TRANSGENIC PLANTS AND INSECTS Assessing the Risk to Nontarget Organisms from Bt Corn Resistant to Corn Rootworms (Coleoptera: Chrysomelidae): Tier-I Testing with Orius insidiosus (Heteroptera: Anthocoridae)

1,2 3 1 1 JIAN J. DUAN, DEBRA TEIXEIRA, JOSEPH E. HUESING, AND CHANGJIAN JIANG

Environ. Entomol. 37(3): 838Ð844 (2008) ABSTRACT A 14-d continuous dietary exposure bioassay using nymphs of the insidious ßower bug, Orius insidiosus (Say) (Heteroptera: Anthocoridae), was conducted to assess nontarget impacts of genetically modiÞed corn event MON 863 expressing the Cry3Bb1 protein for management of corn rootworms, Diabrotica spp. (Coleoptera: Chrysomelidae). Nymphs of O. insidiosus were continuously fed a bee pollen diet inoculated with a maximum hazard exposure dose (930 ␮g/g of diet) of the Cry3Bb1 protein for 14 d. The Cry3Bb1 protein at a concentration of 930 ␮g/g of diet had no adverse effect on the survival and development (to adults) of O. insidiosus nymphs. In contrast, when O. insidiosus nymphs were fed bee pollen diet treated with a hazard dose of the protease inhibitor E64 (53 ␮g/g of diet) or the stomach poison potassium arsenate (8.9 ␮g/g of diet), all nymphs died before developing to adults. Furthermore, statistical power analysis indicated that at levels of 80% power and a 5% type I error rate, the study design would have been able to detect a minimum 30% reduction in survival of test nymphs and a 20% reduction in nymphal development to the adults relative to the buffer control groups. Based on the maximum level (93 ␮g/g) of the Cry3Bb1 protein expressed in MON 863 corn tissues including leaves, roots, and pollen, Þndings from this study indicate that corn hybrids containing the MON 863 event have a minimum 10 times safety factor for nymphs of O. insidiosus and thus pose minimal risk to this beneÞcial insect.

KEY WORDS Cry3Bb1, potassium arsenate, protease inhibitor, biotechnology, nontarget organisms

Genetically modiÞed corn event MON 863 (commer- potential hazard (toxicity) of expressed insecticidal cialized by Monsanto Company, St. Louis, MO) ex- proteins to diverse functional groups of nontarget or- presses a Bacillus thuringiensis (Bt) gene encoding the ganisms in the laboratory under worst-case exposure Cry3Bb1 protein that has insecticidal activity against scenarios (U.S. EPA 1998, 2007, Mendelsohn et al. coleopteran species such as corn rootworms (Di- 2003, Romeis et al. 2008). Currently, various Bt Cry abrotica spp.) and the Colorado potato beetle, Lepti- proteins including the Cry3Bb1 protein have been notarsa decemlineata (Say) (Donovan et al. 1992, U.S. assessed for hazard against several groups of nontarget EPA 2003, Vaughn et al. 2005). Recent Þeld trials have insects including honey bees (Apis mellifera L.), la- shown that MON 863 is highly efÞcacious against lar- dybird beetles [e.g., Hippodamia convergence (Guevin- vae of Diabrotica species and may provide corn grow- Meneville)], ground beetles [e.g., Poecilus chalcites ers an effective alternative to soil insecticides for (Say)], lacewings [Chrysoperla carnea (Stephens)], managing corn rootworms (Ward et al. 2004, springtails [e.g., Folsomia candida (Willem)], and par- Vaughn et al. 2005). Most recently, other transgenic asitic wasps [e.g., Nasonia vitripennis (Walker)] Bt corn cultivars that produce different coleopter- through direct dietary assays (Mendelsohn et al. 2003, an-active Bt proteins (e.g., event DAS 59122Ð7 ex- Ward et al. 2004, OÕCallaghan et al. 2005, Duan et al. pressing Cry34/35Ab1) also have been registered by 2006). Several small-scale Þeld studies also have been other biotechnology companies (Herman et al. 2002, conducted to assess the potential ecological impacts of U.S. EPA 2005). Bt corn cultivars on beneÞcial and pest nontarget An important step in the commercialization of ge- arthropods (Al-Deeb and Wilde 2003, Al-Deeb et al. netically modiÞed Bt crops is the evaluation of the 2004, Bhatti et al. 2005a, b). One group of nontarget organisms that has been 1 Monsanto CompanyÐRegulatory Environmental Technology largely absent from Tier-I laboratory hazard assess- Center/V2C, 800 North Lindbergh Blvd., St. Louis, MO 63167. ments is heteropteran predators such as various spe- 2 Corresponding author, current address: BeneÞcial Insect Intro- cies of pirate bugs in the genus Orius (Heteroptera: duction Research Unit, USDAÐARS, 501 South Chapel St., Newark, Anthocoridae). These are important biological con- DE 19713 (e-mail: [email protected]). 3 Springborn Smithers Laboratories, Massachusetts Research Cen- trol agents of numerous insect pests in many crop ter, Wareham, MA 02571. ecosystems including corn, cotton, soybean, fruit, and

