On-Plant Survival and Inheritance of Resistance to Cry1ab Toxin

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5-2009 On-plant survival and inheritance of resistance to Cry1Ab toxin from Bacillus thuringiensis in a field- derived strain of European corn borer, Ostrinia nubilalis Andre LB Crespo University of Nebraska-Lincoln

Terence A. Spencer University of Nebraska-Lincoln

Analiza P. Alves University of Nebraska-Lincoln

Richard L. Hellmich Iowa State University, [email protected]

LeonFolloawr dothi Cs a. ndMa agddalhitaiones al works at: http://lib.dr.iastate.edu/ent_pubs UnivPerasitrty ofof NtheebrasEnka-Ltomoincololngy Commons, and the Plant Breeding and Genetics Commons TheSee nex tompc page forle addte bitioniblaiol agruthorapshic information for this item can be found at http://lib.dr.iastate.edu/ ent_pubs/125. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the Entomology at Digital Repository @ Iowa State University. It has been accepted for inclusion in Entomology Publications by an authorized administrator of Digital Repository @ Iowa State University. For more information, please contact [email protected]. Authors Andre LB Crespo, Terence A. Spencer, Analiza P. Alves, Richard L. Hellmich, Leonardo C. Magalhaes, and Blair D. Siegfried

This article is available at Digital Repository @ Iowa State University: http://lib.dr.iastate.edu/ent_pubs/125 1071 corn, Cry1Ab- Bt Ostrinia 8 to define values was 50 a O. nubilalis ˜ aes Unit, Iowa State University, Ames, IA e, Agricultural Research Service (DOA) 2009 Society of Chemical Industry c a egfried, Department of Entomology, University of calculated on the basis of LC e to Cry1Ab and survived on transgenic corn D either because they have moderate levels of , the functional recessive inheritance of resistance ) that targets the European corn borer, 9,10 Bt A recent study testing the survival of third-instar larvae n assumptions such as functional recessive inheritance, O.nubilalis 4,11 Leonardo C Magalh United States Department of Agricultur Correspondence to: Blair D Si Corn Insects and Crop Genetics Research 50011, USA Department of Statistics, 340 Hardin Hall,68583-0963, University USA of Nebraska, Lincoln, NE Nebraska, Lincoln, NE 68583-0816, USA. E-mail: [email protected] Department of Entomology, University ofUSA Nebraska, Lincoln, NE 68583-0816, For c Berliner ( February 2009 Published online in Wiley Interscience: 29 May 2009 ∗ c a b Analiza P Alves, resistance or because they have lostas the ability a to use food corndiet. plants source after many generations feeding on artificial resistant strains previously testedcorn lack plants, the ability to survive on has only been tested using Cry1F-expressing corn plants. cessive. No survivors were found on vegetative-stage a gh dose plus refuge; insecticide resistance management 5–7 Bacillus thuringiensis . The authors used a field-derived resistant strain of 2,3 Ostrinia nubilalis Bacillus thuringiensis progeny were able to survive on reproductive corn 15 days after infestation. strain exhibited high levels of resistanc O. nubilalis 1 800-fold resistance to Cry1Ab. Resistance was primarily autosomal and controlled by > ∗ Erin E Blankenship, toxins in laboratory strains a L.) expressing Cry toxins from This strategy is more effective b 1 Bt Terence A Spencer, O. nubilalis ). a Bt Zea mays : 1071–1081 www.soci.org Berliner ( 65 has been shown to increase as concentration 27), indicating that resistance was incompletely re . 2009; 0 progeny. which can be particularly important for this pest ¨ ubner) (Lepidoptera: Crambidae). This strategy was based o Lepidoptera; inheritance; transgenic maize; Cry1Ab; hi 1 = 4 (H ´ eLBCrespo,

45 ( O. nubilalis . 2009 Society of Chemical Industry ¨ Dominance of resistance to ubner) (Lepidoptera: Crambidae) in areas cultivated with 0 c Bacillus thuringiensis genetically modiﬁed corn ( Keywords: by feeding on tissues with lowpolygenic. Cry1Ab Tissue expression. and The on-plant Cry1Ab survival resistance data was indicated primarily that autosomal, dominance incompletely varies recessive depending and on plant stage. Supporting information may be found in the online version of this article. CONCLUSIONS: A ﬁeld derived of RESULTS: The resistant strain exhibited more than one locus or multiple alleles at one locus. The degree of dominance in delaying resistance evolution when the frequencyalleles of is resistance low and resistance is functionally recessive. Pest Manag Sci This variation in Cry1Aband reproductive expression corn levels tissues between suggestsassumption the of vegetative functional need recessive for inheritance testingduring using the both corn corn growing plants stages. High dose plus refugeresistance is management one of plan(H the mandated major components for of the 1INTRODUCTION

