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(2 NOTE Eur. J. Entomol. 112 (4): 000–000, 2015 doi: 10.14411/eje.2015.079 ISSN 1210-5759 (print), 1802-8829 (online)

New set of microsatellite markers for the spotted-wing Drosophila suzukii (Diptera: Drosophilidae): A promising molecular tool for inferring the invasion history of this major insect pest

ANTOINE FRAIMOUT ௘1, ANNE LOISEAU ௘2, DONALD K. PRICE௘3, ANNE XUÉREB௘2, JEAN-F RANÇOIS MARTIN ௘2, RENAUD VITALIS ௘2, SIMON FELLOUS ௘2, ௘ VINCENT DEBAT ௘1, ௘ and ARNAUD ESTOUP ௘2, ௘

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Key words. Diptera, Drosophilidae, Drosophila suzukii , population genetics, microsatellite, invasion routes, invasive species, genetic structure

Abstract. Historical and observational data for invasive species are often sparse and incomplete, so molecular genetic markers are LQFUHDVLQJO\XVHGDQGKDYHSURYHGWREHHI¿FLHQWWRROVWRPDNHLQIHUHQFHVDERXWLQYDVLRQKLVWRULHV+HUHZHUHSRUWWKHGHYHORSPHQW of 28 polymorphic microsatellite markers in the invasive spotted-wing drosophila, Drosophila suzukii (Matsumura, 1931) (Diptera: 'URVRSKLOLGDH GHVLJQHGIURPUHFHQWJHQRPLFVUHVRXUFHVDQGWKHLUFURVVDPSOL¿FDWLRQLQFORVHO\UHODWHG Drosophila species of the suzukii subgroup. The markers, located on autosomal chromosomes, were characterized in two distinct invasive populations from France and Hawaii (USA), and in four sister species of the suzukii subgroup. They all showed substantial polymorphism as well as revealing strong genetic differentiation between the two genotyped populations. These markers represent a promising molecular tool for population genetic studies on D. suzukii , more especially in order to reconstruct the pathways and demographic processes of the world-wide invasion in this major insect pest.

INTRODUCTION DQG LL LWLVUHTXLUHPHQWQHFHVVDU\IRUGH¿QLQJDQGWHVWLQJGLIIHU - ent hypotheses concerning the environmental and evolutionary The spotted-wing drosophila, Drosophilla suzukii (Matsumura, factors responsible for biological invasions in general (Estoup & 1931) (Diptera: Drosophilidae) is an oriental species of the mela- Guillemaud, 2010). nogaster group. It is widely distributed in south-eastern Asia, Most of our knowledge about the introduction routes of inva- IURP,QGLD 3DUVKDG 3DLND WR-DSDQZKHUHLWZDV¿UVW sive species derives from historical and observational data, which described in 1931 (Hauser, 2011). D. suzukii females exhibit a are often sparse, incomplete and, sometimes, misleading. In the large serrated ovipositor which allows them to lay eggs in ripen- case of D. suzukii , this approach suggests there was a single intro- ing fruits, hence causing severe damages to stonefruit and red duction in Europe, located in France in between Spain and Italy, berry crops. The species has drawn particular attention during the ZKHUHWKHSHVWZDV¿UVWUHSRUWHG &LQLHWDO $QDOWHUQDWLYH SDVW¿YH\HDUVEHFDXVHRILWVVWDWXVDVDFURSSHVWDQGLWVUDSLG approach is to use population genetics analyses based on poly- invasion across Europe and the USA (reviewed in Hauser, 2011 morphic molecular markers, such as microsatellites, which have and Calabria et al., 2012). When an invasive species presents im- proved to be powerful tools for reconstructing invasion routes, mediate threats in a new environment (e.g. economical, ecologi- highlighting the complexity and the often counter intuitive na- cal or for human health), most research plans legitimately focus ture of the true story (Estoup et al., 2001; Estoup & Guillemaud, on how to prevent further expansion or to reduce damages caused 2010). Here we report the development of a set of 28 polymor- by introduced populations (Thresher et al., 2014). However, re- phic microsatellite markers in D. suzukii designed from recent constructing the geographic pathways followed by propagules genomics resources, as well as characterizing genetic variation from their original source location to the newly invaded location at these particular loci in two distinct invasive populations along (hereafter referred to as invasion routes) is often overlooked. In ZLWK WKHLU FURVVDPSOL¿FDWLRQ SDWWHUQV LQ FORVHO\ UHODWHG Dros- light of this, even though some crucial aspects of D. suzukii biol- ophila species. ogy and ecology have been already investigated (Kimura, 2004), little is known about the species’ invasion history and more espe- MATERIAL AND METHODS cially the origins of the different introduced populations (but see Adrion et al., IRUD¿UVWDSSURDFK 7KLVLVXQIRUWXQDWHVLQFH Sampling and DNA extraction robust information about the invasion routes of a species is cru- Adult D. suzukii ZHUHFROOHFWHGIURPWKH¿HOGDWWZRGLVWLQFW cial for at least two principle reasons: (i) it facilitates the design of localities of the invasive range between October and November, strategies for controlling present or preventing future invasions; 2013, and stored in ethanol. The Hawaii population consisted of