0046-225X/08/0838Ð0844$04.00/0 ᭧ 2008 Entomological Society of America June 2008 DUAN ET AL.: ASSESSING THE RISK OF Bt CORN TO Orius insidiosus 839 vegetables (Lattin 1999, Rutledge and OÕNeil 2005, rently available on the concentration of Cry3Bb1 in Desneux et al. 2006). Because many heteropteran insect prey, the predominant food source for O. in- predators are also capable of using plant tissues (e.g., sidiosus (Harwood et al. 2005, 2007). pollen grains and young leaves) as supplementary Five pollen-based artiÞcial diet (Duan et al. 2007) food sources when their prey are scarce, there is a treatments were used, including (1) test substanceÑ greater potential for direct oral exposure of Cry pro- Cry3Bb1 at 930 ␮g/g of diet; (2) control sub- teins expressed in Bt plants to this group of predators stanceÑ10 mM sodium carbonate/bicarbonate buffer compared with obligate predators (Pilcher et al. 1997, (pH 10) at 0.248 ml/g of diet; (3) reference control Corey et al. 1998, Lundgren et al. 2008). The paucity substanceÑpotassium arsenate at 8.9 ␮g/g of diet; (4) of Tier-I laboratory studies to evaluate the potential positive control (protease inhibitor) E64 at 53 ␮g/g of hazard to heteropteran predators is mainly caused by diet; and (5) water assay controlÑi.e., pollen diet only a lack of artiÞcial diet-based test systems. This has led (consisting of Ϸ40% water and 60% pollen). The to reliance on costly higher-tier Þeld studies on Bt Cry3Bb1 protein was produced in a heterologous crops for the risk assessment for this group of preda- Escherichia coli fermentation system and prepared in tors (Pilcher et al. 1997, Ponsard et al. 2002, Bhatti et 10 mM sodium carbonate/bicarbonate buffer (the al. 2005b). Recently, Duan et al. (2007) developed a control substance) for storage and dosing. The continuous dietary exposure system that, based on the Cry3Bb1 solution was stored at Ϫ80ЊC before use in abundant and ubiquitous insidious ßower bug, Orius preparation of diet treatment. Potassium arsenate insidiosus (Say), fed a bee pollen diet incorporated (KH2AsO4) and E64 at the selected doses are known with the protease inhibitor E64 or an inorganic stom- to be effective in reducing the survival and develop- ach poison (potassium arsenate). The continuous di- ment of O. insidiosus nymphs (Duan et al. 2007) and etary exposure system was capable of detecting sig- were used as a reference or positive control, respec- niÞcant adverse effects of E64 and arsenate on the tively, to evaluate the efÞcacy of the dietary exposure survival and development of O. insidiosus nymphs in system for detecting toxicity or adverse effects of a dose-dependent manner (Duan et al. 2007). stomach toxins. A total of 25 nymphs (3Ð5 d old) were In this study, we evaluate the potential hazard of the tested for each of the diet treatments, and the test was Cry3Bb1 protein expressed in MON 863 corn to replicated three times. All treatment diets were pre- nymphs of O. insidiosus using the continuous dietary pared on the Þrst test day and stored at Ϫ20ЊC for use exposure system described by Duan et al. (2007). This during the testing period (14 d). study reports the use of a heteropteran predator as a The dietary