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

Omer Hema

Moussa Namountougou

Laurence Mouton

Olivier Gnankiné
email: olgnankine@hotmail.com

Hema Omer

Fabrice Vavre

Impact of pest management practices on the frequency of insecticide resistance alleles in Bemisia tabaci (Hemiptera: Aleyrodidae) populations in three countries of West Africa

Keywords: Pest management programs, insecticides, kdr, Ace1 R, Bemisia tabaci

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

Impact of pest management practices on the frequency

of insecticide resistance alleles in Bemisia tabaci (Hemiptera: Aleyrodidae) populations in three countries of West Africa

Introduction

In western Africa, cotton is an economically important crop providing substantial incomes for farmers. However, the cotton plant is attacked by key pests including the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae) and the whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). Three main spraying programs are recommended to control these pests in many countries of West Africa. They include the Conventional Cotton program (CP), the Biological Program (BP) and the transgenic Bt cotton program (Bt Cotton).

The CP is based on calendar-based applications of insecticides belonging to pyrethroids, organophosphates and neonicotinoids families separately or as a mixture (Gnankiné et al., 2013a;[START_REF] Silvie | Threshold-based interventions for cotton pest control in West Africa : What ' s up 10 years later ?[END_REF]. They are applied with temporal rotations during the whole cotton season (from May to October). The repeated use of such insecticides have imposed strong selection pressures on target pests' populations, resulting in the evolution of field resistances [START_REF] Houndeté | Insecticide resistance in field populations of Bemisia tabaci (Hemiptera: Aleyrodidae) in West Africa[END_REF]. Over $60 millions of chemicals were spent for chemical pests control in Burkina Faso [START_REF] Vitale | The Economic Impacts of Monsanto's Paper, Bollgard II in West Africa: Emprirical Evidence from Burkina Faso[END_REF]. The BP relies on the use of biopesticides and natural fertilizers without utilization of any chemical. The Bt cotton program uses Bt (Bacillus thuringiensis) transgenic cotton plants that express two crystal toxins (Cry1Ac and Cry2Ab) that target some major lepidopteran pests but are harmless to vertebrates and most other organisms [START_REF] Mendelsohn | Are Bt crops safe?[END_REF][START_REF] Sanahuja | Bacillus thuringiensis : a century of research , development and commercial applications[END_REF][START_REF] Pardo-Lopez | Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection[END_REF]. In this program, only neonicotinoids are used at the end of the cotton phenological stages [START_REF] Héma | Efficacy of transgenic cotton plant containing the Cry1Ac and Cry2Ab genes of Bacillus thuringiensis against Helicoverpa armigera and Syllepte derogata in cotton cultivation in Burkina Faso[END_REF]. It was initiated in 2008 in West Africa but only in Burkina Faso. In 2016, the Burkina Faso government suspended the use of Bt technology due to the quality of cotton fiber which is shorter than the fiber of conventional cotton.

One of major threat to cotton plant remains the whitefly B. tabaci. It causes damages directly through phloem feeding and indirectly through the transmission of plant viruses. B. tabaci is a complex of cryptic species whose taxonomy is still not entirely resolved. Based on the current consensus, B. tabaci is mostly represented by the MED species in western Africa, even though AnSL species can also be found (Gnankiné et al., 2013b;Gnankiné et al., 2013c).

Within the MED species, different biotypes are encountered including MED-Q1, MED-Q3 and ASL. Actually, recent analyses suggest that MED-Q1 and ASL do not hybridize in the field and that ASL is thus a different species [START_REF] Mouton | Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: New insights into the systematics of this worldwide pest[END_REF]. Interestingly, these species/biotypes differ in terms of host plants range and insecticide resistance traits.

Generally, MED-Q1 is predominant in cotton areas and sometimes found in sympatry with ASL on vegetables crops (Gnankiné et al., 2013b). It is also associated with higher levels of resistance to some insecticides as pyrethroids, organophosphates and neonicotinoids (Gnankiné et al., 2013a). In B. tabaci, two mutations in the para-type voltage-gated sodium channel gene, L925I and T929V, and one mutation in the acetylcholinesterase enzyme ace1 (F331W: allele Ace1 R ) confer resistance to pyrethroids and organophosphates, respectively [START_REF] Roditakis | Identification of mutations in the para sodium channel of Bemisia tabaci from Crete, associated with resistance to pyrethroids[END_REF][START_REF] Alon | Organophosphates ' resistance in the Bbiotype of Bemisia tabaci ( Hemiptera : Aleyrodidae ) is associated with a point mutation in an ace1 -type acetylcholinesterase and overexpression of carboxylesterase[END_REF][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]. Recent studies showed that the Ace1 R was found in both MED-Q1 and ASL but, while this resistant allele was almost fixed in MED-Q1 (0.99), its frequency was 0.59 in ASL [START_REF] Mouton | Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: New insights into the systematics of this worldwide pest[END_REF]. In addition, while the L925I mutation in the sodium channel gene is almost fixed in MED-Q1 populations, it is rarely detected in ASL. The T929V was never found in B. tabaci populations from West Africa [START_REF] Mouton | Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: New insights into the systematics of this worldwide pest[END_REF]. The objectives of the present study were to perform a first analysis of the impact of the agricultural practices on the B. tabaci biotypes/species composition and diversity, and the frequencies of alleles that confer resistance to pyrethroids and organophosphates. Sampling was performed in three countries of Western Africa:

Burkina Faso, Benin and Togo.

Materials and methods

Management of cotton pests

Three pest control programs are recommended in West African countries:

(i) The Conventional Program (CP) is based on two to four treatments with pyrethroids (PY) plus organophosphates (OP) and 2 other treatments with neonicotinoids (see table 1 for details).

Identification of B. tabaci

Species/biotypes were identified using the Polymerase Chain Reaction-Random Fragment

Length Polymorphism (PCR-RFLP) diagnostic assay based on the mitochondrial cytochrome oxidase 1 gene sequence (mtCO1) described in [START_REF] Henri | Molecular characterization of genetic diversity within the Africa/Middle East/Asia Minor and Sub-Saharan African groups of the Bemisia tabaci species complex[END_REF]. This technique allows discriminating the species/biotypes present in West Africa (Gnankiné et al., 2013b).

Identification of susceptible and resistant alleles of the sodium channel and the acetylcholinesterase ace1 genes

Resistant and susceptible alleles in the para-type voltage-gated sodium channel and ace1 genes were identified using the diagnostic assays developed by [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].

Briefly, ace1-susceptible (F331) and -resistant (W331) alleles, as well as susceptible (L925)

and resistant (I925) para-type voltage-gated sodium channel alleles were detected using PCR-RFLP [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]. Some PCR products were sequenced for each susceptible and resistant allele and each country. We never found the T929V mutation in the sequences.

The frequencies of kdr and ace-1 R mutations were calculated according to the formula p= n♂(R)+2n♀(RR)+n♀(RS)/n♂+2n♀where RR was the number of homozygotes, RS the number of heterozygotes and n the size of specimens analysed.

Statistical analyses

Statistical analysis were performed using the R statistical software (http://www.Rproject.org). The effects of the pest management practices on the proportions of B. tabaci composition and the frequencies of resistance alleles were tested by using Fisher's exact tests.

Results

Geographic distribution of biotypes

All the 170 B. tabaci individuals collected in 5 localities in Burkina Faso, Benin and Togo belonged to MED-Q1 or ASL (Table 2). In Togo and Benin, only ASL was found (except one MED-Q1 individual in Tové), while in Burkina Faso, MED-Q1 and ASL were found in sympatry at variable frequencies: depending on the locality, it was either ASL or MED-Q1 that predominated. In Pô, the frequency of ASL reached more than 80% whatever the control strategy (CP or Cotton Bt). In Saria, MED-Q1 was more common than ASL, but their relative frequencies depended on the management program: on CP areas, 93 % of individuals belonged to MED-Q1 while only 57% of whiteflies were MED-Q1 on Bt Cotton fields (Fisher exact test, p<0.005).

Frequency of the L925I mutation

For the para-type voltage-gated sodium channel gene, we studied the frequency of the L925I mutation that correspond to the allele called r1 by [START_REF] Alon | Organophosphates ' resistance in the Bbiotype of Bemisia tabaci ( Hemiptera : Aleyrodidae ) is associated with a point mutation in an ace1 -type acetylcholinesterase and overexpression of carboxylesterase[END_REF]. We found high variations depending on the country (Fisher exact test, p<0.0005). Indeed, in Burkina Faso, r1

was fixed for both MED-Q1 and ASL in the two localities (Table 3). In Benin, this allele has not been found (Table 4). In Togo, its frequency varied between 0.5 and 0.75 (Table 5).

