A Bt Transgene Reduces Herbivory and Enhances Fecundity in Wild Sunflowers A

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln

Diana Pilson Publications Papers in the Biological Sciences

4-2003 A Bt Transgene Reduces Herbivory and Enhances Fecundity in Wild Sunflowers A. A. Snow Ohio State University, [email protected]

Diana Pilson University of Nebraska–Lincoln, [email protected]

L. H. Rieseberg Indiana University - Bloomington, [email protected]

M. J. Paulsen University of Nebraska State Museum, [email protected]

N. Pleskac University of Nebraska-Lincoln

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Snow, A. A.; Pilson, Diana; Rieseberg, L. H.; Paulsen, M. J.; Pleskac, N.; Reagon, M. R.; Wolf, D. E.; and Selbo, S. M., "A Bt Transgene Reduces Herbivory and Enhances Fecundity in Wild Sunflowers" (2003). Diana Pilson Publications. 1. http://digitalcommons.unl.edu/bioscipilson/1

This Article is brought to you for free and open access by the Papers in the Biological Sciences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Diana Pilson Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors A. A. Snow, Diana Pilson, L. H. Rieseberg, M. J. Paulsen, N. Pleskac, M. R. Reagon, D. E. Wolf, and S. M. Selbo

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Ecological Applications, 13(2), 2003, pp. 279±286 ᭧ 2003 by the Ecological Society of America

A Bt TRANSGENE REDUCES HERBIVORY AND ENHANCES FECUNDITY IN WILD SUNFLOWERS

A. A. SNOW,1,4 D. PILSON,2 L. H. RIESEBERG,3 M. J. PAULSEN,2 N. PLESKAC,2 M. R. REAGON,1 D. E. WOLF,3 AND S. M. SELBO1 1Department of Evolution, Ecology, and Organismal Biology, Ohio State University, Columbus, Ohio 43210 USA 2School of Biological Sciences, University of Nebraska, Lincoln, Nebraska 68588 USA 3Department of Biology, Indiana University, Bloomington, Indiana 47401 USA

Abstract. Gene ¯ow from transgenic crops can introduce novel traits into related species, but the ecological importance of this process is unknown. Here, we report the ®rst empirical evidence that wild plants can bene®t from a bacterial transgene under uncaged, natural conditions. Cultivated sun¯ower (Helianthus annuus) is known to hybridize fre- quently with wild sun¯ower (H. annuus) in the western and midwestern United States. We studied a crop-developed Bacillus thuringiensis (Bt) transgene, cry1Ac, in backcrossed wild sun¯ower populations. Lepidopteran damage on transgenic plants was strongly reduced relative to control plants at our two study sites, while damage by several weevil and ¯y species was unaffected. Our results suggest that reduced herbivory caused transgenic plants to produce an average of 55% more seeds per plant relative to nontransgenic controls at the ®eld site in Nebraska. A similar but nonsigni®cant trend was seen at the site in Colorado (14% more seeds per plant). In a greenhouse experiment the transgene had no effect on fecundity, suggesting that it was not associated with a ®tness cost. If Bt sun¯owers are released commercially, we expect that Bt genes will spread to wild and weedy populations, limit damage from susceptible herbivores on these plants, and increase seed production when these herbivores are common. Key words: Bt transgene; ecological effects; fecundity; ®tness bene®ts and ®tness costs; gene ¯ow, ecological and evolutionary effects; Helianthus annuus; herbivore damage; hybridization; risk assessment; sun¯ower, wild; transgenic crops.

