Evaluation of dietary effects of transgenic corn pollen expressing Cry3Bb1 protein on a non-target ladybird beetle, Coleomegilla maculata

A transgenic corn event (MON 863) has been recently developed by Monsanto Company for control of corn rootworms, Diabrotica spp. (Coleoptera: Chrysomelidae). This transgenic corn event expresses the cry3Bb1 gene derived from Bacillus thuringiensis (Berliner), which encodes the insecticidal Cry3Bb1 protein for corn rootworm control. A continuous feeding study was conducted in the laboratory to evaluate the dietary effect of MON 863 pollen expressing the Cry3Bb1 protein on the survival, larval development, and reproductive capacity of the non-target species, Coleomegilla maculata DeGeer (Coleoptera: Coccinellidae). First instar C. maculata (less than 24 h old) and newly emerging adults (less than 72 h old) were fed individually on a diet mixture containing 50% of MON 863 pollen, non-transgenic (control) corn pollen, bee pollen (a component of normal rearing diet), or potassium arsenate-treated control corn pollen. In the larval tests, 96.7%, 90.0%, and 93.3% of C. maculata larvae successfully pupated and then emerged as adults when fed on MON 863 pollen, non-transgenic corn pollen, and bee pollen (normal rearing) diets, respectively. Among the larvae completing their development, there were no significant differences in the developmental time to pupation and adult emergence among the transgenic corn pollen, non-transgenic corn pollen, and bee pollen diet treatments. All larvae fed on arsenate treated corn pollen diet died as larvae. For tests with adults, 83.3%, 80.0%, and 100% of adult C. maculata survived for the 30 days of the test period when reared on diets containing 50% of MON 863 pollen, non-transgenic corn pollen, and bee pollen respectively. While the adult survival rate on MON 863 pollen diet was significantly less than that on the bee pollen diet, there was no significant difference between the MON 863 and non-transgenic corn pollen treatments. During the period of adult testing, an average of 77, 80, and 89 eggs per female were laid by females fed on the MON 863 pollen, control corn pollen, and bee pollen, respectively; no significant differences were detected in the number of eggs laid among these treatments. These results demonstrate that when offered at 50% by weight of the dietary component, transgenic corn (MON 863) pollen expressing Cry3Bb1 protein had no measurable negative effect on the survival and development of C. maculata larvae to pupation and adulthood nor any adverse effect on adult survival and reproductive capacity. Relevance of these findings to ecological impacts of transgenic Bt crops on non-target beneficial insects is discussed.     

Development of tier-I toxicity assays for Orius insidiosus (Heteroptera: Anthocoridae) for assessing the risk of plant-incorporated protectants to nontarget heteropterans

A 13-d continuous dietary exposure bioassay using nymphs of the insidious flower bug, Orius insidiosus (Say) (Heteroptera: Anthocoridae), was developed to assess the potential dietary effects of insecticidal substances that have little or no contact toxicity. The nymphs were fed a bee pollen diet treated with different concentrations of an inorganic stomach poison, potassium arsenate, and a cysteine protease inhibitor, E-64. The results showed that the test system was capable of detecting the dietary effects of both substances on the survival and development of O. insidiosus from the nymph to the adult stage in a dose-dependent manner. For the potassium arsenate treatments, approximately 25% of the nymphs survived and developed to the adult stage by 13 d of dietary exposure at 3.8 microg/g of diet, whereas no test nymphs survived to adulthood at or above 15 microg/g of diet. The assay time required for a 75% mortality response ranged from approximately 7 d at 30 microg/g of diet to 13 d at 3.8 microg/g of diet. For the E-64 treatments, no test insects survived to adulthood at any of the concentration tested (75-600 microg/g of diet) by 13 d of dietary exposure, and the assay time required for a 75% mortality response ranged from 5 to 11 d at dietary rates of 600 and 75 microg/g, respectively. The research presented here describes a robust test system that is useful for evaluating potential adverse effects (or toxicity) of plant-incorporated protectants on nontarget heteropteran predators such as O. insidiosus.     

