Olivier Gnankine
email: olivier.gnankine@univ-ouaga.bf

Laurence Mouton

Aly Savadogo

Thibaud Martin

Antoine Sanon

Roch Dabiré

Fabrice Vavre

Frédéric Fleury

Olivier Gnankiné

Roch K Dabire

Biotype status and resistance to neonicotinoids and carbosulfan in Bemisia tabaci (Hemiptera: Aleyrodidae) in Burkina Faso, West Africa

Keywords: Bemisia tabaci, insecticide resistance, neonicotinoid, carbamate, biotype

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

Introduction

The sweet potato whitefly Bemisia tabaci (Gennadius) is one of the most serious pests of many agricultural and ornamental crops in all tropical and subtropical regions. B. tabaci is considered as a complex of morphologically indistinguishable species which vary greatly with respect to host range, fecundity, insecticide resistance, ability to transmit plant viruses, and induction of plant disorders [START_REF] Brown | The sweet-potato or silverleaf whiteflies-biotypes of Bemisia tabaci or a species complex[END_REF][START_REF] Beitia | Current status of Bemisia tabaci (Gennadius) in Spain: The presence of biotypes of this species[END_REF][START_REF] Devine | Biotypes of Bemisia tabaci and their relevance to the evolution and management of insecticide resistance[END_REF][START_REF] Perring | The Bemisia tabaci species complex[END_REF][START_REF] Simon | Survey of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in Italy with the description of a new biotype (T) from Euphorbia characias[END_REF]. Although, De [START_REF] Barro | Bemisia tabaci: a statement of species status[END_REF] suggested that many biotypes of B. tabaci are cryptic species rather than races of B. tabaci, the taxonomy and systematics remain controversial. To date, 28 putative cryptic species within the B. tabaci complex were identified [START_REF] Mckenzie | Distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) biotypes in Florida-investigating the Q invasion[END_REF][START_REF] Dinsdale | Refined global analysis of Bemisia tabaci (Hemiptera: Aleyrodidae) mitochondrial cytochrome oxidase 1 to identify species level genetic boundaries[END_REF][START_REF] Shu-Sheng | Species concepts as applied to the whitefly Bemisia tabaci systematics: how many species are there?[END_REF]. Among the 28 putative species, the Middle East-Asia Minor 1 (MEAM1), known commonly as biotypes B and B2 and the Mediterranean (MED), known as biotypes Q, J, L and Subsaharan Africa Silverleaf biotypes species (ASL), are recognized as predominant in many areas [START_REF] Dinsdale | Refined global analysis of Bemisia tabaci (Hemiptera: Aleyrodidae) mitochondrial cytochrome oxidase 1 to identify species level genetic boundaries[END_REF]. In Q biotype, three groups were recently designated as Q1 (Western Mediterranean populations), Q2 (Middle Eastern populations) [START_REF] Chu | Genetic differentiation of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype Q based on mitochondrial DNA markers[END_REF], and Q3, restricted to populations from Burkina Faso [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. Hereafter, we have retained the commonly used term biotype here to link this study with previous literature.

Life-history traits such as resource exploitation, and resistance to insecticides may affect the distribution and frequency between populations of the B. tabaci species complex [START_REF] Pascual | Intra-and interspecific competition between biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) from Spain[END_REF][START_REF] Horowitz | Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance[END_REF][START_REF] Crowder | Mating behaviour, life history and adaptation to insecticides determine species exclusion between whiteflies[END_REF]. In agricultural areas, human activities including cultivation practices, the use of cultivated plants and pesticide treatments, create intense selection pressure on populations and may have a major influence on population demographics and on patterns of species distribution [START_REF] Reitz | Competitive displacement among insects and arachnids[END_REF][START_REF] Crowder | Mating behaviour, life history and adaptation to insecticides determine species exclusion between whiteflies[END_REF].

In West Africa, population outbreaks were reported in 1998 in cotton fields in Burkina Faso, Mali and Côte-d'Ivoire inducing severe crop damages [START_REF] Otoidobiga | Susceptibility of field populations of adult Bemisa tabaci Gennadius (Homoptera: Aleyrodidae) and Eretmocerus sp (Hymenoptera:Aphelinidae) to cotton insecticides in Burkina Faso (West Africa)[END_REF]. The resulting loss severely impacted economic activity of these countries since cotton production and distribution is one of the main financial resources, especially in Burkina Faso. In addition, in this country, monitoring of population dynamics on cotton revealed high levels of B. tabaci at the boll opening stage [START_REF] Gnankiné | Traitements insecticides et dynamique des populations de Bemisia tabaci en culture cotonnière au Burkina Faso[END_REF]. At this stage, B. tabaci causes damages indirectly through honeydew excretion leading to the sticky cotton. Consequently, the recommended pest management strategy at this stage is two foliar neonicotinoid treatments against whiteflies in combination with a pyrethroid against bollworms [START_REF] Gnankiné | Traitements insecticides et dynamique des populations de Bemisia tabaci en culture cotonnière au Burkina Faso[END_REF].

Farmers generally spray a mixture of acetamiprid and cypermethrin. This leads to select resistance particularly observed in Burkina Faso [START_REF] Otoidobiga | Susceptibility of field populations of adult Bemisa tabaci Gennadius (Homoptera: Aleyrodidae) and Eretmocerus sp (Hymenoptera:Aphelinidae) to cotton insecticides in Burkina Faso (West Africa)[END_REF][START_REF] Gnankiné | Évolution de la sensibilité des adultes de Bemisia tabaci Gennadius (Homoptera: Aleyrodidae) vis-à-vis de quelques insecticides dans l'Ouest du Burkina Faso[END_REF]Houndété et al. 2010a). Recently, Houndété et al. (2010a) showed resistance of B. tabaci to pyrethroids, such as deltamethrin and bifenthrin, to organophosphates (OPs), such as dimethoate and chlorpyriphos ethyl, and to neonicotinoids, such as acetamiprid and thiamethoxam. Unfortunately, that study was not related to B. tabaci populations identity collected in Burkina Faso in connection with agricultural practices. In the meantime, the presence of Q1 living in sympatry with the local biotype, Sub-saharan Africa Silverleafing (ASL), has been observed on cotton and vegetables crops [START_REF] Gnankiné | Symbiotic status of Bemisia tabaci (Homoptera: Aleyrodidae) and their prevalence in Western Africa[END_REF][START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. Q1 was shown to be dominant in Burkina Faso but was not detected in Benin and Togo where ASL was the only biotype [START_REF] Gnankiné | Symbiotic status of Bemisia tabaci (Homoptera: Aleyrodidae) and their prevalence in Western Africa[END_REF]. In these ASL populations, pyrethroid and OP resistance was highlighted (Houndeté et al. 2010a). In contrast, no neonicotinoid resistance was detected in populations from Benin.

To prevent outbreaks and to establish sustainable control of this pest, the susceptibility to pesticide used must be surveyed in connection with identification of B. tabaci populations, human activities and environmental factors. In Spain, the Q biotype exhibited a greater degree of pesticide resistance than the B biotype, which had better fecundity and competitive abilities [START_REF] Pascual | Intra-and interspecific competition between biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) from Spain[END_REF]. In Israel, Q excluded B biotype when insecticides were used whereas B rapidly evolved resistance to insecticides in the United States [START_REF] Horowitz | Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance[END_REF][START_REF] Khasdan | DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics[END_REF].

The present study aimed to estimate the proportion of different biotypes in populations sampled from cotton fields not yet investigated and evaluate their susceptibility to three neonicotinoids and one carbamate insecticide. Among them, acetamiprid is the only insecticide usually sprayed on cotton fields in Burkina Faso.

Materials and Methods

Whitefly populations

In 2007, at least 10,000 adults of B. tabaci were collected from cotton fields close to the villages of Sidéradougou, Houndé, Diébougou, Boromo, Solenzo, Datomo, Diabo, Diapaga, Pô and Bittou (Figure 1). The environmental factors and agricultural practices varied considerably in the different zones of the country (Table 1). In each area, after neonicotinoid treatments, B. tabaci adults were collected at random from 50 cotton plants using a mouth aspirator, then confined in a wooden rearing cage (50 by 35 by 35 cm) containing cotton seedlings, and returned to the laboratory within 2-5 h. Whiteflies of both sexes and different ages were tested the same day or the following day. At least, thirty (30) individuals of B. tabaci were collected from fields and conserved in alcohol (80 %) for molecular analysis in laboratory.

Insecticides

The following formulated insecticides were used for the bioassays: imidacloprid provided by Syngenta Agro AG (Dielsdorf, Switzerland). Among the insecticides tested, only acetamiprid at 12 g active ingredient/ha is used by farmers for cotton protection in Burkina Faso during the last treatments in combination with cypermethrin at 36 g a.i./ha (Gnankine et al. 2007). The other insecticides may be used at any time and any dosages for protecting vegetables in growing areas.

