approach is not available for all systems and interpretation of the results depends on the temporal persistence of antibodies following infection [START_REF] Gilbert | Deciphering serology to understand the ecology of infectious diseases in wildlife[END_REF][START_REF] Garnier | Insights from parasite-specific serological tools in eco-immunology[END_REF][START_REF] Metcalf | Use of serological surveys to generate key insights into the changing global landscape of infectious disease[END_REF]). However, recent examples have illustrated that serology could be useful to detect individuals surviving to supposedly highly lethal infectious agents (e.g., [START_REF] Mulangu | Serologic evidence of ebolavirus infection in a population with no history of outbreaks in the Democratic Republic of the Congo[END_REF] for the Ebola virus, [START_REF] Cizauskas | Frequent and seasonally variable sublethal anthrax infections are accompanied by short-lived immunity in an endemic system[END_REF] for the anthrax bacillus). In addition, when serological data are rigorously analyzed or combined with other data sources, they can bring valuable information on epidemiological dynamics and recent advances show that, despite being an old tool, it still show promise [START_REF] Borremans | Estimating time of infection using prior serological and individual information can greatly improve incidence estimation of human and wildlife infections[END_REF][START_REF] Buzdugan | Inference of the infection status of individuals using longitudinal testing data from cryptic populations: towards a probabilistic approach to diagnosis[END_REF]Pepin et al. 2017).

Overall, our results highlight the value of detailed observational and experimental studies of the dynamics of host exposure to infectious agents in the wild, where ecological and evolutionary processes are likely critical for driving disease dynamics, and such knowledge may be useful to inform management plans.

Fig. 1

 1 Fig. 1 Prevalence of anti-Pm antibodies in non-vaccinated adult yellow-nosed albatrosses after egg-laying measured using an MAT and an ELISA. Sample sizes are reported below the bars; bars represent the 95% CI

Fig. 2

 2 Fig. 2 Quantification of anti-Pm antibodies by MAT (a) or ELISA (b) before and after vaccination and individual antibody-level dynamics (c) in adult yellow-nosed albatrosses after injection of NaCl or of an autogenous vaccine. a, c MAT non-null titers were considered positive. b ELISA positivity threshold is denoted by a dashed line; sam-

Fig. 3

 3 Fig. 3 Prevalence of anti-Pm antibodies in the first blood sample of yellow-nosed albatross nestlings (a) and antibody-level decay after hatching (b) measured using MAT. a Sample sizes and mean ± SD nestling age are reported below the boxes. b The gray line represents the fitted value of the generalized additive mixed model linking antibody level to age with nestling identity as a random effect (ESM 1); only nestlings with detectable levels of antibodies in their first blood sample are represented

Table 1

 1 Model selection for survival and resighting probabilities of yellow-nosed albatrossesFixed effects considered: c = constant, trt = treatment (non-injected, NaCl-injected and vaccinated), t = breeding season. Random effect: pair. #Id.Par. is the number of identifiable parameters of the model. Additive effects are denoted by a plus symbol (+) and interactions by a dot(.)

				1.0	MAT (agglutinating Ig targeting the D2C strain)
					ELISA (IgY targeting the H1 serotype)
				0.8
			Seroprevalence	0.4 0.6
				0.2
				0.0	133	131	54	53	28	28	24	22
					2013-2014 2014-2015 2015-2016 2016-2017
	Model	Survival	Resighting		#Id.Par.		Deviance	AICc	AICc weight
	1	trt+pair	trt.t+pair		14		603.03		631.65	0.030
	2	trt+pair	trt.t		13		624.73		651.27	0.000
	3	trt+pair	trt+pair		8		615.17		631.38	0.034
	4	trt+pair	t+pair		8		614.97		631.18	0.038
	6	trt	trt.t+pair		13		604.22		630.76	0.047
	7	c	trt.t+pair		11		604.65		627.04	0.302
	8	c	trt+pair		5		616.67		626.76	0.347
	9	c	t+pair		5		617.76		627.85	0.201

Acknowledgements

We are grateful to Nicolas Giraud, Marine Bely, Romain Bazire, Rémi Bigonneau, Hélène Le Berre, David Hémery, and Marine Quintin for their help in the field, and Cédric Marteau, Camille Lebarbenchon, and Raul Ramos for help at various stages of the work. We also thank Stéphanie Lesceu and Khadija Mouacha (IDvet, France) and Nelly Lesceau and her team (Ceva Biovac, France) for technical help.

