Moufida Rira
email: moufidar@yahoo.fr

Diego P Morgavi

Milka Popova

Carine Marie-Magdeleine

Tatiana Silou-Etienne

Harry Archimède

Michel Doreau
email: michel.doreau@clermont.inra.fr

Ruminal methanogens and bacteria populations in sheep are modified by a tropical environment

Keywords: Rumen microbiota Volatile fatty acids Temperate vs tropical site Sheep breed Forage Methane CH4, methane, VFA, volatile fatty acid, DM, dry matter, H2, hydrogen, CO2, carbon dioxide, H, high, L, low, NDF, neutral detergent fibre assayed organic matter, NH3, ammonia, PBS, phosphate buffer saline, DGGE, denaturing gradient gel electrophoresis, TAE, Tris-Acetate-EDTA, MFS, methylgreen formalin saline

Microbial fermentation of carbohydrates in the rumen is largely responsible for the emission of methane by ruminants. Ruminants fed tropical forages usually produce more enteric methane than ruminants fed temperate forages. The relative influence of forage type, breed and temperate vs tropical environment on rumen microbial populations is not known. This experiment aimed to separate these effects. We designed two parallel experiments in sheep in two sites: temperate (France) and tropical (French West Indies), using in each site two breeds, Texel (temperate origin), and Blackbelly (tropical origin) fed the same temperate forages (C3 carbon fixation, permanent grasslands of high and low quality) and tropical forages (C4 carbon fixation, permanent grasslands of high and low quality). We determined diet digestibility, ruminal end-products of fermentation and microbial groups: total protozoa, methanogens and bacteria, and selected fibrolytic bacteria. Dry matter digestibility coefficient was higher in tropical site (612 vs 580 g/kg on average, P = 0.004) but no difference was observed between C3 and C4 forages. There was no effect of site on total VFA concentration, but the acetate:propionate ratio was higher for the tropical site (4.30 vs 3.93 on average, P = 0.007). The acetate:propionate ratio was also affected by forage type with higher values for C3 than C4 forage (4.24 vs 3.99 on average, P = 0.03). Concentration of total rumen bacteria and methanogens was determined by qPCR targeting, respectively, the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. For both groups, the number of gene copies per gram of DM rumen content was higher in the tropical site (P < 0.001). For cellulolytic bacteria, higher number of rrs copies per gram of DM of rumen content were detected for Fibrobacter succinogenes in the temperate site (P < 0.001), whereas no differences were observed for Ruminococcus flavefaciens or Ruminococcus albus numbers between sites, breeds and forage type. Protozoa numbers determined by counting did not vary between sites, forages or breeds, but a site × forage interaction was observed (P = 0.01): there were more protozoa and R. albus in tropical sites for tropical forages. Our results suggest that rumen microbiota was mainly influenced by environment (temperate vs tropical) and that forage type (C3 vs C4) and breed had minor effects. However, an interaction between environment and forage type was observed for some variables.

Introduction

Enteric methane (CH 4 ) from ruminants accounts for 39% of livestock sector greenhouse gases emissions [START_REF] Gerber | Tackling Climate Change Through Livestock -A Global Assessment of Emissions and Mitigation Opportunities[END_REF]. Several studies have tested different strategies for CH 4 abatement in ruminants [START_REF] Hristov | Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options[END_REF][START_REF] Martin | Methane mitigation in ruminants. From microbes to the farm scale[END_REF]. However, although ruminant diets are based on forage, there is scant information on the effect of forage type on methanogenesis. A meta-analysis [START_REF] Archimède | Comparison of methane production between C3 and C4 grasses and legumes[END_REF] showed that enteric CH 4 production per kg of dry matter (DM) intake may be higher for ruminants fed tropical forages than for those fed temperate forages. These differences in CH 4 production may be due to the constitutional variation in the chemical structure of tropical and temperate forages, which display C4 and C3 carbon fixation pathways, respectively. However, differences could also be due to the ambient environment or to animal breed. Experiments carried out with tropical forages have always been conducted in the tropics with breeds originating from and adapted to a tropical environment; likewise, experiments with temperate forages have been run in temperate areas with breeds originating from these areas.

In the rumen, CH 4 production results from microbial fermentation of carbohydrates. The main end products of this fermentation are volatile fatty acids (VFA), hydrogen (H 2 ) and carbon dioxide (CO 2 ). A large diverse population of microorganisms drives this process, especially bacteria and protozoa. The H 2 produced is mainly used by methanogenic archaea to reduce CO 2 to CH 4 . The activity, diversity and concentration of microbes harboured in the rumen are influenced by diet, breed, and the environment [START_REF] King | Differences in the rumen methanogen populations of lactating Jersey and Holstein dairy cows under the same diet regimen[END_REF]. However, to our knowledge, the relationships linking these three factors and their relative importance have not been studied.

The aim of this experiment was to compare rumen fermentation variables and microbial community structures of two breeds of sheep (Texel vs Blackbelly) fed C3 and C4 forages (permanent grasslands of high and low quality) in a temperate and a tropical site. Special attention was paid to methanogens and hydrogen-producing protozoa, which are associated with methanogenesis increase. Total bacteria were also studied, with a focus on cellulolytic bacteria, which play an important role in cell wall degradation and so can favour CH 4 production.

Materials and methods

Animals, diet, management and experimental design

The study was conducted in parallel in two research sites located in temperate and tropical areas. The temperate site was in Auvergne, France, at 45.70 • North latitude and 3.03 • West longitude. Where the animals were housed, the average daily temperature ranged between 11.2 • C and 15.9 • C, and the average relative humidity ranged between 32% and 46% during the experiment. The tropical area was in the French West Indies at 16.16 • North latitude and 61.30 • West longitude. The average daily temperature ranged between 21.0 • C and 25.0 • C and the average relative humidity ranged between 83% and 88% during the experiment.

In each site, 4 Texel wethers (temperate origin) and 4 Blackbelly rams (tropical origin) were used in two 4 × 4 Latin square designs. Sheep were born in the site where the experiment took place. Sheep were 2 years old and were fitted with a rumen cannula. Their body weight was 60.2 ± 1.5 kg for the Texel sheep and 51.3 ± 4.3 kg for the Blackbelly sheep in the temperate site; and 44.7 ± 0.7 kg for the Texel sheep and 44.4 ± 2.1 kg for the Blackbelly sheep in the tropical site. Management of experimental animals followed the guidelines for animal research of the French Ministry of Agriculture and other applicable guidelines and regulations for animal experimentation in the European Union (European Commission, 2010).

In both sites, sheep were fed the same forage from permanent grasslands, one grown in the temperate area and one grown in the tropical area. For each forage, there were two maturity stages that determined forage quality, high (H) and low (L), so that a total of 4 forages were studied in each site. Temperate forage was a semi-mountain permanent grassland, first cycle, flowering stage harvested in late June (L), and second cycle harvested in late August (H), both from the same parcel. The tropical forage was permanent grassland rich in Dichanthium spp; regrowths of 36 days (H) and 91 days (L), harvested in October. Transport of forage from one site to the other was by ship. Chemical composition of forages is presented in Table 1.

Each experimental period lasted 4 weeks: 2 weeks for adaptation to the forage, 1 week for CH 4 measurements, and 1 week for digestibility and rumen sampling. Detailed results on digestibility and CH 4 enteric productions arising from this study have been reported in a preliminary communication [START_REF] Archimède | Intake, total-tract digestibility and methane emission of Texel and Blackbelly sheep fed C4 and C3 grasses tested simultaneously in a temperate and a tropical area[END_REF] and will be fully published in a second paper (Archimède et al., unpublished). Sheep were fed ad libitum twice daily at 07:00 and 19:00 and were housed in a closed barn in the temperate site and in a semi-open barn in the tropical site. Animals were tied in boxes equipped for separation between urine and faeces in male sheep. They had free access to water and salt block at all times.

Measurement of feed intake and digestibility, and rumen sampling

Feed intake was measured by weighing daily offered and refused forages for 5 consecutive days. A 200-g sample of offered and refused forages was taken, and DM content was determined by oven-drying at 60 • C until constant weight. Digestibility coefficient was measured by total collection of faeces for 5 consecutive days. After weighing and mixing, 10% of daily collection of faeces were taken every day, and DM content was determined by oven-drying at 60 • C until constant weight.

Approximately 250 g of whole rumen contents were collected through the cannula just before and 3 h after the morning feeding. A subsample of exactly 30 g was used for microbial analysis. The remaining subsample was strained through a polyester monofilament fabric (250 m mesh size). The pH was promptly measured using a portable pH-meter (CG840, electrode Ag/AgCl, Schott Geräte, Hofheim, Germany).

Fermentation characteristics analysis

For VFA, 0.8 mL of rumen fluid filtrate was mixed with 0.5 mL of a solution containing 4 mg/mL (w/v) crotonic acid and 20 mg/mL (w/v) metaphosphoric acid in 0.5 mol/L HCl, kept at 4 • C for 2 h and centrifuged (16,500 × g, 10 min, 4 • C). The supernatant was stored at -20 • C until analysis. Ruminal VFA were determined in the rumen liquid phase by gas chromatography [START_REF] Ottenstein | Improved gas chromatography separation of free acids C2-C5 in dilute solution[END_REF]). The gas chromatograph was a Hewlett Packard 5889 equipped with a Stabilwax DA (30 m × 0.53 mm i.d.) column maintained at 125 • C. The carrier gas was H 2 (34.6 mL/min). For ruminal ammonia (NH 3 ), 1 mL of liquid phase was added to 0.1 mL of H 3 PO 4 0.8 M and frozen at -20 • C until analysis. Ruminal ammonia was assayed by colorimetry using the phenol-hypochlorite method [START_REF] Weatherburn | Phenol-hypochlorite reaction for determination of ammonia[END_REF].

Microbial analysis

Thirty grams of whole rumen contents was diluted with 15 mL of ice-cold phosphate buffer saline (PBS) pH 6.8 and homogenized in three 1 min cycles with 1 min intervals on ice, using a Polytron grinding mill (Kinematica GmbH, Steinhofhalde Switzerland). Approximately 0.5 g was transferred to a 2-mL Eppendorf tube and stored at -80 • C until DNA extraction.

Total DNA was extracted from approximately 200 mg of frozen rumen sample using the QIAamp DNA purification kit (Qiagen, Hilden, Germany) [START_REF] Yu | Improved extraction of PCR quality community DNA from digesta and fecal samples[END_REF]. DNA concentration and quality were checked by the A260 and A280 absorbance ratio in a NanoQuant Plate on a spectrophotometer (Infinity, Tecan, Männedorf, Switzerland). The extracted DNA was diluted to 10 ng/L and used as a template for PCR to amplify the rrs gene of total bacteria and the mcrA gene for methanogens. Primers used in this study are listed in Table 2 [START_REF] Edwards | Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen[END_REF][START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[END_REF]. When PCR products were used for denaturing gradient gel electrophoresis (DGGE), forward primers included a GC clamp (∼ 40 nt). All PCR reactions were performed in a T100 Thermal Cycler (Bio-Rad Laboratories, Marnes-la-Coquette, France) [START_REF] Popova | Effect of fibre-and starch-rich finishing diets on methanogenic Archaea diversity and activity in the rumen of feedlot bulls[END_REF][START_REF] Sadet | PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium[END_REF]. PCR products were analyzed by electrophoresis on 20 g/L agarose gel (w/v) to check their size and estimate their concentration using a Low DNA Mass Ladder (Invitrogen, Carlsbad, CA).