Determination of B. tabaci biotypes or genetic groups

Genomic DNA was extracted from each individual adult of B. tabaci in 26 µl of Nonidet P-40 extraction buffer [START_REF] Delatte | A new silverleaf-inducing biotype Ms of Bemisia tabaci (Hemiptera : Aleyrodidarum) indigenous to the islands of the south-west Indian ocean[END_REF]) and stored at -20 °C. Biotypes were identified using a PCR-RFLP based diagnostic assay. Briefly, in this method, a fragment of the mitochondrial marker CO1 (Cytochrome Oxidase 1 gene sequences, mtCOI) gene is amplified by PCR [START_REF] Frohlich | A phylogeographical analysis of the Bemisia tabaci species complex based on mitochondrial DNA markers[END_REF]) using universal COI primers C1-J-2195 (5'-TTGATTTTTTGGTCATCCAGAAGT-3)' and TL2-N-3014 (5'-TCCAATGCAC TAATCTGCCATATTA-3') [START_REF] Khasdan | DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics[END_REF]). The PCRs were composed of 25 µl Platinum PCR SuperMix 0.5 µl forward primer (10 pmol), 0.5 µl reverse primer (10 pmol) and 2 µl DNA template. The PCR products are then digested by the restriction endonucleases XapI (Fermentas) and/or BfmI (Fermentas), which generates clear polymorphism between biotypes B, MS, Q and Q1, Q2 or Q3 genetic groups. The PCR products were incubated with 10 U/μL XapI (Fermentas) at 37 °C for 3 h before loading onto agarose gel (Henri et al. 2012 submitted).

Bioassay

A leaf dip bioassay method was performed based on previous studies [START_REF] Rowland | Evaluation of insecticides in field control simulators and standard laboratory bioassays against resistant and susceptible Bemisia tabaci (Homoptera : Aleyrodidae) from Sudan[END_REF][START_REF] Cahill | Pyrethroid and organophosphate resistance in the tobacco whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae)[END_REF]. For each insecticide, discs (35 mm in diameter) of cotton leaves were immersed for 10 s in seven aqueous solutions of various concentrations of insecticide, or in distilled water (controls). Leaf discs were air dried for 30 min. Discs were then positioned individually on an agar-coated (7 g.l -1 ) in a vial (45 mm diameter). Adults of B. tabaci (30 individuals of mixed sex) were removed from cotton leaves with a mouth aspirator, chilled and transferred into small plastic vials containing the treated leaf discs. Each vial was then sealed with a transparent ventilated lid. When adults recovered from chilling, vials were stored upside down and maintained at 25 °C (± 2), 60% r.h. (± 5) and a 12:12 h light: dark photoperiod. An insect was considered alive if any sign of movement was observed. Mortality was recorded 24 h later. Three replicates were carried out for each concentration of insecticide and untreated controls. Mortality in the control was always <10% and data from all bioassays were corrected for control mortality using Abbott's formula (Abott 1925).

Statistical analyses

Impact of geographical location on the distribution of biotypes

The data collected were subjected to Fisher's exact test with simulated p-values based on 2000 replicates using R statistical software (http://www.R-project.org).

Doses response bioassays

All bioassay replicates were combined for analysis. LC 50 values were calculated by global optimisation by simulated annealing (GOSA), available at http://bio-log.biz. This software tests the linearity of dose-mortality responses and provides the slope, lethal concentrations (LC 50 ) and 95% confidence limits (CL). A population is considered to be significantly (P < 0.05) more (or less) resistant than another population when there is no overlap of the 95% confidence limits of the LC 50 . The resistance ratios (RR) were calculated following the report of various LC 50 on the LC 50 of the most susceptible field population.

Results

Biotype or genetic groups

In our study, all B. tabaci from populations collected belonged to the Africa/Middle East/Asia

Minor group (biotypes B, Q, ASL, Ms) (Figures 1; 2). A significant relationship was found between the geographical range and the biotype distribution (Fisher's Exact Test, P=0.0005).

Q1 biotype was largely predominant according to various areas. It has been observed in Sidéradougou, Diébougou, Solenzo, Datomo, Diabo, Diapaga, and Bittou. The Q1 biotype was found in sympatry with the ASL biotype in populations from Boromo, Houndé, and Pô.

Resistance to insecticides

Populations with only Q1 appeared to be more resistant to acetamiprid, thiamethoxam and carbosulfan (Table 2). Among the ten B. tabaci populations tested, those living in sympatry (ASL and Q1) from Boromo, Houndé and Pô were in most cases more susceptible to the three neonicotinoids (Figure 1; Table 2). On the contrary, the highest neonicotinoid resistant populations were all from pure Q1 such as the populations from Diabo, Solenzo and Bittou highly resistant to acetamiprid, imidachloprid and thiamethoxam respectively. In the absence of reference strain, the B. tabaci population from Houndé was used as reference to calculate the resistance ratio (RR) for neonicotinoids. As for neonicotinoids, the three B. tabaci populations from Boromo, Houndé and Pô, where ASL and Q1 lived in sympatry, were among the most susceptible to carbosulfan (Figure 1;

Table 2). In the absence of reference strain, the B. tabaci population from Boromo was used as reference to calculate the resistance ratio (RR) for carbosulfan. The highest carbosulfan resistant populations were observed in pure Q1 populations such as Datomo and Sideradougou (RR 50 7 to 14). Q1 populations from Houndé, Diebougou and Po exhibited some variation in their response towards carbosulfan compared to Boromo (RR 50 2 to 5).

Discussion

According to the determination of biotypes, the populations of B. tabaci tested belonged to the Africa/Middle East/Asia Minor group (biotypes B, Q, ASL, Ms). Q and Africa-SL biotypes were identified in MED species [START_REF] Boykin | Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of mitochondrial CO1 DNA sequences[END_REF][START_REF] Barro | Bemisia tabaci: a statement of species status[END_REF]. In Q biotype, three groups were recently designated as Q1, Q2 [START_REF] Chu | Genetic differentiation of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype Q based on mitochondrial DNA markers[END_REF]) and Q3 [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]. [START_REF] Gnankiné | Symbiotic status of Bemisia tabaci (Homoptera: Aleyrodidae) and their prevalence in Western Africa[END_REF] showed that the Q1 was predominant in cotton fields and is probably progressively displacing the African ASL biotype. Q1 originating from the Mediterranean region is generally considered to be an invasive biotype like B. It is the real B.

tabaci [START_REF] Tay | Will the Real Bemisia tabaci Please Stand Up?[END_REF], and has now begun its own global invasion spreading from its Mediterranean home range to at least 10 different countries (De [START_REF] Barro | Bemisia tabaci: a statement of species status[END_REF][START_REF] Dalton | Whitefly infestations: the Christmas invasion[END_REF].

Our results confirmed the resistance of B. tabaci populations from Burkina Faso to neonicotinoids (Houndété et al. 2010a) and showed the resistance to one carbamate. The resistance ratios (RR) showed a variation between the populations from different localities and genetic groups. It varied from 4 to 50 for the neonicotinoids and from 2 to 14 for the carbamate. The resistance ratios for the neonicotinoids were much higher than those obtained three years ago by Houndété et al (2010a) in populations collected in Soumousso and Tiara, Burkina Faso (RR = 3 to 8). The increase of the neonicotinoid resistance ratio might be explained by the use of acetamiprid in the two last insecticide sprays on cotton recommended by the ginning companies providing insecticides, at credit, to cotton farmers. However, a part of these insecticides is deflected to the black economy. That is the reason why most of insecticides used for protecting vegetable comes from cotton [START_REF] Ahouangninou | Characterization of health and environmental risks of pesticide use in market-gardening in the rural city of Tori-Bossito in Benin, West Africa[END_REF]). On the contrary, the low resistance level of B. tabaci to carbosulfan may be explained by the nouse of this insecticide for cotton protection. Up to now, to control whiteflies, farmers use acetamiprid that was progressively introduced in 2000 as a substitute for OPs that become inefficient due to acetylcholinesterase resistance (Houndété et al. 2010b).

Our results suggest that the neonicotinoid resistance status of B. tabaci is linked to the biotype, especially to Q1. Indeed, the highest resistance factors to acetamiprid and thiamethoxam were recorded in the Q1 populations. One noteworthy finding is that the correlation between the neonicotinoid resistance and the biotype was not clearly demonstrated. However the most susceptible B. tabaci populations were collected in Boromo and Houndé. These populations were composed by Q1 and ASL individual with quite the same prevalence. In Benin, previous studies have already identified neonicotinoid susceptible populations as belonging to the ASL biotype compared with heterogeneous populations from Burkina Faso that were resistant [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF]Houndété et al. 2010b;[START_REF] Gnankiné | Symbiotic status of Bemisia tabaci (Homoptera: Aleyrodidae) and their prevalence in Western Africa[END_REF]. The susceptibility of B. tabaci population from Diapaga compared to other areas with pure Q1 biotype could be due to the low insecticide pressure coupled with the absence of vegetable crops around. This result suggests that individuals from Q1 biotype should be always susceptible to neonicotinoid. That did not exclude resistance to other insecticides.

