557 r 2017 (Fuller), and Le Conte, have Busseola fusca resistance but larvae of the first generation Up to now, two noctuid , 4,5 6,7 . This noctuid can complete a variable Diabroticavirgiferavirgifera (J. E. Smith) and As a consequence, the Bt that caused the selec- S. nonagrioides 8–10 resistance monitoring plan implemented for MON 810 (Lefèbvre), a key pest in Mediterranean countries. to the Cry1Ab of MON 810 maize. Specific recom- Correspondence to: GP Farinós, Department ofde Environmental Investigaciones , Biológicas, Centro CSIC, Ramiro de MaeztuE-mail: 9, [email protected] 28040 Madrid, . Department of Environmental Biology, CentroCSIC, Madrid, de Spain Investigaciones Biológicas, The widespread cultivation of Bt varieties and the prolonged ∗ areas, with the highest adoptioneastern rate Spain in (Fig. the 2) Ebromaize where, basin hybrids cultivated since in have been north- 2007, MON 810 moredamage to than prevent by recurring 60% ofnumber the of generations per year, tion pressure have decreased efficacy for controlling the target are the most harmful to maize because they tunnelstem throughout during the the larval stage, damaging maize seedlings. exposure to Btfor resistance represent in target a pests. strong selection pressure Spodoptera frugiperda one chrysomelid beetle, evolved resistance inproteins. the field to Bt maize that expresses Cry to the Cry1Ab protein have occurred over time. The reassessment To 1–3 S. nonagrioides S. nonagrioides ), which expresses the insecticidal protein Cry1Ab from nonagrioides (Hübner) (Lep- S. nonagrioides (Lefèbvre) (Lepi- Zea mays Ostrinia nubilalis Pedro Hernández-Crespo, Félix Ortego * Sesamia nonagrioides ), which expresses the insecticidal pro- : 557–568 www.soci.org © 2017 Society of 74 Zea mays 2018; Bt maize; Cry1Ab; ; corn borers; resistance management; monitoring program

Pest Manag Sci tein Cry1Ab from Bacillus thuringiensis (Bt maize),into the was European introduced agricultural landscape in 2003. It targetspests: two key the European corn borer, MON 810 maize ( 1 INTRODUCTION lessons from a long-term harmonized plan to MON 810 maize in the : idoptera: Crambidae), which isMediterranean present corn all borer, over Europe,doptera: ), and which is the restricted to the MediterraneanFor area. 3 years, MON 810 hybrids shared the market withderived maize from hybrids Event 176 (Cry1Ab protein,España, cv. Compa Madrid, CB; Spain), , available in thesince EU 2006 from MON 1998 810 to has 2005, been but themaize only approved insect-resistant transgenic for cultivationMON in 810 the maize EU. across The Europepart adoption have of been rates the unequal, of as differentrelease a positions of result genetically of in modified the crops into EU the Member environment. States on the Abstract BACKGROUND: Use of MON 810 maize ( Gema P Farinós, Keywords: Supporting information may be found in the online version of this article. (wileyonlinelibrary.com) DOI 10.1002/ps.4735 Monitoring of (Bt maize), is a highly effective method to control mendations for improvement are provided, based onof the this knowledge unique and EU-wide experience accumulated harmonized through plan. © the 2017 implementation Society of Chemical Industry Research Article Received: 30 June 2017 Revised: 11 September 2017 Accepted article published: 19 September 2017 Published online in Wiley Online Library: 13 Novembe of this long-term program has identifiedtions some for practical and improvement: use technical constraints, reference allowing strainsshift us instead from to of dose–response provide susceptibility specific bioassays baselines recommenda- to as diagnosticas concentrations; comparators the and for focus Ebro field-collected monitoring basin on populations; in areas Spain. with high adoption rates, such CONCLUSION: There are no signs of field resistance of Monitoring programs to assessEuropean Union the (EU). potential Here we developmentmaize report in of the the EU results resistance between of 2004 of the and target 2015 and pests reassess the toRESULTS: No different major components shifts Bt of in the this maize susceptibility long-term of harmonized are plan. mandatory in the date, eight EU countries(Fig. have 1A). ever However, cultivated Spain MONhybrids is have 810 been hybrids the commercialized continuously only onand a country since large scale, where 2010 Spain MONMON has 810 810 growing accounted area forThe in over majority the 80% of EU Bt of (116 maize the is 867 ha concentrated total in in three 2016) maize-growing (Fig. 1B). and Pedro Castañera ion For The et al. 16 In addi- 21–24 : 557–568 74 Refuges are ) since its first 16,17 • to the insectici- 16,18 2018; It is commonly accepted as no larvae were found in along with the fact that only The IRM strategy applied to 47 larval dispersal by the Bt trait 19 Pest Manag Sci S. nonagrioides 80% since 2009. 14,15 > 50% of the total maize cultivation area > 20 S. nonagrioides S. nonagrioides 60% since 2006, and > A main element in IRM plans is the monitoring for target pests’ Insect resistance management (IRM) strategies are crucial to susceptibility to the Bt proteins into areas detect, with in high a adoption timely rates, manner, any resistance that evolves. reduce the selection pressure andtance to of delay the target evolution pests of to resis- Bt crops. that effective monitoring programs should have well-established single-trait Bt plants areselection cultivated for in resistance. the EU, which might favor MON 810 maize inegy, whose the appropriate EU implementation has is provedmaintaining to called the be the susceptibility vital in high-dose/refuge of dal strat- protein and preserving the durability of this technology. level of expression of Cry1Absents in a MON high 810 plants dose probably for repre- MON 810 in thedefined EU, a the European sampling Food zonewhere Safety MON of 810 Authority occupies high (EFSA) Btfor maize at adoption least three as consecutive a years. zone tion, the compliance of Spanish farmers withhas refuge been requirements commonly deployed as blocks within Bt fields orIn adjacent contrast, the to mixtures them. of Bt maize withseeds conventional (“refuge maize in the bag”) used in otherBelt areas, or such , as are the not US recommended Corn inare the the EU. stimulation The of main reasons and the dispersal capacity of adults, MON 810 stems after many years of field sampling. EU Inter- www.soci.org GP Farinós 13 © 2017 Society of Chemical Industry Spain Southwest Spain Southwest 2003 2004 20052006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 a 80 60 40 160 140 120 100

68 Central Spain a a hectares of Thousands a B Spain Czech Republic, Slovakia and Poland became members of the European Union in May 2004 and 2004 in May Union European of the members became Poland and Slovakia Republic, Czech Romania in January 2007. a A Northeast Spain Northeast and in some areas they are no longer cultivated. Adoption rate of Bt maize between 2004 and 2016 in the Historical status of MON 810 maize in the European Union (EU). (A) EU countries that have ever cultivated MON 810 maize ( 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 0