(TXDODXWKRUVKLS 1 DGXOW ÀLHV FDXJKW DW WKH 6DGGOH 5RDG ORFDOLW\ Ý :HVWXGLHGFURVVVSHFLHV3&5DPSOL¿FDWLRQRIRXUVHWRIPL - Ý: WKH)UHQFKSRSXODWLRQFRQVLVWHGRIDGXOWÀLHV crosatellite loci, using the same PCR conditions as above, on in- FDXJKWQHDU0RQWSHOOLHU Ý1Ý( 7RWDOJHQRPLF dividuals from four species belonging to the suzukii subgroup: D. '1$ ZDV H[WUDFWHG XVLQJ %LR5DG &KHOH[ (VWRXS HW DO biarmipes (Malloch), D. subpulchrella (Takamori & Watabe), D. 1996). lucipennis (Lin) and D. mimetica (Bock & Wheeler). Each sister- species was represented by a single isofemale line from which 12 3ULPHUGHVLJQDQG3&5DPSOL¿FDWLRQ individuals were genotyped (samples obtained from the UC San We used the recently assembled D. suzukii genome (Chiu et al., Diego Drosophila stock center). 2013; project accession number PRJNA213258) as an input for WKH4''SURJUDP 0pJOHF]HWDO WRVFDQJHQRPLFGDWDIRU RESULTS PLFURVDWHOOLWHUHJLRQV%ULHÀ\4''XVHVIRXUVFULSWV 6HTXHQFH A large number of regions containing continuous microsatel- similarity, BLAST, microsatellite detection and markers design) to lites located on different scaffolds of the D. suzukii genome as- analyze raw data and provide a table summarizing, amongst other, the region of the genome containing a potential microsatellite VHPEO\ZHUHLGHQWL¿HGXVLQJWKH4''VRIWZDUH:HHQVXUHGWKDW the chosen loci were not aggregated on the D. suzukii genome by sequence, the repeat motif and the number of repeats. We decided ¿UVW%/$67VHDUFKLQJWKHPXVLQJWKH D. melanogaster genome. WRIXUWKHU¿OWHUWKHUHVXOWVRI4''E\FKRRVLQJRQO\FRQWLQXRXV We then chose 32 loci matching two autosomal chromosomes (i.e. non-interrupted) microsatellite sequences, with at least (the third autosomal chromosome is of very small size in Droso- 10 repetitions of the motif and excluding (CG) and (AT) . To n n phila species) and different regions within each chromosome, maximize the chances to obtain statistically unlinked loci, we excluded microsatellite loci belonging to the same D. suzukii ZLWK D ¿QDO GLVWULEXWLRQ RI ORFL SHU FKURPRVRPH DUP7KLUW\ two pairs of primers, delimiting the 32 chosen loci, were designed scaffold and blasted the selected sequences to the Drosophila melanogaster (Meigen) genome to infer chromosomal location. IRU3&5DPSOL¿FDWLRQV7ZHQW\HLJKWVXFKPDUNHUVZHUHUREXVWO\ sized and genotyped with GENEMAPPERTM 5.0 and used for fur- We used the program PRIMER DESIGNER (version 2.0; ther analyses. 6FLHQWL¿F (GXFDWLRQDO6RIWZDUH WRGHVLJQIRUZDUG and reverse primers for the chosen microsatellite loci. Genetic diversity (DFKSDLURISULPHUVZDVXVHGIRU3&5DPSOL¿FDWLRQLQD/ Table 1 shows that all 28 microsatellite loci were polymorphic ¿QDO YROXPH FRQWDLQLQJ ; 4,$*(1 0XOWLSOH[ 0DVWHU 0L[ with allele numbers ranging from 2 (DS19, DS34, DS11) to 11 0RIHDFKSULPHUDQG/RIJHQRPLF'1$3&5SURJUDPV (DS32) in the pooled dataset (France + Hawaii). Observed het- ZHUHVHWZLWKDQLQLWLDOSHULRGRIGHQDWXUDWLRQDW& V IRO - erozygosity ( Ho ) ranged from 0.19 (DS21) to 0.84 (DS14) in the ORZHGE\F\FOHVRIDGGLWLRQDOGHQDWXUDWLRQDW& V DQ Hawaii population and 0.12 (DS21) to 0.88 (DS05) in the French DQQHDOLQJSKDVHDW& PLQV DQHORQJDWLRQSKDVHDW& SRSXODWLRQ2QO\RQHORFXV '6 VKRZHGVLJQL¿FDQWGHSDUWXUH PLQ DQG HQGLQJ ZLWK DQRWKHU H[WHQVLRQ SKDVH DW & (P < 0.05) from H-W equilibrium in the Hawaii population and min). WZRORFL '6DQG'6 LQWKH)UHQFKSRSXODWLRQ6LJQL¿FDQW :H¿UVWXVHGDJDURVHJHOIRUPLJUDWLRQRIWKH3&5SURG - LD was found for one pair of loci in the Montpellier population XFWV/RFLIRUZKLFKDPSOL¿FDWLRQVSURGXFHGRQO\RQH'1$EDQG and for 14 pairs in the Hawaii population. All pairs of loci pre- corresponding to the predicted size of the PCR product were then VHQWLQJVLJQL¿FDQW/'ZHUHSXWDWLYHO\ORFDWHGRQWKHVDPHFKUR - DPSOL¿HGIROORZLQJWKHSURWRFROGHVFULEHGDERYHEXWZLWKIRU - PRVRPH DUP 2QO\ RQH SDLU '6±'6 VKRZHG VLJQL¿FDQW ZDUGSULPHUVÀXRURSKRUHODEHOOHGRQWKH¶HQGDQGQRQODEHOOHG LD in both populations. UHYHUVHSULPHUV7KHVHORFLZHUH¿QDOO\VL]HGXVLQJDQ$%, Testing for null alleles and selection sequencer (Applied Biosystems, Montpellier, France) with the 500 LIZ TM GeneScan TM size standard and scored with the GEN- )UHH1DSRLQWHGWR¿YHORFLZLWKQXOODOOHOHIUHTXHQFLHV! EMAPPERTM 5.0 software. in at least one population (DS09, DS25, DS06, DS26 and DS21). 7KHVDPHORFLZHUHLGHQWL¿HGDVEHDULQJQXOODOOHOHVLQDWOHDVWRQH Genetic analyses SRSXODWLRQE\0LFURFKHFNHU2IWKHVH¿YHORFLRQO\RQH '6 All loci were checked for null-alleles using FreeNa (Chapuis & showed high null allele frequencies in both populations and was Estoup, 2007; Chapuis et al., 2008) and MicroChecker (van Oost- therefore left out when testing for selection. DetSel did not detect erhout et al., 2004) in the Hawaii and Montpellier populations. DQ\PLFURVDWHOOLWHORFXVWKDWGHSDUWHGVLJQL¿FDQWO\IURPQHXWUDO Levels of polymorphism and departure from Hardy-Weinberg expectations. (H-W) equilibrium were assessed for both populations. Linkage Population differentiation disequilibrium (LD) was tested for each pair of loci using GE- :HIRXQGDVLJQL¿FDQWDQGVXEVWDQWLDOOHYHORIJHQHWLFGLIIHU - NEPOP 4.2 (Raymond & Rousset, 1995). FST values were esti- mated using both GENEPOP 4.2 and FreeNa (with the ENA cor- entiation between the populations from France and Hawaii (F ST rection for null alleles implemented in the program). To test for = 0.206). In agreement with this, the NJ tree using individuals as loci under selection, we used the DetSel package (Vitalis et al., units shows a clear clustering of individuals in relation to their IRU5 5'HYHORSPHQW&RUH7HDP 'HW6HOLGHQWL¿HV geographic origin (Fig. 1). Moreover genetic assignment tests markers showing deviation from the neutral expectation in pair- VLJQL¿FDQWO\DVVLJQHGDOOJHQRW\SHGLQGLYLGXDOVWRWKHLUSRSXOD - wise comparisons of diverging populations (Vitalis et al., 2001). tion of origin (results not shown). We used the following parameters for our analysis (details about &URVVVSHFLHV3&5DPSOL¿FDWLRQ the user’s parameter options can be found in Vitalis et al., 2012): Table 2 shows that most loci (24 out of 28) successfully ampli- number of simulation points = 1.10 6, mutation rate = 5.10 –5 , time ¿HGLQERWK D. biarmipes and D. subpulchrella. Although their of divergence = 5000 and effective size ( N ) = 500,000. We used H࣠ SUR¿OHVZHUHJHQHUDOO\VLPLODUWRWKRVHREVHUYHGZLWK D. suzukii , a personal program to construct a Neighbor-Joining (NJ) dendro- most loci were homozygous and exhibited substantially differ- gram from individual genotypes using a variant of Chakraborty ent allele sizes compared to D. suzukii &URVVDPSOL¿FDWLRQVXF - DQG-LQ¶VDOOHOHVKDUHGGLVWDQFHGH¿QHGLQ)RXUQLHUHWDO cess was lower in D. mimetica and D. lucipennis (16 and 14 loci, respectively), which was expected according to the further ge-

