Milkweed Matters: Monarch Butterfly (Lepidoptera: Nymphalidae) Survival and Development on Nine Midwestern Milkweed Species.

The population of monarch butterflies east of the Rocky Mountains has experienced a significant decline over the past 20 yr. In order to increase monarch numbers in the breeding range, habitat restoration that includes planting milkweed plants is essential. Milkweeds in the genus Asclepias and Cynanchum are the only host plants for larval monarch butterflies in North America, but larval performance and survival across nine milkweeds native to the Midwest is not well documented. We examined development and survival of monarchs from first-instar larval stages to adulthood on nine milkweed species native to Iowa. The milkweeds included Asclepias exaltata (poke milkweed) (Gentianales: Apocynaceae), Asclepias hirtella (tall green milkweed) (Gentianales: Apocynaceae), Asclepias incarnata (swamp milkweed) (Gentianales: Apocynaceae), Asclepias speciosa (showy milkweed) (Gentianales: Apocynaceae), Asclepias sullivantii (prairie milkweed) (Gentianales: Apocynaceae), Asclepias syriaca (common milkweed) (Gentianales: Apocynaceae), Asclepias tuberosa (butterfly milkweed) (Gentianales: Apocynaceae), Asclepias verticillata (whorled milkweed) (Gentianales: Apocynaceae), and Cynanchum laeve (honey vine milkweed) (Gentianales: Apocynaceae). In greenhouse experiments, fewer larvae that fed on Asclepias hirtella and Asclepias sullivantii reached adulthood compared with larvae that fed on the other milkweed species. Monarch pupal width and adult dry mass differed among milkweeds, but larval duration (days), pupal duration (days), pupal mass, pupal length, and adult wet mass were not significantly different. Both the absolute and relative adult lipids were different among milkweed treatments; these differences are not fully explained by differences in adult dry mass. Monarch butterflies can survive on all nine milkweed species, but the expected survival probability varied from 30 to 75% among the nine milkweed species.

Modeling the impact of cross-pollination and low toxin expression in corn kernels on adaptation of European corn borer (Lepidoptera: Crambidae) to transgenic insecticidal corn.

We used a mathematical model with processes reflecting larval mortality resulting from feeding on cross-pollinated ears or Bt ears of corn to analyze the risk of evolution of Cry-toxin resistance in Ostrinia nubilalis (Hübner). In the simulations, evolution of resistance was delayed equally well by both seed mixtures and blocks with the same proportion of refuge. Our results showed that Bt-pollen drift has little impact on the evolution of Bt resistance in O. nubilalis. However, low-toxin expression in ears of transgenic corn can reduce the durability of transgenic corn expressing single toxin, whereas durability of pyramided corn hybrids is not significantly reduced. The toxin-survival rate of heterozygous larvae in Bt-corn ears expressing one or two proteins has more impact on evolution of Bt resistance in O. nubilalis than the parameters related to larval movement to Bt ears or the toxin-survival rate of the homozygous susceptible larvae in Bt ears. Bt resistance evolves slower when toxin mortality is distributed across the first two larval stadia than when only the first instars are susceptible to Bt toxins. We suggest that stakeholders examine toxin-survival rates for insect pests and take into account that instars may feed on different parts of Bt corn.

Transcript analysis and comparative evaluation of shaker and slowmo gene homologues from the European corn borer, Ostrinia nubilalis.

The movement and dispersal of larval Lepidoptera impact their survival and distribution within the natural landscape. Homologues of the Drosophila behaviour-linked genes shaker (shkr) and slowmo (slmo) were identified from Ostrinia nubilalis (Lepidoptera: Crambidae). Onshkr was isolated as a 1610-nucleotide (nt) constitutively expressed transcript encoding a membrane-localized 469-amino-acid (aa) protein with a conserved tetramerization domain and the six-domain architecture necessary for the molecule to fold into an active K(+) channel. Three expressed splice variants of 682, 970 and 1604 nt were identified for the Onslmo gene, and encode predicted 141 and 228 aa proteins with a conserved protein of relevant evolutionary and lymphoid interest (PRELI) domain that may function in mitochondrial protein sorting and perinuclear protein localization. Onshkr and Onslmo protein sequences aligned within monophyletic lepidopteran groups.