11,12 90 80 70 60 50 40 30 20 10

100 B maize %Bt wileyonlinelibrary.com/journal/ps pests of Agri-Biotech Applications (ISAAA). three areas identifiedbeen in significant: Spain where northeasterncentral the Spain Spain penetration (Castile-La (, of(Extremadura Mancha Catalonia Bt and and and maize Andalusia). Madrid)cultivation Navarre), has and Lines area southwestern show with Spain respectregion. the to Data percentage for the of 2015 totalauthors and maize from Bt 2016 data cultivation of maize are the area provisional. MinisterioMedio in Source: de Ambiente, Gobierno Agricultura each compiled y de by España Pesca, Alimentación (http://www.mapama.gob.es/en/). the y Figure 2. Figure 1. deployment in 2003; (B) Area (ha) of MON 810 maize cultivated in Spain and the total in the EU since 2006. Source: International Service for the Adquisit estingly, other Bt crops whichdal express proteins with Cry1Ab the or same othersame mode insectici- species, of can action, also and be whichfor potentially resistance target management affected, the the being use of appropriate Bt traitsby that protein-specific are not resistance. affected

558 559 S. 29 C, ∘ 1 et al. ± that served as a 12 cm diameter) -hydroxybenzoate × p 10% relative humidity by the addition of 1.6 g ± 10% relative humidity and 39 ± C, 70 S. nonagrioides ∘ 1 C, 70 ∘ wileyonlinelibrary.com/journal/ps ± 1 ± S. nonagrioides , a minimum of 100 last-instar larvae were collected 4 cm plastic boxes containing a corn-based artificial × 24 h old) that were not allowed to feed were selected for 16 < × 5% relative humidity and continuous light. Larvae pupated ± Inthelaboratory,thelarvaeweredippedinasolutioncontaining Larvae were collected in each area from a minimum of three nonagrioides per field, resulting in atlarvae least 300 were larvae gathered per from(September area per plants to season. just November, The depending beforeeach on maize year), the harvesting by area cuttingwas and the taken weather maize to avoid stalk. gathering Only siblings.larvae was If one not the reached larva minimum within a number per reasonable of time,field plant the was search discontinued in and that a new field wasThese searched larvae in the were vicinity. kept and,with if needed, those they collected were in laterthan combined the 5 km). nearest field (separated by no more 1% bleach, toin avoid 21 contamination by pathogens, and placed fields per season.except The in fields were theconcentrated separated C-Ib in by a area at relatively becauseout least small in the 50 zone. refuges km Samplings maize andmaize were fields fields crops (MON carried of 810 there maize nontransgenic or Event maize were 176and maize adjacent MON in 810 2004 to and maize Bt 2005, in allselected by other field technicians, years). based The on evidence fields of damage wereby caused previously corn borers (afterAfter visual checking inspection or that enquiries the to observable growers). damage was caused by for details). Every 1 toby 3 the years, addition the reference of straintive field-collected was consequences individuals refreshed of to inbreeding. avoidalways The the originated field-collected from nega- Cry1Ab-susceptible individuals populations collected in non-Bt fields, with levelsexisting of strain susceptibility (verified similar by to dose–response2.4). that of bioassays; To the see avoid Section introducingrated pathogens, into the field reference individuals strain were incorpo- maintained in the laboratory diet modified from Poitout and Bùes of Wesson’s salt mixture and 1 g of methyl and a photoperiod ofinterrupted 16:8 h by L:D. placing When the necessary, larvae diapause under was conditions of 28 covering a pot with five to eightoviposition maize cage plantlets. originated The from males the in same each location withinwhereas an area, the females originatedthat area. from After 7 different days, eggs locationsplaced laid into within on ventilated the plastic plants boxes wereThe containing collected a and eggs wet filter were paper. larvae incubated ( under standardthe conditions. bioassays. Neonate 2.2 Reference strainA of reference susceptible strain of control in this studyfrom different was areas established in Spain with in individuals 1998 (see collected González-Núñez 70 and the processconditions. continued Pupae in were sexed anwere and placed insectarium in five each to under ofventilated 10 the standard methacrylate male/female oviposition cylinders cages, pairs (30 which cm consisted high of (Sigma-Aldrich Co., St. Louis, MO, USA).diapause Most or of the entered larvae diapause were after in when reaching they the were last placed larval inSanyo, instar Osaka, a ) rearing at chamber 15 (Sanyo MLR-350 H; a photoperiod of 12:12 hin of diapause light:dark were reared (L:D). on Those thethe artificial that diet were standard until not pupation conditions: under 25 ]. O. 36 The 16,17,34 and O. nubi- 37,38 were col- resistance and However, the use of S. nonagrioides 31–33 S. nonagrioides S. nonagrioides S. nonagrioides ; Regulation (EC) 1829/2003 populations were carried out in to Cry1Ab following the cultiva- 35 resistance to MON 810 maize in the EU www.soci.org : 557–568 © 2017 Society of Chemical Industry 74 S. nonagrioides Baselines are also essential to establish the nat- S. nonagrioides The only long-term, EU-wide, harmonized resis- is present. The overview of this long-term har- S. nonagrioides 30 and 2018; 25–28 Thereafter, the use of appropriate bioassays would have been conducted at the national level in Spain 30,33,34 17,25,29,30 . O. nubilalis We report here the results of the Insect resistance monitoring plans for Bt maize are mandatory monitoring plan implemented for MONthe 810 EU maize during cultivation the in Harmonised period IRM 2004–2015, plan. Theareas based plan in on covers the the the EU EuropaBio S. four where nonagrioides MON maize-growing 810 hybrids have been grown and monized programme hasand technical provided constraints, allowing insightslines/recommendations us into to for provide its its specific enhancementThis guide- practical information and is improvement. crucial totance the sustainability monitoring of plans, long-term which resis- maize will technology. result in durability of this Bt tion of Bt maize varieties. Thehas plan been was in implemented place since in then. 2003 and enable estimation of changes in the susceptibilitythe of Cry the proteins with to respect to theused baseline. The approaches most commonly are dose–responsechanges in bioassays, susceptibility which at measure cation the of population diagnostic level, concentrations, andbetween resistant which the and susceptible insects. appli- allow discrimination in the EU [Directive 2001/18/EC Pest Manag Sci the three main areas in the EU wherefrom MON 810 2004 has been to cultivated 2015:of Catalonia, northeastern Aragon Spain and (NE-Ib), Navarracentral-eastern Spain that including (C-Ib), fall areas particularly within the the provinceand of Ebro Albacete; southwestern basin; Iberiawestern (SW-Ib), Andalusia in comprising Spain and southern Extremadura Portugalporting (Fig. and Information). 3 Each and area Sup- was monitored at2 least years. once Additionally, every field populations of 2.1 Insect samplingField and samplings rearing of 2 MATERIALS AND METHODS lected from southwestern FrancePoitout-Charentes in (SW-Fr) the in period Midi-Pyrénées 2005–2007ing Information). and No (Fig. further 3 samplings were and performed inbecause Support- this MON area 810 maize cultivation was disallowed in2007 France onwards. from and Germany. Monitoring of baseline susceptibility data, determined before or shortlyBt after the has been establishedsusceptibility. in the field, to assess later shifts in baseline susceptibility can be undermineding in programs long-term by monitor- the unavailability ofbatch and a formulation single of toxin stable to and assure optimal effective performancetime. over Thus, monitoring programs focussed on nubilalis tance monitoring plan wastion proposed for by Bioindustries the (EuropaBio)working European following group the Associa- on directives IRM of andities the submitted of to the Member the States Competent and Author- the European Commission. ural variability of pest populations in theto distinct be geographic assessed. areas In the EU,protein baselines have of been susceptibility established for to both the Cry1Ab lalis purpose of this harmonized planmethodology is to monitor to the develop potential and developmentof of use resistance common et al. : 557–568 74 2018; (NE-Ib) Northeast Spain Bellvís Pest Manag Sci Sidamunt (C-Ib) FRANCE Huerto Ebro Alberuela de Gimenells Torres de Barbués Central-East Spain Zuera Alagón Chinchilla Almudévar Caballeros Ejea de Los Ejea de Alfamén Motilleja Épila Aguas Nuevas La Gineta El Salobral Beire Júcar (SW-Fr) Pozohondo Tafalla Ribaforada Valtierra Tarazona de la Mancha Southwest France Agramonte Barrax Santa Ana La Roda La Herrera Ebro FRANCE Lavaur Guadalquivir SPAIN El Torno Tajo Porzuna Lézat-sur-Lèze www.soci.org GP Farinós Saint Barthélémy d'Agenais Posadas El Calonge Bérat Limalonges Navalvillar Logrosán Los Palacios Grézac © 2017 Society of Chemical Industry El Trobal Orellana Cissé Peñaflor Miradoux Fregouville Mérida Miajadas Alcolea El Bátán between 2004 and 2015 (see Table S1 for more details). Black dots represent the locations where larvae La Rinconada Villar de Rena Villar de Barrameda Sanlúcar de Torremayor Longueira Avis Cavaleiro S. nonagrioides PORTUGAL Guadiana Arraiolos Coruche (SW-Ib) Sampling locations for Southwest Iberia wileyonlinelibrary.com/journal/ps Figure 3. were collected.