Frequency of the Ace-1 R allele

For the acetylcholinesterase gene, we studied the presence of the F331W mutation (R allele).

Globally, the frequency of this allele was more homogeneous among countries than for the L925I mutation and ranged between 0.71 and 1 (Fisher exact test, p=0.08). In Togo, this frequency did not differ between the two localities, Infa and Tové (Table 5; Fisher exact test, p=0.1). In Benin, where sampling was performed in only one locality, the frequency of R varied between 0.7 and 0.9 but did not significantly differ between the fields that were treated either with BP or CP program (Table 4; Fisher exact test, p=0.49).

In Burkina Faso, the frequency of the resistant allele changed in the two localities according to the treatment: it was lower in Bt Cotton areas than in CP fields (Table 3; Fisher exact test, p=0.003 for Pô and p<0.0005 for Saria). In the two localities, R was fixed in MED-Q1 in the CP fields while it had a frequency of 0.5 and 0.875 in Pô and Saria respectively in Bt areas (Fisher exact test, p<0.0005). For ASL, differences were not so important and not statistically significant (Fisher exact test, p>0.05) with a range of frequencies between 0.91 and 1 for CP fields and 0.71 -0.83 for Bt cotton fields.

Discussion

In this paper, we present the distribution of B. tabaci biotypes/species and the frequencies of the mutations in the para sodium channel gene (kdr) and in the Acetylcholinesterase gene (ace-1 R ) associated with resistance to Pyrethroids and Organophosphates respectively of the pest B. tabaci in 3 countries of Western Africa in connection with the pest management programs.

Our results confirmed the diversity of B. tabaci biotypes on cotton in these countries. As previously described by [START_REF] Gueguen | Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex[END_REF] and Gnankiné et al. (2013b), MED-Q1 and ASL were detected in Burkina Faso, frequently in the same areas. Despite this sympatry, population genetics analyses on microsatellite markers suggested that MED-Q1 and ASL do not hybridize in the field [START_REF] Mouton | Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: New insights into the systematics of this worldwide pest[END_REF]. It was also showed that they do not share insecticide resistance alleles. ASL previously classified in MED-species might thus be considered now as putative species [START_REF] Mouton | Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: New insights into the systematics of this worldwide pest[END_REF].

In the current study, the L925I mutation was fixed in MED-Q1 and ASL individuals in Burkina Faso. Regarding ASL, this is in sharp contrast with the situation observed in 2009 and 2010, as this mutation only reached 0.02 at that time in the western part of Burkina Faso [START_REF] Mouton | Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: New insights into the systematics of this worldwide pest[END_REF]. This is also in contrast with the situation found in Benin, where this resistant allele was not found, whatever the control program. Finally, the situation is intermediate in Togo where the frequencies varied between 0.5-0.75 in CP or BP programs.

The fixation of this L925I mutation might be explained by a high selection pressure due to the repetitive pyrethroids treatments applied in western Africa particularly on cotton and vegetables [START_REF] Gnankiné | Traitements insecticides et dynamique des populations de Bemisia tabaci Gennadius en culture cotonnière au Burkina Faso[END_REF][START_REF] Ahouangninou | Évaluation des risques sanitaires et environnementaux des pratiques phytosanitaires des producteurs maraîchers dans la commune rurale de Tori-Bossito (Sud-Bénin)[END_REF]Gnankiné et al. 2013b).

The management of the resistance mutations in the para-type voltage-gated sodium channel gene, L925I but also T929V, seems to be difficult as they prove to be persistent even without selection in laboratory, suggesting a low fitness cost [START_REF] Roditakis | Identification of mutations in the para sodium channel of Bemisia tabaci from Crete, associated with resistance to pyrethroids[END_REF]. This low fitness cost was confirmed by [START_REF] Alon | Multiple origins of pyrethroid resistance in sympatric biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF] where no departures from Hardy-Weinberg equilibrium were observed when the frequency of resistant genotypes was investigated in B.

tabaci populations reared without any insecticide selection for many years. Accordingly, we could not detect any impact of the control program on the frequency of these resistance mutations.

For the F331W mutation (ace1 gene), the frequencies of resistant alleles were very variable.

According to [START_REF] Mouton | Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: New insights into the systematics of this worldwide pest[END_REF], the frequencies of the F331W mutation in the ace1 gene in individuals from Burkina Faso were 0.98 and 0.59 for MED-Q1 and ASL, respectively. Our data indicated that the frequency of resistance may be lower in individuals sampled in fields using Bt cotton strategy than in the fields using CP. This effect was more pronounced in MED-Q1 than ASL, and also more pronounced in Pô than in Saria. The high frequencies observed in Saria could be due to the number of applications done for the pest control. Indeed, near Saria, many farmers cultivate vegetables that are systematically treated with pyrethroids and Organophosphates. On the contrary, in Pô, no vegetables were cultivated and the insecticide pressure was low. Moreover, a drastic lack of resistant homozygous in the Bt cotton fields is consistent with the hypothesis of a high fitness cost associated with the F331W mutation in this species. As 90% of the zone was using Bt in Pô, while it was only 15% in Saria, the refuge zone without treatment is thus much more important in Pô, which could allow a more rapid counter-selection of resistance alleles without treatment. On the other hand, we could not detect any effect of the control program in Benin (CP vs. BP). This may be explained by the fact that only 10% of the surface were cultivated using BP program. In Togo, we did not detect any significant difference in the mutation frequencies in the two areas but they were under the same insecticide pressure.

Other mechanisms of insecticide resistance, like metabolic resistance may be associated to high frequencies of the two mutations in individuals insects conferring a multiple resistance.

Indeed, detoxifying enzymes such as esterases, glutathione S-transferases, and cytochrome P450-dependent monooxygenases are involved in resistance to numerous insecticide classes [START_REF] Alon | Organophosphates ' resistance in the Bbiotype of Bemisia tabaci ( Hemiptera : Aleyrodidae ) is associated with a point mutation in an ace1 -type acetylcholinesterase and overexpression of carboxylesterase[END_REF][START_REF] Rauch | Identification of biochemical markers linked to neonicotinoid cross resistance in Bemisia tabaci (Hemiptera: Aleyrodidae)[END_REF][START_REF] Ma | Pyriproxyfen resistance of Bemisia tabaci (Homoptera: Aleyrodidae) biotype B: metabolic mechanism[END_REF].

In conclusion, our results suggest that different control programs may alter the frequency of resistance alleles. However, the counter-selection of resistance alleles may depend on the one hand on the fitness cost associated with resistance, and the other hand to the relative surface of untreated areas. This clearly advocates for a better integration of control measures against B. tabaci across the different host crops.

(

  ii) The Biological control Program (BP) does not use chemicals for plant protection. Farmers worked under the supervision of technicians from the Beninese Organization for Organic Farming Promotion (OBEPAP) who participated in the implementation and the survey of good agricultural practices on organic cotton.(iii) The transgenic cotton program (Bt Cotton). In this program, pesticides belonging to OP and PYR are not used. Only neonicotinoids are used at boll opening stage to control sucking pests. In this case, farmers worked under the supervision of technicians from societies of textile fibers in Burkina Faso.2.2 B. tabaci samplingSampling was performed in october and november between 2009 and 2015 in three countries of Western Africa: Burkina Faso, Benin and Togo (Figure1,

  Table2). In Burkina Faso, whiteflies were collected randomly in two localities, Pô in 2013 and Saria in 2015, from Bt cotton and CP fields. In Pô, Bt cotton represented 90% of the sampled fields while in Saria it represented 15%. In Benin, sampling was done at Kandi in 2009 in CP and BP areas (BP The origin of the samples (location) and the number of individuals are summarized in

	Table 2.
	2.3 Molecular analysis
	DNA extraction
	For each individual, total DNA was extracted in 25 μl of an extraction buffer containing
	50 mM KCl, 10 mM Tris-base pH 8, 0.45% Nonidet P-40, 0.45% Tween 20 and 50 mg/ml
	proteinase K. After 3 h at 65°C, samples were incubated at 100°C for 15 min. Pure water
	(35 μl) was then added to the extract.

represent around 10% of areas). In Togo, collection was done randomly in two sites in 2009, Infa and Tové, where only CP is used. The collected adult whiteflies were stored in ethanol 6 95%.