Mortality impact of MON863 transgenic maize roots on western corn rootworm larvae in the field

Mortality of western corn rootworm (Diabrotica virgifera virgifera LeConte) due to feeding on MON863 transgenic maize (Zea mays L.) expressing the Cry3Bb1 protein was evaluated at three Missouri sites in both 2003 and 2004 and at one site each in South Dakota, Nebraska and Iowa in 2004. To do this, survivorship relative to survivorship on isoline maize (i.e. the same genetic background, but without Cry3Bb1) was evaluated. Comparisons were made using low (1650–2500 eggs/m) and high (3300–3500 eggs/m) western corn rootworm egg densities. Significantly fewer beetles were recovered from MON863 than from isoline maize. Emergence from MON863 as a percentage of viable eggs ranged from 0.02% to 0.10%, whereas percentage emergence from isoline maize ranged from 1.09% to 7.14%. Survivorship on MON863 relative to survivorship on isoline averaged 1.51% when averaged across all environments and both years, so mortality because of the Cry3Bb1 protein averaged 98.49%. The average time delay to 50% cumulative beetle emergence from MON863 was 18.3 days later than from isoline maize. Females comprised 56% and 71% of total beetles recovered from MON863 in 2003 and 2004, respectively. Results are discussed in relation to insect resistance management (IRM) of western corn rootworm.     

Response of Danaus plexippus to pollen of two new Bt corn events via laboratory bioassay

Laboratory bioassays were conducted to evaluate the response of first instar larvae of the monarch butterfly, Danaus plexippus L. (Lepidoptera: Danaidae), a non‐target species, to pollen from corn, Zea mays L. (Commelinales: Poaceae), from two new corn hybrids genetically modified to express different types of insecticidal proteins derived from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) (Bt). One hybrid expresses both Cry1Ab and Cry2Ab2 proteins (MON 810 × MON 84006), active against lepidopteran pests, and the other expresses Cry3Bb1 protein (MON 863), targeted against coleopteran pests. First instar larvae were placed on milkweed leaves (Asclepias syriaca L.) (Gentianales: Asclepiadaceae) dusted with doses of either Bt pollen or its nonexpressing (isoline) pollen counterpart ranging from 50 to 3200 grains cm^?2 of milkweed leaves, or no pollen at all. Larvae were exposed to pollen for 4 days, then moved to pollen‐free leaves and observed for another 6 days. Survival was observed after 2, 4, and 10 days. Weight gain was estimated after 4 and 10 days, leaf consumption after 2 and 4 days, and larval development after 10 days. Exposure to pollen of the Cry1Ab/Cry2Ab2‐Bt expressing hybrid reduced larval survival approximately 7.5–23.5% at the dose ranges tested relative to a no pollen control. Larval weight gain and consumption were reduced for larvae exposed to pollen of this hybrid and a small minority of larvae (3.1%) never developed past the third instar after 10 days of observation. Exposure to pollen of the Cry3Bb1‐Bt expressing hybrid had no negative effects on larval mortality, weight gain, consumption, or development relative to the consumption of Bt‐free corn pollen. The relevance of these findings to the risk that these Bt corn hybrids pose to monarch populations is discussed.     

Development and survival of Orius insidiosus (Say) nymphs on encapsulated bee pollen-based diet in a Tier-I toxicity assay