exposure procedure, as previously de- surrogate nontarget organism for Tier-I testing of scribed (Duan et al. 2007), involved Þrst encapsulating plant-expressed insecticidal proteins for the purpose the pollen-based diet fortiÞed with the appropriate of ecological risk assessment for genetically modiÞed amount of test material in ParaÞlm domes (Ϸ50 ␮l Bt crops. per dome) and subsequently exposing O. insidiosus nymphs to the feeding dome (one dome per insect) in a 30-ml plastic cup with ventilated cover. Diet was Materials and Methods replenished every 24 h. Test arenas hosting the insects Insects. Nymphs of O. insidiosus (1Ð3 d old) were (one insect per arena) were placed in an environ- obtained from Koppert Biological Systems (Romulus, mental chamber with controlled temperature (25 Ϯ MI). This commercial insect colony had been reared 2ЊC), photoperiod (L:D 16: 8 h), and humidity (RH on natural diet for many generations. On receipt, O. 40Ð60%). Test insects were monitored daily for 14 d insidiosus nymphs were placed in a plastic culture to assess survival and development to adults. container (Ϸ500 ml), provided with washed string Bioactivity Levels of the Cry3Bb1 Protein in the beans as a water source, and freeze-killed Ephestia Test Diet Used for the O. insidiosus Feeding Assay. kuehniella Zeller (Mediterranean ßour moth; Lepi- Three diet samples were taken on the Þrst test day doptera: Pyralidae) eggs as food. A thin layer of shred- (day 0) from the Cry3Bb1 protein treatments at three ded wax paper also was provided at the bottom of the different locations (top, middle, and bottom) in the hatching container for reducing cannibalistic interac- diet container before dispensing for encapsulation in tions among hatching nymphs. The test nymphs were ParaÞlm domes. These diet samples were stored im- held under conditions similar to the testing conditions mediately in a Ϫ80ЊC freezer and analyzed later using for 2 d and observed to be healthy up to test initiation. the sensitive insect bioassay to determine levels of the Dietary Assay. Orius insidiosus nymphs were ex- Cry3Bb1 protein in these samples. An additional diet posed to a maximum Cry3Bb1 hazard dose of 930 ␮g/g sample (from the middle location of the container) of diet using the same dietary exposure procedures as also was taken on the Þrst test day, stored at Ϫ20ЊC for described in Duan et al. (2007). This maximum hazard Ϸ14 d, and analyzed using the same sensitive insect exposure dose was Ϸ10 times greater than the highest bioassay for assessing storage stability of Cry3Bb1 pro- expected environmental concentration of Cry3Bb1 in tein in the test diet. MON 863 corn plant tissue (Dudin et al. 2001, Vaughn The sensitive insect bioassay involved exposing lar- et al. 2005). The concentration of Cry3Bb1 in MON vae of the Colorado potato beetle, L. decemlineata 863 corn plant tissue was used as a conservative esti- (Say), to a series of doses of Cry3Bb1 protein incor- mate of the potential environmental exposure con- porated into an agar-based artiÞcial diet. Larvae of the centration because of the limited information cur- Colorado potato beetle are known to be highly sus- 840 ENVIRONMENTAL ENTOMOLOGY Vol. 37, no. 3