BACKGROUND: The high dose plustransgenic refuge corn is expressing one Cry of toxins the from major components of the resistance management plan mandated for Ostrinia nubilalis Abstract in a field-derived strain of European corn borer, Richard L Hellmich, and Blair D Siegfried Andr to Cry1Ab toxin from (www.interscience.wiley.com) DOI 10.1002/ps.1793 On-plant survival and inheritance of resistance Research Article Received: 2 September 2008 Revised: 15 January 2009 Accepted: 7 although both resistant larvae and their F − because Cry1Ab expression levelsreproductive vary stages, between with vegetative and highto concentrations other in tissues leaves relative such as pollen, silks, ear shank and kernels. decreases, nubilalis which has not been previously tested for the nature of resistance to Cry1Ab toxinand by their examining F the inheritance and on-plant survival of susceptible and resistant insects . ; ). = × ] et al ;B Some ] SKY values) 14 × 50 SKY FIELD : 1071–1081 ] progeny and FIELD the data from × 65 × 1 22 progeny, which × 1 ] moths of the same Reciprocal crosses 2009; FIELD SKY 21 19 populations: FIELD × 1 + SKY SKY progenyfromreciprocal 18 24 h after eclosing) were 1 < × + ) provided by Dow/Mycogen (Migula) (host strain JM103), SKY [SKY , provided by Monsanto Co. (St  ). Because one of the methods × = Pest Manag Sci SKY ] and backcross progeny were also . To estimate the number of loci 1 [FIELD ;C × crosses were pooled and backcrossed kurstaki ] = progeny from reciprocal crosses were 1 1 [FIELD FIELD Genetic Stock Center. Cry1Ab was purified FIELD × 20 × SKY × Escherichia coli subsp. [FIELD × = Bacillus Based on results of tests with F Resistance ratios were estimated on the basis of [SKY by a modification of the method described by FIELD Cry1Ab concentrations in different batches were 20 23 × were backcrossed to resistant moths to produce four = 17 1 and SKY [FIELD . toxins ]; F × E. coli Bt B. thuringiensis SKY et al SKY = used to estimate the numbervariance of of loci backcross to also both requires parental strains, the phenotypic influencing resistance, F with susceptible females andpopulations males (E to produce two backcross survival assays were analyzedPC by to probit regression calculate using the POLO- median lethal concentrations (LC concentrations used to test F D and slopes. sex from both reciprocal F were made bythe mass resistant strain mating (SKY) to the produce susceptible two F FIELD strain and mass crosses between resistant SKY andwere susceptible tested. FIELD strains The sexobservation of of pupae genitalia was under a determined microscope. visually through Co. (Indianapolis, IN).protein, Because the of source limited ofresistant availability core toxin of after was 21 Cry1Ab generations. changed Althoughwere to the obtained resistant a from insects purified diagnosticof trypsin- bioassays with larvae protoxin, on survival Cry1Ab freshly (MON810) indicated excised that resistance cornresistance alleles also to leaves conferred the expressing activatedfrom toxin. truncated This toxin was obtained either Louis, MO), or from provided by the from Lee 2.4 Concentration–mortalityAfter curves mortality was corrected for control mortality, × 2.2 The toxin used in diagnostic assays and selections21 until was generation a Cry1Ab protoxin (CellCap 2.3 Mass crossesToevaluatesexlinkageanddominance,F and bioassays backcross populations (A backcrossed to the susceptible andindirecttestsformodesofinheritanceishigherwhenthebackcross resistant strain. The power of progeny are originated from crosses between F indicated that resistance was mostly recessive,of males and each females F For all crosses, 100 pupae of eachin strain/sex were pooled mating together cages.and Adults eggs were werebioassays. collected allowed Strains to to and provide emergeCry1Ab crosses in neonates survival were and bioassays. Neonates for ( tested mate, subsequent for susceptibility to exposed to Cry1Ab toxin on the surface of artificial diet. the parental strain whichthe toxicant. is more dissimilar in susceptibility to tested in parental strains,number of which loci using is direct and necessary indirect testsmodes for to of distinguish evaluating among inheritance. the standardized by SDS-PAGE/densitometry. intercepts and slopes of probit lines obtained for each strain or FIELD SKY was 13,14 were After 13,14 14 www.soci.org ALB Crespo 2009 Society of Chemical Industry After 7 days, Because this progeny were c 13 O. nubilalis 1 11,15,16 99% mortality at 13,14 . O. nubilalis > 99 selected Kandiyohi) hybrids in Kandiyohi = Bt 12 generation by exposure to corn leaf 8 were based on those developed at the colony originated from a field collection of progeny from reciprocal crosses in greenhouse 1 , trypsin-activated Cry1Ab formulation). Rearing showed that a Dipel-selected strain survived on 2 O. nubilalis − O.nubilalis progeny were not tested. 1 900 individuals from Iowa and Nebraska. The susceptible ≈ O. nubilalis Two different susceptible strains were used for on-plant In 2001, Cry1Ab-resistant individuals of In the present study, a field-derived strain of generation 21, thewas resistant maintained by strain exposure (SKY toapproximately a Cry1Ab 20-fold concentration in that excess was (1000 of ng the cm diagnostic concentration methods for USDA-ARS Corn Insect Research Unit, Ames, Iowa. bioassays and for inheritanceKandiyohi) strain experiments. originated The from the KYresistant same (susceptible field SKY collection as population the second but susceptible strain was originated in never 2005 from aof exposed field collection to Cry1Ab. A identified from a field collection from Kandiyohi, Minnesota,on based increased survival at a diagnostic Cry1Ab concentration. discs cut from(MON810). freshly Individuals that excised survived cornafter exposure 4 leaves days to were corn expressing then leafthat pooled Cry1Ab was discs to further initiate selected anotherconcentration by resistant exposure for strain to 7 days the diagnostic at Cry1Ab each generation of rearing. www.interscience.wiley.com/journal/ps 22.1 MATERIALS AND METHODS Insects andThe rearing resistant 126 diapausing larvae collected from non- Co., Minnesota, in 2001. After diapause was terminated,were 113 pupae placed in rearingmated. Approximately cages, 900 and individuals from emerging the adults F were mass The progeny derived from these survivors werethe further selected laboratory in using leaf discs expressing Cry1Ab. vegetative- and reproductive-stage plants expressing Cry1Ab, but hybrid F of used in diagnostic bioassays ina which concentration neonates of were exposed Cry1Ab to protoxinend of corresponding the to 95% the confidence upper interval of the LC experiments with entire cornwith plants reproductive and corn tissues. in laboratory bioassays strain was initiated withmore insects appropriate means from to the test assumptions field, relatedmanagement to it strategies resistance for may Cry1Ab-expressing provide corn. a used to defineexamining the the inheritance naturewith in of a progeny resistance susceptible fromin to strain. reciprocal Inheritance Cry1Ab bioassays crosses patterns toxindetermine with were the by Cry1Ab examined degree ofsex toxin dominance linkage/maternal of applied effects thenature resistance, and to of possible the the resistance. artifical In monogenic addition,recessive the resistance diet or assumption to Cry1Ab of toxins polygenic functional was to assessed bythe determining survival of Cry1Ab-susceptible andstrains Cry1Ab-resistant and parental the F 14 individual larvaeconcentration that were survived exposure transferredadults to and to used the untreatedfurther to diagnostic selected initiate diet, at the reared the resistant F strain. to This strain was FIELD strain wasexposure reared to for Cry1Ab six beforedetermined bioassays generations to were be in conducted, susceptible the and on was the absence basis of of the previously described diagnostic Cry1Ab concentration.