The insidious flower bug, Orius insidiosus (Say) (Heteroptera: Anthocoridae) is an important surrogate species for assessing potential effects of plant-incorporated protectants (PIPs) on nontarget heterotrophic predators. In this study, a continuous dietary exposure system was optimized by assessing the effect of diet composition and age on the survival and development of nymphs of O. insidiosus. Greater than 85% control survival and an acceptable rate of development from nymph hatching to adult was achieved using 5-d-old nymphs at test initiation and a bee pollen-based diet supplemented with 25% Ephestia eggs. There was an unacceptable level of mortality (>40%) and/or a significantly prolonged development time when nymphs were <5 d old at test initiation. When 5-d-old nymphs were fed a bee pollen diet containing 25% Ephestia eggs and 100 μg/g potassium arsenate, time-dependent mortality was observed with a median lethal time (LT50) of 4.4 d and 100% mortality was observed after 10 d of feeding, indicating the effectiveness of the test system to detect adverse effects by dietary exposure. It is recommended that well-defined 5-d-old nymphs and an encapsulated bee pollen-based diet containing 25% ground Ephestia eggs be used in a Tier-I dietary feeding exposure assay for detecting potential effects of PIPs on O. insidiosus nymphs.     

Consumption of Bt maize pollen expressing Cry1Ab or Cry3Bb1 does not harm adult green Lacewings, Chrysoperla carnea (Neuroptera: Chrysopidae)

Adults of the common green lacewing, Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), are prevalent pollen-consumers in maize fields. They are therefore exposed to insecticidal proteins expressed in the pollen of insect-resistant, genetically engineered maize varieties expressing Cry proteins derived from Bacillus thuringiensis (Bt). Laboratory experiments were conducted to evaluate the impact of Cry3Bb1 or Cry1Ab-expressing transgenic maize (MON 88017, Event Bt176) pollen on fitness parameters of adult C. carnea. Adults were fed pollen from Bt maize varieties or their corresponding near isolines together with sucrose solution for 28 days. Survival, pre-oviposition period, fecundity, fertility and dry weight were not different between Bt or non-Bt maize pollen treatments. In order to ensure that adults of C. carnea are not sensitive to the tested toxins independent from the plant background and to add certainty to the hazard assessment, adult C. carnea were fed with artificial diet containing purified Cry3Bb1 or Cry1Ab at about a 10 times higher concentration than in maize pollen. Artificial diet containing Galanthus nivalis agglutinin (GNA) was included as a positive control. No differences were found in any life-table parameter between Cry protein containing diet treatments and control diet. However, the pre-oviposition period, daily and total fecundity and dry weight of C. carnea were significantly negatively affected by GNA-feeding. In both feeding assays, the stability and bioactivity of Cry proteins in the food sources as well as the uptake by C. carnea was confirmed. These results show that adults of C. carnea are not affected by Bt maize pollen and are not sensitive to Cry1Ab and Cry3Bb1 at concentrations exceeding the levels in pollen. Consequently, Bt maize pollen consumption will pose a negligible risk to adult C. carnea.     

Selection for Cry3Bb1 resistance in a genetically diverse population of nondiapausing western corn rootworm (Coleoptera: Chrysomelidae)

Five short-diapause laboratory lines of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were selected for resistance to MON863, a variety of corn genetically modified with the Bacillus thuringiensis Berliner (Bt) transgene that expresses the Cry3Bb1 delta-endotoxin. Three of the selected lines were developed by incremental increase in the duration of exposure to MON863 over 11 generations (moderate selected lines). Two selected lines were developed from a control group by constant exposure to MON863 for at least 14 d posthatch over seven generations (intense selected lines). At the end of the experiment, survivorship, as measured by adult emergence, was approximately 4 times higher in each of the selected lines reared on MON863 compared with control lines. Estimates of realized heritabilities (h2) were 0.16 and 0.15 for the moderate and intense selected lines, respectively, and are consistent with h2 estimates reported previously from a variety of pest insects. These lines provide data necessary for evaluating the potential for Bt resistance within diabroticite beetles and will be useful for developing improved insect resistance management strategies.     