Author contribution statement TB and RG conceived the idea of this work; TB, RG, AG, JT, KD, HW, and CB designed the study; HG and ET conceived the vaccine and the MAT. JT, TB, AG, AJ, KD, VB, and JBT collected the data; AG ran the ELISA and analyzed the serological data; AG and CB ran the capture-recapture analyses; AG led the writing of the manuscript and all authors contributed substantially to the drafts and gave final approval for publication.

Funding This work was funded by the French Polar Institute (IPEV programs ECOPATH-1151 and ORNITHOECO-109), the Agence Nationale de la Recherche (project EVEMATA 11-BSV7-003), the Réserve Nationale des Terres Australes Françaises, and the Zone Atelier Antarctique. This paper is a contribution to the Plan National d'Action Albatros d'Amsterdam. AG was supported via a Ph.D. fellowship from French Ministry of Research and VB through a CeMEB LabEx post-doctoral fellowship.

Compliance with ethical standards

Conflict of interest We declare no conflict of interest.

Permits

The experimental design was approved by the Comité de l'Environnement Polaire (TAAF A-2013(TAAF A- -71, A-2014(TAAF A- -134, A-2015(TAAF A- -107, and A-2016-80) -80) and the French Ministry of Research (04939.03).

Data accessibility Supplementary data associated with this article can be found on the OSU OREME online repository at https ://doi. org/10.15148 /c8256 ec7-b5e1-4cc4-8429-7bf6c c7926 3c.


Cyrille Lebon

Célestine Aude Benlali

Atyame Nten

Patrick Mavingui

Pablo Tortosa
email: pablo.tortosa@univ-reunion.fr

Célestine Atyame

Construction of a genetic sexing strain for Aedes albopictus: a promising tool for the development of sterilizing insect control strategies targeting the tiger mosquito

Keywords: Aedes albopictus, Genetic Sexing Strain, sex separation, Sterile Insect Technique, Incompatible Insect Technique

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The recent Zika virus (ZIKV) epidemic has put vector borne diseases under the spotlight, and further confirmed the growing concern of emerging vector-borne pathogens [START_REF] Faye | Molecular evolution of Zika virus during its emergence in the 20th Century[END_REF]. This epidemic shows similarities with chikungunya virus (CHIKV) emergence a decade ago: both CHIKV (alphavirus) and ZIKV (flavivirus) were initially known to cause outbreaks limited in space and time before expanding at a global scale. These epidemics tend to remind that we will most certainly experience future epidemics caused by arboviruses mostly unknown to the scientific community. As a consequence, the prophylactic treatments needed for the control of such diseases will be either scarce or absent.

One way to prevent the spread of emerging arthropodborne pathogens is to limit their transmission through the control of their invertebrate vectors. Such control methods, which have been so far mostly implemented through the use of synthetic pesticides, have proven efficient and notably allowed eradicating malaria from several territories worldwide following World War II. However, the recurrent selection of insecticide resistance in vector natural populations [START_REF] Labbé | Evolution of resistance to insecticide in disease vectors[END_REF], together with unwanted effects on non-target species have led to a ban of several synthetic pesticides and in turn stimulated the development of a handful of environmentally friendly methods. The field of innovative vector control development is currently exceptionally dynamic [START_REF] Bourtzis | More than one rabbit out of the hat: radiation, transgenic and symbiont-based approaches for sustainable management of mosquito and tsetse fly populations[END_REF], and several ongoing programs have reached the step of field pilot trial assays [START_REF] Zhang | Combining the Sterile Insect Technique with the Incompatible Insect Technique: III-Robust mating competitiveness of irradiated triple Wolbachia -Infected Aedes albopictus Males under semi-field conditions[END_REF][START_REF] Mains | Female adult Aedes albopictus suppression by Wolbachia-infected male mosquitoes[END_REF][START_REF] Carvalho | Suppression of a field population of Aedes aegypti in Brazil by sustained release of transgenic male mosquitoes[END_REF]. Among the strategies aiming at suppressing mosquito natural populations, sterilizing technologies based on the mass release of sterile/incompatible males have been experiencing a spectacular revival. These technologies use either Gamma-or X-ray irradiation [START_REF] Mastrangelo | A new generation of X ray irradiators for insect sterilization[END_REF], transgenesis [START_REF] Carvalho | Mass production of genetically modified Aedes aegypti for field releases in Brazil[END_REF], Wolbachia symbiotic bacteria [START_REF] Atyame | Comparison of irradiation and Wolbachia-based approaches for sterile-male strategies targeting Aedes albopictus[END_REF] or more sophisticated associations such as the recently proposed boosted Sterile Insect Technique [START_REF] Bouyer | Boosting the sterile insect technique to control mosquitoes[END_REF], in which males are sterilized through irradiation and then used as dispersers of entomopathogenic viruses and/or insecticides [START_REF] Bouyer | Alternative vector control methods to manage the Zika virus outbreak: more haste, less speed[END_REF]. All these technologies rely on the availability of an efficient sexing method allowing the separation of males and females at industrial scales, which is compulsory for mass release of treated males.