2 TABLE 1. Primer sequences and diversity index for 28 microsatellite loci in two invasive populations of Drosophila suzukii. A – allele number, Ho – observed heterozygosity; He – expected heterozygosity, N – QXPEHURIVDPSOHGLQGLYLGXDOV ±VLJQL¿FDQWGHSDUWXUH from H-W equilibrium ( P < 0.05), D.mel. – position of homologous loci on D. melanogaster chromosomes. Hawaii (N = 33) Montpellier (N = 27) Locus Repeat D.mel. 3ULPHUVHTXHQFH ĺ Dye Size range A Ho He A Ho He DS05 (TG)10 Chr 2L AGGATAACGCGCAGCTTGAC Ned 260–300 6 0.8387 0.6967 4 0.8800 0.7584 TATGGAAGCTGGCAAGCAGA DS06 (TG)11 Chr 2L CGGTTCGAGTGCTTGTTAGA Fam 130–190 4 0.6061 0.6056 3 0.3333 0.4856 ACACGTGGAGGACACCTTC DS07 (CA)13 Chr 2L AAGGCTGGAGTGGCAACAA Ned 160–210 3 0.4545 0.5147 7 0.8148 0.8278 GCTAAGGTTCTGTTCGGCTG DS08 (AG)10 Chr 2L CGTTGTTGGCGGTGAGTAAG Vic 110–170 9 0.6970 0.7222 4 0.8148 0.7565 GGCCATCAATCAGTCAGTCA DS09 (AC)15 Chr 2L CACACATGGCGTATGCGTAT Fam 190–250 4 0.4848 0.6772 8 0.6538 0.6649 ACTTGTTGAGCCGTCCTGG DS10 (GT)11 Chr 2L CGAGACTGTGCGAACGAGAG Ned 270–330 5 0.3939 0.5124 6 0.6538 0.6294 CATATGCTGACTGCCTCACA DS11 (CA)11 Chr 2L CGGTGACTCGTGCAGTTGTA Vic 230–290 2 0.4545 0.3512 3 0.4074 0.4753 GCCGACTCTGTCTAGAGCAA DS12 (GT)19 Chr 2R GCTGTTGCTGTTGCTATTGC Fam 320–380 6 0.8438 0.7866 6 0.6538 0.7167 AGAACCGTTAGCTGAGCGAG DS14 (TG)10 Chr 2R AAGAACCGCAACGAGCAA Fam 180–220 7 0.8485 0.7374 6 0.5833 0.5755 GAATTATCCAGCGACACGAC DS15 (GT)11 Chr 2R GGACAGCCGACATAAGAGG Fam 260–320 5 0.5152 0.5505 7 0.8462 0.6553 GAGTTGCTGGCTCGACACTT DS16 (AC)13 Chr 2R TTCGTATGTTAGGCGCCA Pet 100–140 3 0.6061 0.6074 6 0.7308 0.7234 CTGGCTGCTGACCTCAACTC DS17 (GT)10 Chr 2R CATCTCAGGCCACGAATG Fam 80–130 3 0.3939 0.3742 5 0.7407 0.7064 CTCCAGATTCTCGAGTGCAG DS19 (CT)10 Chr 2R CCGTTGGCATCTCTGAGTCT Vic 180–220 2 0.3636 0.3967 5 0.5556 0.5734 GCAGACGGAGAGCAGTTGTT DS20 (AG)12 Chr 3L CAGCCATATGCAATGCACTG Ned 210–270 6 0.6250 0.6973 4 0.7083 0.7405 ATATCCAGCGGAAGTCGAGA '6 (AC)11 Chr 3L GAGACGCGATGGTACCGTTA Vic 310–370 4 0.1935 3 0.1176 CCAATCGAGTGCAAGCGT DS22 (GT)11 Chr 3L TACAGATACGCCGTCGGATT Pet 290–360 6 0.5938 0.6948 5 0.6667 0.6979 AAGACCAAGACGACGGACCT DS23 (AC)10 Chr 3L TGCCACTAAGCTCACACGGT Pet 237–300 6 0.6129 0.6145 3 0.7917 0.7283 CAGTTGCCACTTGCTGTGTA '6 (CA)10 Chr 3L CCTTCTGCTGTCCTGCTTAT Vic 220–270 5 0.6667 0.6644 4 0.4444 CGGTCGAGCATCAAGTGAA DS26 (CA)10 Chr 3L CCTGTGTGCATCTCAGTGTT Vic 60–130 4 0.3548 0.6113 4 0.7037 0.7277 TACAGCACTCCAGCACATGA DS27 (GT)14 Chr 3L CCAGCGACTGCAGAAGTGAC Ned 80–130 6 0.7576 0.7034 5 0.7037 0.6893 GCAATCCTCCACAACACAAC DS28 (TG)11 Chr 3L TTAAGCTGACCTCCTCCTCG Pet 140–195 4 0.5758 0.5822 7 0.8400 0.7896 GCACTCGCACAGATACAAGG DS32 (TG)15 Chr 3R CGGCGTGTTGCAGTTATTC Fam 330–380 4 0.8333 0.8073 10 0.7813 0.6685 ATGCACTGGTCGACATGACA DS33 (AC)10 Chr 3R GTTGGATGCCTGGAGGAATA Pet 150–210 3 0.6364 0.6318 5 0.8462 0.7655 TTGTGCGAGTATGTCAGCCA DS34 (GA)12 Chr 3R AACAACGACGCAGAAGCTCA Fam 240–300 5 0.4848 0.4444 2 0.5417 0.5634 CGACTGTTGCGCTCTGTAAT DS35 (CA)11 Chr 3R TCCGTATTCCGTATCCGTGT Pet 198–240 8 0.4688 0.5171 3 0.8333 0.7925 GGAGTATGGCAGTGTGGCAG DS36 (GT)13 Chr 3R TTGGCAACGTGTGAAGCTG Vic 160–220 4 0.7879 0.7199 4 0.4074 0.5158 GAGACACTGCAATGCTGCCT DS38 (AC)12 Chr 3R CTGTCGGCTACGCAATATTC Ned 130–180 7 0.5758 0.4835 3 0.8148 0.7833 CAACGGATTGTGGCTGATTG DS39 (AC)12 Chr 3R GCCAGGCAGCAACAACATC Ned 310–370 6 0.7188 0.7466 5 0.7407 0.7257 AGATCCGAGAGTTGCGAGTT

3 netic distance of the two species from D. suzukii compared to D. biarmipes and D. subpulchrella (Kopp & True, 2002). Because WKHLQGLYLGXDOVXVHGIRUFURVVDPSOL¿FDWLRQWHVWVRULJLQDWHGIURP stock-center populations (with a single isofemale line for each species), a high level of inbreeding and adaptation to laboratory FRQGLWLRQVLVVXVSHFWHGIRUWKHVHÀLHVWKHUHE\TXHVWLRQLQJWKHUH - liability of the observed allelic pattern in the non- suzukii species. DISCUSSION The inferential methods used for retracing invasion routes ne- cessitates that the studied populations display substantial poly- PRUSKLVPDQGVLJQL¿FDQWJHQHWLFGLIIHUHQWLDWLRQLQRUGHUWRGLV - criminate among potential origins and make inferences about demographic processes (e.g. Estoup & Guillemaud, 2010). The microsatellite markers here developed for D. suzukii FOHDUO\¿W these criteria, at least for the two tested populations. In a recent paper, Adrion et al. (2014) argued that at least 10 5 independ- HQW VHTXHQFH ORFL ZRXOG EH QHHGHG WR FRQ¿GHQWO\ GLVFULPLQDWH among colonization histories in D. suzukii using ABC methods. We believe that this pessimistic conclusion should be taken cau- tiously due to potential methodological issues: (i) the estimation of such ABC power relies on the analysis of pseudo-observed datasets simulated by drawing parameters into prior distributions, hence not conditionally on the observed dataset, and (ii) the QXPEHURIVHTXHQFHORFLQHHGHGWRDWWDLQSRZHU 5 loci) derives from a delicate extrapolation exercise based on pseudo- observed datasets simulated with a particularly small number of Fig. 1. NJ dendrogram of the microsatellite allele shared dis- loci (comprising between 6 and 192 sequence loci, depending on tances between D. suzukii individuals. Mt – French population; the simulations). Interestingly, the same authors underline the fact +±+DZDLLSRSXODWLRQ0)±PDOHRUIHPDOHÀ\ ZKHQNQRZQ that polymorphic markers such as microsatellites should better