560 561 for 100 ® ∼ ), was esti- 99 prior to analysis. were collected from 42 values estimated every 50 and MIC wileyonlinelibrary.com/journal/ps 50 S. nonagrioides -test and a one-tailed probability dis- t 41 ), were tested against the expected value 99 collected in NE-Ib (2009 and 2011), C-Ib (2008, 2010 , respectively. 50 . The leaves were replaced every 2 days if necessary. Sur- or MIC 50 Moult inhibition means, obtained after treatment at the diagnos- were compared with the valuein estimated 2008. for The variability the in reference susceptibility strain determined of by the comparing reference LC strain was tic concentration (MIC 3.1 Insect collection More than 12 0002004 larvae to of 2015 fromplings) the from four NE-Ib, areas 2632samplings) monitored: (six 3605 from samplings) (seven SW-Ib from sam- and(Supporting C-Ib, Information 3336 Table 2724 S1). (three The (seven efficiency samplings) ofof larvae the from varied collection among SW-Fr sampling areas and years.in Collections were, general, more difficult tonumber accomplish of in fields SW-Ib, were where visited a to higher obtain the minimum number of 3RESULTS and 2012) and SW-Ib (2010 andtration 2012). was tested The in validity bioassays of with the this fieldin concen- populations 2013, collected 2014 and 2015. Forcates each of population 32 and neonates each year, three (96 larvae) repli- concentration were tested following at the the diagnostic sameconditions protocol as and in under the the caseinhibition of same was dose–response recorded after bioassays, 7 days. and moult 2.4.3 Larval survivalA on high MON number 810 of tissue larvae were screened from 2011sure to to 2015 MON by expo- 810 maize tissue totant determine individuals if in there were the any field-collected resis- 1500 populations. Approximately leftover neonates perfield population collections per and year, not generated used in from 810 bioassays, leaves. were They exposed were to siblings MON ofthey were those not used allowed in to the feed bioassayslarvae prior and to were the transferred screen. to Groups plastic of boxes810 provided maize with leaves fresh MON without thead libitum central nerve andvival allowed was to recorded 10 feed days later. Likewise,the all dose–response surviving larvae bioassays (mostly from first and secondbut larval instar, also some thirdcentrations instar tested) larvae were were exposed foundmaize to at was MON grown the in 810 lowest the leaves. con- growth greenhouse stages MON and V5–V8 810 leaf was material used in from thethe plant experiment. Cry1Ab The presence protein of in the plants was tested by ImmunoStrip mated using probit analysis by pooling thedose–response results bioassays obtained performed in with the the fieldS. populations nonagrioides of Bt-Cry1Ab/1Ac (Agdia Inc., Elkhart, IN, USA). 2.5 Statistical analysis The significance of changes in susceptibility in thewas field tested population using the 95% confidence limits ofratios lethal (LCRs) or concentration moult inhibition concentration ratios (MICRs)LC at the year with the values measured for the first time in 2004. 2.4.2 Diagnostic concentration A diagnostic concentration (DC),ing defined 99% here moulting inhibition as of the first-instar level larvae (MIC caus- tribution (SPSS Statistics 23,values IBM were Corp., corrected with Armonk, Abbott’s formula NY, USA). Mean of 99% using a one-sample 2 − )of 90 )] was Cuntil ∘ 50 80 S. nonagri- − and MIC )] or moulting which automat- 90 50 40 C. Test concentrations ∘ 20 program, − ) and 90% (LC 50 POLO-PC resistance to MON 810 maize in the EU www.soci.org 5% relative humidity and continuous ) of the reference strain of ± 50 and MIC 50 : 557–568 © 2017 Society of Chemical Industry C, 70 ∘ 74 1 using BAW128 plastic trays (Frontier Agricultural 25%. A bioassay was considered valid if, from all ± < 17 , S. nonagrioides 2018; et al. to Cry1Ab served as the control against which field pop- l of a solution containing different concentrations of Cry1Ab 𝜇 To determine the susceptibility of each population, seven to 10 The second batch (B2) was sent by in 2011 (B2-1) and The susceptibility (MIC Mortality and growth inhibition data from dose–response bioas- darkness. Mortality and moulting inhibition were determined after 7 days of exposure, whereany mortality reaction denoted when larvae prodded not and showing moultingvae inhibition that had denoted either lar- died or not moulted to the second instar. different concentrations between 0.75 and 640 ng Cry1Ab cm Pest Manag Sci were used. Three replicates weretion and performed the for control. Each each replicate concentra- consistedcentration of (64 32 larvae for per controls), con- concentration making tested a (192 total for of controls).ferent 96 Neonate oviposition larvae larvae cages for from each were dif- replicate to selected be included for in each thetality statistical replicate. had analysis, the to For control be a mor- the concentrations tested, there wereand two two below concentrations the above concentrationof for which 50% an mortality average response [50% lethal concentration (LC 2.3 Cry1Ab protein Two batches of Cry1Abfirst protein batch were used (B1) in wasin the provided 2003 bioassays. (concentration by The 2.03 Monsanto mg/mlpH in (St. 10.5; sodium Louis, bicarbonate purity MO, buffer, 95%) USA) were and prepared in stored sodium bicarbonate at bufferAs (50 mmol/l), a pH reduction 10.5. in toxicitythe was reference observed strain, a in new 2010 batch in of bioassays protein with was required. in 2014 (B2-2), at abicarbonate concentration buffer, of pH 10.25, 1.8 mg/ml and purity inconcentration were 91%. 