1072 1073 ) 2 C, Bt ◦ ≈ The event 11 023 30 Bt ≈ events and Bt The two main events. 32). One-tailed C, 80% RH and a 30 Bt = ◦ 32 n 0). > events. Treatment means with the resistant SKY strain Bt 31 ) susceptible 28 48) and silk ( π Bt = − . A single neonate was transferred to 2 n − type and To facilitate comparisons and create a www.interscience.wiley.com/journal/ps www.soci.org 11 (N4242 YG), MON810 (Pioneer 38G17 resistant 30 π Bt : 1 H To homogenize variances, weight values were O. nubilalis 30 or 7.35 mg cm Triton X-100 solution and dispensed onto the surface isolines: 2 1 ) was the number of larvae alive divided by the number Bt − − π gml µ The survival of larvae on silk or pollen was treated as a binomial Unpollinated silk was stripped from ears, wrapped in 8 mm 5 . -tests for two independent proportions were used to determine z whether the proportion of resistant larvaethan the was proportion statistically of higher susceptible larvae surviving onor the isoline tested ( random variable. Then, thewas considered number to of be the larvaewhere number of alive each successes after in larva a 7 wassuccess series days ( of tested trials independently. Theof probability larvae tested of on pollen ( cm segments. One end ofand each placed silk in tube a wasFrenchtown, 32-cell immersed NJ). rearing in A single water tray neonate (product wasof the no. transferred silk to tube, 9074; the and Bio-Serv, a dry vented, end to clear plastic cover laminate lid the was applied cell. Larvae were allowed to feed for 7 days (27 control for natural resistancethree isolines on were united pollen for and comparisons with silk, data from the diameter bundles and inserted throughof a a seam plastic cut drinking into straw. the The side silk-filled tubes were cut into scotophase of 0 : 24 h light : dark cycle. additive effects of loci in polygenic models were alsoexpected assumed. mortality The for each concentration underwas each estimated hypothesis as the cumulative probability fromtolerance the appropriate distributions for each model (PROBNORM). and the susceptible KY0 strains. The pollen was suspended in a average absolute differencemortalities between was calculated observed as theexpected and mean percentage of expected mortality the minus absolutemortality values the for each of observed concentration. percentage 2.9 On-plant assays 2.9.1 Plants andGreenhouse treatments experiments were carried out with(RX a MON810 634, hybrid Yield Guard,isoline (RX 634) YG) in the expressing vegetative (V8-V9) the and reproductive Cry1Ab (R1-R2) toxin and its and Event 176 (Mycogen 2657 2.8 Pollen and2.8.1 silk bioassays Pollen andPollen silk and sources silk bioassays were conducted using three their non- of 1 mL of artificialto diet in air Bio-CV-128 bioassay dry. traysgrains Pollen and cm allowed concentration was estimated to be each well and allowed tomortality feed were for recorded. 7 Trays days, were after which held weight at and 27 factors were were separated using least-squares5% means (LSMEANS) significance tests level. at a square root transformed. Then,way data ANOVA were implemented analyzed in with SAS a PROC two- MIXED. 80% RH and a scotophase ofweight 0 and : mortality 24 were h recorded. light : dark cycle), after which 2.8.2 Data analysis Larval weights obtainedfor for normality pollen ofand data and distribution normal silk using probability wereUNIVARIATE. the plots first Shapiro–Wilk were tested test, constructed in SAS PROC h 21 were Ostrinia nubilalis A chi- values 28 20 the direct 50 21 2009 Society of Chemical Industry c ) contributing to E n 26,27 values for the parental progeny and the four 3 50 X progeny and the resistant 1 of Cry1Ab resistance in the 1 3 − The slopes of the concentra- D X 1 X − and indirect tests of models with 1 − X 4,28,29 20 2 , was calculated. c X 2 h , 28 c = are the logarithms of the LC D 24 3 : 1071–1081 X progeny and the four backcross populations 65 1 05. . , methods described in detail previously and progeny. Stone’s method estimates the degree of E 0 2009; n 1 2 < X the degree of dominance P , 1 25 X populations was estimated through LC 1 On-plant survival and inheritance of resistance to Cry1Ab in 2.7.3 Indirect test In the indirect tests ofbetween monogenic observed and mortality polygenic and models,with mortality the predicted one, fit by two, models approach described five previously. and ten loci was tested. This follows the 2.7.2 Direct test The direct tests use chi-square values calculated from theand observed expected mortalities of the backcross population. followed. 2.7 Number ofThe loci data obtained infrom survival mass bioassays crosses with were used backcross tothree progeny different determine approaches the including Lande’s number method, of loci by backcross populations were compared onand confidence the intervals of basis median lethal of concentrations obtained theby probit slope analysis. tion–mortality lines for the parental strainsmean were of calculated the as the slopes experimentallystrain. determined It for was each parental assumedresistance that (R) each and the locus other conferring had susceptibility (S). one Equal allele and conferring one, two, five and ten loci. test for monogenic inheritance F strains and F Pest Manag Sci To calculate 2.7.1 Lande’s method This procedure assumes equal and additive effectsgenotypic of loci and and uses phenotypic variancesnumber of to independently estimate segregating genes the ( minimum 2.6 Sex linkageThe and maternal maternal influence effects and the sex-linked naturewere of the resistance examined byresponses of comparing the F observed larval concentration where 2.5 Dominance ofTwo resistance methods were usedmethod, to calculate dominance. Using Stone’s cross. This method allows theratios to confidence be intervals estimated. for resistance a quantitative character between two divergent populations. strain. Concentration responses of F for the resistanthomozygotes homozygotes, respectively. In heterozygotes addition, the effective and dominance susceptible at specific concentrations dominance as follows: originated from crosses between the F square value was calculated for each concentration andwith compared a chi-square distributionnull with hypothesis of one monogenic degree inheritance of wasindicated rejected freedom. if The this test . . 1 Bt d d d d d d b and et al types strains FIELD SKY × : 1071–1081 was twofold × 65 The traits used 6 respectively. 37 . 0 The survival data O. nubilalis O. nubilalis (5)(9) 5.2 21.4 26,27 SKY 2009; ± c c 30 (df) RR 6 × . 2 with χ and percentage weight 001). The median lethal . 0 30 22 < 24 4and1 type, the frequency distribution . P Pest Manag Sci 0 1; ),usewasmadeofthequantitative ± p a 0 For each European corn borer type, h . = 8 O. nubilalis progeny of FIELD 2; df (95% FL) . 1 were 3 50 18 = 2 χ FIELD × Ostrinia nubilalis progeny) and corn hybrids (MON810 and isoline) as the main 4346 7376 (5284–9636)2 8 (7–10) 71 (40–98) 32 (22–46) 9.6 (6) 15 (13–18) 1.1 (4) 10.6 (5) 815.4 12.0 (6) – 1.6 (5) 7.2 3.9 2.0 56 52 (38–70) 174 (68–408) 31.7 182.8 SE LC To estimate the dominance of European corn borer resistance to ...... 1 0 0 0 0 0 0 0 ± higher than the median lethal concentration of SKY survival on Cry1Ab corn wasobserved estimated by on adjusting for non-expressing mortality plants, Cry1Ab-expressing plants ( 3RESULTS 3.1 Concentration–mortalitycurves:maternalinfluence,sexlinkage, resistance levels and dominance The analysis of datasusceptible obtained and from reciprocalfrom resistant crosses the between insects, fourand as backcross resistant well combinations insects,autosomal as and indicated between recessive. data that F1 The obtained resistance progeny mortalities was of primarily progeny and susceptible types respectively, whichby were the analyzed same methods as described previously. and crosses areof the presented concentration–mortality in curves Table of 1 FIELD and Fig. 1. The slopes analysis of variance (ANOVA)employing implemented in the SAS RANDOM PROC MIXED statement, The hypothesis thatin these POLO slopes ( are the same was rejected were analyzed as binomial. To facilitate comparisons across events, 95% confidence intervalswere calculated (CIs) using the for modified binomial Wald method. proportions factors. To homogenizeroot variances, transformed, weight and valuesLSMEANS treatment tests were means at square were the separated 5% using significance level. (unselected Kandiyohi, Cry1AbF selected Kandiyohi and hybrid in the calculation ofsurvival, percentage effective weight dominance gain and on thetraits. plants combination of Because were the two Cry1Ab larval these toxins two affect components of both fitnesstheir growth were phenotypic combined and by values. survival, multiplying concentration for F SKY values of three different traits obtained for resistant, hybrid F gain was calculated relative toisoline plants. the For larval each weight ofof each instars type on found onfrequency distribution MON810 of plants instars waschi-square found test. compared on against isoline the plants using a ± ± ± ± ± ± ± 5 5 0 6 5 2 3 ...... ) Slope C 1 ◦ − www.soci.org ALB Crespo and backcross larvae of nd slopes of probit lines obtained for the strain or cross. 1 generations 2009 Society of Chemical Industry n 446559512559 3 2 3 512 1 2 c 10241640 2 1 -Cry1Ab/Cry1Ac ELISA O. nubilalis Bt 05. types – the susceptible KY . 0 05. . = 6 1 1 1 2 2 0 36 F F P = reproductive-stage experiments hybrid progeny. This combination P 1 O. nubilalis diet, with 95% fiducial limits in parentheses. 2 − Twofactorswerecombinedinfactorialdesign: ). The greenhouse was divided into areas with 1 36 − 33,34 C until quantification. The value significant at ◦ 2 (pooled) F SKY χ All plants were dissected 15 days after infestation to Responses to Cry1Ab of susceptible, resistant, F 80 1 (pooled) F FIELD × F − 1 × FIELD (pooled)F F × 34,35 × × RR, resistance ratios estimated on the basis of intercepts a Ratios statistically higher than 1 at Units are ng Cry1Ab cm Indicates The experiments were carried out in a greenhouse at the c SKY (R) F Strain or cross Generation a b d SKY FIELD SKY FIELD SKY FIELD (S) F Table 1. record survival, larval weight andstage instar. The plants injury was to evaluated vegetative- 1–9Guthriescale. on the basis of leaf injury using the a 2 m aisle separatingamong them treatments. to Plants reduce were the risk infested of (30 larvae neonates moving plant by placing a centrifugebelow either tube the containing whorl the (V8–V9stage). neonates stage) one or the leaf primary ear (R1–R2 two corn hybrids and three www.interscience.wiley.com/journal/ps 2.9.2 Cry1Ab expression Corn plants were testedlinked for immunosorbent toxin assay expressionfrom (ELISA). upper with leaves Leaf when an plants samples were enzyme- during at were the the dissections to V13–V14 taken evaluate plant injury stage, and either larvalplants growth on infested during the vegetativeon stage plants or infested before during infestation stored the at reproductive stage. Samples were stages. These stages were chosen to representwith those that infestations coincide of the first and second common to bivoltine populations thattheUScornbelt. occur throughout most of were conducted separately. In each experiment, the sixwere treatments randomly assigned toplants six of areas each in hybrid were theplants utilized greenhouse, treatment for and corn borer 30 infestations (10 yielded six overall treatments. University of Nebraska,(vegetative and Lincoln, reproductive plants) Nebraska, and June 2006plants). in (reproductive Seeds May–June of Yield 2005 RX Guard 634 were hybrid planted RXsoil, in 634 30% a YG peat substrate andseeds moss mixture its and per composed isoline 20% pot. ofsingle sand Two 50% plant in per weeks pot, 18.9 later, which wereas L the then needed watered pots plants until daily and using reaching were fertilized theinfestation. three thinned appropriate The phenological to vegetative- stage and a for 2.9.3 Data analysis The datadesign were with analyzed years (2005 as and a 2006) as randomized block, using complete a block two-way before use. kit was obtained from Abraxis (Warminster, PA) and stored at 4 strain, resistant KY strain and F