INTRODUCTION spread of transgenes coding for other ®tness-related traits could exacerbate weed problems in agricultural Worldwide, many cultivated plants hybridize spon- settings and affect the population dynamics of wild taneously with wild or weedy relatives (Small 1984, relatives in unmanaged areas (Snow and MoraÂn Palma Ellstrand et al. 1999). In the United States, for example, 1997, NRC 2000). This raises fundamental questions this occurs in more than twenty species, including sun- about the extent to which herbivores, diseases, and ¯ower, sorghum, squash, canola, rice, sugar beet, pop- stressful abiotic conditions regulate populations of wild lar, turf grasses, and forage grasses (NRC 2000). In addition, many crops can become naturalized and per- and weedy plants. With regard to herbivores, studies sist as feral weed populations (Ellstrand et al. 1999). of both native and exotic species suggest that herbi- Thus, transgenes conferring novel traits that enhance vores can have a dramatic impact on plant population survival and reproduction may inadvertently disperse dynamics (e.g., Crawley 1997, Rees and Paynter 1997). from cultivated plants to wild or weedy populations However, little is known about how herbivores affect that lack these traits. In the short term the spread of the population dynamics of wild or weedy relatives of transgenic herbicide resistance is likely to pose chal- cultivated plants. Without ®ndings from carefully de- lenges for controlling weeds and unwanted ``volun- signed ®eld experiments, it is dif®cult to estimate the teer'' crop plants (Snow et al. 1999, Hall et al. 2000). net impact that herbivores have on plant survival, Over the longer term we need to know whether the growth rates, and seed production. Sun¯ower (Helianthus annuus) is an attractive study system because the self-incompatible wild sun¯ower Manuscript received 9 September 2002; revised 7 October (also H. annuus) is an agricultural weed that hybridizes 2002; accepted 22 October 2002. Corresponding Editor: C. R. Linder. easily with the crop (Arias and Rieseberg 1994, Snow 4 E-mail: [email protected] et al. 1998). Wild sun¯ower is common along ®eld and 279 Communications ml muto ttxn(at e 10 per (parts affect very toxin a Bt to Ingesting of 1997). amount expected al. small et not (Estruch is taxa toxic insect but is other which species Cry1Ac, lepidopteran protein to Bt Our the date. to involved commercialized study pests, been crop have important none although economically are weevil which and sun¯ower moth larvae, to tested resistance transgenic ®eld with ge- have varieties as wild companies bene®t such Seed to traits potential notypes. novel the have for resistance coding insect transgenes so (Seiler sun¯ower 1992), cultivated as diseases and herbivores areas crop- from more markers. or sampled DNA one speci®c had plant cultivated was wild (1998) sun¯ower al. where every Dakota et Linder that North example, reported In for 1997). Canada, sun¯ower and al. 1994). (USA) wild Rieseberg et into and (Whitton backcross (Arias populations easily away can m genes 1000 Crop hybridization the as of to far levels adjacent detectable as occur but that small plants with wild crop, on 40% as as rates high hybridization crop±wild demonstrated have and ies stud- Previous western 1954). in the (Heiser States as in United areas midwestern well natural as and plants, agricultural other domesticated near margins road 280 oye fwl u¯wrt raeBC ge- create to speci®c sun¯ower used wild we of dispersal, notypes pollen avoid of and crop problem of the the from effects transgene the Bt simulate a of To cross- introgression trans- prevented. the and be of must damage persistence gene(s) insect and dispersal of yet levels ex- pollination, be natural must to plants biosafety posed Uncaged to concerns. due regulatory dif®cult and inherently is transgenes cial nagenos,wtotbigepsdt herbivores. to exposed grown being were without plants greenhouse, when a bene®t in- in or any with cost associated ®tness was herent transgene and the ®eld, the whether in (3) sun¯owers with wild associated in was fecundity transgene increased the con- whether in ®eld (2) uncaged herbivory ditions, under reducing sun¯owers at wild effective backcrossed was transgene a Bt a to move 2000). or Onstad 1997) and die (Davis al. and plant et host feeding (Estruch nontoxic days stop few either a to within insects susceptible es S,t etfr®ns fet ftetaseeunder transgene the of effects ®tness for Colorado, test and to Nebraska ex- USA, in Field out plants). carried were male-sterile Oseto periments avoid and Korman might 1989, and al. 1989] et [Delisle pollen predators on seed feed (some production seed and sterility and male with herbivory of on effects plants the compared determine to also pollen without we transgene, the out eeml trl n ergtdfrtepeec or presence BC the Among transgene. for Bt the segregated of absence and sterile male were idsnoe ouain otmn ftesame the of many host populations sun¯ower Wild eemnn h clgclefcso pre-commer- of effects ecological the Determining h ol fti td eet eemn 1 whether (1) determine to were study this of goals The M xeietlapproach Experimental TRASADMETHODS AND ATERIALS 9 yial caus- typically ) 1 1 rgn with- progeny rgn that progeny .A NWE AL. ET SNOW A. A. fetdpatfcniyi h bec fherbivores. of sterility absence male the or in fecundity transgene plant the affected whether determine used was to experiment greenhouse A conditions. natural n BC and eeoe o oeta omrilzto n a a plant-optimized had a of and copy commercialization single was potential line transgenic for The sterility. developed plants male inbred of by type United produced this seed the with hybrid in as sold sun¯ower is cultivated female States original all the Nearly as parent. sterility male cytoplasmic with olndnr rdcdbt aeseieadmale- and male-sterile Heterozygous BC both fertile entirely. produced them donors lacked pollen or alleles storer aeseiead4.%wr tpstv ( positive Bt were 49.3% were and 44% greenhouse sterile progeny, and of male ®eld group the this Within for experiments. progeny of group single mn.W sdattlof exper- total ®eld the a in used used We not were iment. positive plants Bt were male-fertile that pollen, via transgene experimental ewr bet eetwl olndnr o h F the for donors pollen wild select but to fertility, able male restore were that plants we alleles wild more or Most nu- one restoration. with have plants fertility wild for by alleles sired clear unless sterile parent transgenic male this were from mater- produced progeny inherited so is selectable nally, sterility a male lacked Cytoplasmic and marker. 35S), CaMV (modi®ed moter orbok nClrd.BC and Colorado. Nebraska in in blocks blocks six four include to sub- plots we the differences, weeded. divided microsite not possible a was for to account and To similar grassland was in plot disturbance cultivated natural Nebraska of The typical ®elds. applications sun¯ower nutrient her- and received plot bicide Colorado The planting. to ag- were prior sites tilled an Both Colorado. was Biological Burlington, other in Point ®eld the Cedar ricultural and at Nebraska, area Ogallala, grassy Station, a was plot One u eut eei¯ecdb atclrwl geno- wild particular that by BC probability in¯uenced types. the were minimizes results sires our relatively of This number donors. pollen large as Nebraska, Ogallala, rvd olnfrcosfriiain h treatment The cross-fertilization. and for effects pollen competitive standardize provide to plants included Wild block. were the in 30 plants or between (Nebraska) (Colorado) cm cm 25 either with alternately, planted nptidse,sw n¯t,adselnswr trans- sites were ®eld seedlings two and to ¯ats, planted in sown dishes, petri in curn ee eg,So ta.19,Hlhl tal. et Halfhill 1999, al. et 2002). Snow (e.g., genes naturally as occurring manner same that the expectation in inherited the are with transgenes consistent is Bt-neg- genotypes and Bt-positive ative of segregation 1:1 The plants). ilsthuringiensis cillus eue rngnccliae ie(CMS-PET1) line cultivated transgenic a used We xeietlprotocol Experimental 1 eeain htwr eeoyosfrre- for heterozygous were that generations 1 1 ed rmalcosswr oldit a into pooled were crosses all from seeds fsrn,bt oaodtesra fthis of spread the avoid to but, offspring, rse o BC for Crosses il Experiments Field `B')fsdt osiuiepro- constitutive a to fused (``Bt'') .ÐBC ϳ 4 maati a 1999. May in apart km 245 ϳ 1 5wl eoye from genotypes wild 15 1 n idselnswere seedlings wild and 1 progeny ed eegerminated were seeds cry1Ac clgclApplications Ecological eefrom gene o.1,N.2 No. 13, Vol. N ϭ 740 Ba- 1 April 2003 FECUNDITY OF TRANSGENIC WILD SUNFLOWERS 281