50 mM prepared Aliquots and sodium at kept a in lower a freezer at Monitoring of for two to three generations and discardedof if disease. they presented signs Sciences, Newark, DE, USA). Each tray contained 128 wells,0.5 in ml which of rearing diet50 was placed andor flattened. sodium Once bicarbonate buffer solidified, forface controls of was the diet. applied After drying to of the theone wells sur- under neonate a larva laminar flow was hood, placed inand each the well wells using were covered a with fine a paintbrush breathingFrontier adhesive Agricultural lid Sciences). (BACV16; The trays were incubated inchambers rearing at 25 ically corrects for natural mortality (mortality in controls). inhibition [50% moulting inhibition concentration (MIC ulations were comparedcollected each in 2005 and year. 2006 were MIC compared toerence the strain values in values 2004, of and of the those ref- of the populations population collected in 2009 oides obtained. says were analyzed by probita analysis. response The in LCs 50% and (LC MICs causing each population were estimateddence together intervals (CIs) with using their the 95% confi- 2.4 Bioassays 2.4.1 Susceptibility toThe Cry1Ab susceptibility in to the dose–response Cry1Ab protein bioassays was tested bythe bioassays F1 on progenies obtained from field-collected parents.says The bioas- were carried out inby accordance Farinós with the methods described use. The test concentrations were prepared in sodiumbuffer bicarbonate (50 mmol/l), pH 10.25. et al. 1.163; 0.164). S.nona- = : 557–568 =− P t 74 2; = S. nonagrioides standard error) 2018; ± values (Table 1). The 90 1.287; df =− t Pest Manag Sci , with a 95% CI of 548–1013. This 2 − 10 000) exposed to MON 810 leaves > level (Fig. 6). The ratios ranged between 0.03). Data for NE-Ib in 2015 could not be 50 = 2% for the NE-Ib population collected in 2013; P 1% for the SW-Ib and C-Ib populations, respec- ± 2; ± = ) represents the response of 6646 neonates derived 99 on MON 810 maize leaves. Additionally, there were no 0.183) and SW-Ib in 2014 ( = was 726 ng Cry1Ab cm P values of populations within an area was in all cases equal to 3.886; df 2% and 96 2; 90 ± = =− 10 2012 2014 2007 2009 2010 2012 2014 since 2006 in three EU areas: northeastern Spain (NE-Ib), central-eastern t (Table 3). analyzed because mortality was 100%. 3.2.4 Survival ofBetween larvae on 2011 MON 810 and leaves 2015from (five the seasons), three larvae areas of on monitored MON were 810 screened tissue. by Nonebioassays rearing of with them the Cry1Ab survivors (4871 inad larvae) the libitum survived dose–response 10 days ofsurvivors feeding among the spare neonatedose–response larvae bioassays that ( were not used in However, moult inhibition was significantly( lower in C-Ib in 2014 96 tively, collected in 2014; andlected 100% in for 2015. the Two NE-Ib ofent population these from col- values the were expected not value significantly of differ- 99%: NE-Ib in 2013 ( df value (MIC 0.6- and 3.5-fold in NE-Ib, 0.4- andin 2.6-fold SW-Ib, in C-Ib, and 0.6- 0.9- and 2.6-fold and2004 2.2-fold to 2015. in SW-Fr, with no clear shifts from 3.2.3 Diagnostic concentration The diagnostic concentration (DC) of Cry1Ab estimated for grioides values, respectively (Fig. 5). Likewise, the magnitude ofMIC variation of or lower than that of the corresponding LC from larvae collected inferent locations maize of fields NE-Ib from2012) (2009 2008 and and SW-Ib to (2010 2011), 2012 and C-Ibvalidity 2012). in was (2008, tested After dif- in 2010 estimation the field of and populationsand this collected DC, in 2015. 2013, its 2014 Theobtained moult were 97 inhibition values (mean higher heterogeneity of LC valuessomeofthelarvaeexposedtotheCry1Abproteinareabletosur- in this species occursvive because to the end of the 7-daythey assay, although are they do not not moult expected and Accordingly, to MIC survive values were under used naturaltibility to field of assess conditions. field shifts populations in the torespect to suscep- the the Cry1Ab susceptible reference protein strain, estimated over byin time variations the with MICR at the MIC 90 50 122 was ± levels, values values www.soci.org GP Farinós 50 and LC 50 50 S. nonagrioides 50 15% for NE-Ib, values ranged © 2017 Society of Chemical Industry ± No success and MIC 50 46 and 389 and MIC 50 values between 93 2 ± S. nonagrioides − 90 populations assessed . In general, the mag- 2 NE-Ib C-Ib SW-Ib − to the Cry1Ab protein level (0.8, with a 95% CI 44, 439 and LC 10% and 31 , with a magnitude of varia- 50 ± 2 2 Success ± − − 2007 2009 2011 2013 2015 2006 2008 20 100) (Fig. 4). The percentages mean values of the laboratory strain ranged values was equal to or lower than that 5 0 = S. nonagrioides 10%, 48 20 15 10 50