Recently, Mouton et al. (unpublished data) have demonstrated the presence of kdr and Ace1-R genes in this population. In Israel, [START_REF] Horowitz | Biotype Q of Bemisia tabaci identified in Israel[END_REF] showed that the Q-type was predominant in areas where resistance to pyriproxyfen evolved rapidly. Cases of resistances of B. tabaci Q populations to pyrethroids were demonstrated in Greece by [START_REF] Roditakis | Current status of insecticide resistance in Q biotype Bemisia tabaci populations from Crete[END_REF]. Moreover, in southern Spain, the Q biotype was linked with high and cross-resistance to neonicotinoids [START_REF] Nauen | Toxicological and mechanistic studies on neonicotinoid cross resistance in Q-type Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF][START_REF] Guirao | Biotype determination in Spanish populations of Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF]. After a few years, Q had almost displaced the B, especially in southern Spain [START_REF] Simón | Variación genética de poblaciones de Bemisia tabaci (Gennadius) en la Cuenca del Mediterráneo occidental[END_REF] probably because of the increased use of neonicotinoids against whiteflies. Our results confirmed also the cross resistance between neonicotinoids as Sideradougou population for example was highly resistant to both acetamiprid (23.8-fold) and thiametoxam (24.7-fold). The extreme resistance to acetamiprid in Diabo Q1 populations (50-fold) is probably due to the extensive use of this insecticide. As far as imidachloprid is concerned, Solenzo appears to be the only populations displaying highest resistance (13.8-fold). [START_REF] Karunker | Over-expression of cytochrome P450 CYP6CM1 is associated with high resistance to imidacloprid in the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF] showed that cytochrome P450 CYP6CM1 was implied in the metabolic resistance of neonicotinoids and appears to be specific for imidachloprid [START_REF] Roditakis | Assessment of the Bemisia tabaci CYP6CM1vQ transcript and protein levels in laboratory and field-derived imidacloprid-resistant insects and cross-metabolism potential of the recombinant enzyme[END_REF]. It has been demonstrated that overexpression of CYP6CM1 is associated with high levels of imidacloprid resistance in B. tabaci [START_REF] Karunker | Over-expression of cytochrome P450 CYP6CM1 is associated with high resistance to imidacloprid in the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF]).

The current status of B. tabaci resistance to neonicotinoid insecticides from West Africa requires urgent attention if neonicotinoids really selected the invasive Q biotype. These results

show again the negative effect of using continuously the same molecule, or the same family of insecticides, year after year until inefficiency. Moreover, our results suggested the selection of a new multiresistant phenotype may threaten the production of cotton or vegetable in the future. Indeed, the spread of a potentially more prolific vector for begomovirus virus will surely lead to a crisis in the production of Solanaceae and especially tomatoes by small-scale farmers in West and Central Africa [START_REF] Hanafi | La mouche blanche et les virus des feuilles en cuillère de la tomate (TYLCV)[END_REF].

The use of genetically modified Bt cotton in Burkina Faso since 2008 could be one way to avoid the selection of B. tabaci resistant populations by reducing insecticide use, which naturally enhances the impact of natural enemies. The suppression of insecticide treatments against whiteflies that are systematically applied before harvest could have a significant impact on sticky cotton. But this problem can easily be solved by harvesting cotton not too late after boll opening or by controlling the degree of stickiness before the ginning process.

(

  Confidor 200 SL), provided by Bayer AG (Leverkusen, Germany), acetamiprid (Mospilan 200 SL) provided by Arysta Life Science (Noguères, France), thiamethoxam (Actara 240 SC) from Syngenta Crop Protection AG, (Basel, Switzerland) and carbosulfan (Marshal 25 EC)

  Q1 populations from Diapaga, Boromo andBittou exhibited some variation in their response towards acetamiprid compared to Houndé (RR 50 2 to 9), while highest resistance (RR 50 23 to 50) was observed in the populations from Sidéradougou (Q1), Po (Q1 and ASL), and Diabo (Q1). Q1 and ASL populations from Boromo and Po exhibited some variation in their response towards imidacloprid compared to Houndé (RR 50 1 to 2), while highest resistance (RR 50 5 to 14) was observed in the Q1 populations from Datomo, Diebougou, and Solenzo. Q1 and ASL populations from Boromo and Po exhibited some variation in their response towards to thiamethoxam compared to Houndé (RR 50 2 to 4), while highest resistance (RR 50 13 to 33) was observed in the Q1 populations from Diabo, Solenzo and Bittou.

Acknowledgements:

We thank Zagré Blaise, Yé Moumine and Handé Samou (INERA, Programme Coton) for their technical assistance. This project was funded by Sofitex, Burkina Faso.


Gabriel Terraz
email: g.terrazgabriel.terraz@univ-lyon1.fr

Gwénaelle Gueguen

Frédéric Fleury

Judit Laurence Mouton

Arnó Affiliation

Nuclear and cytoplasmic differentiation among Mediterranean populations of Bemisia tabaci: testing the biological relevance of cytotypes

Keywords: Bemisia tabaci, MED species, hybridization, microsatellites, insecticide resistance gene, bacterial endosymbionts

published or not. The documents may come   L'archive ouverte pluridisciplinaire

INTRODUCTION

Bemisia tabaci is one of the most devastating pests of the world, directly through plant sap feeding and indirectly by virus transmission. [START_REF] Jones | Plant viruses transmitted by whiteflies[END_REF] Historically, based on significant variations between populations, biotypes had been identified using ecological traits like host plant range, virus transmission abilities, insecticide resistance, enzymatic variability and genetic markers such as mitochondrial cytochrome oxidase gene (mtCO1). [START_REF] Boykin | Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of mitochondrial COI DNA sequences[END_REF][START_REF] Horowitz | Biotypes B and Q of Bemisia tabaci and their relevance to neonicotinoid and pyriproxyfen resistance[END_REF][START_REF] Perring | The Bemisia tabaci species complex[END_REF] But, because some of these characters are shared between genetic groups or show within-group variability higher than between group variations, the term biotype is in fact inappropriate. [START_REF] De Barro | Bemisia tabaci: a statement of species status[END_REF] Based on a 3.5% mtDNA sequence divergence criterion, it has been proposed that B. tabaci is actually made up of at least 24 morphologically indistinguishable species [START_REF] De Barro | Bemisia tabaci: a statement of species status[END_REF][START_REF] Dinsdale | Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries[END_REF] and, recently, this number has been increased to 31. [START_REF] Alemandri | Species within the Bemisia tabaci (Hemiptera: Aleyrodidae) complex in soybean and bean crops in Argentina[END_REF][START_REF] Chowda-Reddy | Bemisia tabaci Phylogenetic Groups in India and the Relative Transmission Efficacy of Tomato leaf curl Bangalore virus by an Indigenous and an Exotic Population[END_REF][START_REF] Hu | An extensive field survey combined with a phylogenetic analysis reveals rapid and widespread invasion of two alien whiteflies in China[END_REF][START_REF] Lee | Taxonomic status of the Bemisia tabaci complex (Hemiptera: Aleyrodidae) and reassessment of the number of its constituent species[END_REF] Belonging to 11 major groups, these putative species regroup the previously described biotypes. This view is consistent with some genetic data accumulated on reproductive incompatibilities within the B. tabaci species complex. [START_REF] Xu | Reproductive incompatibility among genetic groups of Bemisia tabaci supports the proposition that the whitefly is a cryptic species complex[END_REF][START_REF] Liu | Species Concepts as Applied to the Whitefly Bemisia tabaci Systematics: How Many Species Are There?[END_REF] Beside the number of species in B. tabaci complex, several questions remain open such as whether the mtDNA sequence divergence threshold used for species boundaries identification (3.5%) is well founded. It has been suggested that this value is more or less arbitrary and probably under-estimated. For example, Lee et al. [START_REF] Lee | Taxonomic status of the Bemisia tabaci complex (Hemiptera: Aleyrodidae) and reassessment of the number of its constituent species[END_REF] proposed to increase this threshold up to 4% because there is a lot of variability within species, particularly within the Mediterranean (MED) species which appears to be genetically diverse, not only because it includes the J, ASL, L and the highly damaging Q biotypes, [START_REF] De Barro | Bemisia tabaci: a statement of species status[END_REF] but also because high genetic variability has been observed within the Q biotype leading to the recognition of 4 mtCOI haplotypes (Q1 to Q4). [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF][START_REF] Chu | Use of mitochondrial cytochrome oxidase I polymerase chain reactionrestriction fragment length polymorphism for identifying subclades of Bemisia tabaci Mediterranean group[END_REF] This variability could be explained by the fact that MED species may regroup several entities with high genetic differentiation and possible separate species, challenging MED species status. We thus clearly need more information to determine what are the true interbreeding limits of the putative species within the B. tabaci complex, and whether some reproductive incompatibilities occurs within groups recently described as putative species.

