Degradation of transgenic Bacillus thuringiensis proteins in corn tissue in response to post-harvest management practices.

Knowledge of the persistence of Cry proteins in transgenic corn residue after harvest is necessary to assess the ecological risk to nontarget organisms. The amount of protein remaining in crop residue declines over time by a combination of microbial decomposition and leaching, both influenced by temperature, precipitation, and the amount of residue-soil contact. Here, we investigated how long biologically active Cry proteins persist in SmartStax corn residue expressing Cry1A.105, Cry1F, Cry2Ab2, Cry3Bb1, and Cry34/35Ab1, when subjected to four post-harvest practices (chisel plow tillage, flail mowing, cover crop planting, and undisturbed residue). Protein activity in residue samples collected up to 25 weeks after harvest was measured by Ostrinia nubilalis feeding bioassays and cross validated with detection frequencies determined by ELISA. All corn residue remained above ground in the flail-mowed and undisturbed treatments, while the cover crop and chisel plow treatments left 88.3 and 39.6% of the residue remaining above ground, respectively. Cry proteins retained biological activity for as long as 24 weeks after harvest when residue was left above ground with less soil contact, typical of no-till corn systems. ELISA detections were positively correlated with results of the feeding bioassays, which revealed the presence of active proteins beyond the point of ELISA detection.

Sweet Corn Sentinel Monitoring for Lepidopteran Field-Evolved Resistance to Bt Toxins.

As part of an insect resistance management plan to preserve Bt transgenic technology, annual monitoring of target pests is mandated to detect susceptibility changes to Bt toxins. Currently Helicoverpa zea (Boddie) monitoring involves investigating unexpected injury in Bt crop fields and collecting larvae from non-Bt host plants for laboratory diet bioassays to determine mortality responses to diagnostic concentrations of Bt toxins. To date, this monitoring approach has not detected any significant change from the known range of baseline susceptibility to Bt toxins, yet practical field-evolved resistance in H. zea populations and numerous occurrences of unexpected injury occur in Bt crops. In this study, we implemented a network of 73 sentinel sweet corn trials, spanning 16 U.S. states and 4 Canadian provinces, for monitoring changes in H. zea susceptibility to Cry and Vip3A toxins by measuring differences in ear damage and larval infestations between isogenic pairs of non-Bt and Bt hybrids over three years. This approach can monitor susceptibility changes and regional differences in other ear-feeding lepidopteran pests. Temporal changes in the field efficacy of each toxin were evidenced by comparing our current results with earlier published studies, including baseline data for each Bt trait when first commercialized. Changes in amount of ear damage showed significant increases in H. zea resistance to Cry toxins and possibly lower susceptibility to Vip3a. Our findings demonstrate that the sentinel plot approach as an in-field screen can effectively monitor phenotypic resistance and document field-evolved resistance in target pest populations, improving resistance monitoring for Bt crops.

Seasonal Abundance and Phenology of Halyomorpha halys (Hemiptera: Pentatomidae) on Different Pepper Cultivars in the Mid-Atlantic (United States).

The brown marmorated stink bug, Halyomorpha halys (Stål), is an invasive stink bug (Hemiptera: Pentatomidae) introduced into the United States in the mid-1990s. Since its initial establishment, it has spread throughout the east coast as far south as Georgia, and as far north as New Hampshire. While information is available regarding H. halys behavior and life history in some crops, relatively little information is available for vegetables such as peppers. Key questions include understanding when H. halys enters pepper fields to feed and how best to predict infestations, what population levels create economic damage, and if peppers that vary in capsaicin levels also vary in susceptibility to attack. To answer these questions, replicated plots were set up across four mid-Atlantic states using three types of peppers: sweet bell, sweet banana, and hot chili. We found that there was no difference in the overall abundance of all life stages of H. halys on all pepper varieties tested. However, there were differences in bug density by site, but these differences did not translate to differences in the proportion of damaged fruit. The presence of adult H. halys is a better predictor of damage in banana peppers, whereas nymphs are a better predictor in bell pepper. In addition, across all sites, the presence of egg masses was low in pepper crops and densities of both adults and immatures tend to peak on pepper plants in early August. Altogether, this information can be used to help develop a pest management program in peppers that will reduce crop losses to this new devastating pest, while reducing the reliance on insecticides to manage this pest at the same time.

Comparison of Two Sampling Methods for Assessing Halyomorpha halys (Hemiptera: Pentatomidae) Numbers in Soybean Fields.