For DGGE analysis, the amount of PCR product loaded on gels was adjusted to 100 ng for both bacterial rrs and methanogen mcrA genes. Gels had a 6-8 (w/v) polyacrylamide gradient and a denaturant gradient of 30-55% for rrs and 10% to 30% for mcrA. Electrophoresis was performed in 0.5× Tris-Acetate-EDTA (TAE) buffer (TAE Buffer 1×, 40 mmol/L Tris base, 40 mmol/L glacial acetic acid, 1 mmol/L EDTA) at 200 V and 60 • C for 5 h. Gels were silver stained using a commercial kit (Bio-Rad Laboratories) and analyzed using GelCompar II (Applied Maths, Kortrijk, Belgium). The peak area of all bands (N) and of each band (n i ) and the number of bands S were used to calculate the community biodiversity using three indices: the Shannon index (H) calculated as H = -(n i /N). ln (n i /N), Simpson's dominance index () calculated as = (n i /N) 2 , and the evenness index (e) calculated as e = H/ln S [START_REF] Heip | Indices of diversity and evenness[END_REF].

The quantitative (q) PCR for total bacteria (rrs gene) and methanogens (mcrA gene) was carried out [START_REF] Morgavi | Fungal secondary metabolites from Monascus spp. reduce rumen methane production in vitro and in vivo[END_REF]. The slope and efficiency for rrs and mcrA primers were: -3.45 and 95.6%, and -3.43 and 95.1%, respectively, with R 2 being greater than 0.99 in both cases. The cellulolytic bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus were quantified with primers targeting the rrs gene [START_REF] Denman | Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen[END_REF][START_REF] Koike | Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens[END_REF]. The slope and efficiency for each primer pair were, respectively, -3.19 and 105.8%, -3.52 and 92.3%, and -3.54 and 91.5%. For each rumen content sample, results were expressed as the mean of three replicates in rrs or mcrA log 10 copies per gram of DM of rumen contents.

For protozoa counting, 3 mL of rumen fluid was added to 3 mL of methylgreen formalin saline (MFS) solution (35 mL/L formaldehyde, 0.14 mmol/L NaCl, 0.92 mmol/L methylgreen) and stored in the dark at room temperature. Rumen fluid/MFS solutions were diluted in an equal volume of PBS before protozoa counts under a microscope ( × 400) in a Neubauer chamber [START_REF] Ranilla | Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro[END_REF]. Data were expressed as log 10 cells/mL. c Breed effect, breed x site and breed x forage interactions were not significant for all measured parameters.

Statistical analysis

To determine effects of site, breed and forage on feed intake, digestibility, pH, VFA concentration and composition, NH 3 concentration, protozoa numbers, gene copy numbers and diversity indices, data underwent analysis of variance using PROC MIXED (SAS, 2008). The model included site (n = 2), forage (n = 4), breed (n = 2), interactions between forage and site, forage and breed and breed and site as fixed effects and animal nested within breed as random effect. Differences between C3 and C4 forages were also analyzed using orthogonal contrasts. Effects were declared significant at P < 0.05. Linear regressions were established between CH 4 emission per kg DM and the different populations of microbes.

Results

Feed intake, digestibility and ruminal fermentation characteristics

Dry matter intake expressed in g/kg BW did not vary between sites and was higher for C3 forages than for C4 forages. Dry matter digestibility differed between sites, and a site x forage interaction was observed, digestibility being higher in tropical site than in temperate site only for C4 forages. Dry matter digestibility differed between forages, but this difference was due to forage quality within C3 and within C4 forages, but did not vary between C3 and C4 forages. No effect of breed was observed on feed intake and digestibility. Ruminal pH and NH 3 concentration did not vary between sites, breeds or forages, and interactions were not significant before (Table 3) or after feeding (Table S1). Total VFA molar concentration before feeding did not vary with site, but was higher with temperate forages than with tropical forages (P < 0.05). After feeding, VFA concentration was higher in the temperate site than in the tropical site (P < 0.05), and the difference between temperate and tropical forages remained. Before and after feeding, the proportion of acetate was higher for tropical forage in the temperate site and lower for temperate forage in the tropical site, and the proportion of propionate and butyrate was higher for sheep fed tropical forage in the temperate site and lower for tropical forage in the temperate site (Table 3 and Table S1). The acetate:propionate ratio was higher for C4 forages in the tropical site, particularly for H forage with an b Large Entodiniomorphs: >100 m, Small Entodiniomorphs: <100 m. c Breed effect, breed x site and breed x forage interactions were not significant for all measured parameters. b Breed effect, breed x site and breed x forage interactions were not significant for all measured parameters. interaction site × forage after feeding (Table S1). No effect of breed was shown for VFA concentration or pattern, except for total VFA molar concentration after feeding, where a breed × site interaction (P < 0.05) was found.

Ruminal microbiota

Protozoa numbers did not vary among sites, forages or breeds, but a site × forage interaction was observed: protozoa population was higher for C3 forage in the temperate site, and for C4 forage in the tropical site (Table 4). Small entodiniomorphs (<100 m) were more abundant in the temperate site and in sheep fed C3 than in those fed C4 (P < 0.05). Large entodiniomorphs (>100 m), Dasytricha and Isotricha were not different between sites, forages or breeds.

Results of qPCR quantification of the bacterial rrs and methanogen mcrA gene copies are summarized in Table 5. The concentration of bacterial rrs copies was higher in the tropical site (P < 0.05) and for H forages. The concentration of F. succinogenes rrs gene copies was higher in the temperate site (P < 0.05). The concentration of R. albus was influenced by neither site nor forage, but a forage × site interaction was observed: higher rrs copies were detected in the temperate site for C3 forages, whereas in the tropical site higher rrs copies were observed for C4 forages. There was no difference in R. flavefaciens numbers between sites, breeds or forages. Methanogen concentration expressed as mcrA gene copy number in rumen contents was higher in the tropical site. Effects of forage and interaction between site and forage were observed: methanogen concentrations were higher in sheep fed C4 forages in the tropical site and in sheep fed C3 forages in the temperate site (Table 5).

DGGE analysis showed no difference within bacterial community structure and methanogenic community structure, and clustering patterns did not differ substantially among individual sheep (data not shown). Diversity indices were calculated from the rrs DNA PCR-DGGE profiles. Shannon, dominance and evenness indices did not vary between sites or breeds (Table S2). However, a tendential site × breed interaction was observed for the Shannon index in rrs DNA PCR-DGGE profiles (P = 0.055). There was no difference between forages in either the number of bands or the values of diversity indices for the rrs DNA PCR-DGGE profiles (data not shown).

Relationships between microbial populations and methane production

Fig. 1 presents relationships between microbial abundances and CH 4 production according to site, breed and forages (mean of 4 animals for each combination). Significant and positive relationships were found between protozoa population and CH 4 production (r = 0.53, P < 0.05), and between R. albus and CH 4 production (r = 0.52, P < 0.05) (Fig. 1). No relationship was found between total bacteria, R. flavefaciens, F. succinogenes and CH 4 production (r = 0.44, 0.27 and 0.05, respectively, P > 0.05). Higher CH 4 production was associated with higher methanogens numbers but the relationship was non-significant (r = 0.28, P > 0.05). Although some relationships were significant, coefficients of determination (R 2 ) were low.

Discussion

Factors other than animal and feed characteristics are known to affect rumen digestion including, among other factors, climatic conditions [START_REF] Decruyenaere | Factors affecting intake by grazing ruminants and related quantification methods: a review[END_REF]. Digestion and CH 4 production by ruminants differ between temperate and tropical conditions. However, as trials are usually carried out using feedstuffs harvested on site and a single, locally adapted breed [START_REF] Archimède | Comparison of methane production between C3 and C4 grasses and legumes[END_REF] it has not been possible to assess the relative importance of these factors. To our knowledge, our study is the first to simultaneously compare in a temperate and in a tropical environment, a temperate and a tropical sheep breed fed the same diet, allowing a finer interpretation of alleged differences between temperate and tropical sites in CH 4 production, rumen fermentation, and microbial ecosystem. Results on intake and digestibility will be thoroughly discussed in a second paper (Archimède et al., unpublished) and are briefly discussed in this manuscript.

Effect of environment on digestive processes

Intake par kg BW was not affected by site. The temperature in the tropical site was certainly not high enough to affect intake as suggested by the review of [START_REF] Morand-Fehr | Alimentation des ruminants soumis à un stress de chaleur[END_REF]. The higher digestibility observed in the tropical site is in accordance with the review of [START_REF] Morand-Fehr | Alimentation des ruminants soumis à un stress de chaleur[END_REF] for a wide range of temperatures, for a same intake. Independently of diet and animal type, there is limited experimental evidence to explain differences in rumen fermentation and methanogenesis between temperate and tropical sites. Such differences can result from differences in temperature or hygrometry. In this study, no difference in VFA concentration between sites was observed before feeding, but after feeding, the concentration was lower for the tropical site than for the temperate site. This may be explained by the difference in temperature, as it was reported by [START_REF] Kelley | Effect of environmental temperature on ruminal volatile fatty acid levels with controlled feed intake[END_REF] in cows fed a constant diet at different controlled temperatures, and by [START_REF] Martz | Forage intake, ruminal dry matter disappearance, and ruminal blood volatile fatty acids for steers in 18 and 32 • C temperatures[END_REF] in cows fed forage diets with moderate differences in intake. Another explanation could be the dilution of VFA in the rumen due to higher water consumption [START_REF] Silanikove | Effects of water scarcity and hot environment on appetite and digestion in ruminants: a review[END_REF]. However, in this experiment, rumen water content was 87.7% on average and was similar between sites, which does not support this hypothesis.

In contrast to our results, increase in total rumen VFA content and a decrease in pH were reported [START_REF] Kadzere | Heat stress in lactating dairy cows: a review[END_REF] when ambient temperature increased because VFA were absorbed less efficiently. In the present study, the tropical site had higher acetate proportion than the temperate site before and after feeding; the same tendency was observed for the acetate:propionate ratio. This result is in line with a previous study showing that high ambient temperature was associated with higher proportion of acetate [START_REF] Kelley | Effect of environmental temperature on ruminal volatile fatty acid levels with controlled feed intake[END_REF]. However, this trend is not general, as another trial showed that an increase in temperature did not change VFA pattern in cattle, and increased propionate in sheep [START_REF] Lippke | Digestibility and volatile fatty acids in steers and wethers at 21 and 32 • C ambient temperature[END_REF].