In addition to the uncertainty of the mtDNA divergence threshold proposed for species recognition, another challenging point concerns mitochondrial DNA marker, which is probably not the best marker to identify the limit of species. As in a number of other arthropods, mtDNA undergoes indirect selection due to vertically transmitted microorganisms invading host populations. [START_REF] Jiggins | Rapid Insect Evolution by Symbiont Transfer[END_REF] Linkage disequilibrium between mtDNA haplotypes and bacterial endosymbionts may blur the clade history and the history of infection. Moreover, horizontal transfers of bacterial endosymbionts followed by their invasion in the host population can result to rapid spread of mtDNA lineages without any reproductive barriers. This is achieved by their ability to manipulate host reproduction through different ways notably by inducing cytoplasmic incompatibility (for review see Engelstädter & Hurst, [START_REF] Engelstädter | The Ecology and Evolution of Microbes that Manipulate Host Reproduction[END_REF] ). In addition to their effect on host reproduction, symbiotic bacteria may also play an important role in host ecology, host evolution and speciation via a wide range of effects (review in Ferrari & Vavre,[START_REF] Ferrari | Bacterial symbionts in insects or the story of communities affecting communities[END_REF] ) such as resistance to natural enemies, [START_REF] Oliver | Facultative bacterial symbionts in aphids confer resistance to parasitic wasps[END_REF] resistance to insecticides and host-plant specialization. [START_REF] Kontsedalov | The presence of Rickettsia is associated with increased susceptibility of Bemisia tabaci ( Homoptera : Aleyrodidae ) to insecticides[END_REF][START_REF] Tsuchida | Host Plant Specialization Governed by Facultative Symbiont[END_REF] They can also increase their host fitness in some contexts. [START_REF] Himler | Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias[END_REF] Moreover, they can be involved in the emergence of nuclear variation which can lead to speciation. [START_REF] Brucker | Speciation by symbiosis[END_REF] B. tabaci, like most phloem-feeding insects, harbours an obligatory maternally transmitted endosymbiont, Portiera aleyrodidarum, which provides necessary nutrients. B. tabaci also hosts a wide diversity of facultative bacteria since seven secondary endosymbionts, all maternally transmitted, were reported. [START_REF] Zchori-Fein | Diversity of Prokaryotes Associated with <I>Bemisia tabaci</I> (Gennadius) (Hemiptera: Aleyrodidae)[END_REF][START_REF] Bing | Diversity of secondary endosymbionts among different putative species of the whitefly Bemisia tabaci[END_REF] This symbiotic community appears species specific but also show variations among species. [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF][START_REF] Chu | Biotype Status and Distribution of Bemisia tabaci ( Hemiptera : Aleyrodidae ) in Shandong Province of China Based on Mitochondrial DNA Markers Biotype Status and Distribution of Bemisia tabaci ( Hemiptera : Aleyrodidae ) in Shandong Province of China Base[END_REF][START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF] For example, the 4 haplotypes identified within the MED species harbour distinct bacterial communities. [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF][START_REF] Chu | Biotype Status and Distribution of Bemisia tabaci ( Hemiptera : Aleyrodidae ) in Shandong Province of China Based on Mitochondrial DNA Markers Biotype Status and Distribution of Bemisia tabaci ( Hemiptera : Aleyrodidae ) in Shandong Province of China Base[END_REF][START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF] Q1 harbours Hamiltonella and Cardinium whereas Q2 contains Rickettsia and Arsenophonus. Wolbachia can be found in both Q1 and Q2 but was never observed in Q3, which hosts a separate Arsenophonus strain and Rickettsia. Q4 is a rare haplotype only detected in Croatia and for which bacterial infection remains unknown. In this paper, we will use the term 'cytotype' to designate a mtCOI haplotype associated with a specific combination of symbiotic bacteria. These cytotypes have different geographic localizations since Q3 was recorded only in Africa whereas Q1 is localized in the large west part of the Mediterranean basin: Morocco, Tunisia, Portugal, France, Greece and Spain. [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF][START_REF] Dalmon | Genetic structure of the invasive pest Bemisia tabaci: evidence of limited but persistent genetic differentiation in glasshouse populations[END_REF][START_REF] Gorsane | Molecular characterization of Bemisia tabaci populations in Tunisia: genetic structure and evidence for multiple acquisition of secondary symbionts[END_REF][START_REF] Tsagkarakou | Biotype status and genetic polymorphism of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in Greece: mitochondrial DNA and microsatellites[END_REF] All data available at the time on the B. tabaci MED species in France and Spain indicated the mere presence of the Q1 cytotype. 27,30- 33 This cytotype has been found in several parts of the world like in China and America, while Q2 is restricted to the Middle East countries of Mediterranean basin: Israel and Cyprus where Q1 is absent. [START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF][START_REF] Chu | Genetic differentiation of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype Q based on mitochondrial DNA markers[END_REF][START_REF] Hsieh | Evidence from molecular markers and population genetic analyses suggests recent invasions of the Western North Pacific region by biotypes B and Q of Bemisia tabaci (Gennadius)[END_REF] Q1 and Q2 cytotypes overlap in neighboring countries such as Turkey, Egypt and Syria (Genbank records). [START_REF] Dinsdale | Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries[END_REF][START_REF] Rua | New insights into the mitochondrial phylogeny of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in the Mediterranean Basin[END_REF] As these two cytotypes harbour very different bacterial communities, MED species provides a good framework to test and understand the role of these communities in the B. tabaci species complex ecology and evolution. This particular situation also gives good opportunities to test whether the 3.5% mtCO1 threshold reflect the true limit of species within the B. tabaci species complex in which bacterial endosymbiont infection is pervasive. This is particularly important for pest management perspectives.

In this study, we report the presence of the Q2 cytotype in Western Europe in sympatry with Q1. We followed the dynamic of colonization of the Q2 cytotype in France and Spain, which coexist locally with Q1 cytotype. These sympatric situations give us the opportunity to test the biological limit of Q1 and Q2 cytotypes by the use of microsatellite markers (expected to be neutrals) and nuclear genetic differentiation analysis. We also analyzed insecticide resistance genes (expected to be under selection) because of extensive use of insecticides to control B. tabaci populations. [START_REF] Alon | Multiple origins of pyrethroid resistance in sympatric biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF] Moreover, we tested whether symbionts are involved in any nuclear differentiation and reproductive barriers. 1). Sampled adults were placed alive in ethanol (96%) and stored at -20°C until DNA extraction. Besides the populations sampled in France and Spain in order to have Q1 and Q2 "reference" cytotypes, we used samples from Greece and Israel where only one Q cytotype had been reported to date, Q1 in Greece and Q2 in Israel. This is confirmed by the recent study of Gauthier et al (this issue).

MATERIAL AND METHODS

Insect lines and rearing

Two lines belonging to Q1 and Q2 were used for crossing experiments. The Q1 line, originated from Sigean (France, Languedoc Roussillon), is infected by Hamiltonella and Cardinium. The Q2 line, originated from Lyon (France, Rhône-Alpes), is infected by Rickettsia and Arsenophonus. Both lines are fixed for bacterial infection. These lines were maintained in the lab for at least one year (about 17 generations) before the experiments. They are reared on Hibiscus plants at 25°C with an LD cycle of 16:8 and 70% relative humidity. Their cytotypes (mtCOI and associated symbionts) are regularly verified by PCR.

Crossing experiments

Q1 and Q2 B. tabaci pupae were isolated and sexed from one to four hours after emergence. All four possible crosses were performed (crosses between Q1 males and females, crosses between Q2 males and females, crosses between Q1 males and Q2 females and reciprocal crosses) with 10 replicates each. Five females and males were placed on 30mm diameter excised Hibiscus leaf disks on 1% agar medium in a 50mm Petri dish for 6 days. All laid eggs and hatched eggs were counted as well as emerging adults, which were also sexed to determine the offspring sex-ratio.

