Spiders from multiple functional guilds are exposed to Bt-endotoxins in transgenic corn fields via prey and pollen consumption

A comprehensive assessment of risk to natural enemies from Bt-endotoxins from bioengineered crops must evaluate potential harm, as well as exposure pathways in non-target arthropod food webs. Despite being abundant generalist predators in agricultural fields, spiders (Araneae) have often been overlooked in the context of Bt crop risk assessment. Spiders and their prey were collected from transgenic corn fields expressing lepidopteran-specific Cry1Ab, coleopteran-specific Cry3Bb1, both proteins, and a non-transgenic near isoline. Spiders and prey were screened for Cry1Ab and Cry3Bb1 using qualitative enzyme-linked immunosorbent assay. Spiders from the three most common functional guilds, wandering sheet-tangle weavers, orb-weavers, and ground runners, tested positive for Cry1Ab and Cry3Bb1 proteins, with the highest per cent positive (8.0% and 8.3%) during and after anthesis. Laboratory feeding trials revealed that Bt-endotoxins were detectable in the Pardosa sp. (Lycosidae)-immature cricket-Bt corn pathway, but not in the Tennesseellum formica (Linyphiidae)-Collembola-Bt corn pathway. Additionally, direct consumption of transgenic corn pollen by Pardosa sp., T. formica, and Cyclosa turbinata (Araneidae) resulted in transfer of both Cry1Ab and Cry3Bb1 endotoxins. This study demonstrates that Bt-endotoxins are taken up by diverse members of a spider community via pollen and prey consumption and should be factored into future risk assessment.     

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.     

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.     

Quantification of Bt-endotoxin exposure pathways in carabid food webs across multiple transgenic events

Despite the reported specificity of Bacillus thuringiensis proteins against target pests, a number of studies have indicated that the uptake of Bt-endotoxins from bioengineered crops could have negative effects on natural enemies. It is therefore essential to quantify exposure pathways in non-target arthropod food webs across multiple transgenic events. Adult ground beetles (Coleoptera: Carabidae) were collected from transgenic corn fields expressing lepidopteran-specific Cry1Ab, coleopteran-specific Cry3Bb1, and both Cry1Ab and Cry3Bb1 (stacked event), as well as a non-transgenic isoline. Carabid gut-contents were screened for Cry1Ab Bt-endotoxin using enzyme-linked immunosorbent assay. Significant numbers of carabids tested positive for Cry1Ab from the lepidopteran-specific field: Harpalus pensylvanicus (39%, 25 of 64), Stenolophus comma (4%, 6 of 136), Cratacanthus dubius (50%, 1 of 2), Clivina bipustulata (50%, 1 of 2), and Cyclotrachelus sodalis (20%, 1 of 5). The highest proportion of Bt-endotoxin uptake was 4–6 weeks post-anthesis. Only one species, H. pensylvanicus (5%, 4 of 75), screened positive for Cry1Ab from the stacked line, despite similar expression of this endotoxin in plant tissue harvested from both lines. This difference in Cry1Ab uptake could be due to changes in the non-target food web or differential rates of Bt-endotoxin decay between genetic events. This study has quantified the differential uptake of Cry1Ab Bt-endotoxin by the carabid community across multiple transgenic events, thus forming the framework for future risk-assessment of transgenic crops.     

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.     

Decomposition rates and residue-colonizing microbial communities of Bacillus thuringiensis insecticidal protein Cry3Bb-expressing (Bt) and non-Bt corn hybrids in the field