The observed differences in VFA pattern can be due to differences in microbial communities between temperate and tropical sites, especially as there was no effect of site on daily feed intake, on average 20.5 g DM/kg body weight for both sites [START_REF] Archimède | Intake, total-tract digestibility and methane emission of Texel and Blackbelly sheep fed C4 and C3 grasses tested simultaneously in a temperate and a tropical area[END_REF]. The qPCR analysis revealed that total bacteria and methanogen numbers were higher for the tropical site. Change in bacterial numbers may be partially explained by the lower concentration of small entodiniomorphs in the tropical site, and hence a decreased predatory activity on bacteria. In contrast to our results, a significant change in the composition but not in the concentration of microbial populations at elevated ambient temperature and humidity was reported [START_REF] Tajima | Influence of high temperature and humidity on rumen bacterial diversity in Holstein heifers[END_REF], this shift being accompanied by a decrease in concentration of VFA in the rumen. The targeted cellulolytic bacteria were not affected by site, except for F. succinogenes, which was more abundant in the temperate site (Table 5). This bacterium is a major fibre-degrading species in the rumen that does not produce H 2 and is not associated with higher CH 4 production [START_REF] Chaucheyras-Durand | Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs[END_REF]. A higher acetate production is generally linked to higher CH 4 production, but there was no site effect on CH 4 production [START_REF] Archimède | Intake, total-tract digestibility and methane emission of Texel and Blackbelly sheep fed C4 and C3 grasses tested simultaneously in a temperate and a tropical area[END_REF] for this experiment (18.7 and 19.4 g/kg DM intake on average for temperate and tropical sites, respectively). Methane was generally produced in higher quantities when H 2 -producing cellulolytic species such as Ruminococci were dominant, because of their association with methanogens, which consume H 2 and produce CH 4 [START_REF] Chaucheyras-Durand | Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs[END_REF][START_REF] Morgavi | Microbial ecosystem and methanogenesis in ruminants[END_REF] resulting simultaneously in higher production of acetate [START_REF] Morgavi | Microbial ecosystem and methanogenesis in ruminants[END_REF][START_REF] Pavlostathis | Cellulose fermentation by continuous cultures of Ruminococcus albus and Methanobrevibacter smithii[END_REF]. This is in line with our results where the number of Ruminococci was also not affected by any of the factors tested and not correlated with CH 4 production (Fig. 1).

In the present work we found little effect of environmental conditions on rumen fermentation and microbial populations. There are very few data in the literature: a change in rumen diversity and structure of microbiota was observed [START_REF] Romero-Pérez | Effect of environmental factors and influence of rumen and hindgut biogeography on bacterial communities in steers[END_REF], suggesting that shifts in microbial populations may have been due to differences in temperature sensitivity among microbial species, but the range of temperature tested by these authors was between -5 • C and -30 • C. The rumen and its contents are thermally stable but a modest change and disturbance within the rumen may occur with changes in ambient temperature. A difference in drinking water temperature can induce a difference in the temporary decrease in rumen temperature after drinking [START_REF] Bewley | Impact of intake water temperatures on reticular temperatures of lactating dairy cows[END_REF]. Another hypothesis is that a difference in ambient temperature (9 vs. 26 • C) results in a difference in mean retention time through hormonal changes, which may influence rumen fermentation [START_REF] Barnett | Low ambient temperature elevates plasma triiodothyronine concentrations while reducing digesta mean retention time and methane yield in sheep[END_REF] and thus perhaps microbiota.

Effect of forage type on digestive processes

To date, studies on tropical forages have largely focused on comparisons of various plant species or physiological stages [START_REF] Assoumaya | Etude Des Facteurs Limitant l'ingestion Chez Les Petits Ruminants Valorisant Des Fourrages Tropicaux[END_REF]. The higher intake of temperate forages compared to tropical ones was independent of site confirming the conclusion of the review of [START_REF] Assoumaya | Etude comparative de l'ingestion et de la digestion des fourrages tropicaux et tempérés[END_REF] comparing data on temperate forages carried out in temperate areas with data on tropical forages carried out in tropical areas. In the present experiment, it was observed a logical reduction in digestibility with poor quality forages but also a noticeable improvement in digestibility of lower quality C4 forages in the tropical site. In C4 forages, digestibility between high and low quality hay is severely reduced compared to C3 and animals on the tropical site seem better adapted to extract nutrients from this poor quality feed resource. The current study shows that grass physiological type (temperate C3 vs tropical C4) had more influence on rumen fermentation than site. Differences between forages in VFA concentration and pattern could be explained by their quality: nature and content of fibre and amount of lignin. In the present study, poor quality forages (H vs L) result in a lower VFA concentration and in a higher acetate:propionate ratio, independently of the site. This confirms literature data on tropical forages: a decrease in propionate relative to total VFA, while acetate increased with advancing maturity of the C4 grass Panicum maximum was observed [START_REF] Assoumaya | Etude Des Facteurs Limitant l'ingestion Chez Les Petits Ruminants Valorisant Des Fourrages Tropicaux[END_REF][START_REF] Relling | An evaluation of Panicum maximum cv. Gatton. The influence of stage of maturity on diet selection, intake and rumen fermentation in sheep[END_REF].There is no direct comparison between C3 and C4 grasses in the literature, so that it is difficult to know whether differences between C3 and C4 forages observed in this study derived from the chemical composition, such as the higher content of secondary metabolites of Dichanthium annulatum [START_REF] Awad | Phytochemical investigation and biological evaluation of Dichanthium annulatum (Forrsk)[END_REF] present in the mixture of Dichanthium in this study. In a meta-analysis [START_REF] Assoumaya | Etude comparative de l'ingestion et de la digestion des fourrages tropicaux et tempérés[END_REF], ruminal OM digestion, which is related to VFA production, was similar between tropical and temperate forages for the same fibre content, suggesting that chemical composition plays a major role in the differences found between tropical and temperate forages. In our experiment, butyrate proportion was higher for L than for H forages, and higher after feeding for temperate than for tropical forages. This suggests that butyrate production is not directly related to chemical composition. Butyrate is generally associated with protozoa [START_REF] Eugène | Effects of defaunation on microbial activities in the rumen of rams consuming a mixed diet (fresh Digitaria decumbens grass and concentrate)[END_REF], but in our study there was no effect of forage type on numbers of protozoa, bacteria or methanogens. However, a significant site × forage interaction between temperate and tropical was found for total bacteria, R. albus, methanogens and protozoa. This shows that these populations were highest with tropical forages in the tropical site, suggesting that degradation of poor quality C4 forages characteristic of the tropics requires a denser microbial community. Forage type (C3 vs C4) had no effect on the population numbers of total or cellulolytic bacteria and methanogens, whereas a global effect of forage is shown for both total bacteria and methanogens. This suggests an effect of quality, i.e. nutritive value of forages, on these populations. By contrast, a strong variation in ruminal bacteria of young steers fed either a C3 or a C4 grass was reported [START_REF] Pitta | Rumen bacterial diversity dynamics associated with changing from bermudagrass hay to grazed winter wheat diets[END_REF].

No difference in methanogen mcrA copy number was detected among forages, although we expected to find more methanogens with tropical forages, because in this trial they generated more CH 4 than temperate forages: 17.5 and 20.7 g/kg DM intake on average for C3 temperate forages and C4 tropical forages, respectively [START_REF] Archimède | Intake, total-tract digestibility and methane emission of Texel and Blackbelly sheep fed C4 and C3 grasses tested simultaneously in a temperate and a tropical area[END_REF]. These results are in line with previous results [START_REF] Danielsson | Methanogenic population and CH4 production in swedish dairy cows fed different levels of forage[END_REF][START_REF] Zhou | Relationship between rumen methanogens and methane production in dairy cows fed diets supplemented with a feed enzyme additive[END_REF] who reported that CH 4 production was not related to total methanogen population. Methane production could be related to methanogen activity rather than their abundance [START_REF] Popova | Effect of fibre-and starch-rich finishing diets on methanogenic Archaea diversity and activity in the rumen of feedlot bulls[END_REF].

Effect of breed on digestive processes

There is little information available on the extent to which rumen fermentation and microbes vary between sheep breeds. In our experiment two contrasting sheep breeds were chosen: one of temperate origin and the other of tropical origin. Overall, rumen fermentation and microbes were not affected by breed for any of the variables measured. The absence of change in ruminal VFA concentration between genotypes agrees with a previous comparison of two sheep breeds: a local one and an improved one (Ile-de-France and Churra-da-Terra-Quente) fed the same diet [START_REF] Lourenc ¸ O | Effects of sheep breed and soybean meal supplementation on rumen environment and degradation kinetics[END_REF]. Very few studies have compared more widely-contrasting breeds. A comparison between local Bos taurus breed and a Bos indicus breed in a tropical environment showed no differences in digestive processes [START_REF] Grimaud | Effects of level of intake and nitrogen supplementation on digestion by cows in a tropical environment[END_REF]. Differences in digestion between breeds may differ more for poor quality feeds, for which local breeds should be better adapted than improved breeds [START_REF] Lopez | Comparative digestion of herbage by two breeds of sheep: effects of grass maturity stage and level of intake[END_REF]. However, breed × forage interaction was not significant in our study, and the absence of breed effect might be due to the adaptation of ruminal microbiota for both Texel and Blackbelly to the environment where the experiment took place, since they were born and raised close to each experimental site. The establishment of the rumen microbiota is influenced by nutritional and/or environmental exposure in young animals after birth. Consequently, induced variations in microbial populations colonizing the rumen can lead to modifications in fermentation and CH 4 production [START_REF] Abecia | Nutritional intervention in early life to manipulate rumen microbial colonization and methane output by kid goats postweaning[END_REF]. The adaptation of Texel and Blackbelly microbiota to the temperate vs tropical environment may have reduced differences between breeds. In addition, climate conditions during this trial were not extreme enough to favor distinct adaptive and behavioral responses between breeds. Unfortunately, the origin and history of experimental animals is not specified in most studies. At the microbial level, qPCR analysis did not reveal any difference between Texel and Blackbelly. These results corroborate previous studies showing that methanogens, bacteria and protozoa population were not significantly different between two cattle breeds [START_REF] Rooke | Hydrogen and methane emissions from beef cattle and their rumen microbial community vary with diet, time after feeding and genotype[END_REF]. In contrast to this study, some authors have shown the impact of breed on potential CH 4 production and rumen microbiota and suggest that the host animal exerts a controlling effect on its own gut microbiota [START_REF] Danielsson | Methanogenic population and CH4 production in swedish dairy cows fed different levels of forage[END_REF][START_REF] King | Differences in the rumen methanogen populations of lactating Jersey and Holstein dairy cows under the same diet regimen[END_REF].

Differences in microbial populations were predominantly attributable to diet, with the host being less influential. In addition to feed composition effects, the environment appears to influence the establishment and development of the rumen microbiota, leading the host to adapt so as to regulate the influence of diet and environment.