TABLE &URVVVSHFLHVDPSOL¿FDWLRQUHVXOWVIRU D. suzukii microsatellite loci in four species of the suzukii subgroup. Cell values indicate allele numbers and size range (in brackets). N – number of individuals genotyped per species. Locus D. biarmipes (N = 12) D. subpulchrella (N = 12) D. mimetica (N = 12) D. lucipennis (N = 12) DS05 3 (276–282) 3 (276–282) – – DS06 3 (154–189) 3 (154–189) 1 (165) 1 (201) DS07 2 (179–181) 2 (179–181) 1 (177) – DS08 – – 1 (128) 2 (140–157) DS09 1 (211) 1 (211) 2 (225–231) 1 (244) DS10 2 (292–302) 1 (292) – – DS11 1 (251) 2 (251–253) 1 (239) 1 (256) DS12 2 (326–338) 2 (326–338) – – DS14 2 (189–195) 4 (189–207) 1 (200) 1 (201) DS15 2 (260–270) 2 (260–270) 2 (316–323) 1 (244) DS16 1 (111) 1 (111) 1 (119) 1 (100) DS17 1 (99) 1 (99) – – DS19 1 (139) 1 (139) – – DS20 3 (232–236) 1 (236) 1 (243) – DS21 – – – – DS22 1 (329) 1 (329) 1 (330) 1 (317) DS23 1 (256) 1 (256) – 2 (241–271) DS25 1 (241) 1 (241) 2 (225–231) – DS26 2 (82–89) 2 (83–89) – – DS27 2 (84–98) 2 (84–98) 1 (93) 2 (85–87) DS28 2 (149–178) 2 (149–178) – 1 (159) DS32 2 (335–337) 1 (335) 1 (335) – DS33 2 (159–165) 2 (159–165) – – DS34 2 (253–257) 3 (251–255) 1 (257) 1 (268) DS35 – – – – DS36 2 (175–182) 2 (175–182) 1 (177) 1 (201) DS38 4 (133–148) 4 (133–148) 2 (126–128) 2 (113–119) DS39 2 (311–334) 2 (311–334) – –

4 discriminate colonization models and that additional D. suzukii ESTOUP A., W ILSON I.J., SULLIVAN C., CORNUET J.M. & MORITZ C. population samples were needed to better discriminate such mod- 2001: Inferring population history from microsatellite and en- els. In agreement with this view, microsatellite markers (often zyme data in serially introduced cane toads, Bufo marinus . — analyzed using ABC methods) have proven invaluable for recon- Genetics 159: 1671–1687. structing invasion routes in many previous studies dealing with FOURNIER D., ESTOUP A., ORIVEL J., F OUCAUD J., J OURDAN H., LE various biological models, and most of such studies were based BRETON J. & K ELLER L. 2005: Clonal reproduction by males and on a larger set of population samples than those used in Adrion et IHPDOHVLQWKHOLWWOH¿UHDQW² Nature 435: 1230–1235. al. (2014) (see for instance Lombaert et al., 2011 and 2014, and HAUSER M. 2011: A historic account of the invasion of Drosophila references therein). It is also worth noting that a lower number suzukii (Matsumura) (Diptera: Drosophilidae) in the continen- of microsatellite markers (compared to the set of 28 microsatel- WDO8QLWHG6WDWHVZLWKUHPDUNVRQWKHLULGHQWL¿FDWLRQ² Pest lites we developed for D. suzukii ) were used in many of these Manag. Sci. — 67 : 1352–1357. other studies. We hence believe that our suite of 28 markers rep- KIMURA 0 &ROG DQG KHDW WROHUDQFH RI GURVRSKLOLG ÀLHV resents a molecular tool with high potential when attempting to with reference to their latitudinal distributions. — Oecologia reconstruct the pathways and demographic processes of invasion 140: 442–449. in this major dipterous pest, in complement to the sequence data KOPP A. & TRUE J.R. 2002: Evolution of male sexual characters in recently produce for D. suzukii populations as detailed in Adrion the oriental Drosophila melanogaster species group. — Evol. et al. (2014). We are currently using our suite of microsatellites Devel. 4: 278–291. to genotype a large number of population samples collected at LOMBAERT E., GUILLEMAUD T., THOMAS C., LAWSON HANDLEY L.J., various locations in the native and invasive range of D. suzukii to LI J., W ANG S., PANG H., GORYACHEVA I.I., ZAKHAROV I.A., J OUS - IXUWKHUUH¿QHWKHLQYDVLRQKLVWRU\RIWKLVVSHFLHVRQDZRUOGZLGH SELIN E., POLAND R., MIGEON A., VAN LENTEREN J.C., DE CLERCQ scale. P., BERKVENS N., JONES W. & ESTOUP A. 2011: Inferring the origin of populations introduced from a genetically structured ACKNOWLEDGEMENTS. 7KLV ZRUN EHQH¿FLDWHG IURP VWDWH native range by approximate Bayesian computation: case study funding by the Agence Nationale de la Recherche, through the of the invasive ladybird Harmonia axyridis . — Mol. Ecol. 20 : LabEx ANR-10-LABX-0003-BCDiv, of the program “Investisse- 4654–4670. ments d’avenir” (ref. ANR-11-IDEX-0004-02). Funding was also LOMBAERT E., GUILLEMAUD T., LUNDGREN J., KOCH R., FACON B., provided by the French Institute of Agricultural Research (INRA- GREZ A., LOOMANS A., MALAUSA T., NEDVED O., RHULE E., SPE). We thank two anonymous referees and H.D. Loxdale for STAVERLOKK A., STEENBERG T. & ESTOUP A . 2014: Complemen- their helpful editorial comments. tarity of statistical treatments to reconstruct worldwide routes of invasion: the case of the Asian ladybird Harmonia axyridis . REFERENCES — Mol. Ecol. 23 : 5931–6205. ADRION J.R., KOUSATHANAS A., P ASCUAL M., BURRACK H.J., MATSUMURA S. 1931: 6000 Illustrated Insects of Japan-Empire . HADDAD N.M., BERGLAND A.O., MACHADO H., SACKTON T.B., Tokohshoin, Tokyo, 1497 pp. [in Japanese]. SCHLENKE T.A., W ATADA M., WEGMANN D. & SINGH N.D. 2014: MEGLÉCZ E., PECH N., GILLES A., D UBUT V., HINGAMP P., TRILLES Drosophila suzukii : the genetic footprint of a recent, world- A., GRENIER R. & MARTIN J.F. 4''YHUVLRQDXVHU wide invasion. — Mol. Biol. 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56 : ! / % 8; !). 2 # . # < 0 ' 6 # "&1 '$ # . L

67 ! S6$ 3 $ D " 2 S6$ 9 |=9*(99*} 9 |=9(99*} =99 |=9F(99 } 9 |=99*(99F} 9 |=99F(99@} |9*(@F} =99 |=9(99@} 9 |=9 (99F} 9 |=9F(99!} 9 |=9!(99@} 3 $ 9** |9!(} 9@ |9@(9} ;@AB;@$4@C 9 |=9(99@} 9 |=99F(99F} D |9F(9@} |9!@(!!} $B@ 4DC =9 |=9(99 } =99F |=9(99 } " 9** |9!(F} 9@! |9F(} 9 |9! ( } @DB@ 4@EEC 9 |=99F(99F} 2 |9!!(@} 9@ |9F(} 9@ |9@( } @(DB@ 4@EEC 9 |9! (!}

$ ! S6$ 3 $ D " 2 S6$ 9 |=9*(9} 9 |=9*(9} 9 |=9 (9} 9 |=9(9} 9 |=9 (9} 9* |9(9!} 9 |=9!(9} 99 |=9 (9@} 9 |=9*(9} 9 |=9 !(9} 3 $ 9 |9(@*} 9* |9(*} 9 |=9(9@} 9 |=9*(9} 9 |=9*(9} D |9 (} 9* |9(} 9F* |9F(F} 9 |=9@(9*} 9 |=9(9 } " 9* |9(9} 9*! |9*(F } 9F |9(9*} 9@ |9(@*} 9 |=9 *(9} 2 9F |9 ( } 9*F |9F(F} 9 |9 (} 9*! |9 (!} |9 (*}