The control of vector populations through the release of sterile males has proven successful for pest species such as Cochliomyia hominivorax or Ceratitis capitata [START_REF] Krafsur | Sterile insect technique for suppressing and eradicating insect populations: 55 years and counting[END_REF][START_REF] Franz | Genetic sexing strains in Mediterranean fruitfFly, an example for other species amenable to large-scale rearing for the Sterile Insect Technique. Sterile Insect Technique: principles and practice in area-wide integrated pest management[END_REF]. By contrast, these vector control strategies have not been implemented at industrial scales for the control of mosquitoes. The availability of an efficient sexing method is indeed one of the main technological locks. Thus far, pilot trials have been using manual or mechanical separation techniques, which are not either up scalable or fully satisfactory [START_REF] Papathanos | Sex separation strategies: past experience and new approaches[END_REF]. Indeed, mechanical separation relies on sexual dimorphism, which can allow efficient sieving using glass plate size sorters as reported for Ae. aegypti [START_REF] Carvalho | Mass production of genetically modified Aedes aegypti for field releases in Brazil[END_REF]. However, sexual dimorphism may not be sharp enough in some species, such as Ae. albopictus, for which sieving is notoriously challenging and results in important reductions in the recovery of male pupae [START_REF] Balestrino | Validation of a new larval rearing unit for Aedes albopictus (Diptera: Culicidae) mass rearing[END_REF][START_REF] Bellini | Pilot field trials with Aedes albopictus irradiated sterile males in Italian urban areas[END_REF].

Among the several methods currently explored for sex separation in Ae. albopictus [START_REF] Gilles | Towards mosquito sterile insect technique programmes: exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes[END_REF], we used a dieldrin resistance conferring marker to construct a Genetic Sexing Strain (GSS), essentially adapting a strategy previously used for Culex tarsalis [START_REF] Mcdonald | A genetic-sexing strain based on malathion resistance for Culex tarsalis[END_REF] and Anopheles arabiensis [START_REF] Curtis | Genetic sex separation in Anopheles arabiensis and the production of sterile hybrids[END_REF]. Sex linkage of the rdl gene, conferring dieldrin resistance, was obtained through X-ray irradiation and selection of the sex biased mosquito lines. The data presented herein provide, to our knowledge, the first GSS reported to date for Ae. albopictus, and hereafter referred as Tikok. We present the performance and some life history traits of Tikok and discuss future improvements that will accelerate the implementation of sterilizing strategies targeting the tiger mosquito.

Methods

Mosquito stocks and rearing

Two Ae. albopictus lines originally sampled in two localities on La Réunion Island (Plaine des Palmistes and La Providence) were selected for this study. These localities correspond to an elevated rural area and a coastal urban park of La Réunion Island sheltering natural mosquito populations displaying low and high rdl R allelic frequencies, respectively [START_REF] Tantely | Insecticide resistance in Culex pipiens quinquefasciatus and Aedes albopictus mosquitoes from La Réunion Island[END_REF]. Larvae were reared at a density of approximately 1000 first instar larvae in white plastic trays (30 × 40 cm with a depth of 6 cm) containing 3 l of distilled water and were fed ad libitum with a mixture of rabbit-(Naturalima, Urcoopa, Saint Paul, Réunion) and fish-food (Tetra GmbH, TetraMin, Heinsberg, Germany). Upon pupation, males and females were separated manually under a binocular loop using terminalia dimorphism and placed into insect rearing cages (30 × 30 × 30 cm) until emergence. Adults were fed with 10 % sucrose solution (w/v) and females were blood-fed using the Hemotek feeding system (Discovery Workshops, Lancashire, UK) and defibrinated cow blood provided by the regional slaughterhouse. Such blood-feeding does not require ethical clearance.

