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.     

A Comprehensive Assessment of the Effects of Transgenic Cry1Ac/Cry1Ab Rice Huahui 1 on Adult Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae)

Micraspis discolor (Fabricius) (Coleoptera: Coccinellidae) is a widely distributed coleoptera predator in southern Asia in rice ecosystem, and adult M. discolor feed on both rice pollen and soft-bodied arthropods. Bitrophic bioassay and tritrophic bioassay were conducted to evaluate the potential impact of Cry1Ac/Cry1Ab-expressing rice Huahui 1 and its non-transgenic counterpart Minghui 63 on fitness parameters of adult M. discolor. The results showed that the survival, and fecundity of this beetle’ adults were not different when they fed on Bt rice or non-Bt rice pollen or Nilaparvata lugens (Stål) reared on Bt rice or non-Bt rice. Toxicity assessment to ensure M. discolor adults were not sensitive to Cry1Ab or Cry1Ac protein independent from the pollen background, M. discolor adults were fed with an artificial diet containing Cry1Ac, Cry1Ab or both protein approximately 10 times higher concentration than in Huahui 1 rice pollen. No difference was detected for any of the life-table parameters tested between Cry protein-containing and pure diet. Artificial diet containing E-64 (N-(trans-Epoxysuccinyl)-L-leucine 4-guanidinobutylamide) was included as a positive control. In contrast, the pre-oviposition and fecundity of M. discolor were significantly adversely affected by feeding on E-64-containing diet. In both bioassays, the uptakes of Cry protein by adult M. discolor were tested by ELISA measurements. These results indicated that adults of M. discolor are not affected by Cry1Ab- or Cry1Ac-expressing rice pollen and are not sensitive to Cry protein at concentrations exceeding the levels in rice pollen in Huahui1. This suggests that M. discolor adults would not be harmed by Cry1Ac/Cry1Ab rice if Bt rice Huahui 1 were commercialized.     

Effect of Stacked Insecticidal Cry Proteins from Maize Pollen on Nurse Bees (Apis mellifera carnica) and Their Gut Bacteria

Honey bee pollination is a key ecosystem service to nature and agriculture. However, biosafety research on genetically modified crops rarely considers effects on nurse bees from intact colonies, even though they receive and primarily process the largest amount of pollen. The objective of this study was to analyze the response of nurse bees and their gut bacteria to pollen from Bt maize expressing three different insecticidal Cry proteins (Cry1A.105, Cry2Ab2, and Cry3Bb1). Naturally Cry proteins are produced by bacteria (Bacillus thuringiensis). Colonies of Apis mellifera carnica were kept during anthesis in flight cages on field plots with the Bt maize, two different conventionally bred maize varieties, and without cages, 1-km outside of the experimental maize field to allow ad libitum foraging to mixed pollen sources. During their 10-days life span, the consumption of Bt maize pollen had no effect on their survival rate, body weight and rates of pollen digestion compared to the conventional maize varieties. As indicated by ELISA-quantification of Cry1A.105 and Cry3Bb1, more than 98% of the recombinant proteins were degraded. Bacterial population sizes in the gut were not affected by the genetic modification. Bt-maize, conventional varieties and mixed pollen sources selected for significantly different bacterial communities which were, however, composed of the same dominant members, including Proteobacteria in the midgut and Lactobacillus sp. and Bifidobacterium sp. in the hindgut. Surprisingly, Cry proteins from natural sources, most likely B. thuringiensis, were detected in bees with no exposure to Bt maize. The natural occurrence of Cry proteins and the lack of detectable effects on nurse bees and their gut bacteria give no indication for harmful effects of this Bt maize on nurse honey bees.     