A novel class of miniature inverted repeat transposable elements (MITEs) that contain hitchhiking (GTCY)(n) microsatellites.

The movement of miniature inverted repeat transposable elements (MITEs) modifies genome structure and function. We describe the microsatellite-associated interspersed nuclear element 2 (MINE-2), that integrates at consensus WTTTT target sites, creates dinucleotide TT target site duplications (TSDs), and forms predicted MITE-like secondary structures; a 5' subterminal inverted repeat (SIR; AGGGTTCCGTAG) that is partially complementary to a 5' inverted repeat (IR; ACGAAGCCCT) and 3'-SIRs (TTACGGAACCCT). A (GTCY)(n) microsatellite is hitchhiking downstream of conserved 5'MINE-2 secondary structures, causing flanking sequence similarity amongst mobile microsatellite loci. Transfection of insect cell lines indicates that MITE-like secondary structures are sufficient to mediate genome integration, and provides insight into the transposition mechanism used by MINE-2s.

Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers.

Transgenic maize engineered to express insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has become widely adopted in U.S. agriculture. In 2009, Bt maize was planted on more than 22.2 million hectares, constituting 63% of the U.S. crop. Using statistical analysis of per capita growth rate estimates, we found that areawide suppression of the primary pest Ostrinia nubilalis (European corn borer) is associated with Bt maize use. Cumulative benefits over 14 years are an estimated $3.2 billion for maize growers in Illinois, Minnesota, and Wisconsin, with more than $2.4 billion of this total accruing to non-Bt maize growers. Comparable estimates for Iowa and Nebraska are $3.6 billion in total, with $1.9 billion for non-Bt maize growers. These results affirm theoretical predictions of pest population suppression and highlight economic incentives for growers to maintain non-Bt maize refugia for sustainable insect resistance management.

Bacillus thuringiensis resistance influences European corn borer (Lepidoptera: Crambidae) larval behavior after exposure to Cry1Ab.

The behavior of pests targeted by Bacillus thuringiensis (Bt) crops has been recognized as an important factor to define resistance management plans. However, most data do not include the possible impact resistance may have on the behavior of pests. To examine whether resistance influences behavior of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), neonates after exposure to dietary Bt, the responses of Cry1Ab-resistant, -susceptible, and hybrid (F1) lines from two populations were compared in laboratory tests by using artificial diet mixed with 10-50% Cry1Ab or non-Bt isoline corn, Zea mays L., tissue. In no-choice tests, resistant (and usually hybrid) lines were less likely to be irritated (i.e., to move away after physical contact with diet containing Cry1Ab) than susceptible larvae after exposure to diets containing 10-50% Cry1Ab leaf tissue. Early in the no-choice tests (8 h), neonate O. nubilalis also were more likely to move off of diets that contained 10% non-Bt tissue compared with diets with 25 or 50% non-Bt tissue. In agreement with results from no-choice tests, choice tests with 10 or 25% tissue indicated that resistant (and sometimes hybrid) larvae were more likely than susceptible neonates to be found on diet with Cry1Ab. For choice tests, differences among lines seemed dependent on the amount of Cry1Ab tissue incorporated into diets. Results suggest differences in behavior are a result of reduced physiological susceptibility to Cry1Ab and are not an independent behavioral component to resistance.

Evaluating popcorn as a potential refuge of Ostrinia nubilalis (Lepidoptera: Crambidae).

Popcorn was evaluated in a series of experiments conducted over four growing seasons for its potential as a refuge for European corn borer, Ostrinia nubilalis (Hübner). Objectives of these studies were to determine whether more larvae were produced in popcorn than in field corn and to determine how popcorn influenced female oviposition and larval distribution in neighboring field corn. Two varieties of popcorn (M140, 105d and M3374Y, 118d), one mixture of popcorn (50% 105d and 50% 118d), and field corn (DK580, 108d) were evaluated. Number of egg masses, eggs per egg mass, and larvae were significantly higher in popcorn compared with field corn. Moth oviposition and larval distribution were evaluated using 105d popcorn embedded in several cornfields across Iowa. The row of field corn adjacent to popcorn had significantly more larvae compared with background field corn. In larger field experiments, O. nubilalis larval survival after overwintering was significantly different, with 2.2-18.7 times more O. nubilalis larvae surviving in popcorn than field corn. The potential use of popcorn as an O. nubilalis refuge for genetically engineered corn is considered.