n ; in both cases, the magnitude of varia- 50

± Number of fields of Number 2 S. nonagrioides − Number of fields sampled for collection of larvae of values: 7- and 21-fold in NE-Ib, 3- and 5-fold in SW-Ib, and . A bridging experiment confirmed that there were no sig- 50 2 − standard error of success in gathering larvae from a field per Concentration ratios (LCR and MICR at the LC 3.2 Susceptibility of in the NE-Ib, C-Ib anding the SW-Ib low populations, number of respectively. larvae Consider- (83 captured and in 59, SW-Ib respectively; in Table 2004 S1),lations and we from 2009 the excluded analysis these of field susceptibility popu- to the Cry1Ab protein. 3.2.1 Susceptibility ofThe the reference susceptibility strain of the reference strain of evaluated by larval mortality and moult inhibition in differentand years using different Cry1Ab batches (Table 1). LC C-Ib and SW-Ib, respectively. On average, the numberlected of larvae per col- area per season was 515 Spain (C-Ib) and southwestern Iberia (SW-Ib). A collection at a field/site was considered successful if at least 100 larvae were gathered. 2- and 2-fold in SW-Fr, respectively (Fig.C-Ib, 5). The with only ranges exception was of variation of 4- and 3-fold for MIC wileyonlinelibrary.com/journal/ps values (3-fold), whichcm ranged between 42 and 120 ng Cry1Ab area per season were 80 larvae required per field ( ± Figure 4. tion was 6-fold (Fig. 5). MIC tion of 6-fold. This variation was twice that obtained with MIC nificant differences in theagainst toxicity the of laboratory B2-1 and strainby (Table B2-2 1, when the season tested 2013), resistance as ratio indicated at the MIC nitude of variation of MIC of LC between 12 and 69 ng Cry1Ab cm and 565 ng Cry1Ab cm of0.5–1.1). ranged between 19 andranged 482 between ng 7 Cry1Ab and cm 63 ng Cry1Ab cm 3.2.2 Susceptibility ofValues field populations of LC and MIC of respectively) were calculated everyvalue year measured with (in respect 2004). to Thewas the maximum observed first difference in in 2008 LCR (3.1-fold),in value and 2015 that (1.5-fold). for However, in MICRover neither was time case observed (Table was 2). a trend observed between 2004 and 2015 are presented in Table 1. LC between 5 and 28 ng Cry1Ab cm

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0 n 0 ehlcnetain (LC concentrations lethal 90% and 50% n hi 5 ofiec nevl Cs r xrse nn r1bcm Cry1Ab ng in expressed are (CIs) intervals confidence 95% their and ) MIC and (MIC concentrations inhibition moult and ) LC and .

− b 2

otesenSan(EI) eta-atr pi CI) otwsenIei S-b n otwsenFac (SW-Fr). France southwestern and (SW-Ib) Iberia southwestern (C-Ib), Spain central-eastern (NE-Ib), Spain Northeastern

a

± ± 8 (178–590) 284 (23–61) 40 5 3.9 0.2 1.5 288 (238–3426) 516 (20–127) 64 05 7 0.3 1.4 288 B1 2007 .

± ± 4 (105–222) 144 (27–50) 38 19 44.6 0.2 2.2 765 (356–3681) 776 (23–95) 53 519 71 0.1 1.1 765 B1 2006 .

± ± WF 05B 6 0.9 766 B1 2005 SW-Fr 8 (155–815) 284 (6–30) 17 19 48.3 0.1 1.0 766 (466–4412) 1021 (19–61) 37 418 35 0.1 .

± ± 6(53–126) 76 022 29 0.1 1.2 957 B2-2 2014 3 (140–569) 236 (23–43) 31 22 36.8 0.2 1.5 957 (402–3744) 898 .

wileyonlinelibrary.com/journal/ps ± ± 5 (101–339) 158 (19–41) 29 37 86.3 0.2 1.7 782 (542–15918) 1558 (57–212) 99 838 88 0.1 1.1 782 B2-1 2012 .

± ± 6(60–141) 86 (11–21) 16 22 48.8 0.1 1.7 960 (119–245) 163 (19–31) 24 622 30 0.1 1.6 960 B1 2010 .

± ± 2 (153–385) 226 (10–25) 17 16 17.7 0.1 1.1 670 (956–4659) 1796 (35–78) 54 119 15 0.1 0.8 766 B1 2007 .

± ± WI 05B 9 1.0 996 B1 2005 SW-Ib 1 (69–229) 110 (5–18) 11 31 87.1 0.1 1.3 996 (183–986) 339 (9–32) 19 531 92 0.1 .

± ± 3 (81–329) 138 (9–21) 15 22 44.4 0.1 1.3 943 (376–6607) 1018 (34–105) 54 222 44 0.1 1.0 943 B2-2 2014 .

± ± 6 (79–608) 160 (8–25) 15 22 106.4 0.1 1.2 954 (358–4870) 891 (30–82) 47 022 56 0.1 1.0 954 B2-1 2012 .

± ± 1 (81–200) 119 (6–14) 10 37 63.2 0.1 1.2 991 (730–9243) 1834 (24–71) 42 217 63 0.1 1.2 991 B1 2010 .

± ± 7 (124–259) 170 (18–38) 28 19 22.3 0.2 1.6 848 (662–2050) 1045 (57–112) 83 119 18 0.1 1.2 864 B1 2008 .

± ± . 34 0.1 0.8 768 B1 2006 2 (156–1360) 321 7(1–17) 19 42.5 0.1 0.8 768 (513–4934) 1137 (14–49) 29 819 .

± ± -b20 1781.1 768 B1 2004 C-Ib 4 (143–588) 248 (5–22) 12 19 30.5 0.1 1.0 768 (299–1278) 523 (18–48) 32 219 36 0.1 .

± ± 4(63–124) 84 (13–21) 17 22 28.5 0.2 1.8 920 (395–3441) 864 (51–119) 73 622 36 0.1 1.2 920 B2-2 2015 .

± ± 6 (108–287) 163 (14–25) 19 30 67.5 0.1 1.4 1274 (526–4313) 1176 (53–129) 78 930 78 0.1 1.1 1274 B2-1 2013 .

± ± 3 (91–232) 135 (14–27) 20 25 70.1 0.1 1.5 1056 (896–3685) 1601 (76–148) 105 925 43 0.1 1.1 1056 B2-1 2011 .

± ± 8 (138–277) 188 (16–28) 22 25 28.2 0.1 1.4 1055 (5028–58507) 12614 (330–802) 482 425 23 0.1 0.9 1055 B1 2009 .

± ± 9(70–158) 99 (8–20) 14 19 23.9 0.2 1.5 862 (704–3597) 1317 (36–89) 60 819 20 0.1 1.0 862 B1 2007 .

± ± 05B 3 1.1 639 B1 2005 6 2269 3 1.4 639 (242–649) 362 (13–36) 23 919 21 0.1 . 0819 20.8 0.2 6(54–117) 76 9(3–15) .

± ± EI 04B 6 1.2 766 B1 2004 NE-Ib 64(0528)761.3 766 (1095–2687) 1614 (110–178) 140 019 11 0.1 7 (333–1318) 570 (34–99) 63 19 39.9 0.1 .

± ± 05B- 6 3.1 863 B2-2 2015 3(213)833.3 863 (72–1135) 93 (30–44) 36 119 50 0.2 7(50–110) 67 (21–36) 28 19 84.5 0.2 .

± ± 04B- 5 1.5 956 B2-2 2014 3 1277 5 1.8 956 (122–717) 232 (21–48) 31 922 73 0.1 1(57–209) 91 (11–25) 17 22 74.1 0.2 .

± ± 03B- 6 1.3 960 B2-2 2013 1 6–2)901.6 960 (69–226) 112 (8–17) 12 822 56 0.1 . 5622 65.6 0.1 2(26–87) 42 5(3–9) .

± ± 03B- 5 1.3 953 B2-1 2013 6 8–2)931.6 953 (88–423) 161 (11–24) 16 822 80 0.1 . 5922 65.9 0.1 8(31–88) 48 7(5–10) .

