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.     

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.     

Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties

We investigated the use of maize pollen as food by adult Chrysoperla carnea under laboratory and field conditions. Exposure of the insects to insecticidal Cry proteins from Bacillus thuringiensis (Bt) contained in pollen of transgenic maize was also assessed. Female C. carnea were most abundant in a maize field when the majority of plants were flowering and fresh pollen was abundant. Field-collected females contained an average of approximately 5000 maize pollen grains in their gut at the peak of pollen shedding. Comparable numbers were found in females fed ad libitum maize pollen in the laboratory. Maize pollen is readily used by C. carnea adults. When provided with a carbohydrate source, it allowed the insects to reach their full reproductive potential. Maize pollen was digested mainly in the insect's mid- and hindgut. When Bt maize pollen passed though the gut of C. carnea, 61% of Cry1Ab (event Bt176) and 79% of Cry3Bb1 (event MON 88017) was digested. The results demonstrate that maize pollen is a suitable food source for C. carnea. Even though the pollen grains are not fully digested, the insects are exposed to transgenic insecticidal proteins that are contained in the pollen.     

Evaluation of adult emergence and larval root injury for Cry3Bb1‐resistant populations of the western corn rootworm

The transgenic maize (Zea mays L.) event MON 88017 produces the Bacillus thuringiensis Berliner (Bt) toxin Cry3Bb1 to provide protection from western corn rootworm (Diabrotica virgifera virgifera LeConte) larval feeding. In response to reports of reduced performance of Cry3Bb1‐expressing maize at two locations in Illinois, we conducted a two‐year experiment at these sites to characterize suspected resistance, as well as to evaluate root injury and adult emergence. Single‐plant bioassays were performed on larvae from each population that was suspected to be resistant. Results indicate that these populations had reduced mortality on Cry3Bb1‐expressing maize relative to susceptible control populations. No evidence of cross‐resistance between Cry3Bb1 and Cry34/35Ab1 was documented for the Cry3Bb1‐resistant populations. Field studies were conducted that included treatments with commercially available rootworm Bt hybrids and their corresponding non‐Bt near‐isolines. When compared with their near‐isolines, larval root injury and adult emergence were typically reduced for hybrids expressing Cry34/35Ab1 either alone or in a pyramid. In many instances, larval root injury and adult emergence were not significantly different for hybrids expressing mCry3A or Cry3Bb1 alone when compared with their non‐Bt near‐isolines. These findings suggest that Cry34/35Ab1‐expressing Bt maize may represent a valuable option for maize growers where Cry3Bb1 resistance is either confirmed or suspected. Consistent trends in adult size (head capsule width and dry mass) for individuals recovered from emergence cages were not detected during either year of this experiment. Because of the global importance of transgenic crops for managing insect pests, these results suggest that improved decision‐making for insect resistance management is needed to ensure the durability of Bt maize.     

Field-evolved resistance to Bt maize by western corn rootworm

Background

Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are planted on millions of hectares annually, reducing the use of conventional insecticides and suppressing pests. However, the evolution of resistance could cut short these benefits. A primary pest targeted by Bt maize in the United States is the western corn rootworm Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).

Methodology/Principal Findings

We report that fields identified by farmers as having severe rootworm feeding injury to Bt maize contained populations of western corn rootworm that displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays than did western corn rootworm from fields not associated with such feeding injury. In all cases, fields experiencing severe rootworm feeding contained Cry3Bb1 maize. Interviews with farmers indicated that Cry3Bb1 maize had been grown in those fields for at least three consecutive years. There was a significant positive correlation between the number of years Cry3Bb1 maize had been grown in a field and the survival of rootworm populations on Cry3Bb1 maize in bioassays. However, there was no significant correlation among populations for survival on Cry34/35Ab1 maize and Cry3Bb1 maize, suggesting a lack of cross resistance between these Bt toxins.

Conclusions/Significance

This is the first report of field-evolved resistance to a Bt toxin by the western corn rootworm and by any species of Coleoptera. Insufficient planting of refuges and non-recessive inheritance of resistance may have contributed to resistance. These results suggest that improvements in resistance management and a more integrated approach to the use of Bt crops may be necessary.     