Conclusion

This study shows that the differences observed in the rumen microbiota of sheep between tropical and temperate sites were intrinsic to the location and could not be attributed to local forages (temperate C3 vs tropical C4) or breeds. Total bacteria and methanogens were more abundant in the tropical site. This study also shows the adaptation capacity of each breed to its environment, suggested by the site × breed interaction. Future work should characterize the microbiota using high-throughput sequencing for phylogenetic and metagenomic analysis to gain a better understanding of the influence of the environment in shaping rumen populations.

a

  Temp H = temperate forage, high quality; Temp L = temperate forage, low quality; Trop H = tropical forage, high quality, Trop L = tropical forage, low quality.

Fig. 1 .

 1 Fig. 1. Relationship between rumen microbes and methane enteric emission according to site (temperate (France, F) vs tropical (French West Indies, FWI)) and origin of forages (Temperate C3 (Temp) vs Tropical C4 (Trop)): high quality and low quality forage fed to Texel and Blackbelly. Each point is the mean of 4 individual values.

Table 1

 1 Average dry matter content and chemical composition of experimental forages a .

		Temp H	Temp L	Trop H	Trop L
	Dry matter, g/kg fresh matter b	875	879	866	878
	Organic matter, g/kg dry matter	878	926	912	929
	NDF, g/kg dry matter	586	623	742	742
	ADF, g/kg dry matter	410	370	470	540
	Crude protein, g/kg dry matter	134	83	120	69

a Temp H = temperate forage high quality, Temp L = temperate forage low quality, Trop H = tropical forage high quality, Trop L = tropical forage low quality. b Dry matter content of forages was on average 1.3 percentage unit higher in temperate site than in tropical site.

Table 2

 2 Oligonucleotides used as primers for PCR-DGGE and qPCR analysis.

	Oligonucleotides	Oligonucleotide sequences	Target	Amplicon	Use in	Primer references
				length (bp)
	534R -	5 -ATT ACC GCG GCT GCT GG	rrs Bacteria	193	PCR-DGGE	Muyzer et al. (1993)
	341f-GC	5 -(GC)-CC TAC GGG AGG CAG CAG
	mcrAr	5'-TTC ATT GCR TAG TTW GGR TAG TT	mcrA Methanogens	460-490	PCR-DGGE	Luton et al. (2002)
	mcrAf-GC	5 -(GC)40-GGT GGT GTM GGA TTC ACA
		CAR TAY GCW ACA GC
	qmcrA-R	5 -GBARGTCGWAWCCGTAGAATCC	mcrA Methanogens	140	qPCR	Denman et al. (2007)
	qmcrA-F	5 -TTCGGTGGATCDCARAGRGC
	799R2	5 -AACAGGATTAGATACCCTG	rrs Bacteria	280	qPCR	Edwards et al. (2007)
	520F	5 -AGCAGCCGCGGTAAT
	FS586-f	5 -GTTCGGAATTACTGGGCGTAAA	rrs Fibrobacter succinogenes	121	qPCR	Denman and
	FS706-r	5 -CGCCTGCCCCTGAACTATC				McSweeney (2006)
	RF96-f	5 -CGAACGGAGATAATTTGAGTTTACTTAGG	rss Ruminococcus flavefaciens	132	qPCR	Denman and
	RF220-r	5 -CGGTCTCTGTATGTTATGAGGTATTACC				McSweeney (2006)
	RA1281f	5 -CCC TAA AAG CAG TCT TAG TTC G	rrs Ruminococcus albus	175	qPCR	Koike and Kobayashi
	RA1439r	5 -CCT CCT TGC GGT TAG AAC A				(2001)

Table 3

 3 Dry matter (DM) intake and digestibility, and rumen fermentation criteria before feeding in Texel (T) and Blackbelly (B) sheep fed four forages in temperate and tropical site. a

		Temp H		Temp L		Trop H		Trop L		SEM	P value c
		T	B	T	B	T	B	T	B		Site	Forage Site x	C3 vsC4
													Forage
	DM intake, g/kg BW b
	Temperate site	28.9	21.8	26.3	25.2	16.8	17.0	12.9	15.1	1.87	0.95	<0.001 0.17	<0.001
	Tropical site	25.8	22.8	21.9	23.7	14.8	22.8	13.8	18.7
	DM digestibility coefficient
	Temperate site	0.622 0.668 0.554 0.564 0.618 0.625 0.476 0.514 0.0209 0.004 <0.001 0.02	0.27
	Tropical site	0.637 0.652 0.542 0.576 0.666 0.662 0.604 0.555
	pH
	Temperate site	6.45	6.38	6.33	6.40	6.53	6.46	6.52	6.37	0.107	0.22	0.73	0.26	0.56
	Tropical site	6.62	6.64	6.56	6.48	6.40	6.44	6.39	6.50
	VFA b , mmol/L
	Temperate site	92.7	90.5	87.5	91.0	73.1	73.8	93.0	94.4	6.72	0.79	0.02	0.30	0.05
	Tropical site	81.3	83.1	78.6	95.5	75.3	89.9	85.9	98.9
	Acetate, mmol/mol
	Temperate site	711	717	751	754	722	718	711	696	12.5	0.02	<0.001 0.02	0.002
	Tropical site	743	748	734	734	747	741	723	716
	Propionate, mmol/mol
	Temperate site	192	177	174	173	185	187	200	187	8.8	0.02	0.07	0.16	0.11
	Tropical site	163	173	179	178	158	170	185	187
	Butyrate, mmol/mol
	Temperate site	76	80	60	60	79	83	101	97	8.0	0.13	0.002	0.08	0.01
	Tropical site	75	69	68	69	74	75	75	79
	Minor VFA b , mmol/mol
	Temperate site	20	25	13	11	14	12	16	20	3.10	0.65	0.30	0.01	0.06
	Tropical site	19	10	19	19	20	14	17	18
	Acetate:Propionate
	Temperate site	3.72	4.06	4.35	4.37	3.94	3.86	3.43	3.73	0.260	0.007 0.02	0.10	0.03
	Tropical site	4.58	4.42	4.24	4.18	4.78	4.40	3.96	3.85
	Ammonia, mmol/L
	Temperate site	4.56	8.12	6.17	8.42	3.89	4.17	4.43	6.41	1.590	0.30	0.28	0.36	0.69
	Tropical site	6.69	5.28	8.49	6.59	7.95	5.99	8.11	5.83

a Temp H = temperate forage, high quality, Temp L = temperate forage, low quality,Trop H = tropical forage, high quality, Trop L = tropical forage, low quality.

b BW: body weight. VFA: volatile fatty acids. Minor VFA are the sum of isobutyrate, valerate, isovalerate and caproate.

Table 4

 4 Protozoal population in Texel (T) and Blackbelly (B) sheep fed four forages in temperate and tropical site. a

		Temp H	Temp L	Trop H		Trop L	SEM	P value c
		T	B	T	B	T	B	T	B	Site	Forage Site x	C3 vs C4
												Forage
	Total, log10 cells/mL
	Temperate site	6.21 6.26 6.13 6.23 6.16 5.97 6.37 6.36 0.109 0.41 0.62	0.01	0.50
	Tropical site	6.46 5.95 6.34 6.16 6.46 6.35 6.31 6.13
	Isotricha, log10 cells/mL
	Temperate site	5.16 4.69 2.68 2.74 4.35 3.46 5.27 3.92 0.970 0.77 0.16	0.41	0.11
	Tropical site	4.11 3.03 4.25 2.48 4.70 4.00 5.23 3.16
	Dasytricha, log10 cells/mL
	Temperate site	5.59 5.25 5.08 3.53 5.56 5.44 5.94 5.49 0.475 0.35 0.05	0.14	0.52
	Tropical site	4.82 4.37 5.33 4.54 5.43 5.46 4.70 5.43
	Large Entodiniomorphs b , log10 cells/mL
	Temperate site	5.85 6.02 6.00 6.16 5.85 5.65 5.87 6.19 0.138 0.14 0.67	0.41	0.30
	Tropical site	6.31 5.76 6.12 6.02 6.34 6.21 6.15 5.68
	Small Entodiniomorphs b , log10 cells/mL
	Temperate site	5.47 5.34 5.04 5.05 5.04 4.87 5.54 5.35 0.085 0.01 <0.001 <0.001	<0.001
	Tropical site	5.01 4.97 5.27 5.08 5.04 5.01 5.17 5.17

Table 5

 5 Quantification of rumen bacteria and methanogens in Texel (T) and Blackbelly (B) sheep fed four forages in temperate and tropical site. a

		Temp H		Temp L		Trop H		Trop L		SEM	P value b
		T	B	T	B	T	B	T	B		Site	Forage	Site x	C3 vs C4
													Forage
	Total bacteria, log10 rrs copy number/g DM
	Temperate site	11.14	11.64	11.41	11.46	11.47	11.49	11.17	11.26	0.089	<0.001	0.03	0.001	0.14
	Tropical site	11.59	11.51	11.80	11.61	11.74	11.71	11.65	11.62
	Fibrobacter succinogenes, log10 rrs copy number/g DM
	Temperate site	9.78	9.67	9.59	9.57	9.54	9.60	9.30	9.42	0.143	<0.001	0.12	0.13	0.11
	Tropical site	9.00	9.06	9.41	9.23	9.27	9.13	9.03	9.10
	Ruminococcus albus, log10 rrs copy number/g DM
	Temperate site	8.76	8.76	7.43	8.19	7.81	8.33	7.67	7.86	0.302	0.99	0.40	<0.001	0.92
	Tropical site	7.62	7.57	8.48	8.07	8.47	8.45	8.17	8.00
	Ruminococcus flavefaciens, log10 rrs copy number/g DM
	Temperate site	8.78	8.57	8.57	8.47	8.50	8.64	8.23	8.19	0.185	0.30	0.10	0.39	0.22
	Tropical site	8.63	8.48	8.77	8.58	8.68	8.72	8.49	8.54
	Total methanogens, log10 mcrA copy number/g DM
	Temperate site	8.93	8.92	8.80	8.91	8.89	8.83	8.65	8.71	0.072	<0.001	0.03	0.01	0.30
	Tropical site	9.22	8.93	9.31	9.20	9.26	9.24	9.09	9.23

a Temp H = temperate forage, high quality; Temp L = temperate forage, low quality; Trop H = tropical forage, high quality. Trop L = tropical forage, low quality.

Acknowledgments

This work has been granted by the French National Agency for Research (ANR) (EPAD project ANR-09-STRA-01), by Region Guadeloupe and other European funding: FEOGA, FEDER, FSE. We thank the staff of UE 1414 Herbipôle, INRA (France), especially Sébastien Alcouffe, André Guittard and Denis Roux, the staff of UE1284 PTEA, INRA (Guadeloupe) especially Fred Periacarpin for animal sampling, and Dominique Graviou for help in microbial analyses.

Conflict of interest

The authors declare that there are not conflicts of interest.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.anifeedsci.2016.08.010.


H Alout

Dr P Labbé

A Berthomieu

P Makoundou

P Fort

N Pasteur

M Weill

High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾ 1 mg l -1 and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1 R allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1 R and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1 V or the duplicated ace-1 D allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.