± ± 02B- 7 1.2 879 B2-1 2012 7 1831 7 1.4 879 (108–351) 175 (11–21) 15 222 31 0.1 . 3922 33.9 0.1 2(41–107) 62 7(5–10) .

± ± 01B- 041.1 1054 B2-1 2011 8 2768 041.5 1054 (247–688) 383 (19–34) 26 625 40 0.1 . 5725 75.7 0.1 8(45–127) 68 9(6–13) .

± ± 00B 000.9 1050 B1 2010 7 2019)15 1.3 1050 (240–1396) 476 (10–27) 17 825 69 0.7 . 0725 40.7 0.1 4(52–117) 74 8(5–11) .

resistance to MON 810 maize in the EU www.soci.org

± ± 08B 6 1.4 863 B1 2008 6 3812)831.6 863 (358–1127) 565 (48–96) 69 819 34 0.1 2 (76–255) 120 (10–30) 19 19 65.0 0.2 .

± ± 07B 6 1.1 765 B1 2007 4 2716)691.7 669 (297–1467) 541 (20–62) 39 119 46 0.1 4(70–149) 94 (12–22) 16 16 23.3 0.2 .

± ± eeec tan20 1611.4 671 B1 2004 strain Reference 7 1335 7 1.7 575 (113–385) 179 (14–32) 23 116 42 0.1 9(66–208) 99 (11–25) 18 13 32.6 0.2 . : 557–568 © 2017 Society of Chemical Industry

74

Population Year batch ..(5 I 9%CI) (95% CI) (95% d.f. SE Slope n SE Slope n ..(5 I 9%CI) (95% CI) (95% d.f.

𝝌 ± 𝝌 ± a 2 2

50 90 50 90 Cry1Ab

LC LC MIC

S. nonagrioides MIC b b c , b c , b 2018;

ucpiiiyt r1bpoeno eeec tanadfil ouain of populations field and strain reference a of protein Cry1Ab to Susceptibility 1. Table eai nonagrioides Sesamia ewe 04ad2015 and 2004 between Pest Manag Sci Monitoring of for et al. : 557–568 (95% CI) a ) 74 MICR 50 (MIC 2018; levels, respectively, mea- 50 Sesamia nonagrioides (95% CI) a LCR ) Pest Manag Sci bition concentrations are signifi- 50 NE-Ib C-Ib SW-Ib SW-Fr and MIC (LC 50 Fluctuations of about 6-fold for both 43,44 However, the toxicity of the formulation of 0.05) from those in the first year of testing (2004) < 17,29 P were found in the laboratory strain in the period Batch of 50 populations from northeastern Spain (NE-Ib), central-eastern Cry1Ab protein Lethal concentration ratio (LCR) and moult inhibition con- and MIC Lethal concentrations or moult inhi 50 9 2010 2011 2012 2013 2014 2015 sured over time in a laboratory strain of Season Table 2. centration ratio (MICR) at LC 20042007200820102011 B12012 B12013 B12013 B1 B2-12014 B2-1 1.72015 (1.1–2.6)* B2-1 1 3.1 (2.2–4.4)*a B2-2 0.8 (0.5–1.1) 1.1 0.9 (0.8–1.6) (0.6–1.3) B2-2 0.7 1.1 (0.5–1.0) (0.7–1.6) B2-2 0.7 (0.5–1.0) 0.4 (0.3–0.6)* 0.5 (0.4–0.7)* 0.5 (0.4–0.8)* 0.4 (0.3–0.6)* 1.4 (1.0–1.9) 1 0.4 (0.3–0.6)* 1.6 (1.2–2.1)* 0.3 (0.2–0.5)* 1.0 (0.7–1.3) 1.5 (1.2–2.0)* cantly different (* if the 95% confidence intervals (CIs) of LCR or MICR do not include 1. were determined. LC the Cry1Ab protein used inthose the used present in study previous was studies.necessary different This changes from finding, to together the with protocol,baselines discouraged some as the comparators use in of oura these study. As reference an laboratory alternative, we strainsame used as conditions and a at comparator, theviduals. tested same Thus, time the under maintenance as of the the a susceptible field-collected referencethe indi- strain duration for of the monitoringtial program in has this proved study. to The bethe laboratory essen- addition strain of was new regularly healthy refreshedto individuals by preserve collected its vigor in and non-Bt to fields ensurelapse. that the Infusion population of did not wild col- strain individuals into is an a established standardand laboratory method to to prevent maintain awith long-term reduction field populations in populations. their genetic diversity compared S. nonagrioides -Fr), with respect to a laboratory susceptible strain, from 2004 to 2015. Data ref- level of 2 50 www.soci.org GP Farinós 2 MIC50 16 © 2017 Society of Chemical Industry 3 mandated by the S. nonagrioides et al. LC50 5 The baseline suscepti- values of 4 collected in 1998 and 1999 23–27 50 3 S.nonagrioides, 2003 2004 2005 2006 2007 2008 200 8 6 4 2 0 and MIC 7 10

In this study, we made a reassessment of 50 50 ) (MIC MICR 23 21 S. nonagrioides 6 6 Variation in LC Ref. strain NE-Ib C-Ib SW-Ib SW-Fr Ratios of moult inhibition concentration (MICR) at the MIC

5 0

25 20 15 10 Variation rate Variation The first step in monitoring programs is to estimate the tar- Spain (C-Ib), southwestern Iberia (SW-Ib)northeastern and Spain in southwestern 2007–2013 France (SW are from Castañera wileyonlinelibrary.com/journal/ps Figure 6. 4IRM DISCUSSION plans are critical toof extending their the main durability elements of is Btity monitoring crops. for to One target the pests’ susceptibil- Btstrategies protein should expressed be in reviewed the andedge crop. refined, acquired based However, through long-term on research,field the experience knowl- together accumulated through with the implementation laboratorymonitoring of plan the and itself. Figure 5. European Commission, after the widespread planting ofmaize MON in 810 the EU. This informationfurther will provide improvement insights that of enable theprotocols. sensitivity and accuracy of current get pest’s baseline susceptibility justthe before release or of immediately a after Btceptibility crop, among to evaluate field the populations. natural variability in sus- the long-term monitoring plan of bility is tosusceptibility. be Accordingly, baselines used of as susceptibilityfield for a populations Spanish comparator of to assess future shifts in erence (Ref.) straincentral-eastern Spain and (C-Ib), southwestern Iberia field (SW-Ib) and southwestern France populations (SW-Fr)] monitored from [northeastern 2004 to 2015. Spaincates Each the value ratio in (NE-Ib), of the the graph highest indi- andarea lowest and values measurement. obtained for that collection