7 ! S6$ 3 $ D " 2 S6$ 9 |=9@(9} 9 |=9(99F} 9 |=9@(9} 9 |=9(9} 9 |=9F(99F} 9F@ |9!*(@} 9 |=9(9} 9 |=9(9} 9 |=9(9} 9 |=9(9} 3 $ 9 |9!F( } 9** |9!(F} 9 |=9(9} 9 |=99F(9} 9 |=9(9} D F |9@(!@} 9F@ |9!( } 9F |9!@( } 9 |=9(9} 9 |=9(9} " |9!@( @} 9*! |9 *(} 9F |9 *(!} 9@F |9 *(} 9 |=9 (9} 2 |9@ (@} 9 |9! (!} ! |9@(!} 9! |9!@( *} |9!@(!!}

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7) RESEARCH ARTICLE Wolbachia in European Populations of the Invasive Pest Drosophila suzukii : Regional Variation in Infection Frequencies

Julien Cattel1☯*, Rupinder Kaur 2,3☯*, Patricia Gibert 1, Julien Martinez 4, Antoine Fraimout5, Francis Jiggins 4, Thibault Andrieux 1, Stefanos Siozios 2,6, Gianfranco Anfora2, Wolfgang Miller 3, Omar Rota-Stabelli 2‡, Laurence Mouton 1‡

1 Université de Lyon, Université Lyon1, Laboratoire de Biométrie et Biologie Evolutive, UMR CNRS 5558, 43 Bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France, 2 Chemical Ecology lab, Department of Sustainable Agro-ecosystems and Bio-resources, Fondazione Edmund Mach, San Michele all'Adige (TN), Italy, 3 Laboratory of Genome Dynamics, Department of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria, 4 Department of Genetics, University of Cambridge, Cambridge, United Kingdom, 5 Institut de Systématique, Évolution, Biodiversité ISYEB - UMR 7205 – CNRS, MNHN, UPMC, EPHE Muséum national d’Histoire naturelle, Sorbonne Universités57 rue Cuvier, CP50F-75005, Paris, France, 6 Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom

OPEN ACCESS ☯ These authors contributed equally to this work. ‡ These authors also contributed equally to this work. Citation: Cattel J, Kaur R, Gibert P, Martinez J, * [email protected] (JC); [email protected] (RK) Fraimout A, Jiggins F, et al. (2016) Wolbachia in European Populations of the Invasive Pest Drosophila suzukii : Regional Variation in Infection Frequencies. PLoS ONE 11(1): e0147766. Abstract doi:10.1371/journal.pone.0147766 The invasive pest Drosophila suzukii is characterized by a specific fresh-fruit targeting Editor: Wolfgang Arthofer, University of Innsbruck, AUSTRIA behavior and has quickly become a menace for the fruit economy of newly infested North American and European regions. D. suzukii carries a strain of the endosymbiotic bacterium Received: October 6, 2015 Wolbachia, named wSuz, which has a low infection frequency and no reproductive manipu- Accepted: January 7, 2016 lation capabilities in American populations of D. suzukii . To further understand the nature of Published: January 25, 2016 wSuz biology and assess its utility as a tool for controlling this pest ’s populations, we investi- Copyright: © 2016 Cattel et al. This is an open gated the prevalence of Wolbachia in 23 European D. suzukii populations, and compared access article distributed under the terms of the our results with those available in American populations. Our data showed a highly variable Creative Commons Attribution License , which permits infection frequency with a mean prevalence of 46%, which is significantly higher than the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are 17% found in American populations. Based on Multilocus Sequence Typing analysis, a sin- credited. gle wSuz strain was diagnosed in all European populations of D. suzukii. In agreement with

Data Availability Statement: All relevant data are American data, we found no evidence of cytoplasmic incompatibility induced by wSuz. within the paper and its Supporting Information files. These findings raise two questions: a) why Wolbachia is maintained in field populations of

Funding: This work was funded partly by CNRS D. suzukii and b) what are the selective forces responsible for the variation in prevalence (IFR41-UMR5558) and Office National de l'Eau est within populations, particularly between European and American continents? Our results des Milieux Aquatiques (ONEMA) (Ecophyto project) provide new insights into the D. suzukii-Wolbachia association and highlight regional varia- for France, and FEM Accordo di Programma tions that await further investigation and that should be taken into account for using Wolba- Provincia Autonoma di Trento and research funds for GrandiProgetti, Project LExEM (Laboratory of chia -based pest management programs. excellence for epidemiology and modeling, http:// www.lexem.eu), Italy. Julien Cattel is the recipient of a PhD studentship from the Rhône-Alpes region ( “ARC Program” Grant) and Rupinder Kaur has obtained a

PLOS ONE | DOI:10.1371/journal.pone.0147766 January 25, 2016 1 / 12 Wolbachia in European Populations of D. suzukii

PhD fellowship from the FIRS>T (FEM International Introduction Research School-Trentino) programme at Fondazione Edmund Mach, Italy. Drosophila suzukii Matsumura (Diptera: Drosophilidae), first described in Japan in the early 1900s [ 1], is an invasive pest of Southeast Asian origin. Since its early detection in California Competing Interests: The authors have declared that no competing interests exist. (USA), Spain and Italy (Europe) in 2008, D. suzukii has rapidly spread through these two continents aided by global trading and absence of niche competitors [ 2,3,4,5,6]. Although the vast majority of Drosophila species are not fruit pests (larvae developing only in damaged or rotten fruits), D. suzukii is able to lay eggs on healthy ripening fruits thanks to female's serrated ovipositor [ 7]. A wide range of soft and stone fruits including raspberry, strawberry, blueberry, plums and grapes come under D. suzukii ’s damage range [ 4,8]. Damage is caused by developing larvae inside the fruit, leading to millions of dollars of annual economic losses worldwide [ 9,10 ]. Control of D. suzukii populations in the field mainly relies on the use of chemical pesticides, a practice with serious drawbacks because of its use close to harvest and the consequent risk of high residues left on fruits. Management agendas are therefore in permanent search for alternative strategies including those based on bio-control [4,11 ,12 ,13 ,14 ,15 ]. Previous studies revealed the presence of the bacterium Wolbachia in D. suzukii (strain named wSuz) [ 16 ,17 ,18 ,19 ,20 ] but of no other heritable bacterial symbionts [ 21 ]. This is not surprising since Wolbachia is widespread among terrestrial arthropods with an estimate of 52% of species infected at variable prevalence [ 22 ]. Wolbachia is a maternally inherited bacterium that has established a wide range of relationships with their hosts, from mutualism to parasitism. It is particularly known for its ability to manipulate host reproduction through different strategies to maximize its spread and maintenance in host populations [ 23 ]. The most common strategy is cytoplasmic incompatibility (CI), a sperm-egg incompatibility expressed in crosses between infected males and uninfected females leading to the death of embryos in diploid species, thereby facilitating Wolbachia to spread throughout host populations [ 24 ,25 ]. However, in the absence of CI or other means of reproductive manipulation, Wolbachia can also provide direct host fitness benefits (e.g. increasing survival time, enhanc- ing reproduction, provisioning nutrition or protection against viruses), which may sometimes allow infected individuals to outcompete uninfected counterparts [ 26 ]. For example, in D. mauritiana , Wolbachia infection leads to a decrease of apoptosis and an increase of mitotic divisions in germ line stem cells of infected female ovaries, resulting in more eggs produced than in uninfected ovaries [ 27 ]. Lack of any of the suggested effects on the host can potentially lead to loss of infection from a population [ 28 ,29 ,30 ]. Overall, the infection dynamics of Wol- bachia depend on the bacterial strain, host species, genetic background, environmental conditions and interactions among these factors [ 31 ]. Recently, a field survey in North America indicated a low prevalence of Wolbachia in field populations of D. suzukii from four localities sampled over two years (prevalence ranged between 7 to 58%), with a mean infection rate of 17% [ 19 ]. This study showed no signs of CI or any other reproductive phenotype induced by wSuz in D. suzukii . Moreover, the imperfect transmission of Wolbachia from wild-caught females to their offspring (20 to 95%) suggested possible direct fitness benefits provided by wSuz to its American D. suzukii host but the phenotypic nature of these benefits has not yet been determined. In order to improve our understanding of the global infection dynamics of wSuz in natural D. suzukii populations and thereby assess the utility of Wolbachia as a tool for controlling this insect pest population in the field, we investigated the prevalence of Wolbachia in 23 localities among eight European countries, typed Wolbachia strains through a Multilocus Sequence Typing approach and performed mating experiments on two different European populations.