INTRODUCTION

In insects, resistance to organophosphate (OP) insecticides is due to increased detoxification by carboxylesterases or cytochrome P450 oxidases (metabolic resistance), or to mutations of the synaptic acetylcholinesterase (AChE1, target-site resistance) that becomes less sensitive to inhibition by these insecticides [START_REF] Labbé | Evolution of resistance to insecticide in disease vectors[END_REF]). In the mosquito Culex pipiens, various amplified esterases (encoded by the Ester super locus) and two substitutions on the AChE1 (encoded by the ace-1 gene) have been identified; they confer resistance to a large variety of OP and carbamate insecticides. Mutations at the ace-1 gene include the G119S and the F290V substitutions (the R and V alleles, respectively, [START_REF] Weill | Insecticide resistance in mosquito vectors[END_REF][START_REF] Alout | A new amino-acid substitution in acetylcholinesterase 1 confers insecticide resistance to Culex pipiens mosquitoes from Cyprus[END_REF], as well as several duplications (D alleles) containing, in tandem, a susceptible copy (S) and a resistant copy (R or V, [START_REF] Labbé | Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens[END_REF][START_REF] Alout | Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations[END_REF].

In 1999, Pasteur et al. described a high resistance to the OP chlorpyrifos (410 000-fold) in populations collected in Tunisia, which was investigated in the Gara strain, homozygous for the ace-1 R allele [START_REF] Pasteur | A new mechanism conferring unprecedented high resistance to Chlorpyrifos in Culex pipiens (Diptera: Culicidae)[END_REF]. Gara displayed the same propoxur resistance as the reference strain SR [START_REF] Berticat | Insecticide resistance genes induce a mating competition cost in Culex pipiens mosquitoes[END_REF], also homozygous for the ace-1 R allele, but differed strongly in resistance to chlorpyrifos, with Gara strain being 1000-fold more resistant than SR. This high chlorpyrifos resistance (HCR) in Gara was not affected by inhibitors of carboxylesterases or cytochrome P450 oxidases. Bioassays with several insecticides suggested that HCR in the Gara strain was due to a single major gene (or a group of tightly linked genes), distinct from ace-1, but epistatic with and/or genetically linked to it. However, as rearing of the Gara strain was discontinued, no further study was undertaken.

In 2003 and 2005, we again identified HCR (mosquito larvae surviving a chlorpyrifos dose of 1 mg l -1 ) in C. pipiens from Tunisia and observed it in another Mediterranean country (Israel) and in Mayotte Island (Indian Ocean). The present study was undertaken to analyze in more details the underlying HCR mechanism(s) with two major questions in mind: (1) is HCR due to the same gene in all regions? and ( 2) what is the precise relationship between the gene(s) coding HCR and the ace-1 gene (genetic linkage or epistasis)? Here we report on (1) the thorough genetic study of HCR based on extensive crossing experiments; (2) the isolation and characterization of the Madra strain that contains only the major genetic factor (T) involved in HCR and lacks all other known resistance alleles at the Ester or ace-1 loci; and (3) the study of HCR expression in mosquitoes with different genotypes at the T and ace-1 loci, obtained by crossing various strains.

MATERIALS AND METHODS

Strains and crossing experiments

Four reference strains were used: Slab, SR, Ducos and HomoVAL. Slab is the susceptible reference strain [START_REF] Georghiou | Carbamate-resistance in mosquitoes: selection of Culex pipiens fatigans Wied ( = Culex quinquefasciatus) for resistance to Baygon[END_REF]) isolated from a Californian population (that is, subspecies Culex p. quinquefasciatus). SR is resistant to OPs and carbamates through insensitive AChE1 and is homozygous for the ace-1 R allele, in the Slab genetic background [START_REF] Berticat | Insecticide resistance genes induce a mating competition cost in Culex pipiens mosquitoes[END_REF]. Ducos is homozygous for the ace-1 D1 allele, a duplicated allele with a susceptible copy and the resistant R copy, in the Slab genetic background [START_REF] Labbé | Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito Culex pipiens[END_REF]. HomoVAL is homozygous for the ace-1 V allele [START_REF] Alout | Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations[END_REF].

Four new strains studied here were derived from field collections performed in Tunisia (Bismuth, collected in the Tunis area in 2003, andSousse, collected in 2005), in Israel (Yatouch, collected in Tel Aviv in 2010) and in Mayotte Island (Mayotte, collected in Tsoundzou in 2010). Strains from Tunisia and Israel are from the C. p. pipiens subspecies, whereas the one from Mayotte is from C. p. quinquefasciatus. A last strain, Madra, was derived from Sousse for the present study. The characteristics of all strains are given in Supplementary Table S1. Strains were reared in standard insectarium conditions (27 ± 1 °C, 70 ± 8% relative humidity and 12:12 light and dark photoperiod). Mass crosses were used in most experiments. Pupae were isolated individually in 10 ml tubes containing 1 ml of tap water. Adults were sorted by sex upon emergence and released in the appropriate cages (25 × 25 × 25 cm). When necessary, larvae were cured of Wolbachia infection to avoid interstrain cytoplasmic incompatibility: they were then reared in water containing tetracycline hydrochloride (20 mg l -1 , Sigma Aldrich, St Louis, MI, USA) during three generations, followed by one generation of rearing in clear water before performing crosses [START_REF] Duron | High Wolbachia density correlates with cost of infection for insecticide resistant Culex pipiens mosquitoes[END_REF].

Bioassays and insecticide selections

Bioassays were conducted on sets of 20 larvae in plastic cups containing 99 ml of tap water and 1 ml of insecticide solution in ethanol. Mortalities were recorded after a 24-h exposure. Each test was replicated at least two times on larvae reared in different tanks. The insecticides tested were of technical grade and included chlorpyrifos-ethyl (99% (active ingredient), Dow Chemical, Midland, MI, USA), chlorpyrifos-methyl (99.5% purity, Dr Ehrenstorfer GmbH, Augsburg, Germany), chlorpyrifos-oxon (10 ng μl -1 , Dr Ehrenstorfer GmbH), propoxur (99.9% (active ingredient), Bayer AC, Leverkusen, Germany), temephos (97% HPLC, Sigma Aldrich), parathion-methyl (98.5% purity, Dr Ehrenstorfer GmbH) and paraoxon-methyl (99% purity, Dr Ehrenstorfer GmbH). Synergists DEF (S,S,S-tributyl-phosphorotrithioate; Interchim, Montluçon, France) and PB (piperonyl butoxide; Fluka AG, Buchs, Switzerland) were used in bioassays at the concentration of 0.8 and 5 mg l -1 , respectively, 4 h before adding the insecticide. DEF is an inhibitor of carboxylesterases and of glutathione-S-transferases; PB is an inhibitor of cytochrome oxidases.

Dose-mortality responses were analyzed using the R free software (http://www.r-project.org/) and the BioRssay v6.01 script of [START_REF] Milesi | BioRssay: a R script for bioassay analyses[END_REF]. The following parameters were analyzed or computed: (1) the linearity and slope of dose-mortality responses; (2) insecticide concentrations inducing 50% mortality (LC 50 ) with their 95% confidence intervals; and (3) the resistance ratio of a given strain as compared with a reference strain at LC 50 (RR 50 ) and their 95% confidence intervals. The effect of synergists on dosemortality responses was evaluated by the synergism ratio (LC in absence synergist/LC in presence synergist) at LC 50 (SR 50 ). Mortality responses were considered significantly different (Po0.05) when their 95% confidence limits did not overlap.

Chlorpyrifos selections of strains were done in the same conditions as bioassays, using ~50 larvae per cup. The field collection from Sousse was selected with 10 mg l -1 chlorpyrifos during three successive generations. Cages were then set with one male and five virgin females. After 3 days, males were stored in liquid nitrogen, and females were allowed to blood-feed and to lay eggs in individual vials. The Ester genotype of each father and of each mother was characterized using starch electrophoresis (see below). The Sousse strain was founded with egg-rafts produced by parents (males and females) lacking any overproduced esterase. Bismuth, Yatouch and Mayotte collections were selected with 1 mg l -1 chlorpyrifos during at least 6 generations, that is, until mortality at 10 mg l -1 chlorpyrifos was ⩽ 50%. All subsequent selections were mass-performed directly in 1-l tanks.

Characterization of overproduced esterases, ace-1 and ace-2 genes Overproduced esterases were characterized from thorax homogenates of single adults analyzed on starch gel electrophoresis in Tris-Maleate buffer-EDTA (pH 7.4), as described by [START_REF] Pasteur | Practical Isozyme Genetics[END_REF]. Esterases were revealed using αand β-naphthyl acetates and Fast Garnett (Sigma Aldrich). Allele identification was performed by comparing the electrophoretic mobility of the observed esterases with those from laboratory strains homozygous for the different overproduced enzymes. This was used to control that any overproduced esterases were removed from the Sousse strain.

AChE1 phenotypes encoded by the ace-1 locus were determined with the témoin-dichlorvos-prpopoxur test on head homogenates of single adults [START_REF] Alout | Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations[END_REF]. This test discriminates between phenotype S (that is, homozygotes for the S allele), phenotype R or V (homozygotes for the G119S or the F290V mutation, respectively) and phenotypes SR or SV that contain at least one S copy and one resistant R or V copy (for example, S/R, S/V, D, S/D or R/D or V/ D genotypes).

The ace-2 gene was characterized by the PCR test followed by HaeIII restriction developed by [START_REF] Bourguet | The acetylcholinesterase gene Ace: a diagnostic marker for the pipiens and quinquefasciatus forms of the Culex pipiens complex[END_REF] to discriminate between the two C. p. pipiens and C. p. quinquefasciatus subspecies.

Genetic linkage

Genetic linkage between two loci was measured as the frequency of recombinant genotypes in the backcrossing experiments (that is, the recombination rate). This frequency is expected to be 50% for independent loci, below 50% for linked loci.

RESULTS

Inheritance and toxicological properties of HCR

In four field collections from Tunisia (Bismuth and Sousse), Israel (Yatouch) and Mayotte Island (Mayotte), we detected larvae that survived 1 mg l -1 chlorpyrifos, a dose that kills larvae of the SR strain (homozygous for the ace-1 R allele), thus displaying HCR. We selected strains derived from these field larvae with increasing doses of chlorpyrifos each generation until they displayed ⩽ 50% mortality at 10 mg l -1 . After selection, all these HCR strains were found homozygous for the R allele (Supplementary Table S1).

A single sex-linked gene is responsible for HCR. We first analyzed how HCR was inherited by crossing HCR strains to the SR strain, and then by backcrossing the obtained F1 to a parental strain (here SR). Under the hypothesis that resistance in HCR strains as compared with SR is caused by a single gene (named T, with a dominant T allele in HCR strains and t allele in SR), the backcross should contain an equal number of larvae with HCR phenotype (identical to the F1 or Tt) and with no HCR phenotype (identical to SR or tt). Compared with the SR strain, Sousse displays RR 50 of 2200-fold (Table 1). The F1 progeny displayed a linear chlorpyrifos dose-mortality response (as did the parental strains) and a RR 50 of 530-fold, indicating that HCR is codominant. Note that heterozygous Tt insects survived 1 mg l -1 chlorpyrifos, the dose used to select HCR strains. In contrast to the F1, linearity of the dose-mortality response of the offspring produced by F1 females backcrossed with SR males was rejected (P = 0.002). The dose-mortality response disclosed a clear plateau close to 50% mortality (Figure 1), indicating the segregation of two main genotypes (Tt as in the F1, and tt as in SR) with different resistant properties, as hypothesized.