types of the BC1 plants (i.e., with or without Bt, with acterize ¯owering times, we recorded the ®rst day of or without pollen) were not known at the time of plant- anthesis for each BC1 plant. ing, so we assumed that the locations of plants within Statistical analyses.ÐData on stem, head, or seed each treatment group were random. damage, number of heads per plant, number of seeds Few plants died, and the Colorado plants grew to be per plant, and days to ¯owering were analyzed using about twice as large as those in Nebraska. These ex- the GLM or CATMOD procedures of SAS (SAS 1990), periments were carried out under USDA-APHIS (An- for continuous and categorical data, respectively. Anal- imal and Plant Health Inspection Service) Permits 99- yses of each site separately included block and treat-

096-01N and 99-095-07N. All wild and BC1 seed heads ment (treatments were Btϩ/male sterile, BtϪ/male ster- were collected, and any sun¯ower seedlings that ap- ile, or BtϪ/male fertile). The block ϫ treatment inter- peared at the sites after tilling in 2000 and 2001 were action was never signi®cant (P Ͼ 0.50 in all cases), destroyed. and was dropped from the analyses reported here. The Identi®cation of transgenic plants.ÐPrior to ¯ow- analyses of both sites together included site (Nebraska ering, we collected two 7-mm-diameter leaf disks from or Colorado), block(site), treatment, and the site ϫ each BC1 plant to determine which plants were trans- treatment interaction. Block and site, and interactions genic. Plants that lacked a PCR-based marker for the involving these effects, were considered random ef- transgene construct and did not produce Bt toxin (de- fects, and signi®cance tests were constructed accord- termined by enzyme-linked immunosorbent assays ingly. Although the site effects in the combined anal- [ELISAs]) were designated as ``Bt negative'' (BtϪ), yses were generally signi®cant, in none of the analyses while those that had the marker and produced the toxin was the interaction between study site and treatment were designated ``Bt positive'' (Btϩ). We bagged the statistically signi®cant (P Ͼ 0.70 in all cases), indi- ®rst ¯ower head of each plant before it opened to check cating that the transgene had similar effects on insect for male sterility. Bt-positive plants that produced pol- damage and plant fecundity at the two sites. To test for the effect of the transgene on damage and fecundity,