Selection of nontarget arthropod taxa for field research on transgenic insecticidal crops: using empirical data and statistical power.

One of the possible adverse effects of transgenic insecticidal crops is the unintended decline in the abundance of nontarget arthropods. Field trials designed to evaluate potential nontarget effects can be more complex than expected because decisions to conduct field trials and the selection of taxa to include are not always guided by the results of laboratory tests. Also, recent studies emphasize the potential for indirect effects (adverse impacts to nontarget arthropods without feeding directly on plant tissues), which are difficult to predict because of interactions among nontarget arthropods, target pests, and transgenic crops. As a consequence, field studies may attempt to monitor expansive lists of arthropod taxa, making the design of such broad studies more difficult and reducing the likelihood of detecting any negative effects that might be present. To improve the taxonomic focus and statistical rigor of future studies, existing field data and corresponding power analysis may provide useful guidance. Analysis of control data from several nontarget field trials using repeated-measures designs suggests that while detection of small effects may require considerable increases in replication, there are taxa from different ecological roles that are sampled effectively using standard methods. The use of statistical power to guide selection of taxa for nontarget trials reflects scientists' inability to predict the complex interactions among arthropod taxa, particularly when laboratory trials fail to provide guidance on which groups are more likely to be affected. However, scientists still may exercise judgment, including taxa that are not included in or supported by power analyses.

Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs).

Genes expressed in lepidopteran midgut tissues are involved in digestion and Bacillus thuringiensis (Bt) toxin resistance traits. Five hundred and thirty five unique transcripts were annotated from 1745 high quality O. nubilalis larval midgut expressed sequence tags (ESTs). Full-length cDNA sequence of 12 putative serine proteinase genes and 3 partial O. nubilalis aminopeptidase N protein genes, apn1, apn3, and apn4, were obtained, and genes may have roles in plant feeding and Bt toxin resistance traits of Ostrinia larvae. The EST library was not normalized and insert frequencies reflect transcript levels under the initial treatment conditions and redundancy of inserts from highly expressed transcripts allowed prediction of putative single nucleotide polymorphisms (SNPs). Ten di-, tri- or tetranucleotide repeat unit microsatellite loci were identified, and minisatellite repeats were observed within the C-termini of two encoded serine proteinases. Molecular markers showed polymorphism at 28 SNP loci and one microsatellite locus, and Mendelian inheritance indicated that markers were applicable to genome mapping applications. This O. nubilalis larval midgut EST collection is a resource for gene discovery, expression information, and allelic variation for use in genetic marker development.

Influence of plant severing on movement of Ostrinia nubilalis larvae in Zea mays hybrid seed production fields.

Genetically engineered corn hybrids that contain a cry gene from the bacterium Bacillus thuringiensis Berliner (Bt) are gaining popularity for controlling the corn pest Ostrinia nubilalis (Hübner). Continuous use of Bt corn, however, could select for O. nubilalis that are resistant to this corn. Monitoring for insect resistance is important, because it could help maintain the Bt technology. A possible monitoring method is to collect larval insects in commercial drying bins after harvest from Bt seed production fields. A drawback to this method is that these collections may be contaminated by insects that moved as later instars from severed non-Bt male rows into the adjacent Bt female rows. These larvae have little to no exposure to Bt toxin, resulting in possible "false positives." The objectives of this study were to first find which combination of planting and severing dates produces the least number of larvae that move from non-Bt male plants to Bt female plants and to assess O. nubilalis larval movement from severed non-Bt male rows to Bt female rows. Field studies in 2002 and 2003 were designed to simulate a hybrid seed production field. Results suggest that movement of O. nubilalis larvae from male corn is minimized when corn is planted early and male plants are severed by 2 wk post-anthesis. This reduces the likelihood of false positives by reducing the number of susceptible larvae moving between Bt and non-Bt plants. Also, larvae moved to all four female rows that were adjacent to the severed rows, but there were significantly more larvae found in the closest row compared with the other three. These results could be used to develop a monitoring program to find O. nubilalis larvae with resistance to Bt corn in field populations of O. nubilalis.