PLOS ONE | DOI:10.1371/journal.pone.0147766 January 25, 2016 2 / 12 Wolbachia in European Populations of D. suzukii

Materials and Methods Field sampling 561 individuals were sampled between 2010 and 2014 in 23 locations from eight different countries (Austria, France, Germany, Italy, Slovenia, Spain, Switzerland and United Kingdom) of the European continent ( Fig 1 and Table 1). Adults were caught directly in orchards or from attractive traps, placed alive in ethanol (96%) and stored at -20°C until DNA extraction. For Wolbachia screening, we gave priority to females because they are known to be responsible for Wolbachia transmission.

Fig 1. Wolbachia prevalence and collection sites for D. suzukii individuals. A number was assigned for each sampling site and details for each locality are given in the Table 1. Reprinted from http://d-maps.com under a CC BY license, with permission from Daniel Dalet, original copyright 2007 –2016. doi:10.1371/journal.pone.0147766.g001

PLOS ONE | DOI:10.1371/journal.pone.0147766 January 25, 2016 3 / 12 Wolbachia in European Populations of D. suzukii

Table 1. Sampling information and percentage of individuals infected by Wolbachia .

Country Locality Collection period Total No. (M+F) No. of infected individuals Infection rate (95% CI) 1 Austria Neustift am Walde Oct, 2014 19 (6+13) 12 0.63 (0.38 –0.84) 2 United Kingdom East Malling Sep, 2014 24 (6+18) 4 0.17 (0.05 –0.37) 3 France Montauban Aug, 2010 9 (7+2) 4 0.44 (0.14 –0.79) 4 France Finestret Sep, 2010 13 (10+3) 8 0.62 (0.32 –0.86) 5 France Santa Maria Poggio Jan, 2011 43 (25+18) 12 0.28 (0.15 –0.44) 6 France Mirabel Aug, 2011 21 (15+6) 6 0.29 (0.11 –0.52) 7 France Bellegarde Oct, 2012 17 (0+17) 0 0.00 (0.00 –0.20) 8a France Mougins Oct, 2012 13 (0+13) 6 0.46 (0.19 –0.75) 8b France Mougins Oct, 2013 28 (0+28) 16 0.57 (0.37 –0.76) 9 France Chaussan Oct, 2013 10 (0+10) 3 0.30 (0.07 –0.65) 10 France Montpellier Oct, 2013 19 (0+19) 11 0.58 (0.33 –0.80) 11 France Sauternes Jan, 2014 30 (0+30) 21 0.70 (0.51 –0.85) 12 France Prigonrieux Jan, 2014 44 (0+44) 16 0.36 (0.22 –0.52) 13 France Concourson sur Layon Jun, 2014 8 (0+8) 4 0.50 (0.16 –0.84) 14 France Gotheron Jul, 2014 17 (5+12) 6 0.35 (0.14 –0.62) 15 France Carri ère sur Seine Jan, 2014 35 (15+20) 8 0.23 (0.10 –0.40) 16 France Saint Germain d ’Esteuil Jan, 2014 22 (0+22) 8 0.36 (0.17 –0.59) 17 Germany Dossenheim Oct, 2013 41 (0+41) 22 0.54 (0.37 –0.59) 18 Italy Vigolo Vattaro Sep, 2013 16 (0+16) 16 1.00 (0.79 –1.00) 19a Italy San Michele Sep, 2013 9 (0+9) 2 0.22 (0.15 –0.59) 19b Italy San Michele Sep, 2014 20 (8+12) 13 0.65 (0.41 –0.85) 20 Italy Bari Feb, 2014 11 (0+11) 8 0.73 (0.39 –0.94) 21 Slovenia Izola Oct, 2013 35 (0+35) 16 0.46 (0.29 –0.63) 22 Spain Girona Mar, 2014 37 (3+34) 29 0.85 (0.62 –0.90) 23 Switzerland Gottefrey Oct, 2013 20 (0+20) 7 0.35 (0.15 –0.59)

The sample number corresponds to the code of the sampling indicated in Fig 1. CI: 95% con ﬁdence interval. doi:10.1371/journal.pone.0147766.t001

DNA extraction and Wolbachia detection by Polymerase Chain Reaction (PCR) DNA from whole individuals was extracted using the Nucleo Spin Tissue kit (Macherey- Nagel). The quality of extraction was checked by PCR targeting the arthropod-specific locus Internal Transcribed Spacer ITS2 gene (Its2U/Its2L) [ 32 ]. Wolbachia detection was performed using Wolbachia specific primers for two different genes: ftsZ, a bacterial cell division gene (F2/ R2) [33 ], and wsp (Wolbachia surface protein) (81F/691R) [ 34 ]. PCR reactions were performed in 25 μL volumes containing 100 μM dNTP, 200 nM primers, 20mM Dream Taq Green Buffer, 0.5 IU Taq DNA polymerase (Eurobio) and 1μL of DNA template. Cycling conditions were 94°C (2 min), 94°C (30 sec), 55°C ( ITS2 and ftsZ primers) or 52°C ( wsp primers) (30 sec), 72°C (45 sec), 72°C (10 min) for 34 cycles. All the primer sequences were indicated in S1 File . PCR products were visualized in 1% agarose gels. An individual was considered infected when the two Wolbachia-specific primers produced fragments of the appropriate size. To reduce the possibility of false negatives, all samples for which we failed to amplify Wolbachia signal with normal PCR were verified by a more sensitive real-time quantitative PCR method using the wsp primers 81F/691R. The 10 μL reaction mixture contained 200 nM of each primer, 5 μL of Light Cycler1 480 SYBR Green I Master (Roche) and 1 μL of DNA sample. The amplification consisted of 10 min at 95°C followed by 40 cycles of 10 sec at 95°C, 20 sec at 53°C and 30 sec at

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72°C. Only for two individuals, Wolbachia infection was not detected by classic PCR but detected by real-time quantitative PCR.

MLST approach PCRs targeting six genes of Wolbachia (wsp , gatB, coxA, hcpA, ftsZ, fbpA) [ 34 ,35 ] were carried out on two randomly selected wSuz-infected samples per locality (Fig 1 and Table 1). All PCR reactions were performed as described before. Annealing temperature was 55°C except for wsp (52°C) and all the PCR products were sequenced. Sequences were aligned using MUSCLE [ 36 ] algorithm implemented in CLC DNA Workbench 6.9.1. (CLC Bio) and inspected by eye. On an average, we sequenced and compared approximately 3200 nucleotides per individual (between 470 and 610 nt per gene). All primers used in this study are presented in S1 File .