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.     

Ingestion and excretion of two transgenic Bt corn varieties by slugs

The release of transgenic Bacillus thuringiensis (Bt) corn expressing various Cry endotoxins has raised concern that these endotoxins are disseminated in the food web and may adversely affect non-target beneficial organisms, such as predators and organisms of the decomposer food web. We therefore investigated in a laboratory study, whether the Cry1Ab and Cry3Bb1 protein from Bt corn could potentially be transferred to such organisms by measuring the Cry protein content in the two common agricultural slug pests Arion lusitanicus and Deroceras reticulatum and their feces. We measured Cry1Ab and Cry3Bb1 protein concentration in leaves, intestines, and feces of corn leaf-fed slugs using ELISA and determined how much of the ingested protein is excreted by the slugs. Cry3Bb1 concentration in leaves of DKC5143Bt corn was significantly higher than Cry1Ab concentration in leaves of N4640Bt corn. While slugs were feeding on corn leaves, the Cry3Bb1 and Cry1Ab proteins were found in intestines and feces of both slug species. Bt protein concentrations in intestines of Cry3Bb1 corn-fed slugs were in both slug species higher than in Cry1Ab corn fed slugs, whereas no differences between Cry3Bb1 and Cry1Ab protein in feces were found. After slugs had ceased feeding on Bt corn, Cry1Ab was detectable in fresh slug feces for a significantly longer time and often in higher amounts than the Cry3Bb1. Our results indicate that both Cry proteins are likely to be transferred to higher trophic levels and to the decomposer food web. Since different Bt proteins seem to vary in their degradation, they have different transfer probabilities. This should be considered in risk assessments for non-target arthropods.     

Greenhouse-Selected Resistance to Cry3Bb1-Producing Corn in Three Western Corn Rootworm Populations

Transgenic corn producing the Bacillus thuringiensis (Bt) toxin Cry3Bb1 has been useful for controlling western corn rootworm, Diabrotica virgifera virgifera LeConte, one of the most economically important crop pests in the United States. However, rapid evolution of resistance by this beetle to Bt corn producing Cry3Bb1 has been reported previously from the laboratory, greenhouse, and field. Here we selected in the greenhouse for resistance to Cry3Bb1 corn in three colonies of WCR derived from Kansas, Minnesota, and Wisconsin, respectively. Three generations of rearing on Cry3Bb1 corn significantly increased larval survival on Cry3Bb1 corn, resulting in similar survival in the greenhouse for selected colonies on Cry3Bb1 corn and isoline corn that does not produce Bt toxin. After four to seven generations of rearing on Cry3Bb1 corn, survival in the field on Cry3Bb1 corn relative to isoline corn more than doubled for selected colonies (72%) compared with control colonies (33%). For both selected and control colonies, survival in the field was significantly lower on Cry3Bb1 corn than on isoline corn. On isoline corn, most fitness components were similar for selected colonies and control colonies. However, fecundity was significantly lower for selected colonies than control colonies, indicating a fitness cost associated with resistance. The rapid evolution of resistance by western corn rootworm to Bt corn reported here and previously underlines the importance of effective resistance management for this pest.     

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.     

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 .     

Effect of Bt maize on the reproduction and development of saprophagous Diptera over multiple generations

A laboratory experiment was used to quantify the effects of Bt maize on Drosophila melanogaster and Megaselia scalaris, representatives of two saprophagous dipteran families (Drosophilidae, Phoridae). Freshly hatched larvae were reared on a diet containing decaying maize leaves. Two transgenic maize varieties, expressing Cry3Bb1 or Cry1Ab, and their corresponding isolines were tested. In an additional treatment, a solution of pure Cry1Ab was added to the maize diet. According to quantitative ELISA analyses, all Bt diets and all larvae feeding on Bt maize contained low concentrations of Cry proteins but Cry proteins were not detected in adults, thus, predators of the larvae are exposed to Cry proteins whereas predators of adult flies are not. Highest concentrations were in larvae feeding on a maize diet supplemented with a Cry1Ab protein solution. The developmental time and fertility (offspring/female) were measured over four generations for D. melanogaster and over three generations for M. scalaris. Only a few significant differences were found between transgenic and non-transgenic treatments but the differences were not consistent and did not indicate any negative effects of Bt proteins. We conclude that D. melanogaster and M. scalaris larvae are not affected in the long term when feeding and developing on decaying Cry1Ab and Cry3Bb1 maize leaves.     