We also performed the reciprocal backcross to determine whether the T gene is linked to sex. Males of each HCR strain (Sousse, Yatouch and Mayotte) were mass-crossed to females SR, and males of their F1 offsprings were backcrossed to females SR (Supplementary Table S2). In C. pipiens, sex is determined by a single locus with two alleles (M and m): males are heterozygous (Mm), whereas females are homozygous (mm) [START_REF] Gilchrist | Sex linkage and sex determination in a mosquito, Culex molestus[END_REF]. Thus, in HCR Heredity strains, most of males are MT/mT and most of females are mT/mT, whereas in the SR strain, males are Mt/mt and females mt/mt. Males of these F1 offsprings were therefore double heterozygous (MT/mt), and they were backcrossed to double homozygous (mt/mt) females of the SR strain (Supplementary Table S2). Under the hypothesis of independent assortment between sex and the T genes, survivors of a 24-h exposure to 1 mg l -1 chlorpyrifos (that is, a dose killing all t/t genotypes) should contain an equivalent number of males and females. Sex ratios were examined in the offspring of each backcross either in the absence of insecticide or after chlorpyrifos exposure (Table 2). In the absence of insecticide selection (control), sex ratios were not significantly different from the 50% expectation (1.01 ⩾ χ 2 ⩾ 1.53, 1 d.f., P40.2), except for the backcross involving the Sousse strain that displayed a significantly higher frequency of females (57%, Po0.001). In all chlorpyrifos-treated offspring, sex ratios were strongly biased in favor of males (87-91%, χ 2 4700, P<<0.0001). This indicates that the T gene is linked to the sex factor in the three strains. The recombination rate between the sex and T loci, estimated from the percentage of females among the survivors (as they are all recombinant; Table 2), varied between 8.7% ± 0.8 and 12.7% ± 0.8, with a mean value of 10.6% ± 0.5 (mean ± s.e.).

HCR is not due to ace-2 gene. As AChEs are the major targets of OP insecticides such as chlorpyrifos, and because the ace-2 gene encoding the AChE2 enzyme is linked to the sex factor [START_REF] Malcolm | A sex-linked Ace gene, not linked to insensitive acetylcholinesterase-mediated insecticide resistance in Culex pipiens[END_REF], we investigated whether the T gene could be ace-2. This was possible because the HCR strains Sousse and Yatouch belong to the C. p. pipiens subspecies, whereas the reference SR strain carries a C. p. quinquefasciatus genetic background (Supplementary Table S1). The two subspecies have different ace-2 alleles [START_REF] Bourguet | The acetylcholinesterase gene Ace: a diagnostic marker for the pipiens and quinquefasciatus forms of the Culex pipiens complex[END_REF] Abbreviations: CI, 95% confidence interval of the studied parameter; LC 50 = dose inducing a mortality of 50%; NA, not applicable; RR 50 , resistance ratio at LC 50 ; s.e., standard error. Bioassays were performed in laboratory strains (Slab, HomoVAL and SR), in strains isolated in this study (Sousse and Madra) and in various F1 crosses (♀Sousse × ♂SR, ♀Madra × ♂Slab and ♀Madra × ♂SR); see text and Figures 1 and2. The following information is given: slope of the mortality (probit) dose (log) regression and its s.e.; RR 50 using Slab (A) or SR (B) strains as reference. Data's departure from the regression was assessed using the χ 2 conformity test and was not significant for any bioassay (P40.05). Heredity produced in the F1 crosses between males Sousse and females SR are BMT/bmt. When backcrossed to SR females (bmt/bmt), all females surviving an exposure to ⩾ 1 mg l -1 chlorpyrifos would result from a recombination between sex and T loci as seen above. The genotype of these surviving females at the ace-2 locus can be either heterozygous (Bb), indicating that no recombination occurred between the ace-2 and T loci, or homozygous (bb) if there was recombination (Supplementary Table S3A). Surviving females derived from the Sousse strain were all bb (N = 21), whereas those derived from the Yatouch strain were bb (97%) and Bb (3%; N = 35; Supplementary Table S3B). The difference between the two strains was not statistically significant (χ 2 = 0.61, 1 d.f., P40.60). Among a total of 56 surviving females, 55 recombined between T and ace-2, indicating that ace-2 is distinct from the HCR T gene. Furthermore, only one recombinant between the sex and ace-2 loci (that is, a Bb female) was found, showing that sex and ace-2 loci are much closer to one another than either is to the T locus.

Isolation of a strain with only the T allele and no resistant allele at the ace-1 locus. The strong linkage between ace-2 and T made it possible to isolate a strain carrying the T allele but lacking alleles of other loci associated with chlorpyrifos resistance, in particular the R allele at the ace-1 locus and overproduced esterases. We used the Sousse strain as the source of T allele and performed a series of crosses with Slab (Supplementary Table S4) to construct the Madra strain. Initially, all Madra mosquitoes were homozygous for the susceptible ace-1 S allele (S/S), the genotypes of males was Mt/mt or Mt/mT and the genotypes of females was mt/mt or mT/mt. To increase the frequency of the T allele, we hypothesized that it should confer to some extent resistance to chlorpyrifos and selected the Madra strain with increasing doses of chlorpyrifos for at least 12 generations (Supplementary Table S4).

We then analyzed the inheritance of the observed resistance to chlorpyrifos in the Madra strain by crossing Madra females (T/T) with Slab males (t/t) and the offspring females (T/t) were backcrossed to Slab males (t/t). The dose-mortality response to chlorpyrifos of the F1 (T/t) was linear, and that of the backcross displayed a clear plateau around 50% mortality, confirming that chlorpyrifos resistance in the Madra strain is due to one major gene (T) (Figure 2). In the absence of the R allele at the ace-1 locus, the chlorpyrifos resistance conferred by the T allele is approximately six-fold in T/t heterozygotes and 20-fold in T/T homozygotes (Table 1).

Resistance characteristics of the T gene. The toxicological characteristics associated with the T gene was investigated by comparing the resistance with a few insecticides between Madra and Slab strains, both homozygous SS at the ace-1 locus, and between Sousse and SR, both homozygous RR at the ace-1 locus (Table 1 and Supplementary Table S5). Presence of the synergist DEF (inhibiting some carboxylesterases and glutathione-S-transferases) slightly increased chlorpyrifos susceptibility in Madra and Slab, but the synergism ratios in the two strains were not significantly different (P ⩾ 0.95). In presence of PB (inhibiting some cytochrome P450), susceptibility to chlorpyrifos was decreased in Madra and Slab (as expected from the inhibition of cytochrome P450 oxidases that metabolize chlorpyrifos into chlorpyrifos-oxon), but this decrease was not significantly different (P X 0.95) between the two strains (Supplementary Table S5). The Madra strain was significantly more resistant to chlorpyrifos-methyl compared with Slab (RR 50 = 35, p<<0.001, Table 1) than to chlorpyrifos (RR 50 = 21). Thus, although the ethyl moiety of the chlorpyrifos radical may influence the level of resistance, it has no major role in the action of the T allele. The resistance to parathion and paraoxon (methyl forms) were also tested in the Madra and Sousse strains (Table 1). As compared with Slab, Madra showed a significant resistance (RR 50 = 2.3 and 4.0, respectively, Po0.001). Similarly, Sousse was more resistant than SR to these two insecticides (RR 50 = 13 and 5, respectively, Po0.001). Although parathion and paraoxon resistances in Sousse are significantly higher than for SR, they are much lower than those recorded for chlorpyrifos (2200-fold vs 13-fold for parathion) or chlorpyrifos-oxon (890-fold vs 5-fold for paraoxon, Table 1). In the presence of temephos, Madra was more susceptible than Slab (RR 50 = 0.54, Po0.001). Finally, Madra and Slab strains displayed no significant difference in their dose-mortality responses to propoxur (P = 0.43).

In conclusion, chlorpyrifos toxicological data confirmed that, when homozygous, the T gene confers the same resistance characteristics in the presence or absence of the synergists DEF and PB in mosquitoes carrying the S allele of the ace-1 gene. Therefore, this indicates that the T gene does not code for increased detoxification by carboxylesterases or glutathione-S-transferases inhibited by DEF and/or cytochrome P450 oxidases inhibited by PB. In addition, these data show that the T allele by itself confers a resistance to chlorpyrifos and to parathion (and to their respective oxidized forms, chlorpyrifos-oxon and paraoxon), but not to temephos or to the carbamate propoxur.

High synergy between the T and ace-1 genes in HCR It is classically admitted that, when present in the same individual, two distinct resistance genes may have an additive or a multiplicative effect on resistance [START_REF] Raymond | Interactions between pesticide genes: model and experiment[END_REF]. We then addressed this issue by comparing RR 50 ratios of the Sousse, SR and Madra strains relative to Slab (Table 1). SR (t/t R/R) and Madra (T/T S/S) showed moderate and similar RR 50 ratios for chlorpyrifos relative to Slab (19 and 21, respectively). In T/t R/R genotype (F1 offspring of females Sousse × males SR) as well as in T/T R/R genotype (Sousse strain), the epistatic interaction of the T allele with the R allele in chlorpyrifos resistance was far higher than expected from the additive hypothesis, and even higher than expected under the multiplicative hypothesis (for example, 10 000-fold observed vs ~110-fold expected in T/t R/R, and 43 000fold observed vs 400-fold expected in T/T R/R; Supplementary Table S6). The same situation also stood for parathion (methyl form): the resistance ratio observed in Sousse (T/T R/R) is clearly higher than expected under the multiplicative hypothesis (430-fold observed vs ~74-fold expected, respectively). As several resistance alleles (other than R) have been selected at the ace-1 locus in C. pipiens populations, the availability of the Madra strain opens the possibility to assess how the presence of the T allele would modify the level of resistance conferred by ace-1 alleles.