Determination of the dieldrin diagnostic dose

A 1000 ppm dieldrin (Dr Ehrenstorfer, Germany) stock solution was prepared in absolute ethanol and used as a stock for further dilutions. Batches of 25 third-instar larvae were placed in 100 ml of water containing 0 to 10 ppm dieldrin. Three replicates were prepared for each insecticide concentration and cups were conserved at room temperature. All surviving and dead larvae were counted in each cup after 24 h and data were analysed using BioRssay 6.2 [START_REF] Milesi | BioRssay: a R script for bioassay analyses[END_REF]. The diagnostic dose was determined by testing homozygous R-Run (RR) and S-Run lines, (SS) together with heterozygous F1 (RS) larvae obtained by crossing R-Run males with S-Run females. For each homozygous susceptible (SS), heterozygous (RS) or homozygous resistant (RR) mosquitoes and each dieldrin dose, three batches of 25 third-instar larvae were placed in 100 ml of water containing 0 to 10 ppm dieldrin, and left for 24 h at room temperature. Survival was plotted for each dieldrin dose and mosquito genotype. As shown in Fig. 1, a dieldrin concentration of 0.1 ppm killed 100 % of S-Run larvae but none of the heterozygous or R-Run larvae, and was thus selected as the diagnostic/selective dose used for all dieldrin selection steps.

Molecular typing

Adult mosquito DNA was extracted as previously described [START_REF] Rogers | Extraction of total cellular DNA from plants, algae and fungi[END_REF]. A PCR-RFLP test [START_REF] Tantely | Insecticide resistance in Culex pipiens quinquefasciatus and Aedes albopictus mosquitoes from La Réunion Island[END_REF] was used to detect the Ala to Ser substitution in rdl conferring dieldrin resistance in Cx. pipiens and Ae. albopictus. PCR was run for 30 cycles (94°C for 30 s, 52°C for 30 s, and 72°C for 1 min), followed by 5 min at 72°C. The PCR product was then digested for 3 h at 60°C with BstAPI restriction endonuclease (New England Biolabs, Evry, France), which selectively cleaves the susceptible allele [START_REF] Tantely | Insecticide resistance in Culex pipiens quinquefasciatus and Aedes albopictus mosquitoes from La Réunion Island[END_REF]. Allelic profiles were then visualized using 2 % agarose electrophoresis of digested PCR product stained with GelRed.

X-Ray irradiation and GSS identification

Chromosomal translocations were induced using a BloodXrad X-ray irradiator (Cegelec, Bretigny sur Orge, France) located at the Institut Français du Sang, Centre Hospitalier Universitaire de La Réunion. For this, 100 rdl homozygous resistant (R-Run) male pupae were placed in a jar (Ø: 11 cm; H: 8.5 cm) containing 40 ml distilled water and were irradiated with one of the two selected irradiation doses: 20 Gy and 25 Gy. Each of the two batches of males, corresponding to the two irradiation doses, were then crossed en masse with rdl homozygous susceptible (S-Run) females, leading to the F0 heterozygous generation. For each irradiation dose, 40 crosses (80 total) were performed using one F0 (heterozygous) male and five S-Run females into a small insect holding cage (12 × 12 × 12 cm). Eggs from each cage were allowed to hatch and up to 50 third-instar larvae were placed at room temperature for 24 h in plastic cups containing 100 ml of water supplemented with 0.1 ppm dieldrin. Sex ratio was determined at pupal stage and all broths displaying > 60 % males following dieldrin selection were allowed to emerge. Males from each progeny were then backcrossed with S-Run females and dieldrin pressure was maintained at each following backcross.

Measurement of life history traits

Hatch rates as well as larvae survival rates and sex ratios following dieldrin treatment were measured for Tikok.

For this, eggs were allowed to hatch for 24 h in closed cups containing 250 ml water supplemented with 1 ml of 7.5% rabbit food solution (Naturalima Urcoopa, Saint Paul, Reunion). Following hatching, filter papers were observed under a binocular microscope and hatched (opened) and unhatched (closed) eggs were counted. Larvae survival was measured by counting the total number of alive larvae before and after dieldrin selection (0.1 ppm, 24 h). We sexed mosquitoes at the pupal stage using a sexual dimorphism on Terminalia. Mean egg hatches were compared using the Student's t-test.

