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.     

转Bt基因抗虫玉米对亚洲玉米螟幼虫取食行为的影响

室内研究了转cry1Ab杀虫蛋白基因的Bt抗虫玉米MON81 0和Bt1 1对亚洲玉米螟Ostriniafurnacalis初孵幼虫和3龄幼虫的取食行为、取食选择性和存活率的影响。在48h的非选择性试验中玉米螟初孵幼虫在MON81 0和Bt1 1玉米心叶组织上的幼虫取食率随时间的增加而下降,在对照玉米上的幼虫取食率随时间的增加而上升,两者间差异极显著。初孵幼虫接虫到MON81 0和Bt1 1玉米叶片48h的累计死亡率分别为67 .5 %和47 .5 % ,而在对照玉米上死亡率均为0. 3龄幼虫在Bt和非Bt玉米穗轴组织上的幼虫取食率随时间的增加呈上升趋势,第48h时在Bt和非Bt玉米上的幼虫取食率分别达到77 5 %和1 0 0 % ,差异极显著。选择性试验中,第4～48h内,初孵幼虫在Bt玉米上的幼虫取食率呈下降趋势,第48h时MON81 0和Bt1 1与各自非Bt对照的组合中初孵幼虫的累计死亡率分别为2 5 .0 %和1 7. 5 % ,二者差异不显著。3龄幼虫在Bt玉米和非Bt玉米上的幼虫取食率均随时间的延长而增加,但在非Bt玉米的幼虫取食率增加速度快,与Bt玉米差异极显著。Bt玉米对玉米螟幼虫取食有抑制和忌避作用。     

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.     

Assessment of prey-mediated effects of the coleopteran-specific toxin Cry3Bb1 on the generalist predator Atheta coriaria (Coleoptera: Staphylinidae)

A laboratory study was carried out to assess the potential prey-mediated effects of Cry3Bb1-expressing Bt maize on the fitness and predatory ability of Atheta coriaria Kraatz (Coleoptera: Staphylinidae), using Tetranychus urticae Koch (Acari: Tetranychidae) as prey. The concentration of Cry3Bb1 toxin through the trophic chain significantly decreased from Bt maize (21.7 μg g(-1) FW) to mites (5.6 μg g(-1) FW) and then to A. coriaria adults (1.4 μg g(-1) FW), but not from mites to A. coriaria L1-L3 larvae (4.1-4.6 μg g(-1) FW). Interestingly, the toxin levels detected in A. coriaria larvae represent more than 20% of the concentration found in Bt maize, and the toxin was detected up to 48 h after exposure. To our knowledge, this is the highest level of exposure ever reported in a predatory beetle to the Cry3Bb1 protein. When A. coriaria larvae were reared on Bt-fed mites, Bt-free mites or rearing food, no significant differences among treatments were observed in development, morphological measurements of sclerotized structures and body weight. Moreover, no negative effects on reproductive parameters were reported in adults feeding on Bt-fed prey after 30 days of treatment, and survival was not affected after 60 days of exposure. Similarly, predatory ability and prey consumption of A. coriaria larvae and adults were not affected by exposure to the toxin. All together, these results indicate a lack of adverse effects on A. coriaria, a species commonly used as a biological control agent. The use of A. coriaria as a surrogate species for risk assessment of GM crops that express insecticidal proteins is discussed.     

Abundance of Coleomegilla maculata (Coleoptera: Coccinellidae) in corn rootworm-resistant Cry3Bb1 maize

Lady beetles (Coleoptera: Coccinellidae) are important polyphagous predators in maize, Zea mays L., fields. Transgenic Cry3Bb1 maize hybrids express a coleopteran-specific insecticidal protein derived from Bacillus thuringiensis (Berliner) subsp. kumamotoensis that is targeted at corn rootworm larvae. This study evaluated impacts of Cry3Bb1 protein-expressing maize, tefluthrin-treated maize, and untreated controls on lady beetle abundance at preanthesis, anthesis, and postanthesis maize-developmental periods near Brookings in eastern South Dakota during 2001 and 2002. The dominant lady beetle species captured on Pherocon AM sticky traps was Coleomegilla maculata De Geer. It comprised 73.5 and 69.9% of all adult Coccinellidae caught in 2001 and 2002, respectively. Numbers of C. maculata captured in Cry3Bb1 maize were not significantly different from those in untreated plots during preanthesis, and adults were more abundant in Cry3Bb1 maize than in tefluthrin-treated and untreated plots during anthesis and postanthesis. Whole-plant sampling confirmed C. maculata predominance with the species representing 89.2 and 91.4% of all adult lady beetles observed in 2001 and 2002, respectively. Whole-plant sampling also indicated a lack of negative effects from Cry3Bb1 maize on abundance of lady beetle eggs, larvae, pupae, or adults. Overall, these findings indicate that Cry3Bb1-expressing hybrids are not likely to impose harmful effects on C. maculata, a species common to maize production systems in the northern Great Plains. This research further suggests that Cry3Bb1 maize has the potential for conservation of these beneficial coccinellids in maize production systems.     