Minnesota field population of western corn rootworm (Coleoptera: Chrysomelidae) shows incomplete resistance to Cry34Ab1/Cry35Ab1 and Cry3Bb1

In the United States of America, the western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is commonly managed with transgenic corn (Zea mays L.) expressing insecticidal proteins from the bacteria Bacillus thuringiensis Berliner (Bt). Colonies of this pest have been selected in the laboratory on each commercially available transformation event and several resistant field populations have also been identified; some field populations are also resistant. In this study, progeny of a western corn rootworm population collected from a Minnesota corn field planted to SmartStax^® corn were evaluated for resistance to corn hybrids expressing Cry3Bb1 (event MON88017) or Cry34/35Ab1 (event DAS‐59122‐7) and to the individual constituent proteins in diet‐overlay bioassays. Results from these assays suggest that this population is resistant to Cry3Bb1 and is incompletely resistant to Cry34/35Ab1. In diet toxicity assays, larvae of the Minnesota (MN) population had resistance ratios of 4.71 and >13.22 for Cry34/35Ab1 and Cry3Bb1 proteins, respectively, compared with the control colonies. In all on‐plant assays, the relative survival of the MN population on the DAS‐59122‐7 and MON88017 hybrids was significantly greater than the control colonies. Larvae of the MN population had inhibited development when reared on DAS‐59122‐7 compared with larvae reared on the non‐Bt hybrid, indicating resistance was incomplete. Overall, these results document resistance to Cry3Bb1 and an incomplete resistance to Cry34/35Ab1 in a population of WCR from a SmartStax^® performance problem field.     

Bt-maize event MON?88017 expressing Cry3Bb1 does not cause harm to non-target organisms

This review paper explores whether the cultivation of the genetically modified Bt-maize transformation event MON?88017, expressing the insecticidal Cry3Bb1 protein against corn rootworms (Coleoptera: Chrysomelidae), causes adverse effects to non-target organisms (NTOs) and the ecological and anthropocentric functions they provide. Available data do not reveal adverse effects of Cry3Bb1 on various NTOs that are representative of potentially exposed taxonomic and functional groups, confirming that the insecticidal activity of the Cry3Bb1 protein is limited to species belonging to the coleopteran family of Chrysomelidae. The potential risk to non-target chrysomelid larvae ingesting maize MON?88017 pollen deposited on host plants is minimal, as their abundance in maize fields and the likelihood of encountering harmful amounts of pollen in and around maize MON?88017 fields are low. Non-target adult chrysomelids, which may occasionally feed on maize MON?88017 plants, are not expected to be affected due to the low activity of the Cry3Bb1 protein on adults. Impacts on NTOs caused by potential unintended changes in maize MON?88017 are not expected to occur, as no differences in composition, phenotypic characteristics and plant-NTO interactions were observed between maize MON?88017 and its near-isogenic line.     

Western corn rootworm (Coleoptera: Chrysomelidae) beetle emergence from weedy Cry3Bbl rootworm-resistant transgenic corn

Three greenhouse experiments were conducted to evaluate western corn rootworm, Diabrotica virgifera virgifera LeConte, beetle emergence from individual pots containing glyphosate-tolerant transgenic corn, Zea mays L., expressing the Cry3Bbl endotoxin from the soil bacterium Bacillus thuringiensis Berliner (MON863), nontransgenic glyphosate-tolerant isoline corn, grassy weeds (giant foxtail, Setariafaberi R.A.W. Herrm; and large crabgrass, Digitaria sanguinalis (L.) Scop.), and combinations thereof infested with 40 neonate larvae. In the first two experiments, pots with corn and weed combinations were treated with glyphosate 5 d after larval infestation to kill the weeds. The third experiment was similar to the first two except that untreated corn-weed combinations were added. In all three experiments beetle emergence varied significantly. Beetle recovery generally did not vary between the nontransgenic, nontransgenic + weeds, and MON863 + weeds. Significantly more beetles were recovered from MON863 + weeds than MON863 alone or weeds alone. Beetle emergence from MON863 + weeds was likely enhanced by larvae that initially survived on weeds before application of glyphosate. Preliminary data indicated that fecundity was highest from beetles reared on nontransgenic isoline corn and fewer eggs were laid by beetles reared on MON863 alone. Egg viability was generally lowest from beetles reared on MON863. The implications of these results in relation to insect resistant management are discussed.     