Bacillus thuringiensis plants expressing Cry1Ac,Cry2Ab and Cry1F are not toxic to the assassin bug,Zelus renardii

Cotton‐ and maize‐producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), have been commercialized since 1996. Bt plants are subjected to environmental risk assessments for non‐target organisms, including natural enemies that suppress pest populations. Here, we used Cry1F‐resistant Spodoptera frugiperda (J.E. Smith) and Cry1Ac and Cry2Ab‐resistant Trichoplusia ni (Hübner) as prey for the assassin bug, Zelus renardii (Kolenati), a common predator in maize and cotton fields. In tritrophic studies, we assessed several fitness parameters of Z. renardii when it fed on resistant S. frugiperda that had fed on Bt maize expressing Cry1F or on resistant T. ni that had fed on Bt cotton expressing Cry1Ac and Cry2Ab. Survival, nymphal duration, adult weight, adult longevity and female fecundity of Z. renardii were not different when they were fed resistant‐prey larvae (S. frugiperda or T. ni) reared on either a Bt crop or respective non‐Bt crops. ELISA tests demonstrated that the Cry proteins were present in the plant at the highest levels, at lower levels in the prey and at the lowest levels in the predator. While Z. renardii was exposed to Cry1F and Cry1Ac and Cry2Ab when it fed on hosts that consumed Bt‐transgenic plants, the proteins did not affect important fitness parameters in this common and important predator.     

Prey-mediated effects of Cry1Ab toxin and protoxin and Cry2A protoxin on the predator Chrysoperla carnea

Laboratory feeding experiments were carried out to study prey-mediated effects of artificial diet containing Bacillus thuringiensis proteins on immature Chrysoperla carnea. Activated Cry1Ab toxin and the protoxins of Cry1Ab and Cry2A were mixed into standard meridic diet for Spodoptera littoralis (Boisduval) larvae at the following concentrations; for Cry1Ab toxin, 25, 50, 100 g g^–1 diet were used; for Cry1Ab protoxin, the concentration was doubled (50 g g^–1 diet, 100 g g^–1 diet and 200 g g^–1 diet) to give relative comparable levels of toxin concentration. Cry2A protoxin was incorporated into the meridic diet at one concentration only (100 g g^–1 diet). For the untreated control, the equivalent amount of double distilled water was added to the meridic diet. Individual C. carnea larvae were raised on S. littoralis larvae fed with one of the respective treated meridic diets described above. The objectives were to quantify and compare the resulting effects on mortality and development time of C. carnea with those observed in two previous studies investigating prey-mediated effects of transgenic Cry1Ab toxin-producing corn plants and the other studying effects of Cry1Ab toxin fed directly to C. carnea larvae. Mean total immature mortality for chrysopid larvae reared on B. thuringiensis-fed prey was always significantly higher than in the control (26%). Total immature mortality of C. carnea reared on Cry1Ab toxin 100 g g^–1 diet-fed prey was highest (78%) and declined with decreasing toxin concentration. Cry1Ab protoxin-exposed C. carnea larvae did not exhibit a dose response. Prey-mediated total mortality of Cry1Ab protoxin-exposed chrysopid larvae was intermediate (46–62%) to Cry1Ab toxin exposed (55–78%) and Cry2A protoxin (47%) exposed C. carnea. In agreement with the previous studies, total development time of C. carnea was not consistently, significantly affected by the Bt-treatments except at the highest Cry1Ab toxin concentration. However, both highest mortality and delayed development of immature C. carnea raised on Cry1Ab toxin 100 g g^–1 diet – fed prey may have been confounded with an increased intoxication of S. littoralis larvae that was observed at that concentration. At all other B. thuringiensis protein concentrations S. littoralis was not lethally affected. Comparative analysis of the results of this study with those of the two previous studies revealed that in addition to prey/herbivore by B. thuringiensis interactions, also prey/herbivore by plant interactions exist that contribute to the observed toxicity of B. thuringiensis – fed S. littoralis larvae for C. carnea. These findings demonstrate that tritrophic level studies are necessary to assess the long-term compatibility of insecticidal plants with important natural enemies.     

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.     

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.     