1074 1075 , 45 b . 11 c 0 h w − a ), where 22 w Ostrinia nubilalis data was − 11 1 w 1.8 0.02 0.01 ( values and calculation 87.2 0.9 0.80 80.8 0.8 0.78 77.8 0.8 0.78 69.1 0.7 0.70 11.0 0.1 0.11 (%) Fitness / ) spectively. The fitness of Survival 50 22 w − 12 1 1 1 1 1 1 blelarvaedividedbythesurvivalof F F F F F F w KY 10.9KY 0.1 0.0KY 0.0 0.0KY 0.0 0.0KY 0.0 0.0 0.0 KY 36.0 0.4 )ofresistancein SKY 97.3 1.0 SKY 100.0SKY 1.0 100.0SKY 1.0 100.0SKY 1.0 98.9SKY 1.0 98.9 1.0 ( cross using LC h exposed to each dose was estimated Strain or = 1 D h www.interscience.wiley.com/journal/ps www.soci.org 80). divided by the survival of resistant larvae. . 1 0 and susceptible larvae re 1 = 01) and partially dominant at low concentration . h are the fitness values at a particular Cry1Ab concentration 0 ; Effective dominance ( 2 22 ) − 27). Effective dominance calculations indicate that = w 2 . − 0 h was estimated as follows: at different concentrations (Table 2). The position of the and ; 2 c = diagnostic concentration) diagnostic concentration) diagnostic concentration) diagnostic concentration) diagnostic concentration) diagnostic concentration) 12 h h The fitness of susceptible larvae exposed to each concentration was − Thelevelofdominanceisillustratedbyconcentration–mortality ) of the resistance based on pooled F as the survival of F 20 (2.8 times lower than 40 (1.4 times lower than 80 (1.4 times higher than 160 (2.8 times higher than 320 (5.5 times higher than a b Table 2. larvae as function of Cry1Ab concentration Concentration (ng cm 10 (5.5 times lower than resistant larvae exposed to Cry1Ab toxin was defined as 1. estimated as the survival of suscepti resistant larvae. The fitness of F w for resistant, F D h 3.2 Number ofResults genes influencing resistance from threeindicated that methods resistance used inthan the one to SKY locus or strain analyze multiple is alleles at controlled backcross one by locus. data more 3.2.1 Lande’sresistance estimate of the minimum number of factors affecting This method usesestimate the the genotypic minimum and number phenotypic of variances independently to segregating dominance increases as Cry1Ab concentration decreases.resistance Indeed, was virtually recessivecm at high concentration (320 ng concentration–mortality curves forcloser the to reciprocal theindicating crosses susceptible was that parent thanin the to SKY inheritance the was of( resistant incompletely the recessive. parent, The resistance degree to of Cry1Ab dominance of ( curves (Fig. 1), calculation of (10 ng cm = 50 5 5 340), 10 10 Ostrinia nubilalis value . )was 0 2 FIELD progeny − 50 1 SKY = ), obtained x × P 2009 Society of Chemical Industry 4 4 + c 6; ) ) 6 (84.6–222.9) 10 10 2 . 2 − − = progeny of reciprocal 140 1 of SKY (188.5 ng cm = 3 3 8; df Expected Mortality . 90 6 10 10 50 FIELD x SKY = . SKY 2 ][LC 20 % mortality of SKY at + χ × + 2 2 ncentrations causing percentage x 10 10 ] exhibited the lowest LC SKY at ] but did not differ significantly from 1 ) 2 × − Backcross (SKY x F1) ) 1 1 : 1071–1081 of FIELD 10 Cry1Ab Concentration (ng cm Cry1Ab Concentration (ng cm Cry1Ab Concentration 10 SKY x FIELD FIELD 65 SKY 90 Observed Mortality × ). Because the concentration–mortality curves × 2 2009; − 0 0 (% mortality of F FIELD 10 10 × (SKY 5 . Expected concentration–mortality curve for monogenic inheri- Concentration–mortality curves obtained with neonate larvae ]. The hypothesis that the slopes and intercepts were the 0 2 ×