Heredity

Interaction between the T allele and resistant alleles of the ace-1 gene. Two main resistant mutations have been selected at the ace-1 locus in Mediterranean mosquito populations [START_REF] Alout | Multiple duplications of the rare ace-1 mutation F290V in Culex pipiens natural populations[END_REF]: the R allele carrying the G119S mutation and the V allele carrying the F290V mutation. When homozygous in a strain, the R allele confers 19-fold and the V allele confers 8.4-fold resistance to chlorpyrifos at LC 50 , respectively (Table 1). Duplicated resistant alleles of the ace-1 gene are currently spreading in C. pipiens natural populations and are already present in regions where the resistant T allele is present, as in Tunisia [START_REF] Alout | High incidence of ace-1 duplicated haplotypes in resistant Culex pipiens mosquitoes from Algeria[END_REF] and in Mayotte [START_REF] Pocquet | Insecticide resistance in disease vectors from Mayotte: an opportunity for integrated vector management[END_REF]. We then analyzed how the T allele interacts with three ace-1 alleles (R, V, D) in different genotypic combinations (Table 3) obtained by crossing the Slab, SR, HomoVAL, Ducos, Madra and Sousse strains. We also analyzed several F2s of the various crosses in order to investigate genotypes that were not available in our strains or in their F1 offspring (see Supplementary Table S7). All analyses are based on the mortality observed 24 h after exposure to 1 mg l -1 chlorpyrifos. For F2s, observed mortality was compared with expected mortality estimated:

(1) by calculating the frequency of each genotype (estimated from Mendelian inheritance of two independent loci) and ( 2) by taking into account the mortality of each genotype from the study of homozygous strains and their F1 (see Supplementary Information). The observed mortality was in good agreement with the expected one, although slightly higher (Supplementary Table S7). We observed that HCR is expressed in mosquitoes either carrying two T alleles (T/T homozygotes) and at least one R copy (R/R homozygotes, R/S or R/V heterozygotes and likely R/D, D/D, and D/S), or carrying one T allele (T/t heterozygotes) and two ace-1-resistant alleles (R/R homozygotes or R/V and R/D heterozygotes and likely D/D). The inability of the V allele to produce HCR in T/T homozygotes is probably because of the low chlorpyrifos resistance it provides as compared with the R allele (see Table 1). Thus, HCR expression is dependent on the number of resistant alleles at the T and ace-1 loci, as well as on the nature of the resistant allele present at the ace-1 locus. These results also support that HCR expression requires at least three resistance alleles at the T and ace-1 loci, two at one locus and one at the other.

DISCUSSION

Using multiple crossing experiments, we evidenced a new and unique gene responsible for the high chlorpyrifos resistance or HCR in several mosquito populations of the C. pipiens complex. HCR is observed through a synergetic effect between one allele of this gene and resistant ace-1 alleles. These results are similar to those obtained on the Gara strain 415 years ago [START_REF] Pasteur | A new mechanism conferring unprecedented high resistance to Chlorpyrifos in Culex pipiens (Diptera: Culicidae)[END_REF] and strongly suggest that the HCRs observed in Sousse and Gara are due to the same genetic mechanism. Although we did not perform as many detailed bioassays with Bismuth, Yatouch and Mayotte strains as we did with Sousse, the sex linkage studies also supported the notion that HCR of these strains involves a similar unique gene, named T. Thus, the T gene has a large geographic distribution and was observed in strains of two subspecies: C. p. pipiens from Tunisia and Israel and C. p. quinquefasciatus from Mayotte, an island in the Indian Ocean.

Using the linkage observed between the T gene and another sexlinked gene (ace-2), we constructed the Madra strain, containing the resistant allele at the T locus and lacking any other known resistance mechanism (insensitive AChE1 or overproduced esterases). This chlorpyrifos resistance was not affected by DEF or PB synergists. We also showed that, in mosquitoes of the C. pipiens complex, HCR is due to positive epistasis between the resistant alleles of the ace-1 and of the T loci. Resistance to chlorpyrifos provided by the T gene alone (T/T S/S, Madra strain) was moderate and equivalent to that of t/t R/R (SR strain) or T/t R/S one (F1 from Madra × SR) genotypes. In contrast, the T/T R/R genotype (Sousse strain) displayed HCR, with a resistance level far higher than that expected if both genes had multiplicative effects: 440 000 instead of ~400-fold. Investigating the epistasis between the two genes showed that the expression of HCR is controlled by a complex interaction between the number of resistant alleles at each locus and the nature of the mutation present at the ace-1 gene.

HCR and ace-1-resistant alleles

Interactions between resistance mechanisms were investigated by [START_REF] Raymond | Interactions between pesticide genes: model and experiment[END_REF] using a model of insecticide flow in a larva with four compartments (Figure 3): the outer, the intermediate, the target and the elimination compartments. Let us consider here the target compartment in which the concentration of the toxic insecticide (here chlorpyrifos-oxon) is C T . Any mechanism that would decrease C T will result in a resistance phenotype. From our bioassays (Table 1), we know that in t/t homozygotes (Slab, HomoVAL and SR strains), the C T able to inhibit AChE1 enzymes is lower for the S allele (wild type) than for the V allele, and lower for the V allele than for the R allele. Resistance to chlorpyrifos in R/S heterozygotes has been shown to be partially dominant under the same experimental conditions [START_REF] Bourguet | Dominance of insecticide resistance presents a plastic response[END_REF], indicating that the C T inhibiting AChE1 molecules is lower in R/S than in R/R insects. In addition, [START_REF] Labbé | Gene-dosage effects on fitness in recent adaptive duplications: ace-1 in the mosquito Culex pipiens[END_REF] showed that chlorpyrifos dose-mortality responses of D/D genotypes are similar to those of R/S genotypes. From these information, genotypes can be ordered in function of increasing C T inhibiting synaptic AChE1: S/SoD/SoR/S = D/DoD/RoR/R and S/SoS/VoV/VoR/VoR/R. In t/t homozygotes, none of the ace-1 genotypes displays HCR (Table 3 and Supplementary Table S7), indicating that C T is high enough to inhibit all AChE1 molecules present in the synapse when insects are exposed to 1 mg l -1 chlorpyrifos. In T/t heterozygotes, HCR was observed in R/R, in R/D, likely in D/D and to a lesser extent in R/V genotypes (Table 3). This indicates that the presence of one T allele decreases C T enough to allow survival. In T/T homozygotes, HCR was observed in R/S and likely in D/S genotypes, but not in V/S or V/V genotypes (Table 3). Thus, the decrease of C T is higher in the presence of two T alleles than in the presence of a single one, indicating that expression of the T resistance allele is a critical parameter. However, the decrease of C T is not large enough to allow survival of V/V genotypes (or to prevent inhibition of the F290V-AChE1). Ordering ace-1 genotypes regarding the proportion of the AChE1 variants and their sensitivity to increasing C T thus becomes: S/SoS/VoV/VoD/SoR/SoD/Do R/VoR/DoR/R (D/V was not tested). Note that for HCR, genotypes R/S and D/D were not found similar (T/t D/D showed HCR whereas T/t R/S did not show HCR), whereas chlorpyrifos resistance was similar between R/S and D/D in the absence of the T allele [START_REF] Labbé | Gene-dosage effects on fitness in recent adaptive duplications: ace-1 in the mosquito Culex pipiens[END_REF].

Overall, in larvae exposed to 1 mg l -1 chlorpyrifos, the decrease of chlorpyrifos-oxon concentration in the synapse (C T ) due to the presence of two T alleles (homozygosity) is large enough to allow survival (HCR) of mosquitoes with at least one copy of the G119S mutation contained in the R or in the D allele (from D/S to R/R in the above sequence), whereas insects with a single copy of the T allele (T/t heterozygotes) survive only when they carry two ace-1 resistance alleles (either two R copies like in D/D, D/R and R/R, or one R and one V, like in R/V).

Mechanisms for decreasing chlorpyrifos-oxon in the target compartment Epistasis between the T and ace-1 loci can be explained by three interacting processes: (1) the different inhibition constants of each AChE1 variants, (2) the number of R copies at the ace-1 locus when one of the allele is duplicated [START_REF] Labbé | Gene-dosage effects on fitness in recent adaptive duplications: ace-1 in the mosquito Culex pipiens[END_REF] and (3) the ability of the T locus to decrease C T in the target compartment. This latter process is more efficient in T/T than it is in T/t genotypes, and is therefore dependent on the number of T alleles present in a mosquito (codominance). We also have to consider that C T increases with time during insecticide exposure. Taking into account the inhibition constants (k i ) evaluated by [START_REF] Alout | Amino-acid substitutions in acetylcholinesterase 1 involved in insecticide resistance in mosquitoes[END_REF], the C T value inhibiting the AChE1-S enzyme is lower, and thus is reached earlier than the C T value inhibiting the AChE1-V enzyme, itself lower and inhibiting earlier than for the AChE-1-R enzyme. The longer the time for chlorpyrifos-oxon to reach the C T value that inhibits AChE1, the longer the time for the T mechanism to metabolize chlorpyrifos-oxon (or/and chlorpyrifos) into an inactive compound.

Diminishing the quantity of chlorpyrifos-oxon available to inhibit AChE1 in the target compartment can be achieved by various mechanisms (Figure 3): (1) decreased penetration of chlorpyrifos inside the body (Figure 3, step (a)), (2) decreased activation of the AChE1-nontoxic chlorpyrifos into its toxic oxon form (step (b)), (3) decreased circulation between the intermediate and target compartments (step (c)) and ( 4) increased elimination of chlorpyrifos-oxon or chlorpyrifos from the intermediate or/and target compartments to a compartment (respectively steps (d) and (d')) where the pesticide is no longer toxic. However, several mechanisms can be eliminated, such as decreased activation of chlorpyrifos by cytochrome P450 oxidases inhibited by the PB synergist, or increased detoxification by carboxylesterases or glutathione-S-transferases inhibited by DEF. Enhanced elimination of the pesticide can be the result of any process that decreases the pesticide concentration, for example, increased metabolism and increased excretion. These processes were not investigated here, but it must be noted that if they were involved they would have to be specific for certain OPs: the T gene does not provide resistance to temephos (this study and Ben [START_REF] Cheikh | Resistance to organophosphorus and pyrethroid insecticides in Culex pipiens (Diptera: Culicidae) from Tunisia[END_REF] and pyrimifos [START_REF] Pasteur | A new mechanism conferring unprecedented high resistance to Chlorpyrifos in Culex pipiens (Diptera: Culicidae)[END_REF], but was shown to induce resistance to chlorpyrifos and parathion. Among proteins potentially Mortality induced by 24-h exposure to 1 mg l -1 chlorpyrifos in mosquitoes carrying different genotypic combinations at the ace-1 and T loci. ace-1 has four alleles (S, R, V and D) and T two alleles (T and t). For each combined genotype tested in a strain or an F1 cross (Supplementary Table S7), the number of studied mosquitoes is indicated (N). For other genotypes, mortality was deduced from the results obtained in F2 crosses (Supplementary Table S7) as explained in the Supplementary Information. implicated in this mechanism are (1) arylesterases (or A-esterases) that are not inhibited by DEF [START_REF] Aldridge | Serum esterases I[END_REF] and known to be involved in paraoxon and chlorpyrifos degradation (see below) and

(2) cytochrome P450 oxidases, as some of them are not inhibited by PB [START_REF] Feyereisen | Insect P450 enzymes[END_REF]. However, a major hurdle to narrow the list of candidate proteins remains the partial assembly of the C. pipiens genome that prevents taking advantage of the observed linkage disequilibrium of T with ace-2.