Acknowledgments

The authors are grateful to the following institute for the samples collection and the technical assistance from Inera and University of Lomé.


Julien Cattel
email: juliencattel@gmail.com

K Nikolouli
email: nikolouli@iaea.org

T Andrieux

F Jiggins

D Lejon
email: dlejon@rovaltainresearch.com

P Gibert

L Mouton

Back and forth Wolbachia transfers reveal efficient strains to control spotted wing drosophila populations

Keywords: Cytoplasmic Incompatibility, Incompatible Insect Technique, Pest control, spotted wing drosophila, Wolbachia, fruit industry, pesticide, bacteria

1. Since its recent invasion of the European and American continents, the spotted wing Drosophila, Drosophila suzukii, has become a burden of the fruit industry. Armed with a highly sclerotized ovipositor, females can lay eggs in a wider variety of ripening and healthy fruits than other Drosophila species. Economic losses due to Drosophila suzukii reach millions of dollars annually and methods to control natural populations in the field mainly rely on the use of chemical pesticides.

2. We tested if Wolbachia bacteria represents a potential ally to control this pest. These symbionts are naturally present in many insects and often induce a form of conditional sterility called Cytoplasmic Incompatibility (CI): the offspring of infected males die, unless the eggs are rescued by the compatible infection, inherited from the mother that protects the embryo. A long-recognised, a strategy called the Incompatible Insect Technique (IIT) makes use of the CI phenotype to control insect populations through the mass release of infected males. To implement this technique in D. suzukii, we used back and forth Wolbachia transfers between D. suzukii and D. simulans to identify Wolbachia strains that can sterilize D. suzukii females despite the presence of wSuz, a natural Wolbachia infection in this species.

3. We identified two Wolbachia strains as potential candidates for developing IIT in D.

suzukii. Both induce a very high level of CI in this pest which is not attenuated by the presence of wSuz in females. Moreover, the newly transferred Wolbachia do not affect the fitness or the mating competitiveness of the sterilizing males.

4. Synthesis and applications. Although several critical steps still need to be tested and developed outside the laboratory to achieve the control of Drosophila suzukii using Incompatible Insect Technique. By an experimental approach in large population cage, we showed that releases of transinfected males limits population size. Thus, we provide in this study the proof of concept that this technique can be a very promising approach to control D. suzukii populations.

Introduction

Drosophila suzukii, the spotted wing Drosophila, has become a major burden for fruit growers since its recent invasion of the European and American continents [START_REF] Goodhue | Spotted wing drosophila infestation of California strawberries and raspberries: Economic analysis of potential revenue losses and control costs[END_REF][START_REF] Calabria | First records of the potential pest species Drosophila suzukii (Diptera: Drosophilidae) in Europe[END_REF][START_REF] Cini | A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management[END_REF][START_REF] Cini | Tracking the invasion of the alien fruit pest Drosophila suzukii in Europe[END_REF][START_REF] Asplen | Invasion biology of spotted wing Drosophila (Drosophila suzukii): a global perspective and future priorities[END_REF].

Although the vast majority of Drosophila species are not fruit pests, D. suzukii is able to lay eggs on a wide variety of healthy ripening fruits, thanks to a sclerotized ovipositor [START_REF] Mitsui | Spatial distributions and clutch sizes of Drosophila species ovipositing on cherry fruits of different stages[END_REF]. Internal larval feeding represents a direct damage that can also facilitate secondary infections by pathogens as fungi, yeasts or bacteria [START_REF] Cini | A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management[END_REF][START_REF] Hamby | Associations of yeasts with spotted-wing Drosophila (Drosophila suzukii; Diptera: Drosophilidae) in cherries and raspberries[END_REF][START_REF] Ioriatti | Drosophila suzukii (Diptera: Drosophilidae) and Its Potential Impact to Wine Grapes During Harvest in Two Cool Climate Wine Grape Production Regions[END_REF]. Most damages are reported on red fruits, with an approximate $500 million annual loss in the US [START_REF] Goodhue | Spotted wing drosophila infestation of California strawberries and raspberries: Economic analysis of potential revenue losses and control costs[END_REF]. In France and in Northern Italy, up to 100% destruction was reported on cranberries, strawberries and sweet cherries [START_REF] Cini | A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management[END_REF]. Control of D. suzukii populations in the field largely relies on chemical pesticides, a practice with serious drawbacks because of its use close to harvest and the consequent risk of high amount of residues left on fruits. In brief, there is an urgent need for developing effective, specific, and environmentally-friendly methods to fight against D. suzukii.

In this context, suppression of pest populations through the mass release of sterilizing males seems relevant, as it is highly specific, not polluting, and does not require the introduction of a new species into the environment [START_REF] Bourtzis | Harnessing mosquito-Wolbachia symbiosis for vector and disease control[END_REF][START_REF] Lees | Back to the future: the sterile insect technique against mosquito disease vectors[END_REF]. In the Sterile Insect Technique (SIT), males are sterilized by irradiation and released for mating with wild females [START_REF] Knipling | Possibilities of insect control or eradication through the use of sexually sterile males[END_REF]. This approach has already shown its effectiveness against agricultural insect pests like the New World screw-worm fly [START_REF] Lindquist | The New World screwworm fly in Libya: a review of its introduction and eradication[END_REF]. Genetic population suppression approaches, such as Oxitec's RIDL technology could also be considered in principle [START_REF] Black | Why RIDL is not SIT[END_REF], but this technique faces serious regulatory issues in European Union. Finally, one can make use of the bacterial endosymbiont Wolbachia pipientis to produce sterilizing males [START_REF] Boller | Incompatible Races of European Cherry Fruit-Fly, Rhagoletis-Cerasi (Diptera-Tephritidae), Their Origin and Potential Use in Biological-Control[END_REF][START_REF] Riegler | Wolbachia infections and superinfections in cytoplasmically incompatible populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae)[END_REF], which is the option explored here.

The genus name "Wolbachia" designates a highly diverse clade of maternally transmitted intracellular symbionts of arthropods and nematodes, belonging to the αproteobacteria [START_REF] Werren | Biology of Wolbachia[END_REF]. It is well known for its ability to manipulate host reproduction through different strategies that maximize its spread and maintenance in host populations. Among these, the most common appears to be Cytoplasmic Incompatibility (CI), a sperm-egg incompatibility occurring in crosses between infected males and uninfected females or between males and females infected by incompatible Wolbachia strains, leading to the death of embryos [START_REF] Werren | Biology of Wolbachia[END_REF]. CI can be understood as resulting from a modification/rescue system (mod/resc) where the mod function modifies the sperm during spermatogenesis and the resc function, expressed in the egg, rescues the embryo through an interaction with the modified sperm. Recent findings indicate that a pair of genes located in a Wolbachia prophage operon, including a deubiquitylating enzyme, are very likely to encode the mod and resc functions [START_REF] Beckmann | A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility[END_REF][START_REF] Lepage | Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility[END_REF]. Depending on Wolbachia and host factors, some strains can both induce and rescue CI, while others express only the resc function, meaning that they do not induce CI but can rescue the embryos against the mod function expressed by other Wolbachia strains [START_REF] Poinsot | Wolbachia Transfer from Drosophila melanogaster into D. simulans: Host Effect and Cytoplasmic Incompatibility Relationships[END_REF]. CI has led to the proposal and development of the Incompatible Insect Technique (IIT), analogous to the SIT, where sterilization of the targeted populations is achieved by the release of Wolbachiainfected males incompatible with the resident females [START_REF] Boller | Incompatible Races of European Cherry Fruit-Fly, Rhagoletis-Cerasi (Diptera-Tephritidae), Their Origin and Potential Use in Biological-Control[END_REF][START_REF] Riegler | Wolbachia infections and superinfections in cytoplasmically incompatible populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae)[END_REF]. The first successful application of IIT was achieved in Burma where the target population of Culex pipiens, vector of the filariasis, was almost eliminated [START_REF] Laven | Eradication of Culex pipiens fatigans through cytoplasmic incompatibility[END_REF].