80 60 40 20 80 60 40 20 −

= (FIELD 100

100

Mortality (%) Mortality Mortality (%) Mortality x = On-plant survival and inheritance of resistance to Cry1Ab in indicating that the fourresponses backcross to Cry1Ab. populations exhibited similar and the resistant parental strain (SKY).tion Expected % mortality at concentra- the backcross with the[A highest contribution of resistant females Pest Manag Sci SKY and to verify ifthe there four backcrosses were (Fig. maternal-related S1The differences and concentration–mortality Table among curves S1, obtained for supplementary insectsbackcross data). from the with the least contribution of resistant females [D (200.9 ng cm However, the LC Figure 2. tance and observed concentration–mortality curve obtained withlarvae neonate from four backcross combinations between moths of F Figure 1. from susceptible and resistant parental strains and F not significantly different from the LC same was not rejected in POLO ( crosses between parental strains. from regression lines of parental strains. ng cm [108.5 (70.3–166.5) ng cm of the fourmortality(Fig.2),onlyCry1Abco backcrosses exhibited a plateau response at 90% mortality between 0 and 90% were used to estimate the LC . Bt a a a a a 03, . et al 9 P 0001) 0001). . and in . 0.001 0.001 0.001 0.001 0.001 0 11 (0.0) 0 = < < < < < 38 Bt < z < : 1071–1081 P P 65 (fresh weight) 49, type and event . 1 − 2 46 11.0 2009; χ 3, 363; 355.7 gg = 2608.7 µ = z 1, 174; 0.8387) (Fig. 5A). 0001) (Table 5). . 0 7549) (Table 5). However, 19 = . . events. Event 176 silk did 0 0 < O. nubilalis progeny and the susceptible 0934)(Table 5).Nevertheless, 35; df . a a a Bt ative- and reproductive-stage . P = 1 ± 0 8 18; df P 4, . P . 96 Pest Manag Sci = 0 = . 0.001 0.001 0.001 P 10 69, F . < < < = 0 = F 32, and non- . z 1 05). =− Bt . = 0 26 and 3 z . 2 z 0 0.11.0 0.822 0.315 13.9 22.4 3.5 0.061 2.3 0.132 < χ 18.6 P 336.5 ± 11 (0.78; Bt 02 . Model 11, however, no significant differences in proportion a a a a a 1) for monogenic and additive polygenic models Bt 6,7,39 = P 0001) or . 0.001 0.001 0.001 0.001 0 < < < < 11 and MON810 caused significantly higher growth inhibition in < 3.5 On-plant assays 3.5.1 Cry1Ab expression The average concentrations of Cry1Ab in leaves of plants (V13–V14stage) from experiments with veget plants were 3 P compared with resistant larvae on MON810 (0.74; Bt resistant individuals (Fig. 4B). Asusceptible significantly higher larvae proportion of failed to survive on MON810 and Figure 4B shows the weight of susceptible and resistantreared individuals on silk ofnot both cause significant growthresistant inhibition larvae, in and either nosurviving susceptible significant larvae or differences were inresistant detected strains proportion (0.94; between of susceptible (1.0) and indicates that thesilk growth varied across inhibition the caused events ( by feeding on 176. For of surviving larvae were detected betweenresistantlarvae(0.98; susceptible (0.89) and (Table 5). 3.4 Silk assays The significant interaction between the lowest proportionthe susceptible of strain survivingresistant on larvae survived larvae on Event Event 176 was 176 (1.0; pollen observed (0.02), for whereas all 3.5.2 Vegetative stage The results of vegetative-stagein plant experiments Table are 6 presented andplants. Fig. 5. On No isolinesurvival survivors compared plants, with were the hybrid the F detected resistant on MON810 strain exhibited lower the field. strain, but the larval weight wasEuropean not corn significantly borer types different ( among Virtually no leaf injury wason detected isoline on MON810 plants, plants, the whereas Guthrie injury ratings averaged 4.5, which respectively. These values werereported either for higher or MON810 similar plants to grown those in the greenhouse 2 9.5 0.002 Bt . b b b b b 46.1 25.5 13.8 54.5 x P 0001). . 0 www.soci.org ALB Crespo 0.0001 0.0001 0.0001 0.0001 0.0001 < < < < < < a a a a a 2009 Society of Chemical Industry P 11 and Event PX 2 c S (pooled) at ,calculatedas Bt χ 0.001 0.001 0.001 0.001 type and event x × < < < < 3,565; = a events. No significant rences between expected and observed mortality ( One locus Two loci Five loci Ten loci 2 Bt 1) 63;df χ . O. nubilalis 05). = . 41 0 % mortality of R < nificant differences between the ) controlling Cry1Ab resistance = P E + n F and non- x ) 2 − at at each concentration Bt 1 x Y Observed Expected (% mortality of F Indirect tests for deviation between observed and expected mortality (df Direct test for deviation between observed and expected ). The estimate of the minimum number of freely ) Dead Alive Dead Alive × E 2 5 − n . 0 = Probability values indicating sig Probability values indicating significant diffe Expected % mortality x observed and expected mortality ( Y Average mortality (%)Mean difference (%)a 35.4 15.7 35.8 17.1 36.3 7.1 35.4 8.8 320225045009000 45.9 23.6 0.3 16.9 0.600 0.6 0.483 59.7 Cry1Ab concentration (ng cm 20 39.2 Table 4. a b 320225045009000 103 119 116 25 117 9 12 65 11 72 88 63 95 56 46.20 40 70.46 33 28.81 20.44 (ng cm 2080 6 46 122 82 12 21 116 107 3.57 37.83 0.0588 Concentration Table 3. mortality for the monogenic model (df 80 8.4 0.004 Figure 4Ashowstheweightofsusceptibleandresistantindividuals reared on pollen of both segregating effective factors ( 3.3 Pollen assays The significant interaction between 3.2.2 Direct andThe indirect tests direct testsbecause indicated in that most resistancewere of was statistically different the not from concentrationslocus monogenic the expected model tested (Table represented 3). the the Themean mortalities difference, five- best with fit three and outexpected value. of exhibited All six other the models points indicated lowest deviating atof least six from four deviating the points from out the expected value (Table 4 and Fig. 3). in SKY was 2.6. Thisa result is small in number agreement of with(Table the loci 1). hypothesis with of major effects on Cry1Ab resistance indicates that the growth inhibition caused by feeding on difference in growthresistant was individuals detected reareddeveloped between better on than susceptible the MON810. susceptible and strain The on resistant strain pollenvariedacrosstheevents( www.interscience.wiley.com/journal/ps genes (