Effect of corn hybrids expressing the coleopteran-specific cry3Bb1 protein for corn rootworm control on aboveground insect predators

Field and laboratory studies were conducted to determine the effect of transgenic Bacillus thuringiensis (Bt) corn, Zea mays L. (YieldGard Rootworm), expressing the Cry3Bb1 protein on aboveground nontarget insect predators (minute pirate bug, ladybird beetles, and carabids). Visual counts of adult and immature Orius insidiosus (Say), Coleomegilla maculata (DeGeer), Hippodamia convergens Gurin-Meneville, and Scymnus spp. occurring in Bt corn and its non-Bt isoline were made at Manhattan, KS, in 2002 and at Manhattan and Scandia, KS, in 2003. No significant differences were found between the Bt corn and non-Bt isoline plots in the abundance (number per plant) of O. insidiosus, C. maculata, H. convergens, and Scymnus spp. Field predation on Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) egg masses was also observed during the silking stage of corn at Manhattan and Scandia in 2003. No significant differences were observed among treatments in predation rate for predators with chewing versus sucking mouthparts. Two laboratory studies determined the effect of Cry3Bb1 protein expressed in Bt corn pollen on C. maculata and carabids. The larvae of C. maculata were reared on Bt pollen, non-Bt pollen, or greenbugs, Schizaphis graminum (Rondani). The duration of larval and pupal stages, developmental time from egg hatch to adult emergence, percentage of survival, and elytra length were compared among treatments. There were no significant differences in developmental time of larvae fed pollen or greenbugs during their first two instars. However, significantly prolonged development of the third (1 d) and fourth instars (2 d) was observed for larvae fed greenbugs only. Total time for larval development was significantly longer for larvae that fed on greenbugs versus larvae fed on pollen. No significant differences were observed among treatments in the percentage of larvae that pupated or pupal stage duration. Larvae that fed on greenbugs had higher pupal and adult weights compared with pollen-fed larvae. However, pupal and adult weights did not vary between the Bt and non-Bt pollen treatments. No significant differences occurred in longevity and elytra length of beetles among all treatments. Two carabid species, Harpalus caliginosus F. and Harpalus pensylvanicus DeGeer, were reared on moistened dog food sprinkled with Bt or non-Bt corn pollen. No significant differences in mortality of H. caliginosus and H. pensylvanicus were detected among any of the treatments. There was no significant effect of Bt pollen on fecundity and egg viability of H. caliginosus. Our studies showed that YieldGard Rootworm had no effect on the selected coleopteran predators; therefore, this Bt corn hybrid could be used in an integrated pest management system.     

The web-building spider Theridion impressum (Araneae: Theridiidae) is not adversely affected by Bt maize resistant to corn rootworms

The growth of genetically engineered maize that produces the insecticidal protein Cry3Bb1 from Bacillus thuringiensis ( Bt ) is an effective method to control corn rootworms ( Diabrotica spp.), which are threatening maize production in North America and Europe. In this study, the risk of Cry3Bb1-expressing maize for the predatory spider Theridion impressum , a common species in European maize fields, was assessed. Quantification of Cry3Bb1 in potential prey species collected in Bt maize plots and prey spectrum analysis revealed that T. impressum ingests Cry3Bb1 in the field. Exposure to the Bt protein, however, was highly variable because some potential prey species, such as phloem-feeding herbivores and predators, contained little or no Cry3Bb1, whereas leaf-feeding herbivores contained high concentrations. Adult and juvenile T. impressum spiders were fed with Cry3Bb1-containing food (prey or maize pollen) for 8 weeks in the laboratory to examine the toxicity of the Bt protein. No differences in mortality, weight development or offspring production were observed between spiders provided with food containing or not containing Cry3Bb1. Retrospective power analysis indicated that the bioassays were sufficiently sensitive to detect meaningful differences if present. Although Cry3Bb1 is ingested by the spider in the field, our data provide no evidence for toxicity. Consequently, the growth of corn rootworm-resistant Bt maize appears to pose no risk for T. impressum .     