Promising results were also obtained more recently in mosquito species under semi-field [START_REF] Chambers | Male mating competitiveness of a Wolbachia-introgressed Aedes polynesiensis strain under semi-field conditions[END_REF][START_REF] Moretti | Male mating performance and cytoplasmic incompatibility in a wPip Wolbachia trans-infected line of Aedes albopictus (Stegomyia albopicta)[END_REF][START_REF] Atyame | Wolbachia-based population control strategy targeting Culex quinquefasciatus mosquitoes proves efficient under semi-field conditions[END_REF] and field [START_REF] O'connor | Open Release of Male Mosquitoes Infected with a Wolbachia Biopesticide: Field Performance and Infection Containment[END_REF] conditions. While initially limited to species where natural Wolbachia infections allowed the expression of CI, this method came to the fore again within the last decade, thanks to the possibility to transfer Wolbachia strains between species (Hughes & Rasgon 2014). This approach has been successfully tested in the agricultural pest Ceratitis capitata, naturally uninfected, where the transfer of two Wolbachia strains from Rhagoletis cerasi led to the expression of a high level of CI (Zabalou et al. 2004b).

Here we aim at developing this promising approach in D. suzukii, where we face an additional challenge linked to the presence of wSuz, a natural Wolbachia strain, found in most D. suzukii populations but at variable frequencies [START_REF] Hamm | Wolbachia do not live by reproductive manipulation alone: Infection polymorphism in Drosophila suzukii and D. Subpulchrella[END_REF]Cattel et al. 2016a).

Data indicated that wSuz is the only natural infection in D. suzukii where it does not induce strong CI [START_REF] Hamm | Wolbachia do not live by reproductive manipulation alone: Infection polymorphism in Drosophila suzukii and D. Subpulchrella[END_REF]Cattel et al. 2016a). These results indicate that the implementation of IIT in D. suzukii requires the introduction of a foreign Wolbachia strain that would induce a strong CI in D. suzukii and, crucially, a CI that would not be rescued by wSuz.

To identify such a strain, we first introduced wSuz in Drosophila simulans where many Wolbachia infections are maintained. This allowed us to assess the rescue capacities of wSuz against multiple CI-inducing strains after a single trans-infection experiment. We thus selected three promising strains that were then introduced in D. suzukii leading to the identification of two strains that induce a nearly complete CI in this background, regardless of the presence of wSuz in females. In addition, the transinfected males showed a similar competitiveness compared to naturally infected or uninfected males and are able to induce a high level of CI during all their life. Finally, we demonstrated that in large population cages, the IIT can be very efficient to limit the increase of D. suzukii populations size. We thus obtained D. suzukii lines combining the properties required for an effective implementation of IIT.

Materials and methods

MICRO-INJECTIONS AND MATERNAL TRANSMISSION MEASUREMENT

Micro-injections were performed between D. suzukii and D. simulans, in both directions, using a micro-capillary needle to transfer the cytoplasm of infected embryos into uninfected ones, at the Fly Facility of the Department of Genetics of the University of Cambridge, following [START_REF] Poinsot | Wolbachia Transfer from Drosophila melanogaster into D. simulans: Host Effect and Cytoplasmic Incompatibility Relationships[END_REF]. Adult females emerging from the injected embryos (G0 females) were crossed with uninfected males of the same genetic background (line STCP in D. simulans and Fr-BE-Ø in D. suzukii) and allowed to lay eggs during 5 days. We checked the presence of Wolbachia by PCR (see Table S1 for the protocol) in all G0 females and kept the offspring of the infected ones. This process was repeated until a perfect maternal transmission of Wolbachia was observed. All injected lines were maintained in the lab for at least 8 generations before beginning experiments.

WOLBACHIA STRAINS, DROSOPHILA LINES AND REARING PROCEDURES

Crossing experiments in D. simulans involved 10 Wolbachia strains, all belonging to the supergroup A [START_REF] Martinez | Should Symbionts Be Nice or Selfish? Antiviral Effects of Wolbachia Are Costly but Reproductive Parasitism Is Not[END_REF], 9 of which have been used in earlier studies [START_REF] Poinsot | Wolbachia Transfer from Drosophila melanogaster into D. simulans: Host Effect and Cytoplasmic Incompatibility Relationships[END_REF][START_REF] Zabalou | Multiple rescue factors within a Wolbachia strain[END_REF][START_REF] Veneti | Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans[END_REF][START_REF] Martinez | Symbionts Commonly Provide Broad Spectrum Resistance to Viruses in Insects: A Comparative Analysis of Wolbachia Strains[END_REF]) (see Table S2).

In D. suzukii, we used one isofemale line, named Fr-BE-Ø, naturally free of Wolbachia, collected in 2012 in Bellegarde (France) and maintained since as a mass population. A line infected by wSuz (Fr-BE-wSuz) was obtained by back-crosses (see Cattel et al. 2016b). The Fr-BE-Ø and Fr-BE-wSuz lines were maintained for respectively ~50 and 40 generations before the crossing experiments. The Fr-BE-Ø line was also the recipient for injections of Wolbachia strains from D. simulans. D. simulans and D. suzukii lines were reared on a cornmeal diet (agar: 1%, dextrose: 8.75%, maize: 8.75%, yeast: 2%, nipagin: 3%) and maintained in an incubator at constant temperature (22°C) and humidity (50%) with a 12-hours light/dark cycle.

CROSSING EXPERIMENTS

To analyze the resc function of wSuz in D. simulans we first performed mass crosses using infected females carrying wSuz with males infected by each of the Wolbachia strains available (see Table S2). Freshly emerged adults were sexed and placed separately into cornmeal diet tubes to ensure the virginity of flies. Ten virgin males (3 to 5-days old) and ten virgin females (5 to 6-days old) were allowed to mate in food vials for 24h. Females were then allowed to oviposit for 48h on grape-juice agar in a petri dish. The total number of hatched and unhatched eggs was recorded 48h after removal of the females. Six such mass crosses were performed for each Wolbachia strain tested.

Based on the results of mass crosses, three candidate strains were identified (wRi, wTei and wHa). To quantify more precisely the CI relationships between wSuz and these candidate Wolbachia strains, individual crosses were performed in D. simulans following the same protocol except that we used only 3-days old virgin males and 5-days old virgin females and that the egg hatch rates were estimated individually.

In D. suzukii, in order to make sure that mating had taken place, mating was either observed, confirmed by the hatching of at least one egg, or by the presence of sperm in the spermathecae. We also confirmed D. suzukii female's virginity before mating by placing them individually in a petri dish for egg-laying during 48h. Only 13 of the 70 females tested laid eggs, but none of them hatched, confirming their virginity. For all individual crosses, at least 20 repetitions were obtained, excluding females that laid fewer than 10 eggs. To account for variation in background embryonic mortality (not related to CI), we used a corrected index of CI (CI corr ) [START_REF] Poinsot | Wolbachia Transfer from Drosophila melanogaster into D. simulans: Host Effect and Cytoplasmic Incompatibility Relationships[END_REF]) calculated as follows : CI corr = [(CI obs -CCM)/(100 -CCM)] * 100, where CI obs is the percentage of unhatched eggs observed in a given incompatible cross, and CCM is the mean mortality observed in the control crosses.

MEASURE OF LIFE HISTORY TRAITS

Wolbachia infection can negatively affect the fitness of its host depending on the Wolbachia strain, the host genotype and the environmental conditions. Because such costs could undermine the effectiveness of the IIT, we measured different life history traits on the lines transinfected by wHa and wTei and compared them to the naturally infected and uninfected lines (Fr-BE-Ø and Fr-BE-wSuz). The larval development conditions were standardized in these experiments by depositing 50 eggs of each line in 2mL of cornmeal diet placed in a tube with agar and sugar (10%). We prepared at least 12 such tubes per line and used the freshly emerged adults for the different measures.

We first measured the survival of the pre-adult stages, that is, from egg to adult. In each tube where 50 eggs were deposited, we counted the number of adults that emerged. We then measured the adult survival for both sexes. For each line, 10 freshly emerged adults were placed into a tube containing sweetened 10% agar (10 replicates per sex) and the mortality was recorded every day. Finally, to measure fecundity and hatch rate, we placed 10 virgin males and 10 females (1-day old) in a cornmeal diet tube during 48h for mating. Females were then allowed to oviposit for 48h on grape-juice agar in a petri dish. The total number of eggs and their hatch rate were recorded 48h after removal of the females. At least 18 repetitions were performed for each line.