Cytoplasmic Incompatibility (CI) assays Two different populations of D. suzukii were used for CI experiments, one from France (Com- piegne) and another from Italy (Valsugana). The French population was sampled in 2011 and reared in mass population in Lyon on a cornmeal diet (agar: 1%, dextrose: 8.75%, maize: 8.75%, yeast extract: 2%, nipagin: 3%) under constant lab conditions of 21±1°C temperature with a 12 hours light/dark cycle at 70% relative humidity. The Italian population was established from individuals collected in Valsugana region in 2011, and subsequently reared in laboratory at San Michele all ’Adige on standard corn meal diet at a temperature of 23±1°C, 65% relative humidity with a 12:12 light/dark cycle. Maternal transmission of Wolbachia in the laboratory-established lines was perfect and thus the infection was stably maintained in the lab. Before starting the CI experiment, both lines were acclimatized under the same laboratory conditions for three generations in Cambridge Lab at a constant temperature of 22°C and 65% relative humidity with 12:12 light/dark cycle, thereby avoiding any bias due to lab environments. To obtain Wolbachia-infected and uninfected fly lines with the same genetic background, antibiotic treatments were performed on infected lines using 0.25mg mL -1 tetracycline for three consecutive generations in mass populations. After treatments, flies were fed on normal standard diet for two further generations to recover them from any side effect caused by antibiotic treatment. Due to different lab practices, flies from Italian populations were further allowed to feed on natural fecal material from infected individuals to re-acquire their loss of gut-associated microbiota but flies from France were not gut-flora restored. Ten isofemale lines were established and the presence of Wolbachia was checked by PCR in mothers after laying eggs. The absence of Wolbachia was re-confirmed by real-time PCR as described above. This was repeated for three generations and then, for each population, one isofemale line was retained for crossing experiments. Their infection status was also checked and confirmed just before the CI assays (n = 20 for each line), which were performed on the 10 th generation after antibiotic treatments were stopped. All types of crosses (infected female x infected male, infected female x uninfected male, uninfected female x infected male and uninfected female x uninfected male) were performed for each genetic background at Cambridge. Freshly hatched individuals were sexed and placed separately into cornmeal diet tubes to ensure the virginity of flies. A 3-days old virgin male and a 5-days old virgin female were allowed to mate in food vial for 24h. Females were then individ- ually allowed to oviposit for 48h on grape-juice agar petri dish. Every 48h, females were trans- ferred to a new petri dish, following 48h period for oviposition, and the total number of eggs along with the number of hatched larvae per female were recorded. Experiment was performed in replicates for each type of cross as mentioned in Table 2. The incompatibility relationship

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Table 2. Cytoplasmic incompatibility assays for D. suzukii from France and Italy.

Origin Female Male N Number of eggs laid Mean hatch rate ( ±sd) France UN UN 21 1113 0.691 ( ±0.041) b UN IN 13 763 0.404 ( ±0.087) a IN UN 15 588 0.573 ( ±0.075) ab IN IN 15 795 0.718 ( ±0.069) b Italy UN UN 28 1199 0.634 ( ±0.059) α UN IN 38 2000 0.502 ( ±0.044) α IN UN 25 1606 0.505 ( ±0.064) α IN IN 27 1370 0.729 ( ±0.045) α doi:10.1371/journal.pone.0147766.t002

was determined by the rate of eggs that hatched. Females that laid less than 10 eggs were not included in the analysis (percentages are indicated in S2 File ).

Statistical analysis We tested the difference in Wolbachia prevalence between American and European continents by fitting a generalized linear model with a quasi-binomial error (data for North America was taken from [19 ]). Samples originated from the same population but collected in several years were pooled. We tested for the existence of differences between samplings by using Fisher ’s exact test. Estimates of the mean prevalence on each continent were transformed from logits into percentages. Statistical analysis of the CI data was done using a generalized linear model (quasi-binomial family). An ANOVA was done on this model and Tukey's (HSD) tests were used for two by two comparisons. R 3.2.2 version [ 37 ] was used to perform all the analysis.

Ethics Statement Sampling of D. suzukii was carried out on private lands with owners ’ permission. All individuals were sampled in experimental stations and did not require specific permission. The field studies did not involve endangered or protected species.

Results Variable wSuz infection frequencies in D. suzukii populations We detected Wolbachia in 22 populations out of 23 surveyed from eight European countries (Fig 1). Out of the 561 D. suzukii flies tested, 258 were found infected with Wolbachia (Table 1 ). We found a highly variable infection prevalence (from 0 to 100%) between localities (χ2 = 192, d.f = 42, P<0.001). The two extreme values (0 and 100) have been observed only in two populations (Bellegarde in France and Vigolo Vattaro in Italy respectively); in all the other localities, prevalence ranged between 15% and 85% with a mean of 46%. In two European localities, Mougins (in France) and San Michele (in Italy), we sampled for two consecutive years at the same period (October, 2012 –13 and September, 2013 –14 respectively) and found an increasing trend of the Wolbachia prevalence over time for both localities (Mougins: from 0.46 to 0.57; San Michele: from 0.22 to 0.65; Table 1) though the trend was not significant (Fisher’s exact test, P = 0.74 and P = 0.05 respectively). Hamm [19 ] screened 929 individuals sampled from four American localities of D. suzukii over two years and found infection frequencies ranging from 7 to 58% with a mean of 17%. Therefore, in both continents, the prevalence of wSuz is highly variable. Overall, the European

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infection frequencies is found almost three times significantly higher than the ones in North America (GLM: t = 4.54, d.f = 26, P<0.001).

European D. suzukii flies carry one single sequence type of Wolbachia “wSuz” MLST analysis, assayed from two individuals per population (Fig 1 and Table 1), indicated that all European D. suzukii individuals carry the same Wolbachia sequence type (100% nucleotides identity for all genes). Sequences were identical to those of wSuz previously found and characterized in D. suzukii individuals originated from Italy [18 ] and North America [ 19 ]. The sequences obtained in the present study are recorded in Genbank as KS308222-7.

Cytoplasmic incompatibility assays To test whether wSuz is able to induce CI, which could facilitate its spread throughout Euro- pean D. suzukii populations, we performed mating experiments on flies originating from France and Italy ( Table 2 ). In diploid species, CI leads to the death of embryos when infected males mate with uninfected females; therefore in case of wSuz induced CI expression, we expected a lower egg hatch rate in these crosses. We found a significant effect of the type of cross on the hatching rates only for the French population (F = 3.86, d.f = 3, P = 0.01; for Ital- ian populations: F = 2.14, d.f = 3, P = 0.10). Globally, the hatching rate was higher in crosses that involved individuals of the same infection status (control crosses) than that of different infection status (infected and uninfected). The “incompatible ” crosses (infected male x uninfected female) tended to have the lowest hatching rate. For the French population, the difference was significant only when the incompatible cross was compared to the two control crosses: infected male and female (Tukey HSD test, P = 0.02) and uninfected male and uninfected female (Tukey HSD test, P = 0.01). For the Italian population there is no significant difference between the incompatible cross and the three others. Therefore, like in the American populations, these results do not allow us to conclude that wSuz can induce CI in European D. suzukii populations. Mean eggs hatch rate (only females that lay at least 10 eggs) ± standard deviation (sd). N: number of crosses. IN: infected with Wolbachia, UN: uninfected. Statistical analysis was performed on lines from the two origins separately. One-way ANOVA indicated that the hatching rate differed significantly between the mating types. One-way ANOVA was followed by Tukey’s test to compare between crossing types (means marked with the same letter are not significantly different; P = 0.05).