Fig. 1. Survivorship of O. insidiosus nymphs during the 14-d period of exposure to different diet treatments. Seventy-Þve nymphs (Ϸ3Ð5 d after hatching) were tested for each diet treatment. ceptible to the Cry three class of Bt proteins, including Monte Carlo simulation was based on a sample size of Cry3Bb1 (Donovan et al. 1992), and have been used 75 insects tested individually for each treatment to determine the level of the biological activity of (Cry3Bb1 protein versus negative buffer control) per Cry3Bb1 in different sample matrices (Duan et al. experiment and run 1,000 times. The mean minimum 2006). Aliquots of each of the test diet samples con- differences at the deÞned level of power and type I taining Cry3Bb1 protein were incorporated into the error rate were estimated from the 1,000 independent Colorado potato beetle artiÞcial diet to achieve a se- runs of the simulation of the experiment. ries of Þve concentrations of the protein (ranging from Probit analysis was used to determine the LC50 0.125 to 2.0 ␮g Cry3Bb1 protein /ml of Colorado po- values and corresponding 95% CIs for each of the test tato beetle diet). This series of Cry3Bb1 protein con- diet samples containing the Cry3Bb1 protein against centrations in the Colorado potato beetle diet was Colorado potato beetle larvae. The range of overlap of expected to elicit a range of doseÐresponses from 95% CIs of the LC50 was used to judge if there were Colorado potato beetle larvae, allowing an estimate of signiÞcant differences for biological activity between the LC50 value for each of the test diet samples. The each of the test Cry3Bb1 diet samples and the pure LC50 is deÞned as the lethal concentration of the Cry3Bb1 protein standard. protein (␮g/ml) required to kill 50% of the test Col- orado potato beetle larvae and was used as a measure Results of the biological activity of the Cry3Bb1 protein in the test diet samples. For comparison, the same dose series Dietary Assay with O. insidiosus. The mean per- of pure Cry3Bb1 protein standard also was tested. In centages of survival (Fig. 1) and development to adult- addition, the control diets containing the sodium car- hood (Fig. 2) for O. insidiosus nymphs at test termi- bonate/bicarbonate buffer were evaluated. Colorado nation were 80 and 92%, respectively, for the Cry3Bb1 potato beetle larvae were placed on these diets and treatment, 77 and 92% for the buffer control, and 79% allowed to feed for 7 d, after which the number of and 92% for the assay control (diet-only). As expected, survivors was recorded. no test nymphs survived the E64 positive control and Data Analysis. The survival and development of O. arsenate reference treatments. ␹2 tests (by excluding insidiosus nymphs among different diet treatments positive control [E64] and reference [arsenate] treat- were compared using likelihood ratio ␹2 procedures ment) detected no signiÞcant differences in either (PROC Freq; SAS Institute 2002Ð2003). A power anal- mean percent survival (␹2 ϭ 0.152, df ϭ 2, P ϭ 0.9267) ysis was performed using Monte Carlo simulation to or development to adulthood (␹2 Ͻ 0.0001, df ϭ 2, P ϭ estimate the minimum differences in percent survival 1.0000) among the Cry3Bb1, buffer control, and assay and development to adults that can be statistically control treatment groups for test O. insidiosus nymphs, detected at an 80% level of power and a preset type I whereas the positive control (E64) and reference (ar- error rate of 0.05 (SAS Institute 2002Ð2003). The senate) treatments (compared with assay control) June 2008 DUAN ET AL.: ASSESSING THE RISK OF Bt CORN TO Orius insidiosus 841