THE EFFET OF MALE AGE ON CI INTENSITY

To quantify the effect of ageing on CI levels in D. suzukii transinfected lines, we used the same protocol as described above for the mass crosses experiments in D. simulans. The effect of male age on CI intensity was tested for three ages, 3-4, 7-8 or 11-12 days (6 replicates per age), by crossing males infected by wHa or wTei with females naturally infected or not by wSuz. Control crosses were performed (between uninfected males and females, and between males and females carrying wSuz) in order to assess the effect of male age on hatch rate regardless of CI. Here again a corrected CI index (CI corr ) was used in our analysis.

MATING COMPETITIVENESS OF TRANSINFECTED MALES

The mating competitiveness was tested for males infected by wHa by mixing 40 virgin females with different ratios of these sterilizing males with males carrying wSuz or uninfected males in cages of 30×30×30 cm. Five ratios were tested (4 replicates per ratio): 1:1 (20♂ø:20♂wHa:40♀ø), 1:5 (7♂ø:35♂wHa:40♀ø), 1:10 (4♂ø:40♂wHa:40♀ø) and two control ratios, 1:0 (40♂wHa:40♀ø) and 0:1 (40♂ø:40♀ø). Similarly, the same ratios were used with males and females carrying wSuz instead of being uninfected. As for previous experiments, we used 5-6 days-old females and 3-4 days-old males. Females were first placed in the cages followed by the simultaneous release of all males, and mating was allowed for 48h, with food and water supply (two recipients containing 50 ml of cornmeal diet, and two sweetened water sources with 10% sugar). Thereafter, females were allowed to oviposit for 48h on grape-juice agar in petri dishes which were then replaced with new ones for another 48h. The total number of hatched and unhatched eggs was recorded 48h after removal the petri dishes from the cage. We computed the competitiveness index (C) [START_REF] Fried | Determination of sterile-insect competitiveness[END_REF] to compare the performance of sterilizing and compatible males, which is defined as follows:

C=(N/S)*[(H c -H i )/(H i -H s )],
where N is the number of "compatible" males, S is the number of incompatible males, H c is the hatch rate in the compatible crosses, H i the hatch rate observed in the different ratios tested and H s is the hatch rate in clutches from females exclusively crossed with incompatible males. Similarly, expected hatch rate values in male competition experiments were calculated as follows: [(N*H c )+(S*H i )]/(S+N).

PROOF OF CONCEPT OF THE IIT EFFECTIVENESS

The IIT can be used to decrease the population size of the targeted species but also to limit the introduction and the population growth, and this is the point we tested here using males infected by wHa. Two cages of 3x3x2 meters were placed separately in climatic chambers with similar conditions of temperature (22°C), humidity (50%), and light (light/dark cycle of 12-hours). In each cage, we placed 6 bottles of 1L of a cornmeal diet with a red fruits mixture (red fruits: 50%, agar: 2.6%, yeast: 12%, maize flour: 18%, sugar: 17%, nipagin: 0.4%) and 10 bottles of 10cL of sweetened water (10%). In the control cage, 20 4-6 days old mated females and 20 males (half uninfected and half infected by wSuz) were introduced at the beginning of the experiment and then again every 7 days. In the second cage, called the "IIT cage", the same protocol was followed except that in addition to the 40 individuals released every 7 days, we introduced simultaneously 260 sterilizing males, corresponding to a ratio of 13:1 (260♂wHa:20♂(10♂ø+10♂wSuz)). This experiment lasted 62 days, that is, about 9 weeks, so that 360 individuals were released in the control cage and 2700 in the IIT cage (among which 2340 were sterilizing males). The aim of the experiment was to follow the evolution of the population size over time. Six nesting sites (grape-juice agar in a petri dish of 9 cm) were placed in each cage and renewed every 48h. For each nesting site, the number of eggs laid was used as a proxy of the population size and the hatch rate was determined. At the end of the experiment, all living flies were captured and counted.

STATISTICAL ANALYSIS

We used generalized linear mixed models (GLMM) (binomial family) to analyse all hatch rates data.

-For the mass and individual crosses in D. simulans and in D. suzukii, the Wolbachia strain was included as a fixed factor and the replicates as a random factor.

-For the mating competitiveness analysis, the factors "ratio" and "female's status" were included as fixed explanatory factors and the replicates as a random factor. Exact binomial tests were then used to compare the observed and expected hatch rates in the mating competitiveness experiment. Survival data were also analyzed by GLMM (gamma distribution and inverse link). The Wolbachia infection was included as a fixed explanatory factor and the replicates vials as a random factor.

-Fecundity data was analyzed using a GLMM (poisson family); the Wolbachia strain was included as a fixed effect and the replicates as a random factor.

-The survival rate from the egg to adult stage was analyzed with a linear mixed-effects model (Gaussian distribution) were the Wolbachia infection was included as fixed explanatory factor and the replicates as a random factor.

-In the last experiment, the number of eggs laid and the hatch rate were analysed using a GLM with a poisson and binomial families respectively.

Analysis were performed in R version 3.3.0 (R Core Team 2016), using the package lme4 for all mixed models [START_REF] Bates | lme4: Linear mixed-effects models using Eigen and S4[END_REF].

Results

wSuz INJECTION AND CROSSING EXPERIMENTS IN D. SIMULANS

The cytoplasm of D. suzukii embryos infected by wSuz was injected into 234 D. simulans Wolbachia-free embryos. Seven isofemale lines proved to be infected by Wolbachia in G0.

Patterns of maternal transmission of wSuz are presented in Fig. S1. One isofemale line showing a 100% transmission from G2 to G3 (20 individuals tested) was selected for CI tests. CI experiments were designed to select potential Wolbachia strains for the sterilization of D. suzukii populations, that is, strains that would induce CI even when females carry wSuz.

To this end, we performed crosses in D. simulans between males infected by candidate strains and females carrying wSuz. In control crosses, i.e. crosses between males and females both infected by wSuz, the hatch rate was 97.5%. Among the 10 Wolbachia strains tested, known to induce CI in this genetic background (STCP, [START_REF] Martinez | Should Symbionts Be Nice or Selfish? Antiviral Effects of Wolbachia Are Costly but Reproductive Parasitism Is Not[END_REF], three did not appear to induce CI against wSuz-infected females. Indeed, hatch rates were not significantly reduced in crosses involving the wStv, wPro and wMelCS strains compared to the control crosses (Fig. 1). This indicates that wSuz does express a functional rescue in D. simulans against these strains, and discards them as potential candidates. On the contrary, reduced hatch rates were observed in crosses involving males carrying the other strains. Following [START_REF] Poinsot | Wolbachia Transfer from Drosophila melanogaster into D. simulans: Host Effect and Cytoplasmic Incompatibility Relationships[END_REF] we computed a CI index (CI corr ) taking into account the basal embryonic mortality, to indicate only the proportion of embryos killed by CI. The highest CI corr levels were observed with wRi (57,99% ±13,73), wHa (82,59% ±11,60) and wTei (84,74% ±11,78) (Fig. 1).

Focusing on these three promising strains, we performed individual crosses between males carrying these strains and females either uninfected (used as control) or infected with wSuz to characterize more precisely the rescue capabilities of wSuz. We thus showed that the presence of wSuz in females partially rescues the CI induced by wRi (with a 29% decrease of CI corr ) (z=3.53, P<0.001) and wTei (with a 23% decrease in CI corr ) (z=3.57, P<0.001) but not wHa (z=0.09, P=0.97) (Fig. 2). At that stage, wHa thus appeared to be the most promising strain, but we injected all the three strains into D. suzukii in case host effects would change the rescue capabilities of wSuz in its natural host.

CI EXPRESSION OF WOLBACHIA CANDIDATES IN D. SUZUKII

The cytoplasm of D. simulans embryos infected by wRi, wHa or wTei was injected into uninfected D. suzukii embryos. As for D. simulans, isofemale lines were created from infected females for 2 more other generations until perfect transmission of Wolbachia was observed (from G2 to G3; see details in Table S3). For each Wolbachia strain, one isofemale line was then selected for crossing experiments.