Bacillus thuringiensis toxin (Cry1Ab) has no direct effect on larvae of the green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae)

Earlier studies have shown that larvae of the green lacewing predator Chrysoperla carnea are negatively affected when preying on lepidopteran larvae that had been fed with transgenic maize expressing the cry1Ab gene from Bacillus thuringiensis. To test whether the observed effects were directly caused by the Cry1Ab toxin, we have developed a bioassay which allows us to feed high concentrations of the toxin directly to the predator. The results of these feeding studies show no direct toxic effect of Cry1Ab on C. carnea larvae. The amount of toxin ingested by first instar C. carnea in the present study was found to be a factor 10,000 higher than the concentration ingested when feeding on Bt-reared lepidopteran larvae, a treatment that was previously shown to have a negative impact on the predator. In addition, feeding first instar C. carnea with the Cry1Ab toxin did not affect the utilisation of subsequently provided prey. Furthermore, the quality of the prey provided to first instars did not affect the sensitivity of second and third instar C. carnea to the Bt-toxin. The presented results strongly suggest that C. carnea larvae are not sensitive to Cry1Ab and that earlier reported negative effects of Bt-maize were prey-quality mediated rather than direct toxic effects. These results, together with the fact that lepidopteran larvae are not regarded as an important prey for C. carnea in the field, led us to conclude that transgenic maize expressing Cry1Ab poses a negligible risk for this predator.     

Uptake of Bt-toxin by herbivores feeding on transgenic maize and consequences for the predator Chrysoperla carnea

Abstract  1. Chrysoperla carnea is an important predatory insect in maize. To assess the ecological effects of Bt-maize, expressing the Cry1Ab protein, on larvae of this predator, the following factors were examined: (1) the performance of three prey herbivores ( Rhopalosiphum padi , Tetranychus urticae , and Spodoptera littoralis ) on transgenic Bt and non‐transgenic maize plants; (2) the intake of the Cry1Ab toxin by the three herbivores; and (3) the effects on C. carnea when fed each of the prey species.
2. The intrinsic rate of natural increase (r_m) was used as a measure of performance for R. padi and T. urticae . No difference in this parameter was observed between herbivores reared on Bt or non‐transgenic plants. In contrast, a higher mortality rate and a delay in development were observed in S. littoralis larvae when fed Bt-maize compared with those fed the control maize plants.
3. The ingestion of Cry1Ab toxin by the different herbivores was measured using an immunological assay (ELISA). Highest amounts of Cry1Ab toxin were detected in T. urticae , followed by S. littoralis , and only trace amounts detected in R. padi .
4. Feeding C. carnea with T. urticae , which were shown to contain the Cry1Ab toxin, or with R. padi , which do not ingest the toxin, did not affect survival, development, or weight of C. carnea . In contrast, a significant increase in mortality and a delay in development were observed when predators were fed S. littoralis larvae reared on Bt-maize.
5. A combined interaction of poor prey quality and Cry1Ab toxin may account for the negative effects observed on C. carnea when fed S. littoralis . The relevance of these findings to the ecological risks of Bt-maize on C. carnea 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 .     

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.     

Growth and development of Bacillus thuringiensis Cry1Ab-susceptible and Cry1Ab-resistant sugarcane borer on diet and conventional maize plants

The sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), is a dominant maize borer pest and a major target of Bacillus thuringiensis (Bt)‐maize in Louisiana and the Gulf Coast area of Texas (USA). Growth and development of D. saccharalis on non‐toxic diet, diet treated with three low concentrations (0.01, 0.05, and 0.1 μg g^?1) of Cry1Ab toxin, and on non‐Bt maize plants were compared for five insect genotypes: a Bt‐susceptible strain (BT‐SS), a Cry1Ab‐resistant strain (BT‐RR), a back‐crossed and re‐selected resistant strain (BT‐R’R’), and two F₁ progeny of the BT‐SS and BT‐R’R’ strains. Fitness of the five genotypes was examined by infesting neonates on diet with/without Cry1Ab toxin in the laboratory and on intact non‐Bt maize plants in the greenhouse. Biological parameters measured were neonate‐to‐pupa development time and pupation rate, larval survival, larval and pupal weight, and sex ratio. Larvae of BT‐SS and BT‐R’R’ on non‐toxic diet and non‐Bt maize plants grew normally and there were no significant differences between the two strains in all measured parameters, suggesting a lack‐of‐fitness cost of the Cry1Ab resistance in D. saccharalis. Except for the development time on non‐Bt diet, all other parameters on both non‐Bt diet and non‐Bt maize plants were similar among the five genotypes. Larval development of BT‐SS was significantly affected on diet treated with Cry1Ab toxin at 0.05 and 0.1 μg g^?1, whereas the effect to BT‐RR and BT‐R’R’ was not significant. Pupal weight and sex ratio reared on Cry1Ab‐diet were similar and there were no significant differences among the five genotypes. Neonate‐to‐pupation rate decreased as Cry1Ab concentrations increased but the decrease was more significant for BT‐SS than for the other four genotypes. The lack‐of‐fitness costs of Bt resistance in D. saccharalis imply a greater challenge in managing Bt resistance for this maize borer species.     

Presence of Cry1Ab in the Bt maize – aphid (Rhopalosiphum maidis) – ladybeetle (Propylea japonica) system has no adverse effects on insect biological parameters

Transgenic Bacillus thuringiensis Berliner (Bt) crops receive particular attention because they carry genes encoding insecticidal proteins that might negatively affect non‐target arthropods. Here, laboratory experiments were conducted to evaluate the impact of Cry1Ab‐expressing transgenic maize [5422Bt1 (event Bt11) and 5422CBCL (MON810)] on the biological parameters of two non‐target arthropods, the aphid Rhopalosiphum maidis (Fitch) (Hemiptera: Aphididae) and its predator the ladybeetle Propylea japonica (Thunberg) (Coleoptera: Coccinellidae). In a long‐term assay (three generations), no significant differences were found between R. maidis fed Bt maize and those fed a near‐isogenic line (5422) when individual parameters were compared, including nymph development time, adult longevity, aphid spawning period, and fecundity. No negative effects were detected throughout the life cycle of P. japonica in aphids’ feeding amount, development (nymphs, pupae, adults, and progeny eggs), fecundity, or egg hatching when they preyed on Bt maize‐fed aphids compared with non‐Bt maize treatments. A tritrophic assay revealed that Cry1Ab was highly diluted through the food chain (Bt maize leaves, R. maidis, and P. japonica), as detected by an enzyme‐linked immunosorbent assay (ELISA). In conclusion, although Cry1Ab concentrations in maize leaves increased as the plants developed, Cry1Ab levels were significantly reduced in the aphid R. maidis, and no traces of Cry1Ab were detected in P. japonica preying on Bt maize‐fed aphids. The two hybrids of Bt maize expressing Cry1Ab had no negative effects on the measured biological parameters of the aphid R. maidis or its predator, the ladybeetle P. japonica.     

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.     

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

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

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.     

Laboratory toxicity studies demonstrate no adverse effects of Cry1Ab and Cry3Bb1 to larvae of <Emphasis Type="Italic">Adalia bipunctata</Emphasis> (Coleoptera: Coccinellidae): the importance of study design

Scientific studies are frequently used to support policy decisions related to transgenic crops. Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) recently reported that Cry1Ab and Cry3Bb were toxic to larvae of Adalia bipunctata in direct feeding studies. This study was quoted, among others, to justify the ban of Bt maize (MON 810) in Germany. The study has subsequently been criticized because of methodological shortcomings that make it questionable whether the observed effects were due to direct toxicity of the two Cry proteins. We therefore conducted tritrophic studies assessing whether an effect of the two proteins on A. bipunctata could be detected under more realistic routes of exposure. Spider mites that had fed on Bt maize (events MON810 and MON88017) were used as carriers to expose young A. bipunctata larvae to high doses of biologically active Cry1Ab and Cry3Bb1. Ingestion of the two Cry proteins by A. bipunctata did not affect larval mortality, weight, or development time. These results were confirmed in a subsequent experiment in which A. bipunctata were directly fed with a sucrose solution containing dissolved purified proteins at concentrations approximately 10 times higher than measured in Bt maize-fed spider mites. Hence, our study does not provide any evidence that larvae of A. bipunctata are sensitive to Cry1Ab and Cry3Bb1 or that Bt maize expressing these proteins would adversely affect this predator. The results suggest that the apparent harmful effects of Cry1Ab and Cry3Bb1 reported by Schmidt et al., Arch Environ Contam Toxicol 56:221–228 (2009) were artifacts of poor study design and procedures. It is thus important that decision-makers evaluate the quality of individual scientific studies and do not view all as equally rigorous and relevant.     