Decomposition rates and residue-colonizing microbial communities of Bacillus thuringiensis insecticidal protein Cry3Bb-expressing (Bt) and non-Bt corn hybrids in the field

Despite the rapid adoption of crops expressing the insecticidal Cry protein(s) from Bacillus thuringiensis (Bt), public concern continues to mount over the potential environmental impacts. Reduced residue decomposition rates and increased tissue lignin concentrations reported for some Bt corn hybrids have been highlighted recently as they may influence soil carbon dynamics. We assessed the effects of MON863 Bt corn, producing the Cry3Bb protein against the corn rootworm complex, on these aspects and associated decomposer communities by terminal restriction fragment length polymorphism (T-RFLP) analysis. Litterbags containing cobs, roots, or stalks plus leaves from Bt and unmodified corn with (non-Bt+I) or without (non-Bt) insecticide applied were placed on the soil surface and at a 10-cm depth in field plots planted with these crop treatments. The litterbags were recovered and analyzed after 3.5, 15.5, and 25 months. No significant effect of treatment (Bt, non-Bt, and non-Bt+I) was observed on initial tissue lignin concentrations, litter decomposition rate, or bacterial decomposer communities. The effect of treatment on fungal decomposer communities was minor, with only 1 of 16 comparisons yielding separation by treatment. Environmental factors (litterbag recovery year, litterbag placement, and plot history) led to significant differences for most measured variables. Combined, these results indicate that the differences detected were driven primarily by environmental factors rather than by any differences between the corn hybrids or the use of tefluthrin. We conclude that the Cry3Bb corn tested in this study is unlikely to affect carbon residence time or turnover in soils receiving these crop residues.     

Assessment of fitness costs in Cry3Bb1‐resistant and susceptible western corn rootworm (Coleoptera: Chrysomelidae) laboratory colonies

Abstract Maize production in the United States is dominated by plants genetically modified with transgenes from Bacillus thuringiensis (Bt). Cry3Bb delta endotoxins expressed by Bt maize specifically target corn rootworms (genus Diabrotica) and have proven highly efficacious. However, development of resistance to Bt maize, especially among western corn rootworm (Diabrotica virgifera virgifera) populations, poses a significant threat to the future viability of this pest control biotechnology. The structured refuge insect resistance management (IRM) strategy implemented in the United States for Bt maize adopts a conservative approach to managing resistance by assuming no fitness costs of Bt resistance, even though these trade‐offs strongly influence the dynamics of Bt resistance within numerous agricultural pest species. To investigate the effects of Bt resistance on fitness components of western corn rootworm, we compared survivorship, fecundity and viability of five Bt‐resistant laboratory lines reared on MON863 (YieldGard Rootworm), a Bt maize product that expresses Cry3Bb1 delta endotoxin, and on its non‐transgenic isoline. Analysis of performance on the isoline maize demonstrated no fitness costs associated with Bt resistance. In fact, resistant lines emerged approximately 2–3 days earlier than control lines when reared on both MON863 and the isoline, indicating that selection for Bt resistance resulted in a general increase in the rate of larval development. In addition, resistant lines reared on Bt maize displayed higher fecundity than those reared on the isoline, which may have significant management implications. These data will be valuable for formulating improved IRM strategies for a principal agricultural pest of maize.     