A Challenge for the Seed Mixture Refuge Strategy in Bt Maize: Impact of Cross-Pollination on an Ear-Feeding Pest,Corn Earworm

To counter the threat of insect resistance, Bacillus thuringiensis (Bt) maize growers in the U.S. are required to plant structured non-Bt maize refuges. Concerns with refuge compliance led to the introduction of seed mixtures, also called RIB (refuge-in-the-bag), as an alternative approach for implementing refuge for Bt maize products in the U.S. Maize Belt. A major concern in RIB is cross-pollination of maize hybrids that can cause Bt proteins to be present in refuge maize kernels and negatively affect refuge insects. Here we show that a mixed planting of 5% nonBt and 95% Bt maize containing the SmartStax traits expressing Cry1A.105, Cry2Ab2 and Cry1F did not provide an effective refuge for an important above-ground ear-feeding pest, the corn earworm, Helicoverpa zea (Boddie). Cross-pollination in RIB caused a majority (>90%) of refuge kernels to express ≥ one Bt protein. The contamination of Bt proteins in the refuge ears reduced neonate-to-adult survivorship of H. zea to only 4.6%, a reduction of 88.1% relative to larvae feeding on ears of pure non-Bt maize plantings. In addition, the limited survivors on refuge ears had lower pupal mass and took longer to develop to adults.     

Cross-Resistance between Cry1 Proteins in Fall Armyworm (Spodoptera frugiperda) May Affect the Durability of Current Pyramided Bt Maize Hybrids in Brazil

Genetically modified plants expressing insecticidal proteins from Bacillus thuringiensis (Bt) offer valuable options for managing insect pests with considerable environmental and economic benefits. Despite the benefits provided by Bt crops, the continuous expression of these insecticidal proteins imposes strong selection for resistance in target pest populations. Bt maize (Zea mays) hybrids have been successful in controlling fall armyworm (Spodoptera frugiperda), the main maize pest in Brazil since 2008; however, field-evolved resistance to the protein Cry1F has recently been reported. Therefore it is important to assess the possibility of cross-resistance between Cry1F and other Cry proteins expressed in Bt maize hybrids. In this study, an F₂ screen followed by subsequent selection on MON 89034 maize was used to select an S. frugiperda strain (RR) able to survive on the Bt maize event MON 89034, which expresses the Cry1A.105 and Cry2Ab2 proteins. Field-collected insects from maize expressing the Cry1F protein (event TC1507) represented most of the positive (resistance allele-containing) (iso)families found. The RR strain showed high levels of resistance to Cry1F, which apparently also conferred high levels of cross resistance to Cry1A.105 and Cry1Ab, but had only low-level (10-fold) resistance to Cry2Ab2. Life history studies to investigate fitness costs associated with the resistance in RR strain revealed only small reductions in reproductive rate when compared to susceptible and heterozygous strains, but the RR strain produced 32.2% and 28.4% fewer females from each female relative to the SS and RS (pooled) strains, respectively. Consistent with the lack of significant resistance to Cry2Ab2, MON 89034 maize in combination with appropriate management practices continues to provide effective control of S. frugiperda in Brazil. Nevertheless, the occurrence of Cry1F resistance in S. frugiperda across Brazil, and the cross-resistance to Cry1Ab and Cry1A.105, indicates that current Cry1-based maize hybrids face a challenge in managing S. frugiperda in Brazil and highlights the importance of effective insect resistance management for these technologies.     

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.     