Molecular Biology

DNA extraction

For each individual total DNA was extracted in 26µL of a buffer containing 50mM KCl, 10mM

Tris-base pH 8, 0.45% Nonidet P-40, 0.45% Tween 20, and 50mg/mL Proteinase K. After 3h at 65°C samples were incubated at 100°C for 15min. A volume of 35μL of pure water was added to this extract which was then stored at -20°C until use.

mtCOI haplotype determination

Cytotypes were identified by using a PCR-RFLP diagnostics tool as described in Henri et al., (submitted), [START_REF] Henri | Development of a PCR-RFLP method for molecular characterization of genetic diversity within the Africa/Middle East/Asia Minor major group of the Bemisia tabaci species complex[END_REF] which allows the discrimination of all cytotypes within the MED species. Briefly, amplification was performed on a part of the mitochondrial cytochrome oxidase 1 gene (mtCO1) using the primers C1-J-2195 (5'-TTGATTTTTTGGTCATCCAGAAGT-3') and L2-N-3014 (5'-TCCAATGCACTAATCTGCCATAATA-3'). [START_REF] Frohlich | A phylogeographical analysis of the bemisia tabaci species complex based on mitochondrial DNA markers[END_REF] PCR reactions were performed in 25μL volumes containing 2.5µL Buffer, 200μM dNTP, 200nM primers, 0.5U DNA polymerase (DreamTaq, Fermentas) and 2μL of DNA template. The cycling conditions consisted of an initial denaturation at 95°C for 5min followed by 30 cycles of 1min at 95°C, 1min at 50°C, 1min at 72°C and a final extension at 72°C for 10min. Some of the PCR products (2 to 20 individuals per population) were also sequenced for confirmation.

Detection of endosymbiotic bacteria

The six most frequent secondary symbiotic bacteria found in B. tabaci (Wolbachia, Cardinium, Hamiltonella, Rickettsia, Arsenophonus and Fritschea) were screened for each individual using specific PCR primers, as described in Gnankiné et al. [START_REF] Gnankiné | Distribution of Bemisia tabaci (Homoptera: Aleyrodidae) biotypes and their associated symbiotic bacteria on host plants in West Africa[END_REF] All samples were tested for the presence of the obligatory symbiont Portiera aleyrodidarum to test for the quality of the DNA extraction.

Primers and PCR conditions are indicated in table 2.

Insecticide-resistant alleles detection

Resistant and susceptible alleles were identified in two genes associated with insecticide resistance in B. tabaci: the para-type voltage gated sodium channel and the ace1 genes. Two mutations in the para-type voltage gated sodium channel gene, L925I and T929V (r1 and r2 alleles) were sought, and one mutation in the acetylcholinesterase enzyme ace1 (F331W, R allele). These mutations confer resistance to pyrethroids (Pyrs) and organophosphates (OPs) respectively. L925I and F331W resistant mutations were identified by using the PCR-RFLP tool developed by Tsagkarakou et al. [START_REF] Tsagkarakou | Molecular diagnostics for detecting pyrethroid and organophosphate resistance mutations in the Q biotype of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF] The T929V resistant mutation was detected by PCR using primer pairs that amplified only one of the two alleles. [START_REF] Tsagkarakou | Molecular diagnostics for detecting pyrethroid and organophosphate resistance mutations in the Q biotype of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF] Screening was performed on 7 to 29 individuals per population (Table 3). Twelve ambiguous PCR products were sequenced for verification.

Microsatellite genotyping

7 microsatellites loci were used as neutral nuclear markers to study genetic differentiation among populations and sympatric cytotypes (Table 4). B. tabaci is a haplo-diploid species, i.e. males hatch from unfertilized eggs and thus are haploids, and therefore genotyping was done on females only.

Since in Greece and Israel, only one Q cytotype has been described yet (Q1 and Q2 respectively), we expected that they have never been hybridized. In the present study, we have considered populations located in these countries as reference for Q1 and Q2 cytotypes. Around 30 individuals were genotyped from each (Table 1). Among the 531 individuals collected in France and Spain between 2006 and 2010, 242 individuals were genotyped from four localities where Q1 and Q2 cytotypes were found in sympatry (2 sites per country). PCR reactions were performed separately for each primer pair in 10μL volumes containing 200µM of dNTP, 200nM of each primer (fluorescently labelled in 5') and 0.5IU Blue Taq DNA Polymerase (Eurobio). Cycling conditions were initial denaturing at 94°C for 2 min, followed by 35 cycles of 30 sec at 94°C for denaturation, 30 sec at 52 or 57°C depending on the primer set (Table 4) for annealing and 1 min at 72°C for elongation, with a final elongation at 72°C for 10 min. Fluorescent amplicons were loaded on an ABIPrism 3100-Avant Genetic Analyser (Applied Biosystem) automated sequencer and allele sizes were calculated using GeneMapper software (Applied Biosystems).

Genetic data analyses

The mean number of alleles per locus (A¯), the observed heterozygosity (HO) and the unbiased expected heterozygosity (HE) were calculated using Genetix. Weir and Cockerham's estimators of F IS within populations and genetic groups were calculated using Genepop, as well as exact tests for Hardy-Weinberg equilibrium. [START_REF] Weir | Estimating F-Statistics for the Analysis of Population Structure[END_REF][START_REF] Raymond | Genepop (Version-1.2) -Population-Genetics Software for Exact Tests and Ecumenicism[END_REF] Linkage equilibrium between all pairs of loci and F estimates of differentiation [START_REF] Weir | Estimating F-Statistics for the Analysis of Population Structure[END_REF] were calculated and tested with 5000 permutations using Genepop. The level of population structure was assessed using the software STRUCTURE [START_REF] Pritchard | Inference of population structure using multilocus genotype data[END_REF] which differentiates mixed populations on the basis of allele frequency at each locus. Following options were used: 500 000 Markov Chain Monte Carlo with a burn-in period of 50 000, we allowed for admixture and correlated allele frequencies; log-likelihood estimates were calculated for K=1 to 15 with 10 replicates each. STRUCTURE assumes Hardy-Weinberg equilibrium (HWE) and linkage equilibrium within each group, that's why both hypotheses were tested subsequently using exact tests implemented in Genepop 4.0. We also conducted a Principal Component Analysis (PCA), which does not make any assumption such as HWE and linkage equilibrium, with R software, [START_REF]R: A Language and Environment for Statistical Computing[END_REF] adegenet package. [START_REF] Jombart | adegenet: a R package for the multivariate analysis of genetic markers[END_REF] In addition, a hierarchical analysis of molecular variance (AMOVA) was performed, using hierfstat package, [START_REF] Goudet | HIERFSTAT, a package for R to compute and test hierarchical F -statistics[END_REF] implemented in R software, to test for geographical, hostplant, cytotype and secondary symbionts effect. Significance was assessed with permutation tests (5 000 permutations). Resistant alleles frequencies were subjected to Fisher's exact test with simulated p-values based on 1000 replicates using R statistical software, [START_REF]R: A Language and Environment for Statistical Computing[END_REF] while HWE was tested by X 2 with R statistical software. [START_REF]R: A Language and Environment for Statistical Computing[END_REF]

RESULTS

Field survey in France and Spain

From 2006 to 2010, a survey of B. tabaci populations was performed in France and Spain to follow the geographical distribution of B. tabaci cytotypes. 511 individuals from 10 localities were collected (Table 5). We also add data from Dalmon et al., [START_REF] Dalmon | Genetic structure of the invasive pest Bemisia tabaci: evidence of limited but persistent genetic differentiation in glasshouse populations[END_REF] and Gueguen et al., [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF] collected from 2003 to 2006 (302 individuals from 14 localities) for reference situation (Figure 2A). Almost all individuals belong to the MED species, except in two localities, where the MEAM1 species has been observed, in a botanical garden in Nice (France) in 2004 and in Cabrils (Spain) in 2007.

However, as this situation is an exception and concern only very few individuals, MEAM1 species will not be taken into account and discussed further in this study. From 2003 to 2006, all the individuals belonged to the Q1 cytotype, as observed in all the western part of the Mediterranean basin. Interestingly, in 2007, for the first time, Q2 cytotype was detected in several localities either in sympatry with Q1 individuals in four locations in France and Spain, or not: only Q2 was found in very high density in a greenhouse in Lyon (France). Co-existence of Q1 and Q2 cytotypes has also been observed in 2010 in all the sampling sites (Figure 2). Proportion of Q2 individuals in the situation of sympatry varied according to the collection from 6% to 83% in 2007. They predominated in three (83%, 71% and 64%) of four localities in 2010 (22% of frequency in the site where Q1 prevails).

Analysis of reference Q1 and Q2 cytotypes

To date, only Q1 cytotype has been detected in Greece and Q2 in Israel. [START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF][START_REF] Tsagkarakou | Population genetic structure and secondary endosymbionts of Q Bemisia tabaci (Hemiptera: Aleyrodidae) from Greece[END_REF][START_REF] Gauthier | Twelve new polymorphic microsatellite loci and PCR multiplexing in the whitefly, Bemisia tabaci[END_REF] In our sampling, the determination of the mtCOI cytotype of individuals collected in three localities in Greece (10 per locality) and one site in Israel (n=29) confirmed these data. We therefore used them as reference samples for Q1 and Q2 cytotypes.