Late-instar European corn borer (Lepidoptera: Crambidae) tunneling and survival in transgenic corn hybrids

Field studies were conducted in 1996 and 1997 to determine injury by and survival of late-instar European corn borer, Ostrinia nubilalis (Hübner), on genetically altered Bacillus thuringiensis Berliner corn, Zea mays L. Cry1Ab events 176, Bt11, MON810, and MON802; Cry1Ac event DBT418; and Cry9C event CBH351 were evaluated. Plants of each corn hybrid were manually infested with two third-, fourth-, or fifth-instar O. nubilalis. Larvae were held in proximity to the internode of the plant above the ear with a mesh sleeve. Larvae were put on the plants during corn developmental stages V8, V16, R1, R3, R4, R5, and R6. This study shows that not all B. thuringiensis hybrids provide the same protection against O. nubilalis injury. Hybrids with B. thuringiensis events Bt11, MON810, MON802, and CHB351 effectively protected the corn against tunneling by late-instar O. nubilalis. Event 176 was effective in controlling late-instar O. nubilalis during V12 and V16 corn developmental stages; however, significant tunneling occurred by fourth instars during R3 and R5. Event DBT418 was not effective in controlling late-instar O. nubilalis during corn vegetative or reproductive stages of development. Whether the B. thuringiensis hybrids satisfied high- and ultra-high-dose requirements is discussed.     

Use of transgenic plants to measure insect herbivore movement

Use of ingested transgenic corn tissue as a marker for measuring movement of adult Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae; western corn rootworm) was investigated. Laboratory observations of beetles feeding on corn foliage, pollen, silks, or soybean foliage provided background on feeding patterns. The interval between food consumption and its appearance in feces (gut passage time) ranged from 102.7 +/- 11 min for soybean foliage to 56.7 +/- 2.9 min for corn silks. In a laboratory assay, protein expression tests identified the presence of Cry3Bb1 protein inside 50% of adult D. virgifera for up to 16 h after they had last consumed Cry3Bb1 protein-expressing corn silks from 'YieldGard Rootworm' corn plants (Monsanto Co.). Cry3Bb1 protein could not be detected by 32 h postfeeding. The proportion of Cry3Bb1 protein-positive beetles declined linearly with increasing time since feeding on 'YieldGard Rootworm' tissue. Approximately 20% of adult D. virgifera collected near 'YieldGard Rootworm' corn plots tested positive for Cry3Bb1 protein, indicating 'YieldGard Rootworm' tissue consumption within the last 16-32 h. Based on a 16- to 32-h postfeeding detection interval for Cry3Bb1 protein and the distance between 'YieldGard Rootworm' sources and sites where Cry3Bb1-positive insects were collected, 85.3% of males and females moved < or = 4.6-9.1 m/d through R2-R3 stage corn. Among Cry3Bb1-positive adults that left corn and were captured in an adjacent soybean field, 86.4% of males and 93.1% of females moved < or = 4.6-9.1 m/d through soybean. Detection of transgenic plant tissues in mobile insect herbivores is a novel application of biotechnology to the study of insect movement.     

Tolerance of Bt corn (MON 810) to maize stem borer, Chilo partellus (Lepidoptera: Pyralidae)

Transgenic corn (MON 810), expressing the Bacillus thuringiensis (Bt) protein, Cry1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Chilo partellus. Bt corn (MON 810) provided effective protection against the stem borer even under a high level of larval infestation in the greenhouse. The observed tolerance is examined and discussed in the light of the susceptibility of C. partellus to the Cry1Ab protein in laboratory bioassays. The implications of the tissue concentrations of Cry1Ab in MON 810, and baseline susceptibility recorded in the current study, for insect-resistance management are discussed.     

DiPel-selected Ostrinia nubilalis larvae are not resistant to transgenic corn expressing Bacillus thuringiensis Cry1Ab