Effects of Bt maize expressing Cry1Ab and Bt spray on Spodoptera littoralis

Various studies have been conducted to assess the damage caused by secondary lepidopteran pests to transgenic Bt maize expressing Cry1Ab. However, to date little is known on the effects of transgenic maize on Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), a polyphagous herbivore which is considered a pest in Mediterranean maize growing areas. Here we present results on the effects of Bt maize (Bt‐11) and Bt spray (Dipel) on the various life stage parameters of this herbivore. We further assess the expression of Cry1Ab in different leaves and leaf parts in maize at a given plant growth stage, and determine whether the feeding damage of 3rd instar S. littoralis is influenced by Bt toxin expression. Contrary to previous literature reporting that S. littoralis is not sensitive to Bt Cry1Ab toxin, our results show that insects fed on either transgenic or Bt sprayed plants were negatively affected. Young S. littoralis larvae (1st and 2nd instars) were found to be the most sensitive to the Bt toxin. This was represented by a higher mortality and a slower developmental time of larvae maintained on transgenic or sprayed plants when compared to insects maintained on control plants. Moreover, Bt maize had a stronger and prolonged detrimental effect on insects when compared to Bt spray in maize. This was revealed by the fact that insects maintained on transgenic plants from 3rd instar to pupation took longer to reach adult emergence compared to insects that were maintained on sprayed plants. This was likely due to the continuous exposure of insects to the toxin when kept on transgenic maize. ELISA results showed a variation in the amount of Bt toxin among different leaf sections in transgenic maize at a given plant growth stage. These differences in Bt toxin were primarily found in the youngest leaf of growing plants. Although the lowest amounts of Bt toxin were detected in the growing leaf section of young leaves, this difference did not appear to influence the feeding behavior of 3rd instar S. littoralis.     

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.     

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.     

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.     

Decomposition dynamics and structural plant components of genetically modified <Emphasis Type="Italic">Bt</Emphasis> maize leaves do not differ from leaves of conventional hybrids

The cultivation of genetically modified Bt maize has raised environmental concerns, as large amounts of plant residues remain in the field and may negatively impact the soil ecosystem. In a field experiment, decomposition of leaf residues from three genetically modified (two expressing the Cry1Ab, one the Cry3Bb1 protein) and six non-transgenic hybrids (the three corresponding non-transformed near-isolines and three conventional hybrids) was investigated using litterbags. To elucidate the mechanisms that cause differences in plant decomposition, structural plant components (i.e., C:N ratio, lignin, cellulose, hemicellulose) were examined. Furthermore, Cry1Ab and Cry3Bb1 protein concentrations in maize leaf residues were measured from harvest to the next growing season. While leaf residue decomposition in transgenic and non-transgenic plants was similar, differences among conventional cultivars were evident. Similarly, plant components among conventional hybrids differed more than between transgenic and non-transgenic hybrids. Moreover, differences in senescent plant material collected directly from plants were larger than after exposure to soil for 5 months. While the concentration of Cry3Bb1 was higher in senescent maize leaves than that of Cry1Ab, degradation was faster, indicating that Cry3Bb1 has a shorter persistence in plant residues. As decomposition patterns of Bt-transgenic maize were shown to be well within the range of common conventional hybrids, there is no indication of ecologically relevant, adverse effects on the activity of the decomposer community.     

Effects of Cry1Ab-expressing corn anthers on the movement of monarch butterfly larvae

Decreased larval feeding and weight of the monarch butterfly, Danaus plexippus L., have been detected after 4 d of exposure in the laboratory to a high density of Bacillus thuringiensis (Bt)-expressing anthers. One hypothesis is that larvae exposed to Bt anthers exhibit increased wandering, resulting in less feeding and lower weight gain. To test this hypothesis, 2-d-old monarch butterfly larvae exposed to milkweed leaf disks with no anthers, anthers that express Bt (Cry1Ab, event MON810), or other non-Bt anthers were observed using a video-tracking system. As had been shown in previous studies, larvae exposed to Bt anthers fed less and gained less weight than larvae exposed to non-Bt or no anthers, yet there was no evidence of feeding on anthers. Total distance moved, maximum displacement from release point, percentage of time spent moving or near anthers, or mean turn angle did not differ across treatments. However, larvae exposed to Bt anthers spent more time off milkweed leaf disks than those exposed to no anthers and were more likely to move off the leaf than larvae exposed to non-Bt anthers. Results suggest that larvae exposed to Bt anthers behave differently and that ingestion may not be the only way Bt can affect nontarget insects like the monarch butterfly.     