Sequence variation in trypsin- and chymotrypsin-like cDNAs from the midgut of Ostrinia nubilalis: methods for allelic differentiation of candidate Bacillus thuringiensis resistance genes.

Midgut expressed alkaline serine proteases of Lepidoptera function in conversion of Bacillus thuringiensis (Bt) protoxin to active toxin, and reduced level of transcript T23 is associated with Ostrinia nubilalis resistance to Dipel Bt formulations. Three groups of trypsin- (OnT25, OnT23, and OnT3) and two chymotrypsin-like (OnC1 and OnC2) cDNAs were isolated from O. nubilalis midgut tissue. Intraspecific groupings are based on cDNA similarity and peptide phylogeny. Derived serine proteases showed a catalytic triad (His, Asp, and Ser; except transcript OnT23a), three substrate specificity-determining residues, and three paired disulphide bonds. RT-PCR indicated all transcripts are expressed in the midgut. Mendelian-inherited genomic markers for loci OnT23, OnT3 and OnC1 will be useful for association of alleles with bioassayed Bt toxin resistance phenotypes.

Growth, development, and survival of Nosema pyrausta-infected European corn borers (Lepidoptera: Crambidae) reared on meridic diet and Cry1Ab.

Transgenic corn, Zea mays L., hybrids expressing crystal protein endotoxin genes from Bacillus thuringiensis Berliner are an increasingly popular tactic for managing the European corn borer, Ostrinia nubilalis (Hübner), in North America. O. nubilalis populations also are often vulnerable to the ubiquitous entomopathogenic microsporidium Nosema pyrausta (Paillot). We examined the effect of feeding meridic diet incorporated with purified Cry1Ab on growth, development, and survival of Nosema-infected and uninfected neonate O. nubilalis. Infected larvae developed more slowly than uninfected larvae. Increasing the concentration of Cry1Ab in diet reduced larval development, and this effect was amplified by microsporidiosis. Infected larvae weighed significantly less than uninfected larvae. The relationship among Nosema infection, Cry1Ab concentration, and larval weight was fitted to an exponential function. The LC50 of infected larvae was one-third that of uninfected larvae, indicating that infected larvae are more vulnerable to toxin. This work has implications for resistance management of O. nubilalis and demonstrates that it is important to determine whether N. pyrausta is present when testing susceptibility of larvae to transgenic corn hybrids.

A Comparison of Maize Stalk Rot Occurrence in Bt and Non-Bt Hybrids.

Stalk rots, caused by a complex of fungal species, are among the most widespread and destructive diseases of maize. Larvae of the European corn borer (ECB) (Ostrinia nubilalis) promote stalk rot development by creating entry points for fungi, serving as vectors of pathogens, and causing physiological stress that may predispose plants to stalk decay. Field experiments were conducted in 1998, 1999, and 2000 to determine whether the use of transgenic Bt hybrids expressing insecticidal proteins would influence stalk rot symptoms (pith disintegration, pith discoloration, and lodging). Five hybrids representing different Bt types (or "Bt events") (176, BT11, MON810, DBT418, and CBH351) were paired with their near-isogenic, non-Bt counterparts and subjected to treatments of manual and natural infestation with ECB larvae. Manual infestation resulted in significantly more ECB tunneling than natural infestation in 1998 and 1999 and significantly more lodging in 1998. There were significant linear correlations between ECB injury and stalk rot symptoms in non-Bt hybrids in 1998 and 1999, but not in 2000. A standard foliar insecticide treatment for ECB did not significantly affect stalk rot symptoms. In 1998, Bt hybrids had significantly less ECB tunneling, stalk discoloration, pith disintegration, and lodging compared with non-Bt hybrids, but these effects depended upon the Bt event and the infestation treatment. Similar but less pronounced effects of Bt events were observed in 1999. The 2000 results were more variable; the amount of pith disintegration was significantly lower but discoloration was significantly higher in the BT11 hybrid compared with its non-Bt counterpart, and the amount of lodging was significantly higher in the event 176 hybrid compared with its non-Bt counterpart. The ratio of stalk strength to grain weight did not consistently differ between Bt and non-Bt hybrids. These results indicate that, although specific Bt events in some years may cause reductions in stalk rot, the overall effect of Bt transformation on stalk rot occurrence is highly variable.