Despite the rapid adoption of crops expressing the insecticidal Cry protein(s) from Bacillus thuringiensis (Bt), public concern continues to mount over the potential environmental impacts. Reduced residue decomposition rates and increased tissue lignin concentrations reported for some Bt corn hybrids have been highlighted recently as they may influence soil carbon dynamics. We assessed the effects of MON863 Bt corn, producing the Cry3Bb protein against the corn rootworm complex, on these aspects and associated decomposer communities by terminal restriction fragment length polymorphism (T-RFLP) analysis. Litterbags containing cobs, roots, or stalks plus leaves from Bt and unmodified corn with (non-Bt+I) or without (non-Bt) insecticide applied were placed on the soil surface and at a 10-cm depth in field plots planted with these crop treatments. The litterbags were recovered and analyzed after 3.5, 15.5, and 25 months. No significant effect of treatment (Bt, non-Bt, and non-Bt+I) was observed on initial tissue lignin concentrations, litter decomposition rate, or bacterial decomposer communities. The effect of treatment on fungal decomposer communities was minor, with only 1 of 16 comparisons yielding separation by treatment. Environmental factors (litterbag recovery year, litterbag placement, and plot history) led to significant differences for most measured variables. Combined, these results indicate that the differences detected were driven primarily by environmental factors rather than by any differences between the corn hybrids or the use of tefluthrin. We conclude that the Cry3Bb corn tested in this study is unlikely to affect carbon residence time or turnover in soils receiving these crop residues.     

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.     

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 .     

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.     

Larval mortality and development for rotation‐resistant and rotation‐susceptible populations of western corn rootworm on Bt corn

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is one of the most economically important insect pests threatening the production of corn, Zea mays (L.), in the United States. Throughout its history, this insect has displayed considerable adaptability by overcoming a variety of pest management tactics, including the cultural practice of annual crop rotation. Since first reported in Illinois in the late 1980s, populations of the rotation‐resistant western corn rootworm have spread over a wide area of the eastern Corn Belt. Currently, little information is available concerning the interaction of rotation resistance with the use of genetically modified corn expressing insecticidal toxins from Bacillus thuringiensis Berliner (Bt), a popular tactic for preventing larval injury and its associated yield loss. The goal of this greenhouse experiment was to determine whether rotation‐resistant and rotation‐susceptible western corn rootworm larvae differ with respect to survival or development when exposed to single‐ or dual‐toxin (pyramided) Bt corn. Individual corn plants were infested with 225 near‐hatch eggs at the V5 (five leaf collar) growth stage. Larvae developed undisturbed on the root systems for 17 days, after which they were recovered using Berlese–Tullgren funnels. Surviving larvae were counted to estimate mortality, and head capsule widths were measured to assess development. Rotation‐resistant and rotation‐susceptible larvae had statistically similar mean levels of mortality and head capsule widths when exposed to both single‐toxin (Cry3Bb1 or Cry34/35Ab1) and pyramided (Cry3Bb1+ Cry34/35Ab1) Bt corn, suggesting that these two populations do not differ with respect to survival or development when exposed to Bt corn. Additionally, the statistically similar mean levels of mortality for larvae exposed to single‐toxin and pyramided Bt corn suggest that pyramided Bt hybrids containing the Cry3Bb1 and Cry34/35Ab1 toxins do not result in additive mortality for western corn rootworm larvae. Implications for management of this economically important pest are discussed.     

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.     

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.     

Protease activities in the midgut of Western corn rootworm (Diabrotica virgifera virgifera LeConte)

The Western corn rootworm is one of the most economically important pests in corn. One possibility for controlling this pest is the cultivation of transgenic corn expressing Bacillus thuringiensis (Bt) toxins, such as Cry3A, Cry34Ab1/Cry35Ab1, and Cry3Bb1. However, widespread cultivation of the resulting Bt corn may result in the development of resistant pest populations. The Bt toxins are processed by proteases in the midgut of susceptible insects. Thus, protease activity studies were conducted using the midgut juice (pH 5.75) from third instars larvae of the susceptible Western corn rootworm. As a result, the activities of the serine endopeptidases trypsin, chymotrypsin, elastase, cathepsin G, plasmin, and thrombin; the cysteine endopeptidases cathepsin L, papain, cathepsin B, and cathepsin H; the aspartic endopeptidase pepsin; the metallo endopeptidase saccharolysin; the exopeptidase aminopeptidase, and the omegapeptidase acylaminoacylpeptidase were detected. These results are of basic interest but also lead to reference systems for the identification of protease-mediated resistance mechanisms in potentially resistant individuals.     

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.     

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.     