len were destroyed, and bags on other plants were re- Communications moved to allow pollination. Treatment groups were in each of these analyses we examined planned con- designated Bt positive and male sterile (Btϩ/male ster- trasts between BC1 plants that were Btϩ/male sterile ile), BtϪ/male sterile, and BtϪ/male fertile (N ϭ 60 and BtϪ/male sterile. Similarly, to test for the effect plants per treatment in Nebraska and 50 per treatment of male fertility we examined planned contrasts be- in Colorado). tween BC1 plants that were BtϪ/male sterile and BtϪ/ Herbivore damage and plant fecundity.ÐHerbivores male fertile. When necessary the response variables of wild sun¯ower produce several characteristic types were transformed (square or cube root, or log, de- of damage (Cummings et al. 1999, Pilson 2000). In pending on the variable) to meet assumptions of the Nebraska only we censused stem tunnel holes produced analyses. by ®rst-generation Suleima helianthana (on 1 July, pri- or to ¯owering) and numbers of ¯ower heads clipped Greenhouse experiment by the weevil Haplorhynchites aeneus. We did not The purpose of this experiment was to test for any quantify leaf damage, which was negligible at both inherent differences in the fecundity of transgenic ver- sites. Seed heads (i.e., in¯orescences with mature sus nontransgenic plants when grown in the absence seeds) were collected prior to seed dispersal and stored of herbivores. This experiment was not carried out in for later analyses. In Colorado, ϳ4% of the seeds were the ®eld because of the dif®culties of excluding insect lost prior to collection but the percentage lost did not herbivores and carrying out hand pollinations on every differ among experimental groups. For each plant, we ¯ower head over a period of 1±2 months. Although the recorded the proportion of heads with damage by Pla- greenhouse environment is very different from ®eld giomimicus spumosum, which leaves a characteristic conditions, we expected major genetic differences ``bald spot'' on the seed head. Each head was then among treatments would be detectable. In addition, we dissected and we recorded the number of good seeds, subjected the plants to nutrient and drought stress to as well as categorical estimates of seeds damaged by test for genotype-by-environment interactions. Isophrictis similiella, Smicronyx fulvus, S. sordidus We used a three-way factorial design to examine ef- (Nebraska only), Cochylis hospes, and Neolasioptera fects of the transgene, male sterility, growing condi- helianthi (Nebraska only). Total plant fecundity was tions, and interactions among these factors on lifetime calculated by adding the number of good seeds per head seed production. Because this experiment was done in for all heads on a plant. We also report the total number the greenhouse, the Btϩ/male fertile plants could be of in¯orescences per plant, which can in¯uence the retained. In November 1999, we grew ϳ80 BC1 plants plant's male reproductive success. Male reproductive in each of three growing conditions in a greenhouse success can also be affected by other characteristics, with high-intensity halogen lights in Columbus, Ohio. however, such as pollen production, pollen viability, Each plant was grown in a 30-cm-diameter pot ®lled pollinator preferences, and ¯owering times. To char- with standard potting soil. Control plants were watered Communications )bcuePRadEIAtsswr oeie in- sometimes were tests ELISA and (Fig. PCR plants stressed because for 2) lower we are male and sizes were Sample plants positive, sterile. which Bt determine to were anthers plants observed samples which experiments, disk ®eld leaf determine on the to methods supplement in ELISA and As to PCR soil. used nutrients we potting soil the any in receive those not needed as did weeks). daily but 12 re- watered for to were week plants per subjected twice Nutrient-stressed were or but (once way, wilting peated same were the plants in amount Water-stressed fertilized standard fertilizer. a slow-release with fertilized of and needed as daily damage² Lepidopteran trait ®tness or species Herbivore T 282 ln,the plant, nrnfre means. untransformed a nldda oait,the covariate, a as included was ABLE aaeb h u¯wrmoth, sun¯ower the by damage td sites. study ed e ln 0 8 .5 071814 2077 0.054 ࿣ 588 909 5.0 6.1 Notes: plant per Seeds plant per In¯orescences helianthi Neolasioptera sordidus Smicronyx fulvus Smicronyx aeneus