We performed CI crosses to assess (i) if wRi, wHa and wTei can induce CI in D.

suzukii (transinfected males crossed with uninfected females) and (ii) if wSuz in its natural host is able to rescue these effects (transinfected males crossed with females infected by wSuz). We found that wHa and wTei induce strong CI when infected males are crossed with uninfected females in D. suzukii (95.57% and 96.46% CI corr , respectively), in contrast to wRi for which the percentage of unhatched eggs was nearly as low as in the control compatible crosses (18.21% CI corr ) (Fig. 3). In addition, the low CI induced by wRi was fully rescued by the presence of wSuz in females, while the strong CI induced by wHa and wTei was not.

When males infected by wHa were crossed with females carrying wSuz, only 34 eggs hatched in total out of the 960 eggs laid (3.54%). Moreover, no egg hatched in 13 of the 30 individual crosses. For the wTei strain, 33 eggs hatched out of 842 (3.92%) with 0% hatch rates in 25 of the 33 individual crosses. In two crosses, higher hatch rates (94.7% and 54.6 %) were seen, suggesting the induction of CI may occasionally fail or that occasionally some males can be uninfected, although an infection rate of 100% has always been observed in the line used here.

LIFE HISTORY TRAITS OF THE TRANSINFECTED LINES

Adult longevity data revealed a significant effect of the infection status on this trait in both males and females (♀: χ 2 = 121.95, d.f=3, P<0.001; ♂: χ 2 = 38.98, d.f=3, P<0.001). However, this effect does not indicate any physiological cost of the wHa or the wTei Wolbachia strain.

On the contrary, males and females infected by wTei had a greater longevity than the other lines (Fig. S2A andB). In females, there was no significant difference between the three other lines (uninfected, wSuz and wHa) (Fig. S2A) while in males, individuals infected by wSuz showed a higher longevity than uninfected ones (Fig. S2B).

The infection status also affects the fecundity with a significantly larger number of eggs laid in 48h in the wTei (26.39 ±12.03) and wSuz lines (26.22 ±8.17) (between which no significant difference was found; GLMM: z=-0.32, P=0.752). No significant difference was detected between the uninfected (21.15 ±10.82) and the wHa (22.29 ±9.14) lines (GLMM: z=-0.74, P=0.456) (Fig. S3A). We also found an effect of Wolbachia infection on hatch rates, irrespective of CI (Fig. S3B): the wTei and uninfected lines show higher basal hatch rates (91.33% ±15.59 and 94.59% ±9.14 respectively) than the wHa and wSuz lines (83.31% ±25.50 and 83.83% ±25.36).

The infection status also appears to impact survival rates at the pre-adult stage, that is, from egg to adult. Uninfected individuals showed a significantly higher survival rate than the other lines (ø: 75.33% ±6.34; wSuz: 64.38% ±12.63; wTei: 64.00% ±8.49; wHa: 62.17% ±3.86), while there was no significant difference between the three infected lines (Fig. S4).

MALE AGE AND CI INTENSITY

In the control crosses, although hatch rates varied slightly between experiments performed with young or old males, there was no trend indicating an increase or decrease of basal hatch rates with male age (Fig. 4).

In contrast, we observed an overall decrease in CI intensity with male age (Fig. 4A andB). In crosses between males infected by wHa and uninfected females, the CI corr dropped from 97.65% (±2.81) in 3-4-days-old males to 79.72% (±11.97) in 11-12-days-old males. In crosses with wSuz-infected females, the wHa CI corr dropped from 97.30% (±2.50) to 78.22% (±9.36) (Fig. 4A).

The decrease in CI intensity was larger in crosses involving wTei-infected males, dropping from 94.77% (±5.00) to 60.68% (±16.35) and from 93.22% (±6.02) to 60.71% (±10.78) in crosses with uninfected and wSuz females, respectively (Fig. 4B). Notably, these experiments also confirmed that females infected by wSuz cannot rescue the CI induced by males infected by wHa or wTei, regardless of male age.

MATING COMPETITIVENESS OF STERILIZING MALES

In this experiment, we selected the wHa strain, one among the two candidates, because its CI effect was less affected by male age (full data is provided in Table S4). We first confirmed that wHa-infected males induced nearly 100% CI in D. suzukii, regardless of the presence of wSuz in females, with an average hatch rate of 0.40% (±1.74) and 0.44% (±0.56) in crosses with uninfected and wSuz females, respectively (Fig. 5A). Accordingly, we observed that hatch rates decrease when the proportion of sterilizing males increases (all ratios tested produce significantly different hatch rates, expect the 1:5 and 1:10 ratios, GLMM: z=0.63, P=0.60; Fig. 5A). No effect of females' infection status on hatch rates was detected (GLMM: z=-0.50, P=0.62). The hatch rates observed in the ratios 1:0 and 0:1 (without transinfected males or only transinfected males, respectively) allowed us to calculate the hatch rate expected under the assumption of a similar mating competitiveness between sterilizing and compatible males. For each ratio tested, the observed and expected hatch rates were very close, with slight significant deviations. In three cases, the C index [START_REF] Fried | Determination of sterile-insect competitiveness[END_REF]) was less than 1 (meaning that the transinfected males are less competitive than the compatible males) but the reverse was observed in 3 cases (Fig. 5B). Overall, these results indicate that uninfected and wSuz males have very similar mating capacity.

PROOF OF CONCEPT OF THE IIT EFFECTIVENESS

We finally aimed at assessing if repeated releases of sterilizing males could mitigate the increase of a D. suzukii population. We performed experiments in two cages of 3x3x2 meters.

In these cages, a small D. suzukii population was introduced (20 mated females and 20 males, half uninfected and half infected by wSuz) and then again every 7 days. In the control cage, where no wHa-infected males were introduced, the number of eggs laid per time unit (48h) increased substantially during the first 38 days, which corresponds to about two generations (Fig. 6A). The increase continued until the end of the experiment, reaching 1408 eggs laid in 48h. In the IIT cage, where wHa-infected males were regularly introduced, the number of eggs laid per 48h remained low and stable. It was between 0 and 49 per time unit until the 53th days, and slightly increased to reach 308 eggs per time unit at the end of the experiment.

The multiple releases of sterilizing males thus allowed to keep the population size 5 times smaller than the control cage and this effect is significant (GLM: z=-60.74, P<0.001).

Accordingly, the hatch rate observed in the IIT cage was significantly different to the one obtained in the control cage (GLM: z=-20.14, P<0.001) (Fig. 6B). Among the 380 eggs laid in the IIT cage in the last 48h, only 120 were viable (39%) compared with the 1150 hatched eggs among 1408 in the control cage (82%). At the end of the experiment, that is, after 62 days, all live individuals were caught. We thus counted 2184 individuals in the control cage (55% females), and 664 individuals in the IIT cage (41% females). At the end of the experiment, there were thus 4.3 times less females in the IIT cage than in the control one.

Discussion

This study aimed at identifying strains of Wolbachia that could be candidates for controlling D. suzukii populations through IIT. We achieved this goal in several steps. We first transferred wSuz, the natural infection of D. suzukii, into D. simulans to test its ability to rescue the CI induced by the many Wolbachia strains maintained in D. simulans. We thereby selected three Wolbachia candidates based on incompatibility with wSuz, injected them into D. suzukii, and validated two as highly promising for the development of IIT.

In the course of these experiments, we confirmed previously described CI patterns, namely a strong host effect affecting compatibility relationships in a strain-specific manner (Reynolds & Hoffmann 2002;[START_REF] Weeks | Wolbachia dynamics and host effects: What has (and has not) been demonstrated?[END_REF]). We will first discuss these elements before highlighting critical future developments for the effective implementation of IIT in D. suzukii. We found strong variation in CI intensity (mod function) depending on host factors: wRi induces only moderate CI in D. suzukii although it is well known to induce strong CI in its natural host D. simulans [START_REF] Hoffmann | Unidirectional incompatibility between populations of Drosophila simulans[END_REF] this study), but also after transfer in D. melanogaster [START_REF] Poinsot | Wolbachia Transfer from Drosophila melanogaster into D. simulans: Host Effect and Cytoplasmic Incompatibility Relationships[END_REF]). On the contrary, wTei does not induce CI in its natural host, D. teissieri (Zabalou et al. 2004a), but induces a high level of CI in both D. suzukii (this study) and D. simulans [START_REF] Martinez | Should Symbionts Be Nice or Selfish? Antiviral Effects of Wolbachia Are Costly but Reproductive Parasitism Is Not[END_REF]this study). The wSuz infection exhibits the exact same pattern in its natural host and in D. simulans: it induces very low albeit significant CI (see Fig. S5A,B; 63.7% hatch rate and 64.2% respectively). It is clear from these data that a given host should not be generally considered as permissive or refractory to Wolbachia infection and CI, given these strong host genotype-by-Wolbachia strain interactions. Moreover, the analysis of the CI relationship between wSuz and other Wolbachia strains in D. simulans and in D. suzukii reveals that not only the induction of CI (mod function) is host dependent, but also the ability to rescue CI (resc function).

