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August 2000

Baseline Susceptibility of the Corn Earworm (: Noctuidae) to the Cry1Ab from thuringiensis

Blair D. Siegfried University of Nebraska-Lincoln, [email protected]

Terrence A. Spencer University of Nebraska-Lincoln, [email protected]

Jessica Nearman University of Nebraska-Lincoln

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Siegfried, Blair D.; Spencer, Terrence A.; and Nearman, Jessica, "Baseline Susceptibility of the Corn Earworm (Lepidoptera: Noctuidae) to the Cry1Ab Toxin from " (2000). Faculty Publications: Department of Entomology. 34. https://digitalcommons.unl.edu/entomologyfacpub/34

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Baseline Susceptibility of the Corn Earworm (Lepidoptera: Noctuidae) to the Cry1Ab Toxin from Bacillus thuringiensis

Blair D. Siegfried, Terrence Spencer, and Jessica Nearman

Journal of Economic Entomology Volume 93, Issue 4 (August 2000) pp. 1265–1268 DOI: 10.1603/0022-0493(2000)093[1265:BSOTCE]2.0.CO;2

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This article is the copyright property of the Entomological Society of America and may not be used for any commercial or other private purpose without specific written permission of the Entomological Society of America.

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT Baseline Susceptibility of the Corn Earworm (Lepidoptera: Noctuidae) to the Cry1Ab Toxin from Bacillus thuringiensis

BLAIR D. SIEGFRIED, TERRENCE SPENCER, AND JESSICA NEARMAN

Department of Entomology, 202 Plant Industry Building, University of Nebraska, Lincoln, NE 68583Ð0816

J. Econ. Entomol. 93(4): 1265Ð1268 (2000) ABSTRACT Susceptibility to Cry1Ab toxin from Bacillus thuringiensis (Bt) was determined for 12 Þeld populations of neonate corn earworm, (Boddie), from the United States. Earworm larvae were exposed to artiÞcial diet treated with increasing Bt concentrations, and mortality and growth inhibition were evaluated after 7 d. The range of variation in Bt susceptibility indicated by growth inhibition was very similar to that indicated by mortality. Although interpop- ulation variation in susceptibility to both was observed, the magnitude of the differences was small (less than or equal to Þvefold). These results suggest that the observed susceptibility differences reßect natural variation in Bt susceptibility among corn earworm populations rather than variation caused by prior exposure to selection pressures. Therefore, corn earworms apparently are susceptible to Bt across most of their geographic range.

KEY WORDS Baccilus thuringiensis susceptibility, geographic variability, bioassay

INCREASING PUBLIC CONCERNS about environmental haz- The insect is a generalist feeder on both weed and crop ards and widespread resistance in pest populations are plants, including , , and tomato (Metcalf threatening the continued effectiveness of conven- and Metcalf 1993). tional and should increase the use of in- Corn earworms are unable to overwinter in the secticidal products derived from Bacillus thuringiensis northern United States. However, in southern states, Berliner in the next decade (Tabashnik 1994). This is where populations are potentially exposed to both particularly true for transgenic Bt crops, which rep- transgenic cotton and corn in alternate generations, resent an important new tool for pest management there is a high level of concern regarding the increased (Fischhoff 1996). However, increased use of B. thu- potential for selection pressure and resistance devel- ringiensis in via transgenic plants and other opment (International Life Sciences Institute 1998). modes of application likely will increase the intensity This is especially true because of the shared binding of selection for Bt resistance in pest populations. site between the Cry1Ab and toxins that are The possibility of resistance highlights the need to expressed in Bt corn and cotton (Tabashnik 1994, develop and implement resistance management strat- Gould 1998), respectively. Furthermore, the expres- egies to prevent or delay the evolution of resistance to sion level in either crop plant is probably insufÞcient B. thuringiensis toxins (Hokkanen and Wearing 1994). to achieve a high dose for this pest species because of These strategies are dependent on the development of generally higher tolerance to the Bt toxins (Interna- effective resistance monitoring programs capable of tional Life Sciences Institute 1998). early detection of resistance that will allow implemen- Variation in susceptibility to the Cry1Ac has been tation of appropriate management decisions in a documented previously among geographically dis- timely manner (Dennehey 1987). The initial steps in tinct H. zea populations (Stone and Sims 1993, Sims et implementing such programs include development of al. 1996) and annual evaluations of susceptibility to appropriate bioassay techniques and establishment of Cry1Ac are conducted for insects collected from cot- baseline susceptibility data among populations across ton. The objective of the current study was to establish the geographic range of the target species. With this a baseline of susceptibility to Cry1Ab toxins from information, potential population susceptibility geographically distinct populations of H. zea collected changes in response to selection with B. thuringiensis from corn, emphasizing areas where there is likely to can be identiÞed (Fischhoff 1996). be exposure to both transgenic corn and cotton. The corn earworm, Helicoverpa zea (Boddie), is a serious pest of , Zea mays L., in the United States Materials and Methods (Mason et al. 1996) and a major target pest for control with transgenic Bt corn (Fischhoff 1996). This pest Insects. Populations of corn earworm were col- causes primary damage to corn plants by feeding on lected from nine states from both northern and south- ears, although whorl feeding does occur in early gen- ern areas of the corn belt and bioassayed to determine erations (Neunzig 1969, Keith and Witkowski 1996). susceptibility to the Cry1Ab Bt endotoxin (Table 1).