Haplorhynchites spumosum Plagiomimicus helianthana Suleima Cochylis similiella Isophrictis ln fecundity Plant a enfrtema muto aaepat u o o h rqec fhaswt damage. with heads of frequency the for not but damage/plant, of amount mean the for seen was aaeb te insects other by Damage ntecmie aast hntenme fi¯rsecswsicue sacvraei nACV fsesper seeds of ANCOVA an in covariate a as included was in¯orescences of number the when set, data combined the In ramnsfloigANOVA; following treatments hs eiotrnhrioe pcaieo oeseiswti h genus the within species some on specialize herbivores lepidopteran These ² aaeo ahi¯rsec a unie sflos 0 follows: as quanti®ed was in¯orescence each on Damage ³ nus ohlshospes Cochylis annuus; ed aae.Frec ln h envleoe l htpatsi¯rsecswsue nAOA sn L procedure GLM using ANOVAs in used was in¯orescences plant's that 1990). all Institute over value (SAS mean SAS the of plant each For damaged. seeds u¯wrmoth)³ sun¯ower ed/auei¯rsec 7 152 176 in¯orescence Seeds/mature aeo nhsso rtin¯ores- ®rst of anthesis of Date e u¯wrse weevil)³ seed sun¯ower red a;ga u¯wrse weevil)³ seed sun¯ower gray dae; aae aeoia data) categorical damage; with stems proportion generation: (1st moth) bud sun¯ower idae)³ yia;snoe edmidge)³ seed sun¯ower myiidae; clipped) heads (proportion weevil) head-clipping sun¯ower dae; damage) with heads (proportion dae) annuus noecne ihmtr seeds mature with In¯orescences aefriepat a in®atymr aaeta aeseie ( male-steriles than damage more signi®cantly had plants Male-fertile § ec n.dy fe h ®rst the BC after days (no. cence .Efcso h ttaseeo h muto netdmg n ieiefcniyo BC of fecundity lifetime and damage insect of amount the on transgene Bt the of Effects 1. 1 e plant per ttesuystsi ersaadClrd,UA nlzdsprtl n together. and separately analyzed USA, Colorado, and Nebraska in sites study the at ) ln oee tec site) each at ¯owered plant o ersateewr 86 lnstetetadfrClrd,4±0pat e ramn.Dt hw are shown Data treatment. per plants 47±50 Colorado, for and plants/treatment 58±60 were there Nebraska For P s.(otiia;banded (Tortricidae; ssp. au o h lne otattsigteB fetws045.We h ubro ed e auein¯orescence mature per seeds of number the When 0.4350. was effect Bt the testing contrast planned the for value (Curculionidae; (Curculioni- P (Tortricidae; (Gelechi- ausrpre eeaefrpandcnrssbtenteBt the between contrasts planned for are here reported values and (Cecido- (Attelabi- (Noctui- uem helianthana Suleima NS ooooaelectellum Homoeosoma P indicates au o h lne otattsigteB fetws0.0106. was effect Bt the testing contrast planned the for value 1720.5 21.7 Bt .0 .7 .0 .0 .5 .4 0.001§ 0.04§ 0.056 0.007 0.006 0.074 0.007 .4 0.179 0.249 .4 0.034 0.040 .0 .5 .0 odt odt ´ ´´´ ´´´ data no data no 0.001 0.550 0.100 .3 .6 .01003019000§0.0001 0.0001§ 0.189 0.003 0.0001 0.465 0.037 .8 0.357 0.387 .7 0.077 0.072 .0 .1 .1§0020.113 0.082 0.011§ 0.110 0.008 sterile ϩ /male . 4.1 5.2 P Ͼ Nebraska Bt 0.10. .A NWE AL. ET SNOW A. A. sterile Ϫ a eeooial motn rppss ewr nbet census to unable were We pests. crop important economically be can /male Prlde,aohrse-edn rpps htwscmo tour at common was that pest crop seed-feeding another (Pyralidae), n ece ie htwr iia oor®eld-grown our to similar vigorously were BC grew that experiment sizes per this reached seeds and in and plants in¯orescences The of plant. numbers mature, counted were seeds that we When unlikely daily overlooked. it were the ¯orets made and any stigmas days, unpollinated Individual several for set. for checks seed receptive maximum are ensure stigmas on to hand basis by by daily pollinated identi®ed were a and easily stigmas pollina- exserted were hand their ¯orets supplement Unpollinated captive to tions. used cross-pollination, were facilitate bumblebees To conclusive. NS NS NS NS NS NS NS NS P ϭ 1 ed aae;1 damaged; seeds 0 lnsi Nebraska. in plants P Bt 05870050.036 0.045 8.7 12.4 10.5 17.8 15.0 16.0 Ͻ odt odt ´ ´´´ ´´´ ´´´ data no data ´´´ no data no data no odt odt ´ ´´´ ´´´ data no data no sterile .5 0.204 0.150 ϩ 1 218 212 .5.For 0.05). /male Helianthus Bt Colorado ϩ .spumosum P. sterile Ϫ ϭ ml trl n Bt and sterile /male /male ±0sesdmgd 2 damaged; seeds 1±30 n r otcmo on common most are and , 1 u¯wr ( sun¯owers nClrd hseffect this Colorado in SNS NS NS SNS NS NS NS NS NS P clgclApplications Ecological 0.024§ § Ϫ o.1,N.2 No. 13, Vol. ml sterile /male ohsites Both combined Helianthus 0.037 0.051 ϭϾ P ࿣ 30 H. April 2003 FECUNDITY OF TRANSGENIC WILD SUNFLOWERS 283