Results

Construction of dieldrin resistant (R-RUN) and susceptible (S-RUN) homozygous mosquito lines

The construction of a GSS through sex linkage of a selectable insecticide resistance gene requires the availability of homozygous resistant and susceptible mosquito lines. A dieldrin sensitive strain was constructed as follows: 20 cages were set with five females and one male in each cage. Adult mosquitoes were left for 48 h and each male was then genotyped at the rdl locus following a previously described procedure [START_REF] Tantely | Insecticide resistance in Culex pipiens quinquefasciatus and Aedes albopictus mosquitoes from La Réunion Island[END_REF]. Only those cages whith homozygous rdl S males were conserved and females were given a blood meal. Two days later, each female was placed in a separate cage and allowed to lay eggs, which were conserved while females were genoytyped. Those eggs laid by rdl S homozygous females were Fig. 1 Survival of R-Run (RR), S-Run (SS) and heterozygous (RS) third instar larvae following 24 h treatment with increasing dieldrin concentrations. The diagnostic dose is circled and corresponds to the dieldrin dose for which all sensitive mosquitoes die while all heterozygous mosquitoes survive poolled and immersed in water for hatching, providing an homozygous sensitive line hereafter named S-Run.

The construction of a homozygous resistant strain required three rounds of dieldrin selection to increase the frequency of rdl R allele in the cages. For this, third-and fourth-instar larvae from La Providence were placed in plastic cups containing 50 ml of water with increasing dieldrin concentration. F0 larvae were placed in 0.05 ppm dieldrin for 24 h and surviving larvae were subsequently fed until emergence. Eggs laid by surviving F0 females were allowed to hatch and third and fourth instar larvae were placed again under 0.05 ppm dieldrin. At F2, larvae were placed under 0.1 ppm dieldrin, surviving larvae were allowed to moult and pupae were sex separated. Isofemale lines were constructed following the same exact procedure as that implemented for the construction of S-Run, and eggs resulting from copulations between homozygous rdl R male and female mosquitoes were pooled. Hatching eggs led to the homozygous rdl R mosquito line hereafter referred as R-Run.

X-Ray irradiation, identification and amplification of Tikok

Following irradiation at 20 or 25 Gy, R-Run male pupae were allowed to emerge and then crossed en masse with virgin S-Run females for 48 h. Females were subsequently blood-fed and allowed to lay eggs.

As expected, hatch rates were low for both irradiation doses (29.6 % and 11.3 % for 20 and 25 Gy irradiation, respectively). Following dieldrin selection, no difference was observed between these two irradiation doses either for larvae survival rate (20 Gy: 47.87 ± 1.19; 25 Gy: 49.61 ± 1.49) or sex ratio (20 Gy: 50.48 ± 1.52; 25 Gy: 54.70 ± 2.29). For each irradiation dose, 40 F1 dieldrin resistant males were individually crossed with S-Run females (one male and five females per cage), blood-fed and allowed to lay eggs. Hatch rates were significantly lower in crosses involving males irradiated at 25 Gy than in crosses involving males irradiated at 20 Gy (82.89 ± 3.33% and 93.38 ± 0.99%, respectively; Student's test, t = 3.53, P = 0.001). Only four males led to progenies exhibiting male biases exceeding 60 % and were further backcrossed. As shown on Table 1, a single male produced strong male bias (100 %) over 4 successive backcrosses. This mosquito line, hereafter referred as Tikok, was maintained and amplified while all other lines were discarded. At each generation, Tikok males were back-crossed with S-Run females with a 1:3 male/female ratio. Hatch and survival rates as well as sex ratio were measured for each generation. As shown in Fig. 2, hatch rate averaged 30 % over the entire experiment, larvae surviving rate following dieldrin treatment averaged 50 % and sex ratio bias was stable at 97.8 % (SD 5.96).

Fecundity of Tikok females obtained following insecticide pressure

Sex bias was not perfect and 2 % females descending from crosses between Tikok males and S-Run females survived the dieldrin treatment at the larval stage. Seven out of these surviving females were genotyped showing that they were all heterozygous at rdl (Table 2). This pattern suggests that these females actually resulted from meiotic recombination or translocation (see discussion) but not from treatment failure. Importantly, when these females were crossed with sensitive S-Run males, hatch rates were high (84.82 ± 6.91%) and sex ratio was not biased as dieldrin treatment of third instar larvae led to 50.33 % (± 18.12) males (see Table 2).

Stability of Tikok

In order to test a possible drift of Tikok in the absence of selective pressure, a part of layers was not treated with dieldrin after the 7th generation. For this, males and females were kept in rearing cages following emergence and the line was maintained without insecticide pressure during all subsequent generations. At each generation, eggs were recovered on two distinct filter papers. One was used to establish the next generation while the other was used to measure the level of dieldrine resistance following the standardized procedure detailed in the Materials and methods section and further discarded. Egg hatch steadily increased from 25.7 % to 92.7 % over 4 generations while larvae survival following dieldrin treatments decreased from 47.14 % to 3.20 % in the meantime. No resistant larvae were observed after 5 generations in the absence of insecticide pressure (Table 3).