Fig. 2. Development to adults by O. insidiosus nymphs during the period of 14 d of exposure to different diet treatments. Seventy-Þve nymphs (Ϸ3Ð5 d after hatching) were tested for each concentration treatment. No nymphs developed to adults in the positive control (53 ␮g E64/g of the test diet) and reference (8.3 ␮g potassium arsenate/g of the test diet) treatments.

showed signiÞcant reductions in both survival (for conÞrming the homogeneity and stability of the pro- E64: ␹2 ϭ 97.25, df ϭ 1, P Ͻ 0.0001; for arsenate: ␹2 ϭ tein in the diet used in the O. insidiosus feeding assays. 123.314, df ϭ 1, P Ͻ 0.0001) and developmental success The buffer control substance (sodium carbonate/bi- (for E64: ␹2 ϭ 127.78, df ϭ 1, P Ͻ 0.0001; for arsenate: carbonate buffer) had no biological activity against ␹2 ϭ 165.16, df ϭ 1, P Ͻ 0.0001). Colorado potato beetle larvae. The statistical power analysis (Table 1) indicated that, at levels of 80% power and 5% type I error rate, Discussion the study design with 75 individuals of O. insidiosus nymphs per treatment would have been able to detect Results from this study showed that the Cry3Bb1 a minimum Ϸ30% reduction or 22% increase in O. protein at 930 ␮g/g of diet, which is equivalent to 10 insidiosus survival at 14 d of dietary exposure and a times the maximum concentration expressed in MON minimum of Ϸ20% reduction in development to adult- 863 corn tissues, has no apparent adverse effect on the hood. survival and development of O. insidiosus and thus is Cry3Bb1 Protein Analysis. Results from the sensi- expected to pose no risk to this nontarget beneÞcial tive insect assay showed that the test diet samples insect. The robustness of the methods used in the containing Cry3Bb1 protein were biologically active study is supported by the extended period (14 d) of against the susceptible insect (Colorado potato beetle continuous exposure, encompassing growth and de- ␮ larvae) with the LC50 ranging from 0.22 to 0.25 g velopment across one life stage boundary (Romeis et Cry3Bb1 protein /ml of Colorado potato beetle diet al. 2008), the high dietary Cry3Bb1 concentration con-

(Table 2). The 95% CIs of the LC50 values for the Þrmed using a sensitive insect (Colorado potato beetle Cry3Bb1 protein diet samples taken from different larva) bioassay, and the sensitivity of the assay design locations of the container and stored for 14 d at Ϫ20ЊC as demonstrated by the statistical power analysis (U.S. overlap with that of the Cry3Bb1 protein standard, EPA 2007).

Table 1. Power analyses of detectable differences in percent survival and completed development (to adults) of O. insidiosus nymphs between the negative (buffer) control and the other treatments with 80% power and 5% type I error rate

Percent differences of No. insects observed Observed means (O ) Treatment means (T ) m m detectable treatment Endpoints for each treatment from negative (buffer) to be detected with means relative to (n) control 80% power control meansa Survival (%) 75 77.3 Ͻ54 or Ͼ94% Ͻ30 or Ͼ22% Percent nymphs developed 75 92.0 Ͻ74% Ͻ20% to adults (%)

a ͓ Ϫ ͔ϫ Calculated as (Tm Om)/Om 100%. 842 ENVIRONMENTAL ENTOMOLOGY Vol. 37, no. 3

Table 2. Biological activity of the Cry3Bb1 protein against larvae of the susceptible insect (L. decemlineata) in the test diet samples used in O. insidiosus feeding assays

Storage 95% CI Dietary LC duration (d) Sampling location 50 treatment (␮g/ml of diet) Lower limit Upper limit (Ϫ20ЊC) (␮g/ml of diet) (␮g/ml of diet) Cry3Bb1 protein 0 Top 0.22 0.13 0.30 0 Middle 0.23 0.15 0.32 0 Bottom 0.25 0.17 0.34 14 Middle 0.24 0.16 0.33 0 Reference stardard 0.24 0.16 0.33