Sampling soybean fields for the brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), can be challenging. Both adults and nymphs have a "startle response" and drop to the ground with even the slightest disturbance. This behavior could reduce the effectiveness of the traditional sweep net and ground cloth sampling methods. In 2013 and 2014, in Virginia, Delaware, and Maryland, we evaluated a visual plant inspection method that consisted of counting the number of brown marmorated stink bug nymphs and adults seen on soybean plants in a 2-min inspection period while walking carefully between two rows. After a 30-min interval, which allowed the stink bugs to reposition in the canopy, the area was resampled using 15 sweeps with a 38-cm-diameter sweep net. In total, 76 soybean fields and 2,042 paired comparisons were used to determine a strong linear relationship between sampling methods (y = 0.984x + 0.4359, R2 = 0.6934, where y = brown marmorated stink bugs/2-min visual count and x = brown marmorated stink bugs/15 sweeps). An average visual count of 5.4 brown marmorated stink bugs in 2 min was estimated as being equivalent to the current economic threshold of 5 stink bugs per 15 sweeps. Visual inspection appears to be an effective method for assessing brown marmorated stink bug populations in soybeans.

Seasonal Abundance, Host Suitability, and Feeding Injury of the Brown Marmorated Stink Bug, Halyomorpha halys (Heteroptera: Penatomidae), in Selected Vegetables.

The invasive brown marmorated stink bug, Halyomorpha halys (Stål) (Heteroptera: Penatomidae), is a major pest of agricultural crops in the Mid-Atlantic States. Here we report the seasonal abundance, host preference, and injury potential of H. halys on tomato, bell pepper, green bean, sweet corn, eggplant, and okra in a small-scale, diversified vegetable farm setting. Life-stage densities, feeding injury, and crop phenology were monitored throughout the growing season in each crop. Populations consisted of both overwintered adults and F1 progeny and encompassed almost two generations over the growing season. H. halys preferred host plants with reproductive structures for feeding and was more abundant and capable to reproduce on vegetable crops that had extended periods of fruiting. Sweet corn, okra, and bell pepper had significant higher abundances of adults and nymphs compared with green bean, eggplant, and tomato. Results showed significant differences in the reproductive suitability of the host plants, as evident by the relative proportion of nymphal stages to adults. Eggplant, okra, and bell pepper were the most suitable host plants for reproduction and development, as evident by higher proportions of nymphs, with abundance trends indicating progressive development to adult eclosion. Crop loss potential due to H. halys feeding injury varied significantly among vegetable host plants. Sweet corn, okra, bell pepper, and tomato were very susceptible and experienced the highest injury rate per stink bug. The implications of these findings with respect to sampling and management of H. halys in vegetable production are discussed.

Does feeding by Halyomorpha halys (Hemiptera: Pentatomidae) reduce soybean seed quality and yield?

The nonnative brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae), has become an abundant pest of mid-Atlantic soybean since its introduction in the mid-1990s. Currently, there is little information indicating how this new pest should be managed in soybean or if economic thresholds developed for native stink bugs should be adjusted. In 2010 and 2011, field cage studies were conducted in Beltsville, MD, and Suffolk, VA, to evaluate H. halys injury to three different soybean reproductive development stages. Cages were infested for 2 wk using densities of zero, one, two, four, or eight stink bugs (fifth instars and adults) per 0.3 row-m. Cage plots were harvested, and subsamples were taken to determine pod losses and seed quality. Feeding injury to soybean caused by H. halys was similar to that of native stink bugs, as evidenced by seed destruction, punctures, and destroyed pods. Densities of four stink bugs per 0.3 row-m resulted in significant seed damage in three of four experiments. The full flowering (R2) soybean development stage was least affected by H. halys feeding. The full pod (R4) and the full seed (R6) stage were similarly sensitive to injury. There was no significant yield loss was associated with stink bug densities at either location, although there were significant differences among stages in two of four experiments. The data do not indicate that threshold densities for H. halys should be different than for the native stink bugs.

Variation in tolerance and resistance to the leafhopper Empoasca fabae (Hemiptera: Cicadellidae) among potato cultivars: implications for action thresholds.

The potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae), is an emerging pest of potato and insecticide applications to control this insect have increased in recent years. Based on field observations of leafhopper-crop dynamics, however, currently recommended action thresholds seem to be overly conservative. As a result, we initiated two experiments designed to quantify the impact of leafhoppers on potato yield, and determine how the magnitude of this effect changes among cultivars. In experiment 1, leafhoppers were manipulated (control versus insecticide-treated plots) on 17 potato varieties. In experiment 2, three cultivars (Superior, Atlantic, and Snowden) were planted representing early-, mid-, and late-season maturing lines, and six insecticide spray regimes were imposed (early-, late-, and full-season applications at high and low rates). In both experiments, leafhopper abundance, plant damage, and potato yield were measured. Overall, leafhoppers reduced yield in control plots by 15.7% relative to insecticide-treated plots. Leafhopper impact, however, varied among cultivars; a significant effect of leafhoppers on yield was detected in 6, 12, and 59% of cultivars tested in each of three trials. Of the 44 cases in which leafhoppers exceeded action thresholds, yield loss was only documented in 13 cases. Data from these experiments provide evidence that such variable effects ofleafhoppers on yield are explained by cultivar-specific resistance and tolerance traits. Our results suggest that potato growers can accept higher leafhopper densities than current thresholds recommend, particularly when cultivating resistant and/or tolerant varieties.

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.

Development, survival and fitness performance of Helicoverpa zea (Lepidoptera: Noctuidae) in MON810 Bt field corn.

Helicoverpa zea (Boddie) development, survival, and feeding injury in MON810 transgenic ears of field corn (Zea mays L.) expressing Bacillus thuringiensis variety kurstaki (Bt) Cry1Ab endotoxins were compared with non-Bt ears at four geographic locations over two growing seasons. Expression of Cry1Ab endotoxin resulted in overall reductions in the percentage of damaged ears by 33% and in the amount of kernels consumed by 60%. Bt-induced effects varied significantly among locations, partly because of the overall level and timing of H. zea infestations, condition of silk tissue at the time of egg hatch, and the possible effects of plant stress. Larvae feeding on Bt ears produced scattered, discontinuous patches of partially consumed kernels, which were arranged more linearly than the compact feeding patterns in non-Bt ears. The feeding patterns suggest that larvae in Bt ears are moving about sampling kernels more frequently than larvae in non-Bt ears. Because not all kernels express the same level of endotoxin, the spatial heterogeneity of toxin distribution within Bt ears may provide an opportunity for development of behavioral responses in H. zea to avoid toxin. MON810 corn suppressed the establishment and development of H. zea to late instars by at least 75%. This level of control is considered a moderate dose, which may increase the risk of resistance development in areas where MON810 corn is widely adopted and H. zea overwinters successfully. Sublethal effects of MON810 corn resulted in prolonged larval and prepupal development, smaller pupae, and reduced fecundity of H. zea. The moderate dose effects and the spatial heterogeneity of toxin distribution among kernels could increase the additive genetic variance for both physiological and behavioral resistance in H. zea populations. Implications of localized population suppression are discussed.

Effects of MON810 Bt field corn on adult emergence of Helicoverpa zea (Lepidoptera: Noctuidae).

A 3-yr study (1996-1998) was conducted to evaluate the effects of MON810 Bt corn on Helicoverpa zea (Boddie) emergence and to determine whether delayed larval development as a result of Bt intoxication results in higher levels of diapause induction and pupal mortality. In the 1997 study, there was no difference in prepupal mortality between corn types, although significantly more prepupae from Bt plots than from non-Bt plots died in emergence buckets before constructing pupal chambers in 1998. In all years, significantly fewer moths emerged from prepupae collected from Bt plots, suggesting that effects of the expressed Cry1Ab extended to the prepupal and pupal stages. Late plantings of corn showed the greatest reductions in moth emergence from Bt corn because environmental conditions were more conducive to trigger diapause at the time H. zea was developing in these plantings. This was supported by a significantly greater proportion of diapausing pupae remaining in the ground in the late plantings of both Bt and non-Bt corn. For April and early May plantings, larval feeding on Bt corn delayed the time to pupation, although there was no significant difference in moth emergence between corn types for those larvae that successfully pupated. Although Bt expression had less impact on the proportion of moths emerging, the actual number of moths emerging from Bt corn was significantly reduced because fewer larvae reached pupation. Delays in adult emergence, along with significant reductions in adult emergence from MON810 Bt corn, should reduce the rates of colonization in soybean and other late host crops but may also result in asynchrony of mating between individuals emerging from Bt and non-Bt corn. This, in turn, may contribute to the evolution of resistance to Bt corn.

Effect of MON810 Bt field corn on Helicoverpa zea (Lepidoptera: Noctuidae) cannibalism and its implications to resistance development.