Discussion

To our knowledge, we report the first GSS for Ae. albopictus. Tikok allows producing 97.8 % males following dieldrin treatment of third-instar larvae. Such a genetic tool may advantageously replace traditional sieving that has proven imperfect for this species [START_REF] Balestrino | Validation of a new larval rearing unit for Aedes albopictus (Diptera: Culicidae) mass rearing[END_REF][START_REF] Bellini | Pilot field trials with Aedes albopictus irradiated sterile males in Italian urban areas[END_REF]. Tikok hence paves the way for the up scaling of incompatible/ sterile insect techniques targeting Ae. albopictus. The measurement of some life history traits shows that the line can be easily maintained in the presence of dieldrin selection. However, Tikok still displays a number of drawbacks that need to be discussed and for which improvements must be considered. The strong bias in sex ratio provided by Tikok could be considered as strong enough for sterile insect technique, although proper modelling will need to be carried out at each future environmental set-up chosen for pilot trials. In addition, since virtually all released females are vectors, the accidental release of females needs to be avoided in any case. Perfect sex separation is particularly critical for the incompatible insect technique [START_REF] Zabalou | Incompatible insect technique: incompatible males from a Ceratitis capitata genetic sexing strain[END_REF] as released females will be fertile with incompatible males and may lead to the invasion of a hetereospecific Wolbachia in Ae. albopictus natural populations [START_REF] Mains | Female adult Aedes albopictus suppression by Wolbachia-infected male mosquitoes[END_REF]. As shown by the characterization of surviving Tikok females following dieldrin selection, all tested females were heterozygous for rdl. In addition, these females were nearly fully fertile when crossed with non-translocated S-Run males. The recovery of a nearly perfect fecundity suggests that these females result from a meiotic recombination/translocation restoring chromosome integrity rather than from a recombination between the male locus and the linked rdl. Anyway, a good fitness of these RS Tikok females will undoubtedly provide any accidentally released females with good chances of laying viable eggs. As a consequence, R-Run translocation will have to be repeated as long as the obtained sex ratio distortion is not close to 100 %.

Alternatively, this genetic sexing tool could be used right away in a combined Wolbachia-irradiation strategy [START_REF] Zhang | Combining the Sterile Insect Technique with the Incompatible Insect Technique: III-Robust mating competitiveness of irradiated triple Wolbachia -Infected Aedes albopictus Males under semi-field conditions[END_REF] where low irradiation doses allow sterilizing the few surviving and accidentally released Tikok females. First, we need to address whether Tikok line is suitable for mass rearing. Cage experiments in which selective pressure was relieved are informative in this respect. It appears that the dieldrin resistance conferring allele disappears from the population within 5 generations in the absence of selective pressure, suggesting that the dieldrin resistance conferring allele and/or its sex linkage have a strong genetic cost. We have shown that the < 2 % Tikok females surviving dieldrin selection are heterozygous and fully fertile, we propose that the rearrangement(s) leading to these few resistant females also lead to fully fertile sensitive males. In this case, such males would have an important fitness advantage over Tikok resistant males displaying affected fecundity (see Table 3). As a result, mass rearing of Tikok will require crossing GSS males with S-Run females every other generation. This would not necessarily need manual sex separation of S-Run females but instead crossing Tikok males with S-Run pupae collected 3 days after the first pupation, since any progeny originating from S-Run males accidentally released in the cage would be killed by dieldrin at larval stage. Lastly, provided Tikok is suitable for mass rearing, any release of irradiated and/or incompatible males will have to follow a thorough examination of mating competitiveness of released males. Indeed, it is absolutely required to demonstrate that the genetic background of Tikok does not alter male competitiveness.