Beyond these CI-relevant results, the main point of this study was to identify Wolbachia strains that could be used as biological control of D. suzukii populations through IIT. By performing back and forth Wolbachia transfers between D. suzukii and D. simulans, we identified two candidates, wHa and wTei. These two strains induce a very high level of CI in D. suzukii, which is not attenuated by the presence of wSuz in females. A number of additional results further confirm that these transinfected lines can be envisaged to implement the control of D. suzukii populations. First, wHa and wTei show a perfect maternal transmission. Second, transinfected males induce a high level of CI throughout their life, despite a reduction with ageing. Finally, transinfected males do not suffer from reduced mating competitiveness or other fitness costs. Accordingly, we showed that the repeated release of sterilizing males can limit the explosion of a D. suzukii large cage population.

Overall, this study provides, at a laboratory scale, a proof of concept that the IIT approach can be powerful to control D. suzukii populations.

An efficient IIT program must rely on efficient methods to avoid the release of fertile females, which could result in population replacement rather than population suppression [START_REF] Bourtzis | Harnessing mosquito-Wolbachia symbiosis for vector and disease control[END_REF]. In case of accidental release, the newly introduced infection would easily spread across D. suzukii populations, since the resident infection induces only very low CI. Notably, the fact that the wHa and wTei strains are mutually incompatible [START_REF] Zabalou | Multiple rescue factors within a Wolbachia strain[END_REF]) means that one strain might still be used for population control in case of accidental release and invasion of the first strain. To circumvent the difficult sexing step, IIT could be coupled with moderate irradiation [START_REF] Brelsfoard | Integration of irradiation with cytoplasmic incompatibility to facilitate a lymphatic filariasis vector elimination approach[END_REF][START_REF] Bourtzis | Harnessing mosquito-Wolbachia symbiosis for vector and disease control[END_REF][START_REF] Zhang | Combining the sterile insect technique with the incompatible insect technique: I-impact of Wolbachia infection on the fitness of triple-and double-infected strains of Aedes albopictus[END_REF] that would be sufficient to sterilize females without affecting the life history traits and competitiveness of males which would be fully sterile thanks to the Wolbachia [START_REF] Calvitti | Wolbachia strain wPip yields a pattern of cytoplasmic incompatibility enhancing a Wolbachia-based suppression strategy against the disease vector Aedes albopictus[END_REF][START_REF] Bourtzis | Harnessing mosquito-Wolbachia symbiosis for vector and disease control[END_REF]. Finally, IIT relies on the massive production of males which seems achievable for such a small and polyphagous insect, but requires additional developments.

The existence of D. suzukii lines transinfected by CI inducing Wolbachia strains represents a critical step toward the implementation of IIT. Our results indicate that these lines carry a number of crucial properties that make them usable in practice to limit population size in large population cages. While additional developments are still needed, we are now much closer to make IIT a credible alternative to pesticides to control D. suzukii populations.

FiguresFig . 1 .

 1 Figures

Fig . 2 .

 2 Fig .2. Assessment of the resc capabilities of wSuz in D. simulans. un: uninfected, wSuz: infected by wSuz, wRi: infected by wRi, wTei: infected by wTei, wHa: infected by wHa. The CI corr index removes the basal embryonic mortality (estimated in control crosses); it is thus a measure of the CI-related mortality. 20 repetitions were performed for each type of cross. ***: P<0.001.

Fig . 3 .Fig. 4 .BFig. 5 .

 345 Fig .3. Measure of the mod function of wRi, wHa and wTei and the resc function of wSuz in D. suzukii. un: uninfected, wSuz: infected by wSuz, wRi: infected by wRi, wTei: infected by wTei, wHa: infected by wHa. The CI corr index removes the basal embryonic mortality (estimated in control crosses); it is thus a measure of the CI-related mortality. ***: P<0.001.

Fig. 6 .

 6 Fig. 6. Evaluation of the IIT effectiveness to limit D. suzukii population growth in a large climatic chamber. A: number of eggs laid per 48h at regular intervals. B: hatch rates at regular intervals.

Acknowledgments

This work was funded by the CNRS (IFR41-UMR5558) and supported by the ONEMA (APR Biodiversité-Ecophyto), the ANR (ANR-15-CE21-0017-01) and the FWF (I 2604-B25).

Julien Cattel was the recipient of a PhD studentship from the Rhône-Alpes region ("ARC Program" Grant). We also warmly thank Yuk Sang Chan at Cambridge University, for his invaluable competence on Wolbachia transinfections, and Aymeric Bellemain and Justine Planche for their technical contribution.

Data accessibility

Data available from the Dryad Digital Repository. DOI: 10.5061/dryad.4c7kq [START_REF] Cattel | Data from: Back and forth Wolbachia transfers reveal efficient strains to control spotted wing drosophila populations[END_REF]).

Author's contributions JC, SC, FV, PG and LM conceived the project and designed its methodology; JC, TA, KN and DL collected the data; JC analyzed the data; JC, SC, FV, PG and LM led the writing of the manuscript. All authors contributed critically to the drafts and gave final approval for publication.

Supporting Information

Table S1. Primers used in this study Table S2. Details of all D. simulans lines used in this study Table S3. Data on microinjections and maternal transmission of Wolbachia strains in D. suzukii Table S4. Data on mating competitiveness of wHa-transinfected males


Yuxin Liu

Abdelkrim Talbi

Bahram Djafari-Rouhani

Lucie Drbohlavová

Vincent Mortet

Houssaine El El

Boudouti

Olivier Bou Matar

Philippe Pernod

Interaction of Love waves with coupled cavity modes in a 2D holey phononic crystal

Keywords: numbers: 68, 60, Bs

The interaction of Love waves with square array of pillars deposited on a cavity defined in a 2D holey phononic crystal is numerically investigated using Finite Element Method. First, the existence of SH surface modes is demonstrated separately for phononic crystals that consist of square arrayed holes, or Ni pillars, respectively in, or on, a SiO 2 film deposited on a ST-cut quartz substrate. The coupling between SH modes and torsional mode in pillars induces a transmission dip that occurs at a frequency located in the range of the band-gap of the holey phononic crystal. Second, a cavity is constructed by removing lines of holes in the holey phononic crystal and results in a transmission peak that matches the dip. The optimal geometrical parameters enable us to create a coupling of the cavity mode and the pillar mode by introducing lines of pillars into the cavity, which significantly improved the efficiency of the cavity. The obtained results will pave the way to implement advanced designs of high-performance electroacoustic sensors based on coupling modes in phononic crystals.

Phononic crystals (PnCs) have received increasing attention in the last two decades as an analogue of photonic crystals (PtCs). [1][2][3][4][5][6][7][8] Coupling modes have been investigated in both PnCs [9][10][11][12][13][14] and PtCs [15][16][17][18][19], exhibiting features such as energy transferring [10,11,16], absorption [9,12], wave confinement [16] and frequency modulation [13]. Most of the research is based on the cavity/waveguide [10,13,16], cavity/cavity [13,18] or waveguide/ waveguide [11] systems. Wang et al. [12] demonstrated the transmission cancellations due to the coupling modes of a 1D locally resonant PnC. Ohta et al. [17] studied the coupling between a PtC nanobeam cavity and a quantum dot. The emission peak of the quantum dot could be tuned by temperature to cross the cavity mode which splits into two peaks. In recent years, peak splitting phenomena have also been discussed in the coupling of two PnC cavities [13], giving rise to a capacity of frequency modulation as well as improved quality factors. We believe that it is conceivable to investigate similar effects in different PnC devices.