FIG. 1. Effects of the Bt transgene and male sterility on relative amounts of sun¯ower seed damage by Cochylis moth Communications species and the number of good seeds (undamaged) per plant in Nebraska and Colorado (USA). Untransformed means and 1 SE are shown; N ϭ 58±60 plants in Nebraska, N ϭ 47±49 plants in Colorado. Levels of statistical signi®cance are based on planned contrasts between adjacent treatment means (see Table 1 for details, including methods for reporting damage levels). *P Ͻ 0.05; **P Ͻ 0.01; ²P ϭ 0.054; ³ P ϭ 0.077; NS, P Ͼ 0.10.

RESULTS AND DISCUSSION damage by the moth Plagiomimicus spumosum was more common in nontransgenic plants at both sites, but Field experiments this effect was only marginally signi®cant in Colorado Transgenic resistance to lepidopterans appears to be (P Ͻ 0.10, Table 1). These three lepidopteran seed a dominant trait because BC1 plants that were hemi- predators generally caused more damage on male-fer- zygous for the cry1Ac gene had very low levels of tile than on male-sterile plants (Table 1, Fig. 1), in- lepidopteran damage at both ®eld sites (Table 1, Fig. dicating that the bene®ts of Bt may be underestimated 1). Halfhill et al. (2002) also reported strong suppres- in male-sterile plants. The Bt toxin had no effect on sion of target herbivores in hemizygous BC1 progeny amounts of damage caused by four non-lepidopteran from wild Brassica rapa and cultivated B. napus with species (head-clipping weevils, red and gray seed wee- a cry1Ac transgene. Further experiments would be use- vils, and the sun¯ower seed midge; Table 1), as ex- ful to determine whether plants that are homozygous pected. Although some of these species are negatively for the transgene produce greater concentrations of Bt affected by competition with lepidopterans (M. J. Paul- toxin and experience less herbivory than hemizygous sen, and D. Pilson, unpublished data), they did not plants. cause more damage on Bt plants than on controls. Flowering times did not differ between transgenic Transgenic plants produced an average of 55% more plants and nontransgenic controls at either site (Table seeds per plant than nontransgenic controls in Nebraska 1), so temporal differences between treatments were and 14% more seeds per plant in Colorado (Table 1, not likely to affect exposure to herbivores. In Nebraska, Fig. 1). In separate ANOVAs for each site, this effect stem damage by ®rst-generation sun¯ower bud moth was signi®cant at P Ͻ 0.054 for Nebraska and was not larvae (Suleima helianthana) was about ®ve times more signi®cant for Colorado (P ϭ 0.262). This difference common in nontransgenic controls as compared to in signi®cance could be due to lower statistical power transgenic plants (Table 1; this type of damage was not for the Colorado data (which had smaller sample sizes), examined in Colorado). Likewise, larvae of Isophrictis differences in herbivore pressure at the two sites, and/ similiella and Cochylis spp. (C. hospes and C. arthuri) or the fact that nonlocal Nebraska genotypes were destroyed more seeds on control plants than on trans- grown in Colorado, whereas local genotypes were genic plants at both sites (Table 1, Fig. 1). Flower head grown in Nebraska. In any event, the same trend was Communications Ͻ trans- that ( found in¯orescences more we signi®cantly produced ANOVA, plants this genic trans- In the 1). of (Table effects it main gene so the examine signi®cant, to statistically appropriate not is was and included transgene site between Bt that interaction ANOVA the the An that showed 1). sites (Fig. both sites both at seen 284 ecs lhuhw i o tep ocomprehen- to damage, attempt internal not sample did sively in¯ores- we fewer Although with plants nontransgenic cences. smaller in damage in be stem resulting may and plants, This root in¯orescences. differ- greater to of to due due numbers largely the were in plant the ences per in seeds differences of Bt that number signi®cant suggesting the disappear, caused to This effect footnote). ANCOVA an 11 in 1: covariate (Table a of as number plant the per used in¯orescences we plants, transgenic of advantage damage plants. lepidopteran be- male-sterile greater than presumably to experienced 1), compared they Table as 1, cause (Fig. plants plants male-fertile male-sterile in signif- was lower production icantly seed plants, nontransgenic the ed ( seeds .Pusn esnlosrain.Frhroe sec- and Furthermore, Pilson ond-generation observation). (D. site personal study Paulsen, Nebraska M. the at common be P ayboi n boi ifrne ewe edand are ®eld between there conditions. differences greenhouse recognizing abiotic and while biotic many possibility, this ex- to amine experiment greenhouse Bt a her- lepidopteran performed the We of bivores. absence with the in associated occur can effects transgene useful whether is it determine Thus, pleiotropy. to or link- genes) crop (e.g., other by effects to position age or as construct such transgenic mechanisms, the other fe- by greater caused (e.g., are effects cundity) phenotypic whether clear not to below. discussed due as was mechanisms, observed unanticipated we other advantage fecundity the that eentafce ytetasee(.Plo n M. and Pilson and (D. study transgene Paulsen, our the J. by in affected mild not were were How- be symptoms transmission. also disease disease might ever, reduced plants with Bt in associated compounds damage 1998). (Agrawal herbivore seeds anti-herbivore Lower fewer the produce that to of plants showed caused radish production wild bene®t. of ®tness induced study a in- a afford of example, might levels For which lower with defense, associated duced was plants genic may this in¯orescences. and fewer with opening, plants before in abort result to them causing ofe ihntese Calt1983), (Charlet stem the within feed to onana oepoeteudryn asso h fecundity the of causes underlying the explore To .3)ta otasei otos(al ) Among 1). (Table controls nontransgenic than 0.037) Ͻ naysuyo igetasomto vn,i is it event, transformation single a of study any In ti osbeta eue ebvr edn ntrans- on feeding herbivore reduced that possible is It .5) oei¯rsecsta rdcdmature produced that in¯orescences more 0.051), P ed nteros(oes18) n ohcan both and 1985), (Rogers roots the on feeds Ͻ .3) n oeval ed e ln ( plant per seeds viable more and 0.036), nulse data unpublished .helianthana S. .Aohrpsiiiyis possibility Another ). .similiella I. tak oe buds, ¯ower attacks uom wom- Eucosma sknown is .A NWE AL. ET SNOW A. A. P Ͻ a ea iha 5 nraei edproduction, seed in a as increase high 55% as a bene®t possibly as and this high that as found be We can herbivores. prevalence the susceptible on of depending 2000). vary seasons, to and al. likely sites is et among bene®t fecundity Barry the 1997, of magnitude Peferoen The Bt (e.g., of studies plants with crop consistent is which sun¯owers, wild rmtcefcso ebvr n eudt nBC in fecundity and herbivory on effects dramatic abortion. embryo increased or fertilization decreased fewer to ovule due produced perhaps plants, plants male-fertile than male-sterile seeds treatment, that treatment. water-stressed In the in male-sterile plants of for except that plants, to similar was fecundity plants The the male-fertile of that herbivores. showed lepidopteran also experiment from greenhouse fe- was protection ®eld the the to that in due suggest plants transgenic results of our in advantage but cundity events study, of transgenic of type several range this wider and a employ conditions to growing preferable ben- be or would cost ®tness It inherent e®t. an with transgene associated the not that indicates was This fertile 2). male (Fig. were sterile male they or whether of condi- regardless and non-stressed tions, or greenhouse, wa- under nutrient-stressed, the grown were ter-stressed, in plants the plant whether of per regardless seeds or in¯orescences aefriiy( fertility male h edpouto fBC of production seed the ramn;Lwwtr1,2,1,ad2 lns n Low and per plants. plants; plants 12 24 27 and and 14, and 12, 11, 16, nutrientsÐ14, 27, 24, waterÐ17, ControlÐ19, (from Low sizes were: treatment; Sample right) plant). per to heads left ¯ower of number the of he odtos(oto,Lwwtr o uret)i a in 1 and nutrients) means Low are water, Data Low greenhouse. (Control, conditions three aeseiepat nte®l.Alkl esnfrthis for reason than likely A plant ®eld. per the in seeds plants more fewer male-sterile had and plants male-fertile damage that lepidopteran is ®nding estimate the higher on based The plants. male-sterile the to transgenic compared are plants male-fertile nontransgenic if fet natrewyAOAwr rwn odto ( condition growing were ANOVA three-way a in effects F .01 n h neato ewe rwn odto and condition growing between interaction the and 0.0001) hssuysosta igeB rngn a have can transgene Bt single a that shows study This of number the on effect no had transgene Bt The IG .Efcso h ttaseeadml trlt on sterility male and transgene Bt the of Effects 2. . P Ͻ rehueexperiment Greenhouse .02 o in®ati iia analysis similar a in signi®cant not 0.0042; Conclusions 1 doubling u¯wrpat rw under grown plants sun¯ower SE h nysigni®cant only The . fse production seed of clgclApplications Ecological o.1,N.2 No. 13, Vol. P 1 April 2003 FECUNDITY OF TRANSGENIC WILD SUNFLOWERS 285 discrepancy is that pollen-producing plants were more Next, we plan to determine whether transgenic wild attractive to lepidopteran herbivores (Delisle et al. sun¯owers that produce more seeds are likely to create 1989, Korman and Oseto 1989). larger populations, more populations, and/or more ex- The fecundity bene®t we observed suggests that se- tensive seed banks. Experimental manipulations of lo- lection favoring an increase in the frequency of a Bt cal population dynamics and models of metapopulation transgene has the potential to be quite strong. A more dynamics are needed to understand these processes. In complete measure of plant ®tness would include esti- addition, to gain a better understanding of how lepi- mates of male reproductive success, which was not dopteran herbivores affect wild sun¯ower populations, addressed in this study. It is possible that transgenic experiments similar to those we have reported here plants produced more pollen per anther or were more should be repeated over several study sites and seasons attractive to pollinators relative to control plants, or using advanced backcrossed generations. Fitness