Overexpression of a novel Cry1Ie gene confers resistance to Cry1Ac-resistant cotton bollworm in transgenic lines of maize

Although transgenic crops expressing either Cry1Ab or Cry1Ac, both derived from Bacillus thuringiensis (Bt), have been used commercially, the evolution of insects resistance to these CRY proteins has become a challenge. Thus, it has been proposed that co-expression of two Bt proteins with different modes of action may delay the development of resistance to Bt. However, few Bt proteins have been identified as having different modes of action from those of Cry1Ab or Cry1Ac. In this study, transgenic lines of maize over-expressing either Cry1Ie or Cry1Ac gene have been developed. Several independent transgenic lines with one copy of the foreign gene were identified by Southern blot analysis. Bioassays in the laboratory showed that the transgenic plants over-expressing Cry1Ie were highly toxic against the wild-type cotton bollworm (Heliothis armigera), producing mortality levels of 50 % after 6 days of exposure. However, the mortality caused by these plants was lower than that caused by the Cry1Ac transgenic plants (80 %) and MON810 plants expressing Cry1Ab (100 %), which both exhibited low toxicity toward the Cry1Ac-resistant cotton bollworm. In contrast, three transgenic maize lines expressing Cry1Ie induced higher mortality against this pest and were also highly toxic to the Asian corn borer (Ostrinia furnacalis) in the field. These results indicate that the Cry1Ie protein has a different mode of action than the Cry1Ab and Cry1Ac proteins. Therefore, the use of transgenic plants expressing Cry1Ie might delay the development of Bt-resistant insects in the field.     

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.     

Independent Action between DvSnf7 RNA and Cry3Bb1 Protein in Southern Corn Rootworm,Diabrotica undecimpunctata howardi and Colorado Potato Beetle,Leptinotarsa decemlineata

In recent years, corn rootworm (CRW)-resistant maize events producing two or more CRW-active Bt proteins have been commercialized to enhance efficacy against the target pest(s) by providing multiple modes of action (MoA). The maize hybrid MON 87411 has been developed that produces the CRW-active Cry3Bb1 Bt protein (hereafter Cry3Bb1) and expresses a RNAi-mediated MoA that also targets CRW. As part of an environmental risk assessment for MON 87411, the potential for an interaction between the CRW-active DvSnf7 RNA (hereafter DvSnf7) and Cry3Bb1 was assessed in 12-day diet incorporation bioassays with the southern corn rootworm (SCR, Diabrotica undecimpunctata howardi). The potential for an interaction between DvSnf7 and Cry3Bb1 was evaluated with two established experimental approaches. The first approach evaluated each substance alone and in combination over three different response levels. For all three response levels, observed responses were shown to be additive and not significantly different from predicted responses under the assumption of independent action. The second approach evaluated the potential for a fixed sub-lethal concentration of Cry3Bb1 to decrease the median lethal concentration (LC₅₀) of DvSnf7 and vice-versa. With this approach, the LC50 value of DvSnf7 was not altered by a sub-lethal concentration of Cry3Bb1 and vice-versa. In addition, the potential for an interaction between the Cry3Bb1 and DvSnf7 was tested with Colorado potato beetle (CPB, Leptinotarsa decemlineata), which is sensitive to Cry3Bb1 but not DvSnf7. CPB assays also demonstrated that DvSnf7 does not alter the activity of Cry3Bb1. The results from this study provide multiple lines of evidence that DvSnf7 and Cry3Bb1 produced in MON 87411 have independent action.     

Susceptibility of field populations of sugarcane borer from non‐Bt and Bt maize plants to five individual Cry toxins