Pairs of Helicoverpa zea (Boddie) larvae reared on diet-incorporated MON810 transgenic leaf tissue of field corn (Zea mays L.) were observed in the laboratory to characterize effects of sublethal levels of Bacillus thuringiensis variety kurstaki (Bt) Cry1Ab endotoxins on cannibalistic behavior and mortality. Feeding on sublethal levels of Bt corn reduced the frequency of cannibalistic behaviors exhibited by H. zea when uneven instars were paired together. Exposure to the Bt endotoxin had no significant effect on when cannibalistic mortality occurred or the level of mortality as a result of cannibalism. Assuming that H. zea larvae reared on nonBt corn tissue behaved in a similar way that resistant larvae would if feeding on Bt tissue, sublethal effects of Cry1Ab intoxication may reduce the chances of successful cannibalism by susceptible larvae and thus play a disproportionate role in the survival of multiple ear infestations. Furthermore, cannibalistic encounters could result in partially resistant larvae feeding on nontoxic food, thus temporarily providing an escape from exposure to the Bt endotoxin. These behavior alterations could increase the selective differential between susceptible individuals and those carrying resistance genes.

Response of potato tuber yield to stem injury by European corn borer (Lepidoptera: Crambidae) in the Mid-Atlantic United States.

The response of potato, Solanum tuberosum L., tuber yield to stem injury by European corn borer, Ostrinia nubilalis (Hübner), larvae was investigated in the Mid-Atlantic region of the United States for 3 yr. This response was described for 'Superior', 'Atlantic', and 'Snowden' potato, which are early-season, midseason, and late-season maturing cultivars, respectively. To model the yield/injury relationship, a range of corn borer injury levels was established in the field by augmenting the natural infestation with varying densities of laboratory-reared larvae. Linear and nonlinear regression analyses (plateau and second-order polynomial models) were used to describe the relationship between yield of U.S. No. 1 grade tubers and the percentage of stems injured by corn borer larvae. The maturity of the cultivar did not affect the response of potato yield to stem injury. In nine of 14 experiments, potato tolerated high levels of corn borer injury (55-90% of stems injured) without yield loss, suggesting that control of corn borer may not be necessary. Yet, in one of five Superior tests, in two of four Atlantic tests and in two of five Snowden tests, corn borer injury significantly reduced yield. Of the five data sets in which corn borer injury reduced yield, the plateau model fit two data sets and the quadratic model fit one data set. In two of the three cases, these models accounted for nearly identical amounts of total variation in yield as that accounted for by the linear model. The linear model fit four of the five data sets, but the R2 values were low for three of the four tests (0.10, 0.18, and 0.31). The parameter or parameters that interact with corn borer injury to cause tuber yield reduction should be identified before economic injury levels and thresholds are developed.

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.

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.

Baseline susceptibility to imidacloprid and cross resistance patterns in Colorado potato beetle (Coleoptera: Chrysomelidae) populations.

During 1995-1998, we tested 134 geographically discrete populations of Colorado potato beetle, Leptinotarsa decemlineata (Say), from the United States, Canada, Germany, France, and Poland for susceptibility to imidacloprid. Neonates were assayed on potato-based agar diet incorporated with imidacloprid and exposed on filter paper to esfenvalerate, azinphosmethyl, and carbofuran to characterize cross-resistance. In all 4 yr, Long Island populations were the most tolerant to imidacloprid, with LC50s ranging up to 29 times higher than the most susceptible populations. Responses to imidacloprid did not change significantly on farms where populations were assayed over time, except for those from Long Island, which doubled in overall tolerance to imidacloprid since 1995. Much of this tolerance was already present before imidacloprid was used on Long Island. Correlative analysis of the populations tested over the 4 yr indicated positive cross-resistance patterns with esfenvalerate and azinphosmethyl. This response was probably caused by preexisting metabolic and excretion mechanisms selected by previous exposure. There was no significant pattern of cross-resistance with carbofuran or bensultap. Regression slopes were also significantly negatively correlated with LC50 values for imidacloprid, indicating higher heterogeneity, which could lead in further resistance development. We discuss the relative sensitivity of diet-incorporated assays with neonates compared with other bioassay studies. Based on a pooled group of susceptible populations tested in 1995, a baseline LC50 of 0.39 ppm and a discriminating concentration of 8 ppm were suggested to detect early stages of resistance in "suspect" populations. We also suggest application strategies for imidacloprid that reduce selection pressure.

Isolation of dieldrin resistance from field populations of Drosophila melanogaster (Diptera: Drosophilidae).

High levels (about 4,000-fold) of resistance to dieldrin were isolated by screening field-collected populations of Drosophila melanogaster (Meigen). The resistance was made homozygous following 2-4 generations of selection. A single, major gene mapping to the left arm of chromosome III was solely responsible for resistance. The implications of the recovery of resistant mutants from field populations of D. melanogaster are discussed.