564 565 16 and ,the How- 54 To rule 47 Helicov- In the case 33,61 values in the 55 50 suggesting that susceptibility to S. nonagrioides who concluded that, 16,17,29 59 ,incornintheUSA S. nonagrioides estimates for assessing (Hübner) in . values by dose–response bioas- in our study the use of MICs gave 50 susceptibility to Bt proteins. 7 wileyonlinelibrary.com/journal/ps 50 O. nubilalis The widespread planting of Bt crops has or MIC The features of dose–response bioassays H. armigera 48–53 50 there is insufficient historical data on popula- estimates seemed to be a better fit of the linear to the Cry1Ab protein in the three main areas was 2-fold. The observed changes in susceptibil- 33,56,60 50 50 Although the two measures (growth inhibition and Heliothis virescens and 10 km within a geographic area where the Bt maize adop- × and LC 7,24,56–58 50 S. nonagrioides, Our results indicate no shifts over time in the susceptibility of The estimate of LC Cry1Ab. This is reflected in the lowerfor magnitude MIC of values variation when found compiling data from 2004similar to 2015. Something was observed by Ali and Luttrell, a more consistent assessment of Moreover, our results show that, at least for says on a regular basis is oneto of the detect most frequently shifts used methods intime. the susceptibility to the Cry1Ab protein over erpa zea S. nonagrioides in the EU2015. where We MON found variation 810 of has up to been 7-fold cultivated for MIC from 2004 to mortality) are often correlated, been associated with the declineof since the 2002 European of corn population borer, levels make them inefficient atsurvivors detecting that resistant have individualsthus been among being insensitive exposed to the to small changesquency sublethal in that resistance concentrations, take allele place fre- in the first stages of resistance. ever, there are different situations thatalways make this feasible. requirement One not of themin concerns the northeastern agronomical Spain. practices Fielders, rotation with is only 55% common of for theconsecutive maize area being years farm- cultivated in with the maize for period two 2007–2010 in the Ebro Valley. ity to thethose Cry1Ab previously protein reported in for different this species, years are comparable to dose–response model than LC these changes could betion. attributable These to differences are commontarget also pests natural of in varia- Bt the cropsbeen range in detected. which of evolution those of found resistance has in not out the presence of000 resistant larvae from individuals the dose–response in bioassays this performed2011 between study, and nearly 2015 15 and sparesays larvae that were were screened not used by in rearing the bioas- them on MON 810 leaves, but the bollworm at 7 days, MIC population from NE-Ib, and(4-fold) and lower SW-Ib variation (3-fold). for Inhybrids the those were case cultivated in from of the France, period C-Ib 2005–2007, whereMIC MON variation 810 in both recommendation of predefined samplingaccomplish, locations as is the difficult yield to offrom larvae year collected to in year maize infields fields all in varied areas southwestern Iberia sampled. in Thus, 2012, despite welarvae sampling could from not 19 collect any enough offield them samplings to for perform the bioassays.sampling Ebro The efforts Valley were efficiency also was of common higher, inthe but that collection region. unsuccessful of The larvae success can of including be influenced the by expertise a of number ofsearch the factors, of people infested involved fields in and(natural the collection enemies, process of of competition, larvae, as etc.)ing well and date, as etc.) abiotic factors. biotic (weather, plant- of tion dynamics to support the ideamaize. of area-wide suppression by Bt been suggested that, in ordersampling to locations capture should variability be in predefined10 sensitivity, km areas of approximately tion rate and the target pest pressure are high to very high. b 4500 3000 3000 larvae ∼ ∼ ∼ Leftover 44 . This focus fedonMON810 a Ideally, one single batch C) and the fact that the ∘ C and small aliquots were S. nonagrioides ∘ 20 60% of the cultivated maize, 80 − 30,33,34 > − However, as adoption of MON 46 in the EU to reduce the detection resistance to MON 810 maize in the EU www.soci.org Sesamia nonagrioides (data not shown), making it necessary to Survivors of bioassays : 557–568 © 2017 Society of Chemical Industry 74 S. nonagrioides as different batches and formulations of Cry1Ab can S. nonagrioides 2018; O. nubilalis 2011 2012 2013 2014 2015 Total Total 45 c Number of larvae of Leftover neonates, generated from field collections and not used in All surviving larvae from the dose–response bioassays with Cry1Ab Northeastern Spain (NE-Ib), central-eastern Spain (C-Ib) and south- Standardization of Cry protein preparations is critical for accu- Periodic insect collection is another key element in monitoring maize to detect resistant individuals in thepopulations progenies of from field-collected 2011 to 2015.sure None to MON of 810 these maize larvae leaf tissue survived expo- western Iberia (SW-Ib). c a b NE-Ib 721 - 1003 - 574 2298 Population Table 3. C-IbSW-Ib - - 491 432 - - 803 847 - - 1294 1279 protein. bioassays. rately assessing possiblepopulations, changes in the susceptibility of field Pest Manag Sci 2004–2015, but no trends weretions observed over have time. been Similar noted varia- intheir other susceptibility laboratory to strains or when insecticidal studying proteins. Monitoring of have significant impacts on toxicity. 810 has progressed differently in(increasing each up of to the about four 80%and areas in assessed SW-Ib, NE-Ib, staying and belowfocus being 40% future in discontinued monitoring C-Ib in effortsern France), in Spain, the we and Ebro moving propose from Valley,area, to biennial in MON to northeast- 810 annual hybrids sampling. represent In this of protein should be usedtoring program. throughout In the this study, duration the of firstwas batch the used of moni- Cry1Ab for seven protein (B1) seasons (2004–2010).a However, decrease after this in time effectivenessried was out observed with in a parallel study car- use a second batch (B2). The losscaused of by activity of the B1 might storage have conditions been ( batch was not subdivided when first received,to thus multiple being exposed freeze/thaw cycles prior tothe its case use of in the later second batch, bioassays. supplied In and at B2-2), two the different times protein (B2-1 was kept at and this is therefore anresistance development EU to area Bt with maize an in increased probability of prepared when each supply wasto received, so be the thawed protein only needed Bridging one experiments more could only time be performed when betweenB2-2, used B2-1 as B1 and for had lost its the activity by bioassays. theresults time highlight B2-1 was the provided. These importance of properdling maintenance of the and Cry han- protein supplies so thatmany they seasons. function optimally for programs. To ensure thatlocal the population, samples we aimed are to representative collectleast once a of every minimum the 2 of years 300 fromin larvae representative at the maize-growing areas EU, with thefor purpose resistance of allele achieving frequency. a detection limit of 5% would permit allocation of availablelecting resources higher and numbers efforts of insects to frompopulation col- the of most Bt-exposed field limit for resistance allele frequency below the current 5%. It has J Nat et al. Tome :57–61 :45–69 PLoS ONE :510–521 :385–398 23 22 : 557–568 31 30 74 Nat Biotechnol J Econ Entomol S Afr J Plant Soil 2018; : Lessons from Puerto Sesamia nonagrioides Food Policy NatBiotechnol (Lepidoptera: Noctuidae) Biotechnol Adv Nat Biotechnol :1031–1038 (2010). Pest Manag Sci 103 :294–300 (2012). : 477–483 (2011). Spodoptera frugiperda 40 110 , ed. by Balachowsky AS, Masson et Cie, Paris, , Early detection and mitigation of resistance to Spodoptera frugiperda :854045, (2012). (Lepidoptera: Noctuidae) from Vaalharts, South :1–12 (2016). (Fuller) to Bt-transgenic maize. :e22629. doi:https://doi.org/10.1371/journal.pone J Econ Entomol et al. 109 6 :374 (2000). 