Abstract Sugarcane borer, Diatraea saccharalis (F.), is a major target of transgenic maize expressing Bacillus thuringiensis (Bt) proteins in South America and the US mid‐south region. Resistance development in target pest populations is a major threat to the sustainable use of Bt crops. In our field trials in 2009, a significant number of live borers and plant injury from D. saccharalis were observed in an experimental SmartStax? maize line. The objective of this study was to assess the relative susceptibility of two field populations of D. saccharalis collected from non‐Bt and Bt maize plants containing SmartStax? traits to five individual Cry proteins. The five Bt proteins included two proteins (Cry1A.105 and Cry2Ab2) that were expressed in SmartStax? maize plants and three other common Bt proteins (Cry1Aa, Cry1Ab and Cry1Ac) that were not produced in SmartStax?. Larval mortality and growth inhibition on Bt diet of the fourth generation after field collections were evaluated 7 days after release of neonates on the diet surface. The laboratory bioassays showed that 50% lethal concentration (LC₅₀) values for Cry1A.105 and Cry2Ab2 for the population originated from Bt plants were 3.55‐ and 1.34‐fold greater, respectively, than those of the population collected from non‐Bt plants. In contrast, relative to the population from non‐Bt plants, the LC₅₀ of the population sampled from Bt plants were 3.85‐, 2.5‐ and 1.64‐fold more sensitive to Cry1Aa, Cry1Ab and Cry1Ac, respectively. The results did not provide clear evidence to conclude that the observed field survival of D. saccharalis on Bt plants was associated with increased levels of resistance.     

Bt Crops Producing Cry1Ac,Cry2Ab and Cry1F Do Not Harm the Green Lacewing,Chrysoperla rufilabris

The biological control function provided by natural enemies is regarded as a protection goal that should not be harmed by the application of any new pest management tool. Plants producing Cry proteins from the bacterium, Bacillus thuringiensis (Bt), have become a major tactic for controlling pest Lepidoptera on cotton and maize and risk assessment studies are needed to ensure they do not harm important natural enemies. However, using Cry protein susceptible hosts as prey often compromises such studies. To avoid this problem we utilized pest Lepidoptera, cabbage looper (Trichoplusia ni) and fall armyworm (Spodoptera frugiperda), that were resistant to Cry1Ac produced in Bt broccoli (T. ni), Cry1Ac/Cry2Ab produced in Bt cotton (T. ni), and Cry1F produced in Bt maize (S. frugiperda). Larvae of these species were fed Bt plants or non-Bt plants and then exposed to predaceous larvae of the green lacewing Chrysoperla rufilabris. Fitness parameters (larval survival, development time, fecundity and egg hatch) of C. rufilabris were assessed over two generations. There were no differences in any of the fitness parameters regardless if C. rufilabris consumed prey (T. ni or S. frugiperda) that had consumed Bt or non-Bt plants. Additional studies confirmed that the prey contained bioactive Cry proteins when they were consumed by the predator. These studies confirm that Cry1Ac, Cry2Ab and Cry1F do not pose a hazard to the important predator C. rufilabris. This study also demonstrates the power of using resistant hosts when assessing the risk of genetically modified plants on non-target organisms.     

The interaction of two-spotted spider mites, <Emphasis Type="Italic">Tetranychus urticae</Emphasis> Koch,with Cry protein production and predation by <Emphasis Type="Italic">Amblyseius andersoni</Emphasis> (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize

Crops producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), are an important tool for managing lepidopteran pests on cotton and maize. However, the effects of these Bt crops on non-target organisms, especially natural enemies that provide biological control services, are required to be addressed in an environmental risk assessment. Amblyseius andersoni (Acari: Phytoseiidae) is a cosmopolitan predator of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), a significant pest of cotton and maize. Tri-trophic studies were conducted to assess the potential effects of Cry1Ac/Cry2Ab cotton and Cry1F maize on life history parameters (survival rate, development time, fecundity and egg hatching rate) of A. andersoni. We confirmed that these Bt crops have no effects on the biology of T. urticae and, in turn, that there were no differences in any of the life history parameters of A. andersoni when it fed on T. urticae feeding on Cry1Ac/Cry2Ab or non-Bt cotton and Cry1F or non-Bt maize. Use of a susceptible insect assay demonstrated that T. urticae contained biologically active Cry proteins. Cry proteins concentrations declined greatly as they moved from plants to herbivores to predators and protein concentration did not appear to be related to mite density. Free-choice experiments revealed that A. andersoni had no preference for Cry1Ac/Cry2Ab cotton or Cry1F maize-reared T. urticae compared with those reared on non-Bt cotton or maize. Collectively these results provide strong evidence that these crops can complement other integrated pest management tactics including biological control.