The main drawback of this GSS is certainly the used marker, as dieldrin has been banned for decades in a number of countries. It must be emphasized that in no circumstances the insecticide should be used in the field. Larvae or even eggs [START_REF] Yamada | Genetic sex separation of the malaria vector, Anopheles arabiensis, by exposing eggs to dieldrin[END_REF] should be treated exclusively in the laboratory for sexing purposes. However, traces of dieldrin may persist in adults as previously reported in Anopheles arabiensis GSS [START_REF] Yamada | Anopheles arabiensis egg treatment with dieldrin for sex separation leaves residues in male adult mosquitoes that can bioaccumulate in goldfish (Carassius auratus auratus)[END_REF]. Although the overall amount of dieldrin residues remaining in the mosquito body has been overestimated in previous studies [START_REF] Yamada | Anopheles arabiensis egg treatment with dieldrin for sex separation leaves residues in male adult mosquitoes that can bioaccumulate in goldfish (Carassius auratus auratus)[END_REF], their main conclusions remain valid: sterile males released in the frame of SIT or other population suppression projects using rdl-based GSS would still carry dieldrin residues which can bio-accumulate in the environment and contribute to the increasingly worrying phenomena of insecticide resistance. This could be circumvented by different ways. First, dieldrin could be replaced by authorized pesticides, such as fipronil, for which cross-resistance with GABA receptor mutants has been previously reported [START_REF] Hosie | Actions of the insecticide fipronil, on dieldrin-sensitive and -resistant GABA receptors of Drosophila melanogaster[END_REF][START_REF] Matsuda | Cross-resistance with dieldrin of a novel tricyclic dinitrile GABA receptor antagonist[END_REF][START_REF] Brooke | Resistance to dieldrin + fipronil assorts with chromosome inversion 2La in the malaria vector Anopheles gambiae[END_REF]. Second, the present work shows the feasibility of genetic translocation for the generation of a sex separation tool in Ae. albopictus. The mutation conferring dieldrin resistance is thus far one of the few available robust selectable marker for this mosquito species. However, given the medical importance of this mosquito species, it is likely that other insecticide resistance markers (such as the recently reported kdr mutants [START_REF] Chen | First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid insecticides in Aedes albopictus populations from Haikou City, Hainan Island. China[END_REF] or carboxylesterase gene amplifications [START_REF] Grigoraki | Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus[END_REF]) will be fully characterized in the next future and will be made available for the development of a GSS avoiding the use of banned insecticides. Lastly, sterile insect strategies are appealing as they are both highly specific and environmentally friendly. Therefore, a high throughput screen allowing the identification of "clean" markers may be a profitable investment. Temperature sensitive lethal mutations have been previously discovered for other Culicinae species [START_REF] Baker | Genetic sexing technique for a mosquito sterile male release[END_REF] and used at industrial scales for the control of medflies [START_REF] Franz | Genetic sexing strains in Mediterranean fruitfFly, an example for other species amenable to large-scale rearing for the Sterile Insect Technique. Sterile Insect Technique: principles and practice in area-wide integrated pest management[END_REF][START_REF] Robinson | Genetic sexing strains in medfly, Ceratitis capitata, sterile insect technique programmes[END_REF][START_REF] Augustinos | Ceratitis capitata genetic sexing strains: laboratory evaluation of strains from mass-rearing facilities worldwide[END_REF]. Hence, the present work shows that the identification or the development of such a marker by random mutagenesis would pave the way to the implementation of fully clean sterile insect strategies that are urgently needed for the control of future emerging epidemics involving the tiger mosquito.

Conclusions

We report the construction of the first Genetic Sexing Strain (GSS) for Ae. albopictus. This strain, obtained through the translocation of rdl R , conferring resistance to dieldrin, allows selecting 98% males following insecticide pressure on third instar larvae. Although Tikok appears to be easily maintained in insectaries provided insecticide pressure is maintained, this study stimulates further investigations allowing identifying "clean" markers that could be translocated following the methods described herein. Such a GSS will accelerate the implementation of environmentalfriendly strategies aiming at controlling natural populations through mass release of sterilizing males, such as the Sterile or Incompatible Insect Techniques.

Fig. 2

 2 Fig. 2 Tikok life history traits. The following parameters were collected over 12 generations under dieldrin pressure applied at each generation. The expected larvae survival rate is depicted as a hatched bar. Abbreviations: a hatch rate; b total number of surviving adults; c sex ratio; d larvae surviving rate

Table 1

 1 Aedes albopictus, dieldrin selection of genetics sexing strain candidates. Egg hatch and sex ratios are provided for each candidate. Egg hatch is given for S-Run as a control (MD: missing data).