Paraoxonases (named as PONs for their ability to degrade paraoxon) may represent interesting candidates, although arthropods genomes, including insects, do not contain orthologs to vertebrate PONs [START_REF] Estin | Paraoxonase 1, quorum sensing, and P. aeruginosa infection: a novel model[END_REF]. However, PONs were also shown to be lactonases, enzymes that metabolize endogenous and exogenous lactones, ubiquitously found in nature. PON activity against OPs is thought to be promiscuous and to have arisen during their evolution. PONs belong to the class of A-esterases described by [START_REF] Aldridge | Serum esterases I[END_REF] and were shown later to degrade chlorpyrifos-oxon at a much higher rate than paraoxon. PONs are not inhibited by OP or DEF, and are also able to detoxify the nerve agents sarin and soman. Because PONs are not inhibited by OPs, they are able to hydrolyze them in much larger quantity than carboxylesterases, ∼ 1000 times more in humans (see [START_REF] Billecke | Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters[END_REF]Harel et al., 2004;[START_REF] Draganov | Lactonases with oragnophosphatase activity: structural and evolutionary perspectives[END_REF][START_REF] Elias | Divergence and convergence in enzyme evolution: parallel evolution of paraoxonases from quorum-quenching lactonases[END_REF].

Another mechanism could involve the ATP-binding cassette (ABC) transporters such as the P-glycoproteins that transport toxic substances outside the cell, thus reducing their intracellular concentration. P-glycoproteins have been associated with resistance to cancer therapy drugs, conferring a multi-drug resistance phenotype when overproduced [START_REF] Wang | Gene amplification and multidrug resistance induced by the phosphatase-inhibitory tumour promoter okadaic acid[END_REF]. ABC transporters have been shown to participate in insecticide resistance in various species, including mosquitoes [START_REF] Porretta | Defence mechanisms against insecticides temephos and diflubenzuron in the mosquito Aedes caspius: the P-glycoprotein efflux pumps[END_REF][START_REF] Gahan | An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin[END_REF][START_REF] Bariami | Gene amplification, ABC transporters and cytochrome P450s: unravelling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti[END_REF]. ABC transporter mRNAs were also found overexpressed in Plutella xylostella strains resistant to chlorpyrifos [START_REF] You | A heterozygous moth genome provides insights into herbivory and detoxification[END_REF]. However, inhibition of some ABC transporters had no effect on C. pipiens mortality exposed to chlorpyrifos [START_REF] Buss | Evidence for p-glycoprotein modification of insecticide toxicity in mosquitoes of the Culex pipiens complex[END_REF]. Even if HCR relies on activity of an ABC transporter, it may prove difficult to identify a candidate, as ABC transporter families are made of 52 members in Anopheles gambiae [START_REF] Dermauw | The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance[END_REF] and 70 in C. pipiens members.

CONCLUSION

In this study, we have shown that high chlorpyrifos resistance (HCR) observed in several C. pipiens populations collected worldwide requires a particular combination of allelic variants of ace-1, the major target of OPs and carbamates, and of an unknown T gene, linked to the sex factor and ace-2. By crossing various strains, we evidenced that ace-1 and T act in strong synergy, as they elicit a resistance level 100 higher than expected from a simple multiplicative effect. This strongly suggests that the T gene has the capacity to reduce the concentration of chlorpyrifos in the synapse. We were able to anticipate the synergy of the T allele with other ace-1 alleles, in particular the duplicated alleles, which are currently spreading in natural populations [START_REF] Labbé | Gene-dosage effects on fitness in recent adaptive duplications: ace-1 in the mosquito Culex pipiens[END_REF]. Finally, the availability of the Madra strain opens a way to test whether the T allele may influence resistance to other insecticide classes, and constitutes a biological material suitable for identifying the T gene by differential transcriptomic analysis.

  , namely B in C. p. pipiens and b in C. p. quinquefasciatus. Thus, males

Figure 1

 1 Figure 1 Chlorpyrifos dose-mortality responses observed in Sousse and SR parental strains, their F1 (♀Sousse × ♂SR) and the offspring of ♀F1 backcrossed to ♂SR. The dotted line represents the expected dose-mortality response of the backcross offspring and black dots the observed mortality. Note that the backcross dose-mortality response shows a clear plateau at 50% mortality, indicating the segregation of two alleles. The arrow indicates 1 mg l -1 chlorpyrifos, the concentration used to discriminate HCR mosquitoes.

Figure 2

 2 Figure 2 Chlorpyrifos dose-mortality responses observed in Madra and Slab parental strains, their F1 (♀Madra × ♂Slab) and the offspring of ♀F1 backcrossed to ♂Slab. The dotted line represents the expected dosemortality response of the backcross offspring and black dots the observed mortality. As in Figure 1, the plateau observed around 50% mortality in the backcross offspring indicates the segregation of two alleles.

Figure 3

 3 Figure 3 Compartment model of insecticide flow in a larva (modified from Raymond et al., 1989). (I) Outer compartment, where the concentration of chlorpyrifos is C O ; (II) intermediate compartment within the larvae, where the concentration of chlorpyrifos is C J and that of chlorpyrifos-oxon C I ; (III) target site compartment, where the concentration of chlorpyrifos-oxon is C T ; (IV) compartment where the insecticide is no longer toxic. (a), (c) and (d) correspond to constants k 1 , k 2 and k 3 described by Raymond et al. (1989), (b) is the constant of transformation of chlorpyrifos to chlorpyrifosoxon and (d') the elimination of chlorpyrifos before it becomes toxic. These constants represent the rate of each transformation. CHL, chlorpyrifos; CHO, chlorpyrifos-oxon.

Table 1

 1 Insecticide dose-mortality responses in Culex pipiens strains

	Insecticides	Strains or F1 crosses	LC 50 in mg l -1 (CI)	Slope (s.e.)	RR 50 (CI) (A)	RR 50 (CI) (B)
	Chlorpyrifos-ethyl	Slab	0.00048 (0.00041-0.00054)	10.14 (2.00)	1	-
		HomoVAL	0.0040 (0.0032-0.0049)	2.99 (0.38)	8.4 (6.2-11)	-
		SR	0.0092 (0.0077-0.014)	6.99 (2.66)	19 (13-30)	1
		Sousse	20 (18-24)	3.42 (0.31)	43 000 (27 000-90 000)	2200 (1300-3800)
		Madra	0.0101 (0.0089-0.012)	5.68 (0.90)	21 (12-33)	1.1 (0.66-1.8)
		♀Sousse × ♂SR	4.9 (3.7-6.27)	2.61 (0.41)	10 000 (5800-18 000)	530 (280-990)
		♀Madra × ♂Slab	0.0028 (0.0024-0.0032)	5.71 (0.89)	5.9 (4.0-8.7)	-
		♀Madra × ♂SR	0.0078 (0.0067-0.0095)	4.14 (0.63)	16 (12-22)	0.85 (0.56-1.3)
	Chlorpyrifos-methyl	Slab	0.00076 (0.00061-0.0010)	7.54 (2.18)	1	-
		Madra	0.027 (0.019-0.034)	4.28 (0.77)	35 (22-54)	-
	Chlorpyrifos(methyl)-oxon	SR	0.020 (0.016-0.26)	2.66 (0.29)	-	1
		Sousse	18.0 (11-60)	1.97 (0.50)	-	890 (310-2600)
	Parathion-methyl	Slab	0.0037 (0.0035-0.0039)	8.87 (0.90)	1	-
		Madra	0.0085 (0.0078-0.0094)	4.14 (0.40)	2.3 (1.7-3.0)	-
		SR	0.12 (0.011-0.13)	4.07 (0.38)	32 (19-52)	1
		Sousse	1.58 (1.437-1.77)	4.59 (0.34)	430 (39-4500)	13 (1.2-148)
	Paraoxon-methyl	Slab	0.0080 (0.0075-0.0086)	9.85 (1.46)	1	-
		Madra	0.032 (0.025-0.040)	4.10 (0.68)	4.0 (2.4-6.6)	-
		SR	0.83 (0.70-0.97)	8.21 (2.28)	100 (NA)	1
		Sousse	4.18 (3.1-5.4)	3.09 (0.53)	524 (230-1200)	5.0 (NA)
	Temephos	Slab	0.0027 (0.0019-0.0036)	6.18 (1.30)	1	-
		Madra	0.0014 (0.0013-0.0025)	6.98 (0.82)	0.54 (0.38-0.76)	-

Propoxur Slab 0.18 (0.16-0.20) 4.09 (0.46) 1 -Madra 0.20 (0.11-0.37) 3.24 (0.83) 1.12 (0.49-2.56) -SR 493.3 (436-559) 8.71 (1.53) 2787 (2208-3519) -

Table 2

 2 Sex linkage of HCR

	Strain of the		Control			Survivors
	heterozygous #
		#	~% ~χ2	#	~% ~χ2
	Sousse	501	673 57% 25.2 ***	1374 147 10% 990 ***
	Yatouch	465	428 48%	1.53 NS 1052 100 9%	787 ***
	Mayotte	135	152 53%	1.01 NS 1500 218 13% 957 ***
	Total	1101 1253 53%	9.81 *** 3926 465 11% 3560 ***

Table 3

 3 Chlorpyrifos mortality in various ace-1 and T genotypic combinations

	ace-1 locus	T locus	% Mortality	N
	SS	tt	100	277
	SS	Tt	100	4300
	SS	TT	100	268
	RS	tt	100	4300
	RS	Tt	100	152
	RS	TT	5.5	235
	RR	tt	100	242
	RR	Tt	3.7	219
	RR	TT	2.4	296
	VS	tt	100 (19)	-
	VS	Tt	100	106
	VS	TT	100 (19)	-
	VV	tt	100	50
	VV	Tt	100 (19)	-
	VV	TT	100 (20)	-
	VR	tt	100 (20)	-
	VR	Tt	29.7	145
	VR	TT	⩽ 29.7 (20)	-
	DD	tt	100	56
	DD	Tt	Low (21, 22)
	DD	TT	Low (21, 22)
	DS	tt	100 (21)
	DS	Tt	100	113
	DS	TT	Low (22)
	RD	tt	100 (21)
	RD	Tt	2.3	133
	RD	TT	Low (21)

Abbreviations: HCR high chlorpyrifos resistance; NS, not significant (P40.05). Sex ratio observed among the offspring of ♂MT/mt from various strains backcrossed to ♀mt/mt, in the absence of chlorpyrifos selection (control) or after 24 h of exposures to chlorpyrifos concentrations ⩾ 1 mg l -1 . The χ 2 tests the sex ratio deviation and has one degree of freedom (significance levels are indicated *Po0.05, ** Po0.01 and *** Po0.001).

ACKNOWLEDGEMENTS

We are very grateful to Sandra Unal and Raja Ben Cheikh for technical assistance and to Ariel Herold and Nicolas Pocquet for providing the Israel and Mayotte strains, respectively. This work was funded by ANR Morevol Santé-Environnement, ANR SilentAdapt (Ministère délégué à la Recherche) and by the Comité de Coopération Marseille-Provence-Méditerranée supervised by Robert Bismuth. Contribution 2015.187 of the Institut des Sciences de l'Evolution de Montpellier (UMR CNRS-UM2 5554).

DATA ARCHIVING

Bioassay data available from the Dryad Digital Repository: http://dx. doi.org/10.5061/dryad.vv46k.

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CONFLICT OF INTEREST

The authors declare no conflict of interest.

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