1076 1077 t 1 0001). In isoline plants, . 4 4 0 10 10 < P a 32 silk assays) on tissues derived Ten loci Two loci = a n 3 3 2, 354; 10 10 types were signiﬁcantly different for = b c www.interscience.wiley.com/journal/ps www.soci.org progeny and susceptible larvae feeding 0; df . 1 typebycornhybridinteractionindicatesthat 2 2 12 Silk (B) b 10 10 = O. nubilalis ) c 2 F − larvae exhibited similar weights on MON810, hybrid 48 pollen assays and 1 a O.nubilalis 1 1 = larvae exhibited the highest larval weight, followed by Expected Mortality a n 1 10 10 Non-Bt Bt11 M810 E176 0 0 5 0 80 60 40 20 80 60 40 20 progeny and the susceptible strain. The percentage mortalities progeny and susceptible larvae feeding on MON810 were 32.6, SKY 20 15 10 100 100 strains ( 1 1 resistant strain showed lower survivalF compared with the hybrid on MON810 were 43.5, 82.2 andsigniﬁcant 99.4% respectively (Table 7). The larval weights of each plant type ( hybrid F progeny and resistantand larvae the exhibited susceptible(Fig. similar 5B). strain The larval percentage exhibited reductions weights, in theF weight of lowest resistant, hybrid larval weight 63.0and75.0%respectively(Table 7).Therefore,althoughresistant andhybridF of resistant, hybrid F susceptible and resistant larvae (Fig. 5B). In MON810, hybrid F 4 4 10 10 c 19; KY d Ostrinia nubilalis = Five loci Cry1Ab Concentration (ng cm One locus 3 3 Ostrinia nubilalis bc O. nubilalis 2009 Society of Chemical Industry 10 45; df 10 type by corn . 0001), which bc Observed Mortality . c 1 0 = < t near-isoline plants. Treatment means were separated using least-squares means (LSMEANS) tests a ab Pollen (A) P 2 2 c Corn Hybrids Corn Hybrids 10 10 O. nubilalis a progeny caused more injury 2, 174; 1 ab = Non-Bt Bt11 M810 E176 1 1 types were detected on MON810 10 10 5 0

20 15 10

0 0 Weight (mg) Weight 93; df . 80 60 40 20 80 60 40 20

4 100 100

SE) of larvae from SKY and control Mortality (%) Mortality = ± O. nubilalis : 1071–1081 F 65 progeny and the resistant parental strain (SKY). 1 2009; Mean weight ( Observed versus expected mortality at each of the five Cry1Ab concentrations tested on neonate larvae from four backcross combinations 081) (Table 6). The resistant strain exhibited the highest . 0 = On-plant survival and inheritance of resistance to Cry1Ab in survival rates on MON810 plants. In contrast, on isoline plants, the P from Cry1Ab-expressing events and combination of a 5% significance level. Pest Manag Sci corresponds to plants exhibiting several leaves with shot-holeelongated and lesions. There was a significant Figure 4. Figure 3. between moths of F indicates differences among injury ratingstypes. Both caused susceptible by and hybrid F 3.5.3 Reproductive stage The results of reproductive-stage plant experiments are presented in Table 6 and Fig.plants, 5. survivors In of all contrast to results from vegetative-stage to isoline plants than the resistant strain (Table 6). plants, although survival rates of susceptible(0.3%) larvae and were minimal not statistically higher than zero ( hybrid interaction ( . ses et al 37 a Cry1Ab 40 O. nubilalis : 1071–1081 65 2009; Survival (%) (95% CI) A maternal influence asso- Cry1Ac (162-fold), 41 9 e ctive transgenic corn MON810 and Pest Manag Sci d 0 c 0.0 (0.0–1.3) 0.3 (0.0–1.2) 1 c 0.0 (0.0–1.3) 18.3 (15.9–22.2) 0 c 0.0 (0.0–1.3) 11.0 (8.7–13.8) 6 a 12.7 (9.4–16.9) 56.7 (52.7–60.6) 6 a 11.0 (7.9–15.0) 61.3 (57.4–65.1) 4 b 2.7 (1.4–5.2) 33.3 (29.7–37.2) ...... 0 0 0 0 0 0 ± ± ± ± ± ± d 0 2 0 6 7 4 ...... 1 4 strain exhibiting some degree of survival on b to Cry1Ab (SKY) and progeny from reciprocal cros 1 1 a and Cry1F (3000-fold). F F SKY 3 SKY 1 4 Injury and survival of larvae from European corn borer types c O. nubilalis Confidence intervals estimated by the modified Wald method. 5 0 1000-fold) The SKY strain exhibited an intermediate resistance level (815- MON810 KY 1 Table 6. released on vegetative and reprodu MON810’s isoline Cornevent Strain or GuthrieIsoline injury cross rating (1–9) KY Vegetative 5 Reproductive a 30 25 20 15 10 > ( fold) (Table 1 andstrains Fig. selected 1) with Dipel among (65-fold), those reported for 4 DISCUSSIONThe present AND study CONCLUSION indicates that thehigh resistant SKY levels strain of exhibited resistancetransgenic to corn Cry1Ab by and feeding the onThe ability tissues resistance to is of survive primarily low autosomal on Cry1Ab andand cannot expression. incompletely be recessive, explained by a single locus withthe two alleles. first SKY was reproductive-stage corn expressing Cry1Ab whenused neonates to were infest plants. ciated with inheritance ofin Cry1Ab analysis of resistance concentration–mortality was curves also ofbetween reciprocal susceptible identified crosses and resistant strains (Table 1 and Fig.ever, 1). the How- similarities among concentration–mortality responses of the four backcross combinations [the hypothesis that the slopes SKY F1 KY larvae 0151), . )from 37 1 0 www.soci.org ALB Crespo 12 larvae, the = MON 810 810 MON a 1 bbb 2009 Society of Chemical Industry P c 2; a = ested were susceptible to Cry1Ab (KS), resistant Corn Hybrids Corn Hybrids Vegetative (A) Reproductive (B) a ). Error bars represent standard errors. Bars in a chart followed by the same letter are statistically similar 1 Isoline Isoline 81; df . 7 a types grown on isoline and Survival (%) (95% CI) = 2 χ