Endosymbiotic community

Individual detection of the seven main bacteria infecting B. tabaci revealed that these cytotypes harbour a specific symbiotic community as already reported. [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF][START_REF] Chiel | Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci[END_REF] The most common bacteria are Hamiltonella for Q1 and the association Arsenophonus ⁄ Rickettsia for Q2 (Figure 1).

Detection of sodium channel and ace1 resistant mutations

Resistance mutations in ace1 (F331W) and para-type voltage gated sodium (L925I and T929V) genes were identified by PCR-RFLP or PASA-PCR. [START_REF] Tsagkarakou | Molecular diagnostics for detecting pyrethroid and organophosphate resistance mutations in the Q biotype of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF] . The tree mutations were detected in both cytotypes: F331W (R allele) is fixed in Q1 and Q2 while L925I (r1 allele) has intermediate frequencies in the two cytotypes (0.70 and 0.74 respectively). Q1 and Q2 cytotypes significantly differ only in the frequencies of the T929V mutation (r2 allele ; 0.02 and 0.29 respectively; Fisher's exact test: p < 10 -4 ).

Microsatellite analysis

The genetic analysis of the 59 individuals genotyped with 7 microsatellites loci revealed a higher allelic richness in the Q1 than in the Q2 cytotype (4.3 versus 3.4 alleles; Table 5). Analysis carried out using STRUCTURE revealed a very high genetic differentiation between Q1 and Q2 cytotypes, which is confirmed by PCA (Figure 1). Pairwise F ST value reaches 0.27, which is very high for two groups that belong to the same putative species.

Nuclear and cytoplasmic differentiation of sympatric Q1 and Q2 cytotypes

Endosymbiotic community

Fritschea was never detected and will not be further discussed. The bacterial screening did not reveal any new association between a mitochondrial haplotype and the symbiotic community compared to reference populations (Israeli and Greek), meaning that the cytoplasmic associations are stable (Figure 3). In Q1 cytotype, Hamiltonella is almost fixed (93% of individuals infected) while Wolbachia and Cardinium are present at intermediate frequencies (respectively 28% and 38%), with a high frequency of bi-infections (62%). In Q2 cytotype, Rickettsia is also almost fixed (95%) while Wolbachia and Arsenophonus are found at a lower frequency (respectively 69% and 62%). In Lyon Wolbachia is absent.

Detection of sodium channel and ace1 resistant mutations

In the French and Spanish sympatric populations sampled in 2010 (242 females tested), all the three resistant mutations were detected in both Q1 and Q2 cytotypes in all the localities with a high variability in frequency (Table 3). All individuals were homozygous for F331W (R allele) of ace1 gene. L925I (r1 allele) was highly frequent (0.56 to 1) and frequencies of T929V (r2 allele) were low (0 to 0.30). These frequencies didn't differ between Q1 and Q2 cytotypes within all the four localities (Fisher's exact tests: p > 0.05). No significant deviations from Hardy-Weinberg equilibrium were detected between genotype frequencies within three localities (regardless of the cytotype involved) for both kdr mutations (Fréjus, Saint-Laurent du Var, and Viladecans, X 2 : p>0.05). Only the population from Cabrils deviates slightly from HWE for T929V (X 2 : p=0.01).

Microsatellite analysis

The 242 females previously used for resistant mutation detection, were genotyped based on 7 microsatellites loci (Table 5). A significant deviation from HWE (p< 0.001) was observed within all localities caused by a systematic heterozygous deficiency which explain the high F IS values within localities (0.243 to 0.357). This deficiency is not due to a particular locus, since all the observed heterozygosity is inferior to the expected heterozygosity in all loci. Within the four localities, the F IS is not higher when all individuals are consider than when cytotypes are considered separately (Table 5) thus suggesting that high F IS are not explained by a genetic structure that differ among cytotypes. This result is confirmed by STRUCTURE software analysis, which include sympatric Q1 and Q2 as well as the Q1 (Greek) and Q2 (Israeli) reference cytotypes since three clusters have been identified. The first cluster regroups all the Q2 reference individuals (from Israel). All Greek Q1 individuals are assigned to a second cluster. Individuals of sympatric population whatever Q1 or Q2 cytotypes belong to a third cluster but for the majority of them they are assigned with different probabilities to the Greek cluster and this third one. The probability of individual assignment to each cluster is shown in figure 4. Sympatric Q1 and Q2 cytotypes thus show similar genetic composition that probably results from the hybridization of two entities one being close to Greek population. The principal components analysis (PCA, Figure 5) of allele frequencies confirms the STRUCTURE software Bayesian analysis. The first axis clearly separates the three groups: mixed Q1-Q2 (Western part), reference Q1 (Greece) and reference Q2 (Israel). The second axis weakly discriminated between Greek Q1 and mixed populations. Pairwise comparisons between localities of these geographic groups are very high (0.09<F ST(Mixed-Greece) <0.14; 0.29<F ST(Mixed-Israel) <0.33). The point is that Q1 and Q2 cytotypes are totally homogeneous when they are sympatric.

We have successively tested for the effects of plant and symbiotic compartment on genetic structuring within the four localities. The AMOVA was not significant (p > 0.1) for all these factors. F-statistics show little but significant differentiation between pairs of these four French and Spanish localities (F ST ranging from 0.01 to 0.02, p < 0.01), meaning a weak geographical differentiation as the only structuring factor. Allelic richness is quite homogeneous among the four localities where sympatric cytotypes were observed (values ranging from 4.3 to 4.9), which is not significantly higher than allelic richness detected within localities from reference Q1 (Greek) and Q2 (Israeli) cytotypes.

Crossing experiments

Viable offspring were obtained in all the crosses performed suggesting there is neither nuclear nor cytoplasmic incompatibilities between Q1 and Q2 cytotypes. The mean number of eggs laid and the percentage of non-hatched eggs are indicated in table 6. The Q1♀XQ1♂ cross produces more eggs than the three others (Tukey HSD test: p < 0.001), but the percentage of hatched eggs is lower (24%, Tukey HSD test: p < 0.001). The number of offspring is slightly higher in the Q1♀XQ2♂ cross than in the others. We have verified that F1 individuals (males and females) were fertile and thus able to produce viable offspring.

DISCUSSION

Cytotype distribution in Western Europe

B. tabaci is widely distributed in France and Spain and putative MED species largely predominates in these areas as in the most part of the Mediterranean basin. MED species has been found in all Spanish and French localities where we performed our sampling from 2003 to 2006. In very few areas, MEAM1 putative species had been observed in France (Nice) in 2004 and in Spain (Cabrils) in 2007 but this presence is anecdotal and will not be discussed further. Until 2007, Q1 was the only cytotype of the MED species found in France and Spain, and more generally in the large West part of the Mediterranean basin. [START_REF] Dalmon | Genetic structure of the invasive pest Bemisia tabaci: evidence of limited but persistent genetic differentiation in glasshouse populations[END_REF][START_REF] Moya | Genetic diversity of Iberian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) based on random amplified polymorphic DNA-polymerase chain reaction[END_REF] In 2007, we detected for the first time the Q2 cytotype in four out of the six localities considered in France and Spain. In 2010, Q2 was found in four other localities, always in sympatry with Q1. Because the initial situation in these countries were well known, we interpret this observation as a recent introduction of Q2 cytotype, which has also been observed in other localities in the Western Mediterranean basin (Gauthier et al., in this issue). This introduction is probably due to trade, which occur with high intensity among Mediterranean countries. Indeed, in France, ornamental plants grow up in greenhouses, but the sowing and cloning are mostly performed in others countries such as Israel, Kenya, Ivory Coast, Germany, Spain, Italy (Terraz, pers. Com). We can suppose that Q2 was introduced from the Eastern part of the Mediterranean basin (middle east) since it has always been detected over there, [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF][START_REF] Rua | New insights into the mitochondrial phylogeny of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in the Mediterranean Basin[END_REF][START_REF] Chu | Genetic differentiation of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype Q based on mitochondrial DNA markers[END_REF] and now is spreading in the basin. These results raise two questions: how the Q2 cytotype had been able to settle in these localities while Q1 was already installed and at which geographic scale this situation occurs in the Mediterranean basin? Extensive surveys of B. tabaci populations at a larger geographical scale would give important insights to answer to these questions.