2012 18 Environ Entomol Psyche J Invertebr Pathol :2011–2025 (2009). Busseola fusca :e0154200. (2016). :498–500 (2013). :147–151 (2007). Biotechnol II, Lépidoptères, vol. 2 France, pp. 1389–1401 (1972). (Lef.) (Lepidoptera: Noctuidae) on theBasin. edges of the Mediterranean ment in GM crops: past, present and future. (2005). resistance to Bt crops: Definition,102 theory, and data. in Puerto Rico. (2013). maize in the EU hotspot: Why has resistance not evolved? fication on country image ofmarkets: imported Perceptions of food channel products members. in European JW and Huckaba RM, Discoverytance and to characterization Bt of Maize: field resis- 11 (2005). maize means for31 science-based risk assessment. 24 Field-evolved resistance to BtPLoS maize by ONE . . TW, LJ Meinke Bt maize by Western corn rootwormEcon (Coleoptera: Entomol Chrysomelidae). resistance to Bt maize in Rico. Busseola fusca ment strategies for(2003). transgenic Bt corn. lessons from the first billion acres. .0022629 (2011). in 1 Peerenboom E, German Health Minister calls time out for Bt maize. 5 Eizaguirre M and Fantinou AA, Abundance of 6 Bates SL, Zhao JZ, Roush RT and Shelton AM, Insect resistance manage- 7 Tabashnik BE, Van Rensburg JBJ and Carrière Y, Field-evolved insect 2 Knight JG, Mather DW and Holdsworth DK, Impact of genetic modi- 8 Storer NP, Babcock JM, Schlenz M, Meade T, Thompson GD, Bing 3 Kuntz M, Davison J and Ricroch AE, What the French ban of Bt MON810 4 Anglade P, Les Sesamia, in Entomologie appliquée à l’, 9 Van Rensburg JBJ, First report of field resistance by the stem borer, 16 Castañera P, Farinós GP, Ortego F and Andow DA, Sixteen years of Bt 10 Gassmann AJ, Petzold-Maxwell JL, Keweshan RS and Dunbar MW, 12 Andow DA, Pueppke SG, Schaafsma AW, Gassmann AJ, Sappington 13 Storer NP, Kubiszak ME, King JE, Thompson GD and Santos AC, Status of 14 Glaser JA and Matten SR, Sustainability of insect resistance manage- 15 Tabashnik BE, Brevault T and Carrière Y, Insect resistance to Bt crops: 11 Kruger M, Van Rensburg JBJ and Van Den Berg J, Resistance to Bt Maize companies (2004), Monsanto Agricultura España SLgenta and Seeds Syn- SA (2005–2007),Europe SA Monsanto (2008–2011), Int. Sarl andThe Monsanto and authors Europe Monsanto gratefully SA acknowledge the (2012–2015). oratory support of technicians, field who and lab- search have of maize contributed fields, collection substantially of larvae,insect to and maintenance colonies of the the through time. Larvaeby from Mr. Khalid France Koubaiti were (Fredon collected personnel Poitou-Charentes) of in Fredec 2006 Midi-Pyrénées and by in(St. Louis, 2007. MO, We USA) for thank providing Monsanto grateful the to Cry1Ab protein. Dr. We Graham are Headand also editing for this kindly manuscript. reading, commenting on SUPPORTING INFORMATION Supporting information may be found inarticle. the online version of this REFERENCES S. www.soci.org GP Farinós S. nona- The use by using 63,64 and there are © 2017 Society of Chemical Industry 32,60,64 , 99 as is the case for An advantage of the DC However, this method has 33 67 45,65 S. nonagrioides value from a larger pool of mortal- year combinations tested. Our results . We have determined a candidate DC 99 . Moreover, it would be very convenient − ), corresponding to the MIC 2 − has provided insights into the most appropriate mon- S. nonagrioides S. nonagrioides The use of this approach also permits a higher number 66 . 7,62 Another approach for detecting shifts in susceptibility to Bt suggest that a refinementin of the the DC monitoring is program,limit needed of for probably its the by future estimated using use MIC the 95% upper proteins in monitoring programs is the use of DCs. wileyonlinelibrary.com/journal/ps ACKNOWLEDGEMENTS This work was supported byical various support R&D made contracts with of private technolog- companies: a consortium of eight 5The CONCLUSIONS reassessment of the continuing monitoring program of nonagrioides itoring techniques. Thus, iting is programs very to maintain useful a susceptible for referenceagainst laboratory long-term which strain monitor- the susceptibility of thecan field-collected be populations compared. In contrast,as comparators the is use limited of because susceptibilityCry it baselines requires protein the formulation use of overbe the accomplished. many same We years, have which alsothan found LCs cannot for that always MICs are less variable to shift from dose–responsetic concentration bioassays and to to the focus(northeastern insect use Spain), sampling of on which a the contains diagnos- Ebro810 maize the basin cultivation in highest the EU. levels Both changes would ofhigher permit numbers testing MON of insects from the mosttion, Bt-exposed thus field popula- decreasing the detectionquency to limit below for the resistance current allelemonitoring 5% plan. fre- and considerably improving the no survivors weremaize found. is These still results efficiently confirm controlling that MON 810 no signs of field resistance toresults the Cry1Ab provide protein. Moreover, these conclusivehigh evidence dose that for this species, MON killinglarvae. more 810 than 99.99% qualifies of susceptible as grioides ity data analyzed by probit analysis. data that represent the response of more thandose–response 6500 larvae in bioassays seven with populationsto 2012. collected When from this 2008 DCulations was collected tested from 2013 against to neonates 2015,mortality of the obtained field was results pop- significantly showed lower that than the in the only expected one 99% of the four area technique is the fact that alltion individuals at are which tested the at percentage atance; of concentra- thus, mortality it is is correlated more withdetecting efficient resis- resistance than at low dose–response frequencies, bioassays for of field individuals to befor dose–response tested bioassays, with thereby helping the to decrease samerent the 5% effort cur- detection as limit required for resistancetion, allele however, frequency. is One that limita- those individualsresistance heterozygous for alleles recessive will not surviveThe at the frequency diagnostic of concentration. recessiveby Bt-resistant F2 screens alleles in can natural populations. be estimated not been used forextremely long-term labor-intensive. monitoring programs because it is of DCs is suitablesents “high in dose” conditions against where the810 target the against pest, resistant as trait is(726 the repre- ng Cry1Ab case cm for MON

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