0022-0493/00/1265Ð1268$02.00/0 ᭧ 2000 Entomological Society of America 1266 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 93, no. 4

Table 1. Source description of European corn borer popula- tein used for bioassays consisted of puriÞed Cry1Ab tions used to establish baseline susceptibility to the Cry1Ab toxin obtained from the B. thuringiensis kurstaki strain from B. thuringiensis HD1Ð9, which produces only Cry1Ab (pro- Parental Generation Sample vided by the Novartis, Research Triangle Park, NC). Collection site Population Bioassayed size The crystal protein preparation was obtained by den- sity gradient centrifugation and contains Ϸ98% crystal Loxley, AL 200 F1 784 Salisbury, MD 199 F 748 protein as determined by phase contrast microscopy. 2 Ͻ Upper Marlboro, MD 152 F2 1,120 Individual neonate larvae ( 24 h after hatching) were Fayetteville, AK 150 F1ÐF2 896 placed in wells, and mortality and individual larval Aurora, NE 236 F1 784 Ͼ weight recorded 7 d later. Control treatments con- Plymouth, NC 100 F1 672 Leland, MS 117 F1ÐF2 672 sisted of wells treated with 0.1% Triton-X 100. When Stoneville, MS 196 F2 672 mortality was recorded, larvae that had not grown Loxley, AL 200 F1ÐF3 1,120 beyond Þrst instar and weighed Յ10 mg were consid- Washington County, MS (Bt) 175 F1ÐF2 748 Ͼ ered to be dead. As a result, the criterion for mortality Washington County, MS 200 F1ÐF3 896 Ͼ Rosemont, MN 100 F1 896 used in this study accounts for both severe growth a Laboratory Colony ÑF5 672 inhibition and death. Statistical Analyses. Bioassays were conducted in a Colony initiated in 1997 from Stratford, TX, Þeld collection. duplicate on three different dates and included at least Þve Bt concentrations that produced signiÞcant Populations were obtained by Novartis personnel as growth inhibition and mortality as deÞned previously. late instars from both Þeld corn and sweet corn and Because of difÞculties in obtaining enough larvae from sent to the University of Nebraska. Additional collec- a single generation, results from multiple generations tions were obtained as larvae or pupae from corn were combined for some populations. Individual larval plants by the Company and provided to weights were recorded after 7-d exposures and trans- Agripest Crop Consulting, Zebulon, NC, for rearing. formed to percentage of growth inhibition relative to Field-collected insects were reared to adults and al- the controls, and these data were analyzed by non- lowed to mate using standardized rearing techniques linear regression (SAS Institute 1988, Marc¸on et al. (Stone and Sims 1991). Eggs from the mated females 1999). Mortality data were analyzed by probit analysis were collected and allowed to hatch. Neonate larvae (Finney 1971) using POLO-PC (LeOra Software obtained from the Þeld-collected parents were used 1987). for subsequent bioassays or used to initiate another generation if insufÞcient numbers were available for Results and Discussion bioassay. A laboratory colony initiated from a 1997 Þeld collection was also bioassayed. Susceptibility data for H. zea populations exposed to Corn Earworm Bioassays. Bioassay of corn earworm puriÞed Cry1Ab protein are presented in Table 2. 2 neonates involved exposure to Bt solutions applied to LC50 values ranged from 70.3 ng/cm (laboratory col- 2 the surface of single wells of artiÞcial diet (Marc¸on et ony) to 221.3 ng/cm (Plymouth, NC). EC50 values al. 1999). Bioassays were performed in 128-well trays (i.e., concentrations that caused 50% growth inhibi- (each well 16 mm diameter by 16 mm high; CD In- tion of corn earworm larvae) ranged from 2.67 ng/cm2 ternational, Pitman, NJ). Dilutions of B. thuringiensis (Marlboro, MD) to 13.31 ng/cm2 (Plymouth, NC). were prepared in 0.1%Triton-X 100 to obtain uniform Differences between the most susceptible and most spreading of Bt solution on the diet surface. The pro- tolerant populations were three- and Þvefold at the