Q1 and Q2 differentiation and hybridization

Cytotypes are identified on the basis of mitochondrial DNA and their symbiotic bacteria. [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF] Until 2007, Q1 and Q2 cytotypes were considered allopatric in the Mediterranean basin with Q2 only known in Israel, but also possibly present in border countries, while Q1 had a larger distribution. This separate distribution is consistent with the very high nuclear genetic differentiation observed between Q1 reference cytotype (Greece) and Q2 reference cytotype (Israel), about 29% F ST for microsatellite markers. Such a value is comparable with those obtained between distinct B. tabaci species such as MED and MEAM1. [START_REF] Saleh | Spatial and host-plant partitioning between coexisting Bemisia tabaci cryptic species in Tunisia[END_REF][START_REF] Tahiri | Evidence of gene flow between sympatric populations of the Middle East-Asia Minor 1 and Mediterranean putative species of Bemisia tabaci[END_REF] This divergence indicates that they probably not hybridized recently, as they had not in contact, leading us to consider these as reference cytotype, even if the nuclear compositions observed in these countries are probably not representative of all the diversity that can exist in each cytotypes. Moreover, this small sampling could explain high differentiation values and the detection of Israeli Q2 specific alleles (many alleles from Q2 reference cytotype are not found anywhere else). Our Q2 reference is then certainly not the population source of the Western Q2 cytotypes as suggested by microsatellites analysis comparison of allopatric and sympatric populations. Populations experiencing strong bottleneck during colonisation process or pesticide treatments inducing reduced population effective size could also explain this high differentiation.

This new situation with Q1 and Q2 cytotypes observed in sympatry in France and Spain gives the opportunity to test their interfertility and the limits of the MED species, within the framework of the mtCOI phylogeny of the B. tabaci complex species. [START_REF] De Barro | Bemisia tabaci: a statement of species status[END_REF][START_REF] Lee | Taxonomic status of the Bemisia tabaci complex (Hemiptera: Aleyrodidae) and reassessment of the number of its constituent species[END_REF] The microsatellite analysis revealed three highly differentiated clusters: Israeli Q2 reference, Greek Q1 reference and the Western mixed cytotypes. These results indicated that Q1 and Q2 are homogeneous in sympatry (i.e. in France and Spain) and clearly demonstrates their full hybridization and the absence of any reproductive barrier amongst them. They also do not differ in the insecticide resistant-alleles frequency for the two genes we considered, i. e. the ace1 (resistance is fixed) and the para-type voltage gated sodium channel genes. Further studies could focus on other resistance mechanisms such as over-expression of cytochromes. [START_REF] Karunker | Over-expression of cytochrome P450 CYP6CM1 is associated with high resistance to imidacloprid in the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF][START_REF] Alon | Organophosphates' resistance in the B-biotype of Bemisia tabaci (Hemiptera: Aleyrodidae) is associated with a point mutation in an ace1-type acetylcholinesterase and overexpression of carboxylesterase[END_REF] In addition to hybridization in the field, crossing experiments in the lab confirm that Q1 and Q2 individuals can interbreed and produce fertile offspring, indicating that there is no nuclear incompatibility between them and that, as indicated by phylogenetic, 5 they belong to the same species.

Biological relevance of Q1 and Q2 cytotypes

Cytotypes stability

Q1 and Q2 freely interbreed but harbour different symbiont communities even when they coexist in sympatry (Q1 is infected by Hamiltonella and Cardinium, while Q2 is infected by Rickettsia and Arsenophonus). This means that CO1 haplotype and bacterial associations are relatively stable at time scale of several years, thus giving biological relevance to cytotypes under the hypothesis of a role of bacteria on B. tabaci phenotype. The absence of new mitochondrial haplotype/bacterial community associations can be explained either by the absence or rare events of horizontal transfers or the impossibility of these bacteria to colonize a new cytoplasmic and/or nuclear environment.

Symbiotic vertically-bacteria are known to be horizontally transferred. Horizontal transmission can occur through the diet between individuals that feed on the same host plant as demonstrated in aphids and fruit flies, [START_REF] Darby | Elucidation of the transmission patterns of an insect-borne bacterium[END_REF][START_REF] Haine | Horizontal transmission of Wolbachia in a Drosophila community[END_REF] or during mating. [START_REF] Gonella | Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae)[END_REF][START_REF] Moran | Sexual acquisition of beneficial symbionts in aphids[END_REF] Transfers can also occur between hosts and parasitoids. [START_REF] Vavre | Phylogenetic evidence for horizontal transmission of Wolbachia in host-parasitoid associations[END_REF][START_REF] Chiel | Almost there: transmission routes of bacterial symbionts between trophic levels[END_REF] Phylogenetic analysis revealed the existence of such transfers in B. tabaci. [START_REF] Mouton | Evidence of diversity and recombination in Arsenophonus symbionts of the Bemisia tabaci species complex[END_REF] Moreover, lab experiments demonstrated that Rickettsia can be horizontally transmitted between B. tabaci individuals through the host plant. [START_REF] Caspi-Fluger | Horizontal transmission of the insect symbiont Rickettsia is plantmediated[END_REF] Therefore a total absence of horizontal transfer is unlikely but since the contact of Q1and Q2 cytotypes occurred recently, lateral transfer between cytotypes didn't occur yet (at the time scale of this study). Another explanation could be that new mtCOI/bacteria associations are unstable either because of a maladaptation of the transferred bacteria in the new environment or exclusion by the recipient cytotype. This exclusion can be achieved by the host nuclear genes or by the symbionts already present. As the two cytotypes interbreed and that we didn't detect any new bacterial infection neither in Q1 nor in Q2, we can suppose that their nuclear compartments are homogeneous. Therefore the existence of nucleocytoplasmic incompatibilities when a bacteria integrates the other cytotype is unlikely; even if we can't exclude that some nuclear genes specific to each cytotype could be involved in bacterial exclusion. Within-host, multiple infections could select for competitive symbionts monopolizing resources at the expense of the other, allowing their own vertical transmission. There are some examples of such cytoplasmic competition between bacteria (Goto, 2006; Oliver, 2006). [START_REF] Goto | Asymmetrical interactions between Wolbachia and Spiroplasma endosymbionts coexisting in the same insect host[END_REF][START_REF] Oliver | Costs and benefits of a superinfection of facultative symbionts in aphids[END_REF] The stability of the mtCO1/symbiotic community associations indicated that the concept of cytotype has a real biological significance.

Effects of bacteria

Q1 and Q2 hybridize despite different symbiotic communities infection, suggesting that bacteria don't induce cytoplasmic incompatibility in B. tabaci, or weakly, which is consistent with the study of Dalmon et al., [START_REF] Dalmon | Genetic structure of the invasive pest Bemisia tabaci: evidence of limited but persistent genetic differentiation in glasshouse populations[END_REF] on Wolbachia and Cardinium. This is confirmed in our study by laboratory reciprocal crosses. However, as symbionts necessarily induce a physiological cost on their host [START_REF] Oliver | Costs and benefits of a superinfection of facultative symbionts in aphids[END_REF][START_REF] Fleury | Physiological cost induced by the maternally-transmitted endosymbiont Wolbachia in the Drosophila parasitoid Leptopilina heterotoma[END_REF] , they are probably involved in another phenotypic effects that can cause their spread and maintenance into the host populations (review in Ferrari & Vavre,[START_REF] Ferrari | Population genetic structure ans secondary symbionts in host-associated populations of the pea aphid[END_REF] ). Some symbiotic bacteria manipulate host reproduction and increase their transmission but apparently it is not the case in B. tabaci. Others have a mutualistic strategy and confer direct fitness benefit to their host such as protection against natural enemies, 18,67 thermal tolerance, [START_REF] Chen | Fitness effects of two facultative endosymbiotic bacteria on the pea aphid, Acyrthosiphon pisum, and the blue alfalfa aphid, Akondoi[END_REF][START_REF] Montllor | Facultative bacterial endosymbionts benefit pea aphids Acyrthosiphon pisum under heat stress[END_REF] or host plant specialisation. [START_REF] Tsuchida | Host Plant Specialization Governed by Facultative Symbiont[END_REF] In B. tabaci, there is little information about the phenotypic effects of the symbionts. It has been demonstrated that Rickettsia enhance host fitness by increasing fecundity and survival to adulthood, [START_REF] Himler | Rapid spread of a bacterial symbiont in an invasive whitefly is driven by fitness benefits and female bias[END_REF] Rickettsia is also suspected to increase sensibility to insecticides, [START_REF] Kontsedalov | The presence of Rickettsia is associated with increased susceptibility of Bemisia tabaci ( Homoptera : Aleyrodidae ) to insecticides[END_REF] and Wolbachia could protect B. tabaci from parasitoids. [START_REF] Xue | Inactivation of Wolbachia reveals its biological roles in whitefly host[END_REF] In Q1 and in Q2, one symbiont is fixed in natural populations, Hamiltonella and Rickettsia respectively, while another is present at intermediate frequencies (around 38% for Cardinium in Q1 and 60% for Arsenophonus in Q2). It is possible that Rickettsia increases host performance in the Q2 populations of France and Spain, however, this trait has only been demonstrated in Q2 populations in USA, and has not been observed in Q2 populations in Israel. Future studies should focus on the phenotypic effects of the bacteria present in Q1 and Q2 in these mixed populations of France and Spain.