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.     

Assessing the Effects of Bt Maize on the Predatory Mite Neoseiulus cucumeris

The investigation of Neoseiulus cucumeris in the context of the ecological risk assessment of insect resistant transgenic plants is of particular interest as this omnivorous predatory mite species is commercially available and considered important for biological control. In a multitrophic feeding experiment we assessed the impact of Bt maize on the performance of N. cucumeris when offered spider mites (Tetranychus urticae) reared on Bt (Bt11, Syngenta) or non-Bt maize (near isogenic line) and Bt or non-Bt maize pollen as a food source. Various parameters including mortality, development time, oviposition rate were measured. Spider mites were used as a prey for N. cucumeris, since these herbivores are known to contain similar levels of Cry1Ab toxin, when reared on Bt maize, as those found in the transgenic leaf material. In contrast, toxin levels in pollen of this transgenic cultivar are very low. No differences in any of the parameters were found when N. cucumeris was fed with spider mites reared on Bt and non-Bt maize. Pollen was shown to be a less suitable food source for this predator as compared to spider mites. Moreover, subtle effects on female N. cucumeris (9% longer development time and 17% reduced fecundity) were measured when fed with pollen originating from Bt maize as compared to non-Bt maize pollen. Our findings indicate that the predatory mite N. cucumeris is not sensitive to the Cry1Ab toxin as no effects could be detected when offered Bt-containing spider mites, and that the effects found when fed with Bt maize pollen can be assigned to differences in nutritional quality of Bt and non-Bt maize pollen. The significance of these findings is discussed with regard to the ecological relevance for risk assessment of transgenic plants.     

Western corn rootworm and Bt maize: challenges of pest resistance in the field

Crops genetically engineered to produce insecticidal toxins from the bacterium Bacillus thuringiensis (Bt) manage many key insect pests while reducing the use of conventional insecticides. One of the primary pests targeted by Bt maize in the United States is the western corn rootworm, Diabrotica virgifera virgifera LeConte. Beginning in 2009, populations of western corn rootworm were identified in Iowa, USA that imposed severe root injury to Cry3Bb1 maize. Subsequent laboratory bioassays revealed that these populations were resistant to Cry3Bb1 maize, with survival on Cry3Bb1 maize that was three times higher than populations not associated with such injury. Here we report the results of research that began in 2010 when western corn rootworm were sampled from 14 fields in Iowa, half of which had root injury to Cry3Bb1 maize of greater than 1 node. Of these samples, sufficient eggs were collected to conduct bioassays on seven populations. Laboratory bioassays revealed that these 2010 populations had survival on Cry3Bb1 maize that was 11 times higher and significantly greater than that of control populations, which were brought into the laboratory prior to the commercialization of Bt maize for control of corn rootworm. Additionally, the developmental delays observed for control populations on Cry3Bb1 maize were greatly diminished for 2010 populations. All 2010 populations evaluated in bioassays came from fields with a history of continuous maize production and between 3 and 7 y of Cry3Bb1 maize cultivation. Resistance to Cry34/35Ab1 maize was not detected and there was no correlation between survival on Cry3Bb1 maize and Cry34/35Ab1 maize, suggesting a lack of cross resistance between these Bt toxins. Effectively dealing with the challenge of field-evolved resistance to Bt maize by western corn rootworm will require better adherence to the principles of integrated pest management.