Temporal and spatial overlap between monarch larvae and corn pollen.

To assess the likelihood that monarch larvae will be exposed to Bacillus thuringiensis (Bt) pollen, we studied milkweed and monarch densities in habitats which comprise much of the land available to breeding monarchs, e.g., cornfields, cornfield edges, other agricultural fields, and nonagricultural areas, in four regions of the monarch breeding range. We found that monarchs use milkweed in cornfields throughout their breeding season, and that per plant densities are as high or higher in agricultural habitats as in nonagricultural habitats. As a result of the prevalence of agricultural land, most of the monarchs produced in the upper Midwest are likely to originate in cornfields or other agricultural habitats. There was a greater temporal overlap between susceptible monarchs and corn anthesis in the northern than the southern part of the summer breeding range, because of earlier pollen shed in the south. The importance of agricultural habitats to monarch production suggests that, regardless of the impact of genetically modified crops, agricultural practices such as weed control and foliar insecticide use could have large impacts on monarch populations.

Assessing the impact of Cry1Ab-expressing corn pollen on monarch butterfly larvae in field studies.

Survival and growth of monarch larvae, Danaus plexippus (L.), after exposure to either Cry1Ab-expressing pollen from three Bacillus thuringiensis (Bt) corn (Zea mays L.) events differing in toxin expression or to the insecticide, lambda-cyhalothrin, were examined in field studies. First instars exposed to low doses ( approximately 22 grains per cm(2)) of event-176 pollen gained 18% less weight than those exposed to Bt11 or Mon810 pollen after a 5-day exposure period. Larvae exposed to 67 pollen grains per cm(2) on milkweed leaves from within an event-176 field exhibited 60% lower survivorship and 42% less weight gain compared with those exposed to leaves from outside the field. In contrast, Bt11 pollen had no effect on growth to adulthood or survival of first or third instars exposed for 5 days to approximately 55 and 97 pollen grains per cm(2), respectively. Similarly, no differences in larval survivorship were observed after a 4-day exposure period to leaves with 504-586 (within fields) or 18-22 (outside the field) pollen grains per cm(2) collected from Bt11 and non-Bt sweet-corn fields. However, survivorship and weight gain were drastically reduced in non-Bt fields treated with lambda-cyhalothrin. The effects of Bt11 and Mon810 pollen on the survivorship of larvae feeding 14 to 22 days on milkweeds in fields were negligible. Further studies should examine the lifetime and reproductive impact of Bt11 and Mon810 pollen on monarchs after long-term exposure to naturally deposited pollen.

Corn pollen deposition on milkweeds in and near cornfields.

The density of corn pollen on leaves of milkweed plants inside and outside of cornfields was measured in several studies from different localities. The purpose was to obtain a representative picture of naturally occurring pollen densities to provide a perspective for laboratory and field studies of monarch larvae feeding on milkweed leaves with Bt corn pollen. Pollen density was highest (average 170.6 grains per cm(2)) inside the cornfield and was progressively lower from the field edge outward, falling to 14.2 grains per cm(2) at 2 m. Inside the cornfield, and for each distance from the field edge, a frequency distribution is presented showing the proportion of leaf samples with different pollen densities. Inside cornfields, 95% of leaf samples had pollen densities below 600 grains per cm(2) and the highest pollen density observed was 1400 grains per cm(2), which occurred in a study with a rainless anthesis period. All other studies had rainfall events during the anthesis period. A single rain event can remove 54-86% of the pollen on leaves. Leaves on the upper portion of milkweed plants, where young monarch larvae tend to feed, had only 30-50% of the pollen density levels of middle leaves.

Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen.

Laboratory tests were conducted to establish the relative toxicity of Bacillus thuringiensis (Bt) toxins and pollen from Bt corn to monarch larvae. Toxins tested included Cry1Ab, Cry1Ac, Cry9C, and Cry1F. Three methods were used: (i) purified toxins incorporated into artificial diet, (ii) pollen collected from Bt corn hybrids applied directly to milkweed leaf discs, and (iii) Bt pollen contaminated with corn tassel material applied directly to milkweed leaf discs. Bioassays of purified Bt toxins indicate that Cry9C and Cry1F proteins are relatively nontoxic to monarch first instars, whereas first instars are sensitive to Cry1Ab and Cry1Ac proteins. Older instars were 12 to 23 times less susceptible to Cry1Ab toxin compared with first instars. Pollen bioassays suggest that pollen contaminants, an artifact of pollen processing, can dramatically influence larval survival and weight gains and produce spurious results. The only transgenic corn pollen that consistently affected monarch larvae was from Cry1Ab event 176 hybrids, currently <2% corn planted and for which re-registration has not been applied. Results from the other types of Bt corn suggest that pollen from the Cry1Ab (events Bt11 and Mon810) and Cry1F, and experimental Cry9C hybrids, will have no acute effects on monarch butterfly larvae in field settings.

Impact of Bt corn pollen on monarch butterfly populations: a risk assessment.

A collaborative research effort by scientists in several states and in Canada has produced information to develop a formal risk assessment of the impact of Bt corn on monarch butterfly (Danaus plexippus) populations. Information was sought on the acute toxic effects of Bt corn pollen and the degree to which monarch larvae would be exposed to toxic amounts of Bt pollen on its host plant, the common milkweed, Asclepias syriaca, found in and around cornfields. Expression of Cry proteins, the active toxicant found in Bt corn tissues, differed among hybrids, and especially so in the concentrations found in pollen of different events. In most commercial hybrids, Bt expression in pollen is low, and laboratory and field studies show no acute toxic effects at any pollen density that would be encountered in the field. Other factors mitigating exposure of larvae include the variable and limited overlap between pollen shed and larval activity periods, the fact that only a portion of the monarch population utilizes milkweed stands in and near cornfields, and the current adoption rate of Bt corn at 19% of North American corn-growing areas. This 2-year study suggests that the impact of Bt corn pollen from current commercial hybrids on monarch butterfly populations is negligible.

Dispersal of adult European corn borer (Lepidoptera: Crambidae) within and proximal to irrigated and non-irrigated corn.

The European corn borer, Ostrinia nubilalis (Hübner), causes economic damage to corn, Zea mays L., throughout the Corn Belt. Because this insect has become the primary target of Bacillus thuringiensis Berliner (Bt) transgenic corn, current efforts addressing the management of O. nubilalis resistance to Bt corn require information on adult European corn borer dispersal and factors affecting its dispersal. In 1998 we conducted mark-release-recapture, release-recapture, and caged-mating studies to directly measure and compare local dispersal patterns of O. nubilalis adults within and proximal to irrigated and non-irrigated cornfields. Releases of marked adults were made corresponding to the first and second flight of O. nubilalis in eastern Nebraska. Adult dispersal was significantly different between irrigated and non-irrigated cornfields. Released adults tended to remain in and near irrigated cornfields, but dispersed out of and away from non-irrigated cornfields. When released at the edge of the cornfield, neither male nor unmated female O. nubilalis displayed an initial tendency to move out of irrigated corn and into the mixed smooth bromegrass (Bromus inermis Leyss) and broadleaf-weed field edge. Mating efficiency in a late-season cornfield was not significantly different than in dense foxtail (Setaria spp.). Generally, we found that adult O. nubilalis dispersal may vary depending on variables such as action-site availability and agronomic practices and their interaction with O. nubilalis life history.

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.