Table 2. Susceptibility of corn earworm neonates exposed to the Cry1Ab protein from B. thuringiensis as measured by growth inhibition and mortality

a,b Ϯ b b ␹2 Populations EC50 (95% CI) Slope SE LC50(95% CI) LC90 (95% CI) df Nebraska 8.72 (6.20Ð12.3) 3.29 Ϯ 0.43 157.0 (137.2Ð177.8) 384.2 (321.5Ð494.9) 1.90 5 Garden City, KS 6.95 (5.37Ð8.89) 2.45 Ϯ 0.22 131.6 (110.1Ð155.8) 444.3 (535.9Ð599.0) 2.73 5 Leland, MS 5.47 (3.63Ð8.08) 2.70 Ϯ 0.31 201.3 (153.9Ð250.5) 599.9 (442.3Ð1050.1) 7.11 5 Upper Marlboro, MD 5.83 (4.95Ð6.82) 2.63 Ϯ 0.21 149.5 (130.7Ð169.1) 389.3 (389.3Ð568.2) 2.29 5 Salisbury, MD 4.93 (3.55Ð6.55) 2.04 Ϯ 0.23 121.1 (72.6Ð173.9) 512.4 (333.3Ð1137.9) 9.03c 5 Plymouth, NC 13.31 (8.58Ð20.8) 3.51 Ϯ 0.47 221.3 (191.4Ð258.6) 512.7 (407.8Ð736.4) 4.94 5 Stoneville, MS 5.34 (4.97Ð5.75) 1.43 Ϯ 0.15 189.9 (147.9Ð252.9) 1490.0 (923.2Ð2990.1) 2.37 5 Rosemont, MN 3.00 (2.14Ð4.00) 3.17 Ϯ 0.31 157.2 (128.8Ð187.5) 850.6 (593.4Ð1542.1) 5.54 5 Loxley, AL 7.55 (5.91Ð9.62) 1.80 Ϯ 0.13 153.4 (118.6Ð201.0) 792.6 (522.7Ð1513.3) 11.58c 5 Washington County MS 5.89 (4.45Ð7.75) 2.13 Ϯ 0.22 129.9 (97.7Ð169.7) 517.9 (359.7Ð921.2) 7.21 5 Washington County, MS(Bt) 3.46 (2.43Ð4.71) 2.51 Ϯ 0.27 128.8 (83.9Ð174.8) 416.7 (290.0Ð826.6) 22.0c 5 Fayetteville, AK 3.04 (2.65Ð3.48) 1.84 Ϯ 0.19 96.24 (59.2Ð136.4) 480.4 (316.3Ð984.9) 8.22 5 Laboratory Colony 3.44 (2.30Ð5.01) 2.86 Ϯ 0.36 70.3 (49.5Ð91.6) 197.3 (146.6Ð317.8) 5.46 5

a Concentration of Cry1Ab that produces 50% growth inhibition relative to untreated controls. Calculated by nonlinear regression Þtted to a probit model. b ng CryIA(b)/cm2 of treated artiÞcial diet surface. c Chi-square signiÞcant (P Ͻ 0.05). August 2000 SIEGFRIED ET AL.: BASELINE SUSCEPTIBILITY TO BTINCORN EARWORM. 1267