Currently, the potential negative effects of geneti- as pollen. The dietary-exposure assay reported in this cally modiÞed insecticidal products on nontarget or- study did not investigate the potential exposure of O. ganisms are evaluated in regulatory risk assessments insidiosus through trophic interactions with its prey using surrogate test organisms and a tiered approach feeding on transgenic plants or reared on artiÞcial diet (Mendelsohn et al. 2003, Romeis et al. 2006, 2008, U.S. fortiÞed with Bt Cry proteins (Zwahlen et al. 2000, EPA 2007). Because testing all taxa in an exposed Harwood et al. 2005, Gonzalez-Zamora et al. 2007, ecosystem or environment is impossible, appropriate Torres and Ruberson 2007). In the direct, dietary- surrogate species are selected and tested in tier-I lab- exposure assay reported here, the concentration of oratory studies to serve as indicators of risk to other Cry3Bb1 in the test diet was 10 times greater than the nontarget arthropods. Tier-I toxicity testing has been maximum level of the Cry3Bb1 protein expressed in a Þrst critical step in this assessment and is typically MON863 corn plant tissue, and thus likely ensures a conducted in the laboratory using high doses of pu- conservative margin of safety in concluding that there riÞed proteins to represent worst-case exposure sce- are no adverse effects on O. insidiosus even when a narios (Mendelsohn et al. 2003, Romeis et al. 2006, reasonable level of season long exposure is assumed. 2008). Data generated by tier-I testing often help sci- Data from recent small scale Þeld studies (Bhatti et al. entists determine whether higher-tier Þeld studies are 2005b) and laboratory testing with MON 863 corn warranted for speciÞc nontarget organism communi- pollen (J. Lundgren, personal communication) seemed ties. Under the current EPA regulatory risk framework to support a conclusion of no signiÞcant adverse effects for plant incorporated protectants (e.g., Cry proteins of the MON 863 corn event on heteropteran insects produced in Bt plants), if no adverse effects are de- including O. insidiosus. tected under the worst-case conditions of the tier-I toxicity tests, minimal risks are assumed, and no fur- ther data are required (U.S. EPA 1998, 2007). Acknowledgments Because of its high abundance and role in biological control in many crops, including corn, O. insidiosus We thank D. Carson, T. Nickson, T. Vaughn, T. K. Ball, S. may be an appropriate surrogate species for assessing Starter, R. Sidhue, and P. Eppard (Monsanto Company), and J. Lundgren (USDAÐARS) for providing comments on ear- potential impacts of genetically modiÞed crops on lier drafts of the manuscript. M. Paradise (Monsanto Com- predatory guilds of Heteroptera that have similar eco- pany) helped with the logistics of sending test and control logical and biological characteristics. In addition, sur- materials to Springborn Smithers Laboratory. prisingly little is known about the digestive physiology and toxicology of both beneÞcial and pest heterope- tran insects (Knop-Wright et al. 2006). Heteropteran References Cited insects use a complex feeding process that involves cell rupture, feeding with piercing/sucking mouth- Al-Deeb, M. A., and G. E. Wilde. 2003. Effect of Bt corn parts, and extraoral digestion through their salivary expressing the Cry3Bb1 toxin for corn rootworm control glands (Askari and Stern 1972, Cohen 1995, Habibi et on above ground nontarget arthropods. Environ. Ento- mol. 32: 1164Ð1170. al. 2008). The signiÞcant adverse effect of E64 (a Al-Deeb, M. A., G. E. Wilde, J. M. Blair, and T. C. 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Entomol. 34: 1325Ð1335. corporated protectants to nontarget organisms, but Bhatti, M. A., J. J. Duan, G. Head, C. Jiang, J. M. McKee, T. E. they are only one aspect of the risk-analysis frame- Nickson, C. L. Pilcher, and C. D. Pilcher. 2005b. Field work. In nature, O. insidiosus nymphs are omnivorous evaluation of the impact of transgenic Bt corn resistant to and feed on other insects as well as plant tissues such corn rootworm (Coleoptera: Chrysomelidae): effect on June 2008 DUAN ET AL.: ASSESSING THE RISK OF Bt CORN TO Orius insidiosus 843

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