The survival of KS-SC DiPel-resistant and -susceptible European corn borer, Ostrinia nubilalis (Hübner), was evaluated on different tissues from corn, Zea mays L., hybrids, including a nontransgenic and two transgenic corn plants (events MON810 and Bt11) expressing high doses of Bacillus thuringiensis (Bt) Cry1Ab. The survival of Bt-resistant and -susceptible third instars was similar after a 5-d exposure to transgenic plant tissues. Survivors eventually died when returned to Bt corn tissues, but many were able to continue development when transferred to non-Bt corn tissues. Survival of resistant and susceptible larvae also was evaluated in bioassays with dilutions of leaf extracts from the three corn hybrids incorporated in an artificial diet. In these assays, survival was significantly higher for resistant O. nubilalis neonates at three of the five dilutions compared with the susceptible strain, but the resistance ratio was only 2.2- and 2.4-fold for MON810 and Bt11, respectively. The data demonstrate that Bt-resistant and unselected control O. nubilalis larvae were similar in susceptibility to MON810 and Bt11 event corn hybrids. Although we were unable to evaluate the Cry1Ab protein that larvae were exposed to in the transgenic tissue because of company restrictions, Cry1Ab protoxin produced in Escherichia coli was incubated with extracts from non-Bt corn leaves to simulate the in planta effect on the transgenic protein. Cry1Ab protoxin was hydrolyzed rapidly by enzymes in the corn extract into peptide fragments with molecular masses ranging from 132 to 74 kDa, and eventually 58 kDa. Overall, these data suggest that plant enzymes hydrolyze transgenic toxin to one that is functionally activated. Therefore, resistant insect populations with reduced proteinase activity do not seem to pose a threat to the efficacy of commercial MON810 and Bt11 corn hybrids.     

Sensitivity of the cereal leaf beetle Oulema melanopus (Coleoptera: Chrysomelidae) to Bt maize-expressed Cry3Bb1 and Cry1Ab

The sensitivity of the cereal leaf beetle, Oulema melanopus (Coleoptera: Chrysomelidae), to maize-expressed Bacillus thuringiensis (Bt) proteins was investigated in the present study. Neonate larvae of O. melanopus were caged on leaves of Cry3Bb1-expressing (MON88017) or Cry1Ab-expressing (MON810) Bt maize, the corresponding near-isolines, or two non-related, conventional maize varieties. Larval survival was reduced on Cry3Bb1-expressing, but not on Cry1Ab-expressing maize compared with conventional varieties. Differences among conventional varieties were also present. The amount of eaten leaf material, developmental time to prepupal stage, and prepupal weight did not differ between Bt maize varieties and their corresponding near-isolines. In an additional feeding study with newly emerged adults, survival and beetle weight did not differ when leaves of Cry3Bb1-expressing maize or the near-isoline were offered as food over 3 weeks. ELISA measurements revealed that larvae feeding on Bt maize contained rather high Cry3Bb1 or Cry1Ab levels, which were in the same order of magnitude as the leaves. In contrast, concentrations in feces were one order, and concentrations in prepupae and adults two orders of magnitude lower.     

Insecticidal activity of Cry3Bb1 expressed in Bt maize on larvae of the Colorado potato beetle, Leptinotarsa decemlineata

Environmental risk assessment for genetically modified crops producing insecticidal Cry proteins derived from Bacillus thuringiensis (Bt) includes the evaluation of adverse effects on non-target organisms. Although ELISA concentration measurements indicate the presence of Cry proteins, sensitive insect bioassays determine whether there is biological activity. The insecticidal activity of the coleopteran-active Cry3Bb1 expressed in different tissues of Bt maize, contained in maize-fed herbivores, and in spiked soil was measured in sensitive insect bioassays using larvae of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Biological activity was confirmed of Cry3Bb1 contained in pulverized Bt maize pollen, roots, leaves, silk, and Bt maize-fed spider mites and western corn rootworm adults. When test substances were incorporated into artificial diet at the same concentrations of Cry3Bb1 (measured by ELISA), maize pollen and leaf litter exhibited lower toxicity than fresh plant material and maize-fed arthropods. This suggests that nutritional quality of food and degradation of Cry proteins may influence toxicity to insects. When soil was spiked with Cry3Bb1, the Bt protein was highly adsorbed and retained its full biological activity. Because toxicity of Cry proteins contained in different matrices cannot always be determined from ELISA values alone, sensitive insect bioassays can improve hazard and exposure assessments in environmental risk assessment of Bt crops.