5 0

30 25 20 15 10 Weight (mg) Weight O. nubilalis 3383). In contrast, for hybrid F . a 5% significance level). 0 = P KY 97.9 (94.1–99.3) 94.8 (88.4–97.8) SKYSKY 97.9 (89.1–99.6)SKY 91.7 (80.4–96.7) 78.1 (61.2–88.9) 100.0 (92.6–100.0) 74.2 (56.8–86.3) 93.5 (79.2–98.2) SKY 95.8 (91.1–98.1) 94.1 (89.8–96.7) 2; = Survival of two strains of European corn borer (F Larval weight of European corn borers types after 15 days feeding on vegetative and reproductive stages of the MON810 plant (RX 634 YG) 17; df . 2 events Bt Bt = 11 KY 89.1 (77.0–95.3) 0.0 (0.0–11.3) larvae exhibited a twofold reduction in weight caused by Confidence intervals estimated by the modified Wald method. 2 MON810Event 176 KY KY 97.9 (89.1–99.6) 2.1 (0.4–10.9) 0.0 (0.0–11.0) 100.0 (87.9–100.0) Bt Kandiyohi County, Minnesota, after 7of days feeding on pollen and silk a Corn eventNon- Strain Pollen Silk Table 5. 1 χ which indicates a major developmental delay of hybrid F feeding onMON810.Calculationsofdominanceofresistanceusing relative phenotypic values obtained forthe combination survival of and these weight two and which variables indicates varied that from 0.17 resistance(Table to in 7). 0.31, SKY is incompletely recessive www.interscience.wiley.com/journal/ps Figure 5. and its isoline (RX 634): European corn borer types t between susceptible and resistant parents (F F (Fisher’s protected LSD, at frequency distribution of instars exhibited afrom significant the departure expected frequency ( feeding on transgenicweight plants of compared resistant larvae. withof Analysis the instars of from the reduction these frequency in two distribution MON810 plants revealed similar trends. The frequency distribution of instars of resistant larvaeto found on the MON810 frequency plants was distribution( similar of instars found on isoline plants