Cytotypes coexistence

The sympatry observed in France and Spain is relatively new (only since 2007): Q2 cytotype has spread in Western Europe without excluding Q1 until now. The question is thus how this situation will evolve and whether these two cytotypes will coexist or if one will be excluded. Coexistence of several B. tabaci cytotypes is uncommon and generally a transient situation which lead to the displacement and/or exclusion of one of the competing cytotypes. [START_REF] Moya | Genetic diversity of Iberian populations of Bemisia tabaci (Hemiptera: Aleyrodidae) based on random amplified polymorphic DNA-polymerase chain reaction[END_REF][START_REF] Khasdan | DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics[END_REF][START_REF] Horowitz | Biotype Q of Bemisia tabaci identified in Israel[END_REF] Indeed, each greenhouse can provide an environment that is more or less suitable to one cytotype, that exclude the less adapted cytotype. In this case, we should have a mosaic of situations where only one cytotype will be present in a greenhouse but not necessarily the same cytotype in the different greenhouses located in the same locality. Situations evolve according to the effect of bacteria on host phenotype, such as host-plant preference or resistance to insecticides. We didn't find any difference in term of host plant preference between cytotypes. Most of resistance mechanisms are nuclear while the two cytotypes freely interbreed and reference Q1 and Q2 population show similar composition of resistance genes. However, Rickettsia (associated to Q2 cytotype) could increase susceptibility to some insecticides (Kontsedalov et al., 2008) and thus disadvantage Q2 cytotype in such environment.

However it is possible that these two cytotypes coexist in the long term. Mixed populations could be maintained through plant rotation and recurrent introductions of cytotypes. This high flow between glasshouses is supported by the fact that observed geographical differentiation is very low, only 1% F ST between localities within country, and 2% between countries. This low differentiation is in agreement with previous results, [START_REF] Dalmon | Genetic structure of the invasive pest Bemisia tabaci: evidence of limited but persistent genetic differentiation in glasshouse populations[END_REF] or more recently Gauthier et al., (2013, this volume). This is probably due to commercial exchanges of vegetable and ornamental crops (see above and Byrne et al., [START_REF] Byrne | Localized migration and dispersal by the sweet potato whitefly, Bemisia tabaci[END_REF] ). Clearly, temporal survey with analysis of the nuclear compartment is required to bring more insights into the evolution of the Q1 and Q2 distribution in Europe.

CONCLUSION

We report here, for the first time, the detection of the Q2 cytotype of B. tabaci MED species in Western Europe (France and Spain). A short survey over a period of 5 years indicated that Q2 seems to increase in frequency but without exclusion of the Q1 cytotype. Both cytotypes are always found in sympatry. Genetic analysis showed complete hybridization of the two cytotypes in the field despite a very high differentiation observed among allopatric sampling. This interfertility is confirmed by crossing experiments in the lab. These results are consistent with the phylogenies based on mtCOI, which regroup Q1 and Q2 cytotypes in the same MED species and indicated that symbionts are probably not involved in reproductive isolation. Our data also showed that the stable associations of mitochondrial haplotypes with bacteria confer a biological reality to Q1 and Q2 cytotypes. * means significantly different from others (Tukey HSD tests, p < 0,01). The number of replicates is ten in each cross.

TABLES AND FIGURES

(

  ± SD), mean percentage of hatched eggs (± SD). * means significantly different from others (Tukey HSD tests, p < 0,01). The number of replicates is ten in each cross.

Figure 1 .Figure 2 .

 12 Figure 1. Characteristics of Greek Q1 and Israel Q2 cytotypes. a) Factorial map of the principal components analysis. b) Infection status of Q1 individuals c) Infection status of Q2 individuals.

Figure 3 .

 3 Figure 3. Infection status of Bemisia tabaci individuals

Figure 4 .

 4 Figure 4. Clustering results from STRUCTURE for all samples.

Figure 5 .

 5 Figure 5. Factorial map of the principal components analysis with the entire dataset.

3 *: populations used for microsatellite analysis 4 ¤

 34 : the first two or three letters of the acronyms correspond to the locality; the last capital letter indicates the 5 host plant.

Figure 1 .

 1 Figure 1. Characteristics of Greek Q1 and Israel Q2 cytotypes. a) Factorial map of the principal components analysis. b) Infection status of Q1 individuals c) Infection status of Q2 individuals

Figure 2 .

 2 Figure 2. Map of cytotype distribution in France and Spain by year of sampling. Red: Q1 cytotype (MED species), blue: Q2 cytotype (MED species), yellow: MEAM1 species.

Figure 3 .

 3 Figure 3. Infection status of Bemisia tabaci individuals.

Figure 4 .

 4 Figure 4. Clustering results from STRUCTURE for all samples.

Figure 5 .

 5 Figure 5. Factorial map of the principal components analysis with the entire dataset. 1

2.1 Bemisia tabaci field sampling 531

  individuals from 10 localities were collected in France and Spain from 2006 to 2010 for a temporal survey. Adults were collected in greenhouses on different hosts including ornamental plants and vegetables (Table

Table 1 .

 1 Characteristic of the sample site locations;

	N: number of sampled individuals.
	*: populations used for microsatellite analysis
	¤: the first two or three letters of the acronyms correspond to the locality; the last capital letter indicates the
	host plant.

Table 2 .

 2 : Characteristics of primers used for endosymbiont screening

Table 3 .

 3 Frequencies of sodium channel and ace-1 resistant mutations in B. tabaci per host plant,

	cytotype and locality

Table 4 .

 4 Characteristics of the primers used for microsatellite genotyping

Table 5 .

 5 Genetic characteristics of the populations. N. number of individuals; Ho. observed heterozygosity; He. unbiased expected heterozygosity; A¯. mean number of alleles per population per locus

Table 6 .

 6 Results of crossing experiments. Mean eggs laid per replicates (± SD) , mean hatched eggs

Table 1 .

 1 Characteristics of sample site locations 1 2

N: number of sampled individuals.

Table 2 .

 2 Characteristics of primers used for endosymbiont screening

	6
	7
	8

Table 3 .

 3 Frequencies of sodium channel and ace-1 resistant mutations in B. tabaci per host plant, cytotype and locality 1

							Sodium channel						ace
							r1 allele					r2 allele		R allele
	Country	Locality	Cytotype	r1 genotypes		resistance frequencies	r2 genotypes		resistance frequencies	Genotypes	resistance frequencies
				n r1r1 r1s1 s1s1		n r2r2 r2s2 s2s2		n RR RS SS
	Greece	Various	Q1	29 16 11	2	0.74	29	0	1	28	0.02	29 29 0	0
	Israel	Hof-Carmel	Q2	23 12	8	3	0.70	27	3	11 13	0.29	27 27 0	0
	Spain	Cabrils Viladecans	Q1 Q2 Q1 Q2	7 25 28 12 14 3 4 6 16 9 6 2	0 3 2 1	0.71 0.56 0.68 0.78	7 25 28 9	0 0 0 0	3 15 10 4 6 22 3 6	0.21 0.30 0.11 0.17	7 25 25 0 7 0 28 28 0 9 9 0	0 0 0 0
		Saint-Laurent du	Q1	12 11	1	0	0.96	12	0	0	12	0	12 12 0	0
	France	Frejus Var	Q2 Q1 Q2	15 12 8 8 7 6	3 0 1	0 0 0	0.90 1 0.93	15 8 7	0 0 0	2 0 0	13 8 7	0.07 0 0	15 15 0 8 8 0 7 7 0	0 0 0
	2
	3
	4
	5
	6
	7

Table 4 .

 4 Characteristics of the primers used for microsatellite genotyping

	Locus	Primer name	Primer sequence

Table 6 .

 6 Results of crossing experiments. Mean eggs laid per replicates (± SD) , mean hatched eggs (± SD), mean percentage of hatched eggs (± SD).

	Cross	Q1♀XQ1♂	Q1♀XQ2♂	Q2♀XQ1♂	Q2♀XQ2♂
	Eggs	127* ± 43	75± 13	40± 13	65± 9
	Hatched eggs	32 ± 15	48 ± 9	32 ± 12	39 ± 8
	Hatched eggs (%)	24* ± 14	65 ± 13	79 ± 5	62 ± 16

Acknowledgement:

This work was funded by CNRS (IFR41-UMR5558) and by the Arimnet agency (SWIPE project).

We are grateful to G. Bout, J. Arno, A. Tsagkarakou, E. Zchori-Fein, J. Vendeville and M.

Peterschmitt for sample collection. Gabriel Terraz is recipient of a PhD studentship from the Rhône-Alpes region ("Program Cible" Grant).