Discussion We found that the Wolbachia infection in D. suzukii is highly variable in European populations with a mean prevalence of 46%. This infection rate differs significantly from the one found in the North America (17% in average) [ 19 ]. Several parameters could explain this variability such as the efficiency of vertical transmission or the costs/benefits of infection, which might differ between continents. Moreover, variability can also be due to differences in the host and/ or Wolbachia genetic backgrounds as indicated in other insect species by trans-infection experiments using a given Wolbachia strain injected into different hosts (for example, see [ 38 ]). Apart from different host- Wolbachia association dynamics, the variability can also be dependent on environmental factors ( i.e. temperature, diet, larvae density . . . ) [ 39 ,40 ,28 ,41 ,42 ] that might vary locally and across time with their colonization dynamics, hence providing higher fitness advantage for Wolbachia persistence in European D. suzukii populations than in the American ones. If this is true, we should expect persistence at most/all localities to a certain

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infection rate representing the fitness advantage based on that particular locality ’s environmental and habitat specificity. Although we found a (non-significant) increasing trend for the Wol- bachia prevalence in two localities, more sampling time points over longer durations are needed to draw a clearer picture. Infection dynamics of vertically transmitted symbionts depend on the phenotypic effect induced on the host, the fitness cost and/or benefits of the infection, and how reliably symbionts are inherited. Our results clearly showed that wSuz is not fixed in D. suzukii populations as otherwise expected in cases of obligatory mutualism. We did not detect any significant evidence for CI induction by wSuz in Italian or French D. suzukii population. An imperfect vertical transmission of endosymbiotic bacteria (20 to 95% in American populations) [ 19 ] associated with an absence of strong reproductive manipulation would theoretically lead to elimination of the symbiont from host populations unless it provides the other host fitness advantages [29 ]. The stable presence of wSuz in all but one of the populations studied in Amer- ica and Europe would thus suggest that wSuz may provide direct benefits to its host allowing its maintenance in natural populations of D. suzukii. Another possibility would be frequent intraspecific horizontal transfers of Wolbachia [43 ]. However, we favored the first hypothesis because the second one would imply that horizontal transfers occur in all populations from two continents, which seems unlikely. In addition, Wolbachia are also known as a mutualist in insects [44 ] providing beneficial effects to their native hosts, such as an increase of host survival [45 ] or fecundity under nutritional stress [ 46 ], or protection against viral, microbial, and fungal pathogens [47 ,48 ,49 ,50 ,51 ,52 ]. While no fitness benefits have been detected in American populations [19 ], a recent study suggested a beneficial effect of Wolbachia infection on female fecundity in an Italian population of D. suzukii [53 ]. Since both of the studies used different protocols to perform the assays, different lab practices might impair the significance of this difference. For example, Hamm [19 ] crossed native Wolbachia infected and uninfected flies from the wild: thereby we cannot exclude the confounding effect of different host genetic backgrounds on the fly fecundity. On the other hand, Mazzetto [ 53 ] compared infected individuals to uninfected ones that were immediately obtained after antibiotic treatments. Therefore the reduced fecundity of uninfected females can also be explained by side effects of the antibiotics on mitochondria and/or host metabolism [ 54 ]. Although with caution, excluding any of these confounding factors suggest that the beneficial effect induced by wSuz may depend on the fly genetic background and/or that D. suzukii from Europe and North America are infected by different Wolbachia strains. Highly sophisticated approaches, for example, Next Generation Sequencing (NGS) based tools are needed to have more insights into the Wolbachia strain diversity in D. suzukii populations. Moreover, future experiments involving exchange or intro- gression of European and American Wolbachia strains to study the life history traits of host organisms in the presence and absence of Wolbachia would help to unveil whether differences in wSuz-mediated fitness benefits in different continental D. suzukii populations are Wolbachia strain-dependent or host dependent. There is a growing interest in exploring symbiotic microorganisms for biocontrol management programs [55 ,56 ,57 ]; in the case of Wolbachia, the idea is to exploit its capability to induce CI to control natural populations of arthropod pests in an Incompatible Insect Tech- nique (IIT) fashion [58 ,59 ], where males infected with a CI-inducing strain of Wolbachia are released in the field to mate with incompatible females leading to embryo mortality and ulti- mate population suppression. This procedure is somewhat analogous to the Sterile Insect Tech- nique (SIT), a species-specific method of insect control that relies on the release of large numbers of sterile males instead of incompatible males [ 60 ], but the advantage is that insects do not have to be irradiated or genetically modified before release. The inconvenience is, however, that IIT and SIT require solid methods allowing efficient males and females separation,

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but D. suzukii males are easily recognizable because of their spotted wings [ 1]. Considerable research efforts have already demonstrated that Wolbachia-inducing CI could be used as a tool for population control [61 ,62 ,63 ]. However, it might be more complicated if Wolbachia natu- rally infects the insect pest like in the case of D. suzukii since it would require finding an incompatible CI-inducing Wolbachia strain, which is not rescued by wSuz in its original host. Hamm [19 ] and our results are concordant in showing that D. suzukii is infected by only one strain of Wolbachia, which is present in almost all populations screened and does not induce CI. Trans- infection experiments using closely related Wolbachia strains such as the highly CI-inducing wRi strain from D. simulans will thus be needed to assess modification and rescue capabilities of different strain combinations towards a bidirectional CI-based control strategy.

Supporting Information S1 File. Primers used in this study. (PDF) S2 File. Percentage of excluded females that laid less than 10 eggs in crosses experiment. (PDF)

Acknowledgments We thank Fabrice Vavre and Sylvain Charlat for useful discussion and Hélène Henri for help in molecular biology. We thank Adriana Escudero (Spain), Gabrijel Seljak (Slovenia), Marco Valerio Rossi Stacconi (Italy), Darren Obbard (UK), Traude Kehrer (Austria), Heidrum Vogt, Serge Fischer, Lionel Delbac, Nicolas Ris, Christophe Plantamp and the CTIFL from Lanxade and Balandran for providing us D. suzukii from the field. We are also grateful to the INRA cen- tre of Gotheron and to the landowners who allowed us to collect insects in their orchards.

Author Contributions Conceived and designed the experiments: JC RK PG JM FJ SS WM ORS LM. Performed the experiments: JC RK JM TA. Analyzed the data: JC RK JM FJ ORS. Contributed reagents/materials/analysis tools: PG FJ SS WM ORS LM AF GA. Wrote the paper: JC RK PG JM FJ SS WM ORS LM.

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PLOS ONE | DOI:10.1371/journal.pone.0147766 January 25, 2016 12 / 12 CORRECTION Correction: Wolbachia in European Populations of the Invasive Pest Drosophila suzukii : Regional Variation in Infection Frequencies

Julien Cattel, Rupinder Kaur, Patricia Gibert, Julien Martinez, Antoine Fraimout, Francis Jiggins, Thibault Andrieux, Stefanos Siozios, Gianfranco Anfora, Wolfgang Miller, Omar Rota-Stabelli, Laurence Mouton

The image for Fig 1 is incorrect. Please see the corrected Fig 1 here.

OPEN ACCESS

Citation: Cattel J, Kaur R, Gibert P, Martinez J, Fraimout A, Jiggins F, et al. (2016) Correction: Wolbachia in European Populations of the Invasive Pest Drosophila suzukii : Regional Variation in Infection Frequencies. PLoS ONE 11(2): e0150050. doi:10.1371/journal.pone.0150050

Published: February 19, 2016

Copyright: © 2016 Cattel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PLOS ONE | DOI:10.1371/journal.pone.0150050 February 19, 2016 1 / 2 Fig 1. Wolbachia prevalence and collection sites for D. suzukii individuals. A number was assigned for each sampling site and details for each locality are given in the Table 1. Reprinted from http://d-maps.com under a CC BY license, with permission from Daniel Dalet, original copyright 2007 –2016. doi:10.1371/journal.pone.0150050.g001 Reference 1. Cattel J, Kaur R, Gibert P, Martinez J, Fraimout A, Jiggins F, et al. (2016) Wolbachia in European Popu- lations of the Invasive Pest Drosophila suzukii: Regional Variation in Infection Frequencies. PLoS ONE 11(1): e0147766. doi: 10.1371/journal.pone.0147766 PMID: 26809119

PLOS ONE | DOI:10.1371/journal.pone.0150050 February 19, 2016 2 / 2

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