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.     

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.     

Using field-evolved resistance to Cry1F maize in a lepidopteran pest to demonstrate no adverse effects of Cry1F on one of its major predators

Spodoptera frugiperda (JE Smith) represents the first documented case of field-evolved resistance to a genetically engineered crop expressing an insecticidal protein from Bacillus thuringiensis (Bt). In this case it was Cry1F-expressing maize (Mycogen 2A517). The ladybird beetle, Coleomegilla maculata, is a common and abundant predator that suppresses pest populations in maize and many other cropping systems. Its larvae and adults are polyphagous, feeding on aphids, thrips, lepidopteran eggs and larvae, as well as plant tissues. Thus, C. maculata may be exposed to Bt proteins expressed in genetically engineered crops by several pathways. Using Cry1F-resistant S. frugiperda larvae as prey, we evaluated the potential impact of Cry1F-expressing maize on several fitness parameters of C. maculata over two generations. Using Cry1F resistant prey removed any potential prey-mediated effects. Duration of larval and pupal stages, adult weight and female fecundity of C. maculata were not different when they were fed resistant S. frugiperda larvae reared on either Bt or control maize leaves during both generations. ELISA and insect-sensitive bioassays showed C. maculata were exposed to bioactive Cry1F protein. The insecticidal protein had no effect on C. maculata larvae, even though larvae contained 20?C32?ng of Cry1F/g by fresh weight. Over all, our results demonstrated that the Cry1F protein did not affect important fitness parameters of one of S. frugiperda??s major predators and that Cry1F protein did not accumulate but was strongly diluted when transferred during trophic interactions.     

Effects of adult emergence timing on susceptibility and fitness of Cry3Bb1‐resistant western corn rootworms

Field‐evolved resistance by the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte to the Cry3Bb1 trait expressed in maize, has been documented in areas of Nebraska USA. Currently, only limited information is available on life‐history traits of Cry3Bb1‐resistant field populations. Therefore, the Gassmann on‐plant bioassay was used to investigate the potential variability among four Cry3Bb1‐resistant WCR field collections made in 2011–2012 by focusing on the key parameters: larval survival, developmental stage and weight with specific emphasis on the impact of adult emergence timing on these parameters in subsequent progeny. Key results: In three of four collections, the susceptibility of larval progeny from adults that emerged early or late within a generation from Cry3Bb1 plants was similar. Each of the three collections exhibited complete resistance; that is, survival on Cry3Bb1 plants was greater or equal to survival on non‐Bt isoline plants. Bioassays from an additional field collection from one site 2 years (2013) after the original collection (2011) (both from Cry3Bb1 maize) indicated that resistance to Cry3Bb1 was maintained over time at the site despite Bt trait rotation in 2012. In general, comparative WCR life‐history parameter data from Cry3Bb1 and isoline maize indicate that fitness of field collections exhibiting complete resistance was similar on each hybrid. The mean proportion of larvae in third instar and mean weight of larvae recovered in bioassays from progeny of early‐ and late‐emerged adults was not significantly affected by emergence period. This suggests that delays in development and associated mean adult emergence commonly observed in populations that are susceptible to Cry3Bb1 may become smaller as populations become resistant to Cry3Bb1. Results from this article will inform Cry3Bb1 resistance mitigation efforts and contribute to the development of sustainable WCR management programmes.     

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.     

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

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

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.     

Baseline susceptibility of western corn rootworm (Coleoptera: Crysomelidae) to Cry3Bb1 Bacillus thuringiensis toxin

Susceptibility to Cry3Bb1 toxin from Bacillus thuringiensis (Bt) was determined for western corn rootworm, Diabrotica virgifera virgifera LeConte, neonates from both laboratory and field populations collected from across the Corn Belt. Rootworm larvae were exposed to artificial diet treated with increasing Cry3Bb1 concentrations, and mortality and growth inhibition were evaluated after 4-7 d. The range of variation in Bt susceptibility indicated by growth inhibition was similar to that indicated by mortality. Although interpopulation variation in susceptibility was observed, the magnitude of the differences was comparable with the variability observed between generations of the same population. In general, the toxin was not highly toxic to larvae and estimated LC50 and EC50 values were several times higher than those reported for lepidopteran-specific Cry toxins by using similar bioassay techniques. These results suggest that the observed susceptibility differences reflect natural variation in Bt susceptibility among rootworm populations and provide a baseline for estimating potential shifts in susceptibility that might result from selection and exposure to Cry3Bb1-expressing corn hybrids.