	F0 Irradiation	Generation Starter	Eggs	Tikok Hatch	S-Run Hatch	Number of larvae	Survival rate following	Number of	Number	Male
	dose (Gy)		male	number	rate (%)	rate (%)	exposed to dieldrin	dieldrin selection (%)	male pupae	of pupae	%
	20	F2	A	169	95.27	MD	138	48.55	40	66	60.61
	20	F2	B	139	93.53	MD	122	51.64	37	56	66.07
	25	F2	C	143	90.21	MD	114	41.23	24	33	72.73
	25	F2	D	121	30.58	MD	18	44.44	6	6	100.00
	20	F3	A	192	88.02	73.2	117	62.39	44	70	62.86
	20	F3	B	352	86.93	73.2	251	51.79	64	116	55.17
	25	F3	C	486	74.07	73.2	316	47.78	68	139	48.92
	25	F3	D	149	24.16	73.2	31	29.03	7	7	100.00
	20	F4	A	1242	25.93	87.1	284	50.00	62	141	43.97
	25	F4	D	929	13.46	87.1	104	46.15	36	36	100.00
	25	F5	D	1465	34.61	93.7	390	47.95	125	132	94.70
	25	F6	D	6462	28.04	87.0	1540	47.14	500	509	98.23
	25	F7	D	2288	31.42	79.8	538	37.55	80	82	97.56
	25	F8	D	482	26.56	MD	95	37.89	30	30	100.00
	25	F9_a	D	1218	32.02	89.5	317	48.90	102	115	88.70
	25	F9_b	D	1747	27.48	89.5	429	48.25	113	116	97.41
	25	F10	D	1499	34.89	91.7	350	49.71	164	166	98.80
	25	F11	D	3241	39.46	MD	1648	46.84	744	750	99.20
	25	F12	D	2461	35.19	86.6	759	47.56	340	345	98.55

Table 2

 2 Life history traits of larvae resulting from Tikok dieldrin resistant females crossed with S-Run males.

	Generation Female ID Hatch rate (Total	Genotype Number of larvae	Survival rate following	Number of	Number of	Male %
			number of eggs)		exposed to dieldrin	dieldrin selection (%)	male pupae	female pupae
	F10	A	100.00 (4)	RS	NT	NT	NT	NT	NT
	F10	B	98.90 (91)	RS	NT	NT	NT	NT	NT
	F10	C	92.86 (14)	RS	NT	NT	NT	NT	NT
	F11	D	48.28 (29)	RS	9	22.22	2	0	100.00
	F11	E	74.31 109)	RS	58	48.28	14	14	50.00
	F11	F	92.96 (71)	RS	39	38.46	2	12	14.29
	F11	G	86.44 (59)	RS	52	53.85	10	17	37.04

Table 3

 3 Tikok performance without dieldrin selection.

	Generation Egg number Hatch rate (%) Number of larvae	Survival rate following	Number of male pupae Number of pupae Male %
				exposed to dieldrin	dieldrin selection (%)
	F1	1219	25.68	280	47.14	70	70	100.00
	F2	2147	40.06	627	29.98	159	160	99.38
	F3	1373	64.02	850	8.35	64	65	98.46
	F4	328	92.68	250	3.20	7	7	100.00
	F5	1292	93.73	1102	0.00	/	/	/

Acknowledgements

We wish to thank Hafsah Limbada, Emmanuelle Clervil and Mathilde Pagès for their help in the construction of R-Run and S-Run mosquito lines. Institut Français du Sang is warmly acknowledged for giving permanent access to the irradiator facilities. We thank Jean-Sébastien Dehecq and Abdoul Rutee for providing larvae from Plaine des Palmistes that allowed the establishment of the S-Run mosquito line.

Funding

This study was supported by the International Atomic Energy research contact no. 17933 as part of the Coordinated Research Project "Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes", by a Credit Impôt Recherche provided by the French Ministry of research and higher education to SymbioTIC. The bank Crédit Agricole is acknowledged for financial support provided to SymbioTIC and allowing the purchase of equipment used is this study. Publication costs for this study was provided by the International Atomic Energy Agency as part of the Coordinated Research Project "Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes".

Availability of data and materials

The data supporting the conclusions of this article are included within the article. The raw data will be shared with legitimate investigators providing an appropriate request documenting the study objective and investigator affiliations.

About this supplement

This article has been published as part of Parasites & Vectors Volume 11 Supplement 2, 2018: Exploring genetic molecular, mechanical and behavioural methods of sex separation in mosquitoes. The full contents of the supplement are available online at https://parasitesandvectors.biomedcentral.com/ articles/supplements/volume-11-supplement-2.

Authors' contributions

Conceived and designed the study: PT, CA and PM. Performed the experiments: CL, AB, CA and PT. Wrote the paper: all authors. All authors read and approved the final manuscript.

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No specific ethics approval is required for the manipulation of invertebrate organisms.

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