in B. thuringiensis susceptibility may reßect nonge- netic variation or sampling error, because the popu- lations tested represented a small sample, taken at one time, of considerably larger multivoltine populations. Similar levels of variability were also observed among European corn borer susceptibility to Cry1Ab and Cry1Ac (Marc¸on et al. 1999) that included popula- tions of different voltine ecotypes and pheromone strains. The variation in baseline susceptibility to B. thurin- giensis toxins may reßect differences in vigor among parental populations (Rossiter et al. 1990), attributes that are the product of both genotype and the mater- Fig. 1. Comparisons of doseÐresponse curves for mor- nally determined nutritional status of the egg. Varia- tality and growth inhibition of corn earworm neonate larvae tion in susceptibility to B. thuringiensis toxins has been exposed to increasing concentrations of the Cry1Ab B. thu- reported among repeated bioassays against larvae ringiensis toxin. Each point represents the mean response from single strains of Colorado and di- from all 13 populations tested. amondback (Robertson et al. 1995) and Euro- pean corn borer (Marc¸on et al. 1999). Intrapopulation LC50 and EC50, respectively. Similar levels of suscep- variation in response to chemical or microbial insec- tibility were observed in collections that originated ticides is a common phenomenon when any bioassay from Bt corn and non-Bt hybrids (Washington is repeated (Robertson et al. 1995), and the extent of County, MS). both inter-and intrapopulation natural variation in Sigmoid curves were observed for both mortality susceptibility to a given should be investi- and growth inhibition responses (Fig. 1). The con- gated before biologically important changes can be vergence of the two curves noted at the upper end of identiÞed with any certainty. Ideally, this should be the response distribution is not unexpected given that done before the product is used commercially rather mortality included both extreme growth inhibition than after resistance is already widespread. Because and true mortality. Growth inhibition data (estab- the commercialization of both transgenic cotton and lished by weighing individual larvae) were more sen- corn preceded the data collection in the current study, sitive than mortality data for detecting the sublethal it is possible that signiÞcant selection may have al- effects of Cry1Ab. The differences between LC50 and ready occurred. However, based on the limited vari- EC50 values ranged between 18- and 36-fold, indicat- ation seen in this initial comparison, it appears unlikely ing that the toxin was having signiÞcant effects on corn that the small differences in response are the result of earworm growth and development at concentrations prior selection and are more likely to have resulted much lower than those that caused mortality. The from natural variability in susceptibility among geo- implications of such sublethal effects are unknown, graphically distinct corn earworm populations. but could be important to Þeld exposures and the Development of baseline susceptibility data repre- development of corn earworms on Bt transgenic corn sents the Þrst step toward the development of a mon- not expressing a high dose. If development is delayed itoring program designed to detect changes in suscep- in response to sublethal exposure, mating synchrony tibility that may result from repeated and prolonged between insects developing on Bt corn versus non- exposure to Bt toxins. These data also may provide transgenic hybrids might be disrupted. information that will allow development of diagnostic Apart from the increased sensitivity observed in the bioassays that would be more efÞcient in detection of growth inhibition data, there was no clear advantage resistant populations. However, it should be noted in using growth inhibition instead of mortality data. that corn earworm neonates are generally less sensi- The range of variation in susceptibility was similar tive to Cry1Ab than other Lepidopterans such as the between the two response criteria, and because mor- European corn borer (Marc¸on et al. 1999). As a result, tality data are easier and faster to collect, this response designation of diagnostic concentrations equivalent to should be more appropriate for large-scale suscepti- an LC99 may be difÞcult to achieve because of the bility monitoring efforts. large amounts of puriÞed crystal toxins that would be Although variation in susceptibility to Cry1Ab was required for large scale testing of Þeld populations. observed, the magnitude of the difference was small (i.e., less than Þvefold) and similar to other estimates of baseline variability among geographically distinct Acknowledgments populations of other insect species. Stone and Sims (1993) found considerable interpopulation variation The authors acknowledge the contributions of J. Van- Duyne (North Carolina State University), Don Parker (Mis- in B. thuringiensis suceptibility to Cry1Ac and formu- sissippi State University), and T. Horner and G. Dively (Uni- lated Dipel among U.S. populations of both corn ear- versity of Maryland) who generously provided their time in worm and cotton budworm (16- and fourfold, respec- collecting H. zea for inclusion in this study. A. Anderson tively). These data were reexamined by Sims et al. (Agripest Crop Consulting) conducted initial rearing of (1996), who suggested that interpopulation variation some Þeld populations. M. Shcarf and L. Meinke provided 1268 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 93, no. 4 critical review of an earlier draft of the manuscript. Funding Mason, C. E., M. E. Rice, D. D. Calvin, J. W. Van Duyn, W. B. for this work was provided by Novartis Seeds and Monsanto Showers, W. D. Hutchinson, J. F. Witkowski, R. A. Hig- Company. This is journal series number 12765 of the Uni- gins, D. W. Onstad, and G. P. Dively. 1996. Eur. corn versity of Nebraska Agricultural Experiment Station and con- borer ecology and management. 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