stud- that they were less successful for unknown reasons, but ies are an essential ®rst step in understanding the eco- we have no reason to expect the latter. If herbivores logical and evolutionary effects of gene ¯ow because cause less damage to wild genotypes than to BC1 sun- it is important to know the magnitude of presumed ¯owers, it is possible that the fecundity advantage as- fecundity effects of a given transgene. This knowledge, sociated with Bt would diminish with subsequent gen- together with an evaluation of the ecological effects of erations of backcrossing (Cummings et al. 1999). Even transgenes, is critical for biosafety risk assessments. if this occurs, we may have underestimated the fecundity advantage of Bt in this study, and we have ob- ACKNOWLEDGMENTS served higher levels of lepidopteran damage on wild We thank R. Meyer, D. Shanahan, G. Cole, G. Seiler, and plants in other years (Pilson 2000; D. Pilson and M. J. R. Peterson for logistical support and advice. Dow Agro- Sciences and Pioneer Hi-Bred provided the cultivated trans- Paulsen, unpublished data). Therefore, we expect that genic line, and C. Scelonge at Pioneer Hi-Bred performed the subsequent generations of Bt wild plants would pro- ELISA and PCR analyses. D. Adamski and R. J. Gagne as- duce more seeds per plant than nontransgenic individ- sisted with insect identi®cations. H. Alexander, J. Antonovics, uals in many locations and growing seasons. Because M. Cohen, P. Curtis, L. Gibbs, R. Linder, and M. Miriti pro- Communications the rate of spread of an allele is controlled largely by vided helpful comments on the manuscript. Funding was provided by Dow AgroSciences, Pioneer Hi-Bred, and grants its selective advantage, a Bt transgene could spread from the United States Department of Agriculture. The data quickly across wild sun¯ower populations. In addition, analyses, interpretation, and conclusions are solely that of the wild populations that occur near cultivated Bt sun¯ow- authors and not of Dow AgroSciences, LLC, or Pioneer Hi- er would be exposed to repeated episodes of gene ¯ow Bred International, Inc. from the crop. The rapid spread of Bt transgenes could LITERATURE CITED potentially lead to a reduction in population size of Agrawal, A. A. 1998. Induced responses to herbivory and susceptible, native lepidopterans that feed on wild sun- increased plant performance. Science 279:1201±1202. ¯ower. It is possible that specialist herbivores would Arias, D. M., and L. H. Rieseberg. 1994. Gene ¯ow between eventually evolve resistance to transgenic Bt toxins, cultivated and wild sun¯owers. Theoretical and Applied but this has not been reported yet in target pests of Genetics 89:655±660. transgenic Bt cotton or corn (e.g., Carriere et al. 2001). Barry, B. D., L. L. Darrah, D. L. Huckla, A. Q. Antonio, G. S. Smith, and M. H. O'Day. 2000. Performance of trans- In summary, our study is the ®rst to demonstrate that genic corn hybrids in Missouri for insect control and yield. a transgene derived from a crop has the potential to Journal of Economic Entomology 93:993±999. increase the ®tness of wild plants, and thus increase in Carriere, Y., C. Ellers-Kirk, Y. B. Liu, M. A. Sims, A. L. frequency in wild populations. The large effect of a Patin, T. J. Dennehy, and B. E. Tabashnik. 2001. Fitness costs and maternal effects associated with resistance to constitutively produced Bt toxin on lepidopteran dam- transgenic cotton in the pink bollworm (Lepidoptera: Ge- age, and consequently also on fecundity, is noteworthy lechiidae). Journal of Economic Entomology 94:1571± and suggests that lepidopteran herbivory within stems, 1576. roots, and ¯ower heads can be extensive. We are not Charlet, L. D. 1983. Insect stem fauna of native sun¯ower aware of other cases in which a gene derived from a species in western North Dakota. Environmental Ento- mology 12:1286±1288. crop, transgenic or otherwise, has been clearly shown Crawley, M. J. 1997. Plant ecology. Blackwell Science, Ox- to increase the ®tness of wild plants under realistic ®eld ford, UK. conditions. For transgenes to have important ecological Cummings, C. L., H. M. Alexander, and A. A. Snow. 1999. effects in wild populations, they must also alter inter- Increased pre-dispersal seed predation in sun¯ower crop- wild hybrids. Oecologia 121:330±338. actions between the wild plant and its biotic and abiotic Davis, P. M., and D. W. Onstad. 2000. Seed mixtures as a environment. A cry1Ac gene that becomes common in resistance management strategy for European corn borers wild sun¯ower populations will clearly have negative (Lepidoptera: Crambidae) infesting transgenic corn ex- effects on the suite of native lepidopteran herbivores pressing Cry1Ab protein. Journal of Economic Entomology that use wild sun¯owers as their primary host plant. 93:937±948. Delisle, J., J. N. McNeil, E. W. Underhill, and D. Barton. Moreover, other types of Bt genes could potentially 1989. Helianthus annuus pollen, an oviposition stimulant reduce populations of coleopteran and dipteran herbi- for the sun¯ower moth, Homoeosoma electellum. Ento- vores. mologia Experimentalis et Applicata 50:53±60. 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