1078 1079 c 1 h 11 Bt larvae which hybrid 6 1 Diatraea progeny, 1 O. nubilalis among all isolines Ostrinia furnacalis MON810 and 42 In both studies, the F Susceptible insects died 45 larvae establishment and 5 Calculations of degree of Two factors may contribute on reproductive-stage corn. 8 7 Event 176 exhibits relatively 11 caused 100% mortality to and Cry1Ab-resistant D. saccharalis Bt 8 5,42 11. O. nubilalis Bt O. nubilalis www.interscience.wiley.com/journal/ps www.soci.org O. nubilalis In addition, results from pollen and silk assays strains or crosses were based on the percentage 5–7 Similar results with on-plant studies were obtained 44 (F.) (Lepidoptera: Crambidae). In addition, the second generation of To account for genetic differences, fitness comparisons 43 45 ´ ee) (Lepidoptera: Crambidae), which exhibited some ability O. nubilalis On reproductive-stage isoline plants, the resistant and F Greenhouse tests indicate that dominance may vary depending to increased survival of The suitability ofthe the plant matures, corn favoring survival. plant as a larval host changes as were engineered with a promoter to expressplant, toxin throughout although the at low levels in pollen. on pollen of Eventcontrast, 176, silk whereas from SKY MON810 exhibitedsusceptible and insects high but survival. very low In mortalityresults to SKY were (Table obtained 5). Similar with(Guen Asian corn borer, to survive after 3plants days but no feeding survival on on leaf silk tissue. and kernels of MON810 with Cry1F-resistant which exhibited higher fitnesshybrid vigor than indicates either genetic differences parentalsusceptible between strain. strains resistant that and This mayor selection. have been caused by genetic drift suggests that resistant insectslower likely used levels other of plantand Cry1Ab parts ear with expression shank. such as pollen, silk, kernels dominancebasedonreductioninweightofsurvivinglarvaeandon larval survival and larval weight combined revealed thatwas resistance incompletely recessive, as estimates were between 0.17 and corroborated on-plant assays,were suggesting able that to resistant surviveThe insects results on are tissues consistent withreproductive with lower the tissues Cry1Ab Cry1Ab of expression expression. Event various profile 176, of Cry1Ab MON810 corn and hybrids including of reduction in weight andexpressing plants survival in of relation larvaefor to feeding these each phenotypic on type characteristics Cry1Ab- feeding on isolines. are exposed to a mixturemay of favor leaves the and survival of reproductivelow-Cry1Ab-expressing resistant tissues tissues. insects that that initiate feeding on saccharalis hybrid larvae exhibited higher survival onthis isoline pattern plants, was although not consistent for tested. on plant stage. Vegetative-stage MON810 plants infested withtended SKY to exhibit highersurvivors were Guthrie found ratings on this (Table stage. 6),on However, reproductive-stage corn although larvae were that found no feeding survived on silk,ear ear shanks, tips, kernels andand pollen S3 supplementary accumulated data). in A seasonal leafexpression analysis axils of indicated tissue-specific (Tables that S2 plants Cry1Ab were levels not reduced in during leaves the reproductive of stage, MON810 indicated that dominance was dependent on toxinwith concentration, resistancecorresponding being to nearly 6 timesmonitoring recessive for the resistance diagnostic in at field concentration populations. the used in concentration More importantly, the calculations of effective dominance high levels of Cry1Ab expression innon-green pollen tissues and such as poor the expression pith in and kernel. larvae exhibited the lowest(Table and 6 the and Fig. highest 5B). fitnessa It respectively consequence is unclear of whether resistancewith the or reduced repeated fitness whether is selection inbreedingImportantly, this fitness associated may deficit was not apparent have in the F adversely affected fitness. = 50 c p h Ostrinia nubilalis SKY 8; df . × 6 = 2009 Society of Chemical Industry b 2 c χ Fitness a calculated on the basis of LC strains selected under laboratory D 6.6 0.2 0.17 27), indicating that resistance was . 17.837.0 0.3 0.31 0.5 0.28 0 = Percentage of survival Percentage of weight gain were only 7.2- and 3.9-fold respectively, in : 1071–1081 O. nubilalis h resistance to transgenic corn MON810 expressing ) controlling Cry1Ab resistance in SKY was 2.6, 65 E n 45 ( . SKY phenotype value of susceptible)/(phenotype value of Larval survival and weight gain combined 0 2009; × − − phenotype value of susceptible). Phenotype values, relative fitness and effective dominance 1 − It is important to mention that results from direct tests 340)] suggesting that resistance was primarily autosomal . 4 0 Ostrinia nubilalis = Each fitness value is the phenotype value of the strain divided by the Phenotype values for each trait are measures of genotype perfor- The dominance at each concentration was calculated as: (phenotype 1 1 1 All methods used to analyze the number of genes involved The degree of dominance P KYF 0.6 0.0 c b a Cry1Ab toxin during the reproductive stage Strain or cross Phenotype value of Table 7. phenotype value of the fittest (i.e. resistant) strain. SKY 38.1 1.0 value of F KYF 0.2 0.0 SKYKYF SKY 56.5 25.3 67.4 1.0 0.4 1.0 resistant mance on MON810 relative to theon the performance isoline. of the same genotype On-plant survival and inheritance of resistance to Cry1Ab in in resistance on theindicate basis of that the more response thanSKY. of The one backcross estimate locus progeny of contributed theeffective minimum factors to number ( resistance of freely in segregating (Fig. S1 and Tablebe S1, concluded that supplementary the data). resistance isthat Although autosomal, the it it appears maternal cannot unlikely influencessignificantly observed to in Cry1Ab resistance. this study contributed 6; incompletely recessive. The resistance ratios of FIELD which is in agreementResults with of the indirect tests polygenic indicate model thatby of resistance as may inheritance. be many controlled as fivetwo loci Cry1Ab-resistant (Table 4 and Fig. 3). A previous study with conditions indicated that Cry1Ab resistance was polygenic in these strains. used to rule outmutations at monogenic the inheritance same locus do thatresult not might in impact consider deviations susceptibility and other fromalleles. the In addition, model results from with polygenic models a arebecause single approximate one locus of and the two assumptionsmodels used is to that generate inheritance the ofwas polygenic resistance is shown additive, to yet resistance bepresent incompletely results indicate recessive that inis Cry1Ab SKY. complex resistance Therefore, and in the cannot thealleles. be SKY explained strain by a single locus with two Pest Manag Sci and FIELD values was and intercepts were the same was not rejected ( contrast to a resistance ratio of 815-folddifferences in the between resistant strain. the The twosmall considering reciprocal the crosses magnitude were of resistance therefore in SKY (815-fold). . CRC, et al :9–14 JEcon Bacillus Bacillus Ostrinia 40 :703–712 :541–553 crops safe? 84 99 Appl Environ : 1071–1081 Bombyx mori Bt (Lepidoptera: Sci Agric Sinica 65 J Econ Entomol delta-endotoxin. . LeOra Software, Bull Entomol Res Pyrausta nubilalis Iowa J Sci ´ ee). Genetics 2009; :1046–1055 (1992). in diamondback moth 85 (Guen Cry1Ab. :171–184 (1919). delta-endotoxins. J Econ Entomol for standardization of insect :614–621 (2002). 18 :1588–1595 (2000). 95 toxin (Dipel ES) in the European :130–135 (2008). llies, with description of larvae, nsgenic Bt corn and its effects Ostrinia nubilalis 93 74 epidoptera: Crambidae) on Bt and :925–930 (2000). :325–326 (1968). Pest Manag Sci Bacillus thuringiensis JAgrRes 93 38 larvae are not resistant to transgenic Pesticide Bioassays with Arthropods. J Econ Entomol orn borer egg masses. :965–967 (1999). :208–214 (2007). Ostrinia furnacalis J Econ Entomol 53 284 Bacillus thuringiensis :82–87 (2007). 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(2006). resistance to the Cry1Ab hybrid progeny from Cry1Ab-resistant 1 These results should be interpreted with caution because The present study is the first attempt to evaluate the assumption 2 Gould F, Potential and problems with high-dose strategies for 3 Gould F, Sustainability of transgenic insecticidal cultivars: integrating 1 Federal Insecticide, Fungicide, an 4 Alves AP, Spencer TA, Tabashnik BE and Siegfried BD, Inheritance of delayed on themuch higher reproductive-stage proportion of plants, larvaedevelopment than would it reflected be by the unable is 15 to day survival. complete likely thatnubilalis a Therefore, to test themore studies assumption are of necessary to recessive verify theproduced inheritance number of by fully, fertile progeny moths thatplants. Other complete resistance-delaying development effects not on testedstudy in transgenic the present may ameliorate the risk of Cry1Ab. Reproductive fitness costs and lowinsects, survival of as diapausing observed in(Say) overwintering (Coleoptera: Chrysomelidae) of functional recessive inheritance fornubilalis resistance to Cry1Ab in the resistance identified in the SKYTherefore, strain progeny is from functionally the recessive. second generation,completed which may development have onsurvive transgenic on plants, high-Cry1Ab-expressing areplants. tissues This unlikely does of not to preclude vegetative-stage resistance an alleles increased for frequency the of second generation, Cry1Ab does where not the appear resistance to bealso functionally recessive. be However, it notedinfestation should and that does not survivalBecause reflect the survivorship was to development the measured of adult the at stage. F 15 days after resistance was functionallyreproductive stages. recessive for both vegetative and exhibited higher survivalexpressing than corn plants. susceptible insects on Cry1Ab- www.interscience.wiley.com/journal/ps REFERENCES Supporting information may be found in thearticle. online version of this SUPPORTING INFORMATION ACKNOWLEDGEMENTS The authors thank the Monsanto Co. and Dan Ziegler ( Genetic Stock Center)the for bioassays. providing Funding theAgricultural for Cry1Ab this Biotechnology toxin research used Stewardshipand was in the Technical provided National Committee Science byManagement. Foudation the Center for Integrated Pest 0.31 (Table 7). The presentfor findings F are similar to those reported the effects ofreproductive-stage plants. incomplete recessive inheritance observed for (Saunders) (Lepidoptera: Gelechiidae),

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