Recently, cavity modes for Love waves, which is a shear horizontal (SH) surface acoustic waves (SAW), are demonstrated in a holey PnC (h-PnC) that consist of square arrayed holes in a silica (SiO 2 ) film deposited on a ST-cut quartz [20]. This letter investigates the interaction of Love waves with a Ni pillar-introduced cavity in the h-PnC. Love waves are analyzed separately in the h-PnC and the pillared PnC (p-PnC). A mode-coupling induced transmission dip for the p-PnC is found in the a) Electronic mail: yuxin.liu@phd.ec-lille.fr band-gap region of the h-PnC. An optimally sized cavity in the h-PnC gives rise to a cavity mode that matches the transmission dip. The cavity mode is coupled to the pillar mode by introducing the pillars into the cavity, which significantly improved the sharpness of the transmission peak. These effects are used to design the microelectromechanical resonators with highly confined cavity modes. The band structures and the transmission spectra are calculated with the finite element method(FEM, COMSOL Multiphysics ). , where Rayleigh waves cannot be generated [21] due to a zero electromechanical coupling factor in the substrate. The surfaces of the PnCs coincide with the plane z = 0. The wavelength normalized energy depth (NED) [20] is calculated to select the surface modes:

NED = D 1 2 T ij S * ij (-z)dxdydz nλ D 1 2 T ij S * ij dxdydz , ( 1
)
where T ij is the stress and S ij the strain. The star symbol (*) means the complex conjugate. -z denotes that we integrate in the whole domain of the unit cell D. λ is the wavelength. For a relatively small k (with wave speed greater than the SH waves velocity of the substrate), λ is fixed to 2a that is resulting from k = π a and k = 2π λ . n = 1 for the h-PnC and n = 1.3 for the p-PnC because of the pillar height, which equals to 0.6a = 0.3λ. Surface modes have a NED < 1. The NED can filter out the bulk modes as well as the plate modes that appear in our finite-depth substrate which is supposed to be semiinfinite for Love waves.

SAW include SH type SAW and Rayleigh type SAW. The SH polarization ratio is calculated to distinguish these two polarizations:

SH ratio = D u SH u * SH dxdydz D (u x u * x + u y u * y + u z u * z )dxdydz , (2)
where u x , u y and u z are respectively the displacement along the x, y, z directions. u SH is the SH displacement component that can be expressed as u x cos θ -u y sin θ, which is perpendicular to the wave vector k. θ is the The transmission spectra is calculated by simulating the same SAW device using our preceding approaches [20,22], which has been validated by experiments. The model consists of two parts of aluminum inter-digital transducers (IDTs) with the PnC located between IDTs. The input IDT is given a V 0 = 1V harmonic voltage signal. The output is measured by averaging the voltage difference between odd and even electrodes. The width of the electrodes of IDTs L IDT is updated for every frequency in the spectrum according to the relation The coupling between the torsional mode p2 and SH waves has induced a sharp transmission dip at 508 MHz, denoted as χ. The origin of this dip is a narrow band gap between 507.6 and 510.2 MHz, formed by the coupling of p2 and p3 [12]. The slight frequency shift is mainly due to the mesh construction of the FEM.

L IDT = λ 4 = v 4f . v
By removing lines of holes along the y direction in the h-PnC, a cavity is formed perpendicularly to the waves propagation direction. Here we set the cavity width W to 4.5a h , since a cavity mode is predicted [20] at almost the same resonant frequency as f χ . The resonator is realized by introducing the p-PnC into the cavity. A supercell containing 1×(6 + W a h ) unit cells is constructed, as shown in Fig 4, with periodic boundary conditions applied along the y direction. Since the crystal size only slightly affects the cavity mode frequencies [13,20], the transmissions are calculated with 4 holes on each side of the cavity.

The Love waves normalized transmission spectra and band structures for the cavity without and with pillars are shown in Fig 5(a) and (b), respectively. On the bare cavity (without pillars), two cavity modes C1 and C2 appear inside the band-gap of the perfect h-PnC, giving rise to two transmission peaks at 438.9 MHz and 507.5 MHz, respectively. The 2 nd peak matches the transmission dip χ at 508 MHz that is denoted by the dotted line (f C2 ∼ = f χ ). After introducing the pillars, the fundamental cavity modes C1 and C2 are coupled to the pillars and become two new modes Cp1 and Cp2, while new pillar modes ∀p appear above. Due to the injected pillars mass, Cp1 at 433.4 MHz has a slight down frequency shift compared with C1, whereas Cp2 has shifted down to 490.5 MHz to show a well separated peak from ∀p. The frequency shift of Cp2 results from both the mass loading and the peak splitting effect that is attributed to a degeneracy of the cavity mode. [13] Meanwhile, the transmission peak of Cp2 is significantly sharper than that of the mode C2, as a result of the matched frequencies between f C2 and f χ . The pillar modes ∀p are different combinations of pillars vibrating in mode p2 (since f p2 f χ ), with the maximum displacements existing in different pillars. The number of pillar modes equals to the number of pillars, i.e., 18 pillar modes with adjacent frequencies around f p2 , which can be adjusted by the p-PnC parameters. The dispersion curves of ∀p are zoomed on the right of Fig 5(b). The first pillar mode gives rise to a sharp transmission peak at 507.3MHz. The last mode, with a less smoothing and homogeneous dispersion curve, only arouses a small resonance at 517.5 MHz. Other pillar modes disappear in the transmission spectrum because they are not coupled to the SH waves. This transmission spectrum results from a coupling between the p-PnC and the bare cavity-containing h-PnC. The differences in sharpness of the transmission peaks for modes C2, Cp2 and the 1 st of ∀p could be explained after observing their displacement fields shown in Fig 5(c). When Love waves propagate through the cavity, the displacement of C2 is concentrated in the center of the model and is confined in the silica guiding film. The mode Cp2 is actually a coupling of p2, p3 and C2 modes. The pillars vibrating in mode p2 and p3 are alternated along the propagating axis. This pillar-coupling mode is simultaneously coupled to the fundamental cavity mode C2 guiding in the film. Nevertheless, the maximum displacement amplitude is in the pillars, leading to an energy more confined to the surface (a smaller NED). This is proved by the dispersion curve of Cp2 whose red color is more intense than that of C2. As for the 1 st collective pillar mode of ∀p, only tiny displacement exists in the guiding layer, leading to the most confined energy and the sharpest transmission peak. The magnification of these three peaks is shown aside. The quality factor (Q) of the mode Cp2 is 6 times greater than that of the mode C2. The Q of the sharpest peak is 80 times greater than that of C2, with a transmission peak at almost the same frequency. This means that the efficiency of the cavity could be significantly enhanced by introducing the optimally sized pillars.

In summary, we investigated the SH surface modes in two phononic crystals that consist of square arrayed holes/Ni pillars in/on a silica film deposited on a 90STcut quartz substrate. The h-PnC gives rise to a band gap within which two pillar modes of the p-PnC are coupled, leading to a sharp transmission dip. An optimally sized cavity is introduced into the h-PnC by removing lines of holes and arouses a cavity mode that matches the pillar-coupling frequency. Pillar-containing cavity for Love waves is first demonstrated by introducing the pillars into the cavity. A coupling of the cavity mode to the pillar mode is created with significant improvement on the cavity efficiency. The sharpest transmission peak observed at the original cavity mode frequency has a 80 times greater Q. This study could be used for potential PnC applications such as filters and bio-sensors.

FIG. 1 :FIG. 2 :FIG. 3 :

 123 FIG.1: Unit cells of the PnCs with square arrayed cylindrical holes, or Ni pillars, respectively in, or on, the silica film deposited on a 90ST-cut quartz. r h =0.2a h , r p =0.2a p , H h =H p =2.4µm, h p = 0.6a p , a h = 4µm, a p = 2µm.

FIG. 4 :FIG. 5 :

 45 FIG. 4: Supercell of pillar-introduced cavity in the h-PnC. W = 4.5a h , a h = 4µm, a p = 2µm.

  is the velocity of Love waves for H h = 0.6a h , resulting from the basic dispersion relation of Love waves. That is, each frequency corresponds to a single wavelength, and this model simulates the dispersive SAW devices. The frequency responses are then nor-malized by that of the matrix, referred to as normalized or relative transmissions ∆S 21 . Fig 3(b) shows the normalized transmission spectrum of the h-PnC around the band-gap region. The attenuation appears clear and is consistent with the band structure prediction. Fig 3(d) is the normalized transmission spectrum of the p-PnC.

f (MHz) Ni 0 1 SH polarisation ratio

ACKNOWLEDGMENT

The authors thank RENATECH, the French national nanofabrication network, the J.E. Purkyn fellowship awarded to V. Mortet by the Czech Academy of Sciences and Centrale Initiative Foundation for their financials supports.