Absence of non-target effects of two Bacillus thuringiensis coleopteran active δ-endotoxins on the bulb mite, Rhizoglypus robini (Claparède) (Acari, Acaridae)

Transgenic crops with plant‐incorporated protectants are often more specific than synthetic insecticides and have the potential to reduce impacts on non‐target organisms. In this study we assessed the impact of Cry3Aa and Cry3Bb1 coleopteran‐active δ‐endotoxins on the bulb mite, Rhizoglypus robini. The effect of Cry3Aa prototoxin in solutions of the biopesticide Novodor^® on mite survival was assessed in laboratory studies. Survival of R. robini exposed to Cry3Aa in a short‐term contact and ingestion experiment was not affected, although R. robini was significantly affected by the insecticide Fipronil^® used as a positive control. Similarly, R. robini exposed in a longer duration feeding trial to the Cry3Aa toxin in artificial diet were also not significantly affected. When Cry3Aa was tested on the positive control insect, Leptinotarsa decemlineata, reduced weight of larvae and increased mortality was recorded. The effect of Cry3Bb1 toxin in transgenic corn tissues on R. robini food choice was assessed in a laboratory study. In no‐choice tests a greater proportion of R. robini were found on garlic roots than on Cry3Bb1 transgenic corn and a near‐isogenic non‐transgenic corn. In a choice test, more R. robini was recovered on garlic roots than on either corn variety, and on Cry3Bb1 corn than on non‐transgenic corn. In large field plots using specific mite traps across the growing season, R. robini mite populations were not significantly different between Cry3Bb1 corn and non‐transgenic corn alone or non‐transgenic corn treated with different combinations of two insecticides. Our results, combined with results from other studies, suggest that transgenic plants expressing the Cry3Aa or Cry3Bb1 Bacillus thuringiensis toxins are likely to have negligible impact on R. robini mite populations.     

Bt抗、感种群亚洲玉米螟幼虫取食Bt玉米后Cry1Ab杀虫蛋白在其体内的组织分布与含量

采用ELISA方法检测了实验室汰选的对Cry1Ab产生107倍抗性的亚洲玉米螟Ostrinia furnacalis (Guenée)种群与敏感种群3龄幼虫取食表达Cry1Ab杀虫蛋白的Bt玉米心叶后,杀虫蛋白在幼虫体内的分布情况。结果表明：Cry1Ab杀虫蛋白在抗性种群幼虫中的组织分布情况与敏感种群相近,主要存在于中肠组织和血淋巴中。抗、感种群中均以含有内含物的中肠组织中含量最高,分别为277.2 ng/g 和104.9 ng/g;其次为血淋巴,分别为93.7 ng/g 和69.5 ng/g;不含内含物的中肠组织中52.7 ng/g 和40.1 ng/g;在丝腺和马氏管组织的含量很低,丝腺中分别为8.5 ng/g和11.7ng/g,而马氏管中分别为6.7 ng/g和6.5 ng/g。脂肪体、生殖器官中未检测到杀虫蛋白。抗性种群中肠组织(含有内含物和不含内含物)中Cry1Ab的含量显著高于敏感种群。幼虫期取食过Bt玉米的亚洲玉米螟发育的蛹、成虫及其卵中均不含杀虫蛋白,说明Bt杀虫蛋白不会通过幼虫取食向蛹、成虫及卵传递。     

Transfer of Cry1Ac and Cry2Ab proteins from genetically engineered Bt cotton to herbivores and predators

With the cultivation of Bt cotton, the produced insecticidal Cry proteins are ingested by herbivores and potentially transferred along the food chain to natural enemies, such as predators. In laboratory experiments with Bollgard II cotton, concentrations of Cry1Ac and Cry2Ab were measured in Lepidoptera larvae (Spodoptera littoralis, Heliothis virescens), plant bugs (Euschistus heros), aphids (Aphis gossypii), whiteflies (Bemisia tabaci), thrips (Thrips tabaci, Frankliniella occidentalis), and spider mites (Tetranychus urticae). Tritrophic experiments were conducted with caterpillars of S. littoralis as prey and larvae of ladybird beetles (Harmonia axyridis, Adalia bipunctata) and lacewings (Chrysoperla carnea) as predators. Immunological measurements (ELISA) indicated that herbivores feeding on Bt cotton contained 5%–50% of the Bt protein concentrations in leaves except whiteflies and aphids, which contained no or only traces of Bt protein, and spider mites, which contained 7 times more Cry1Ac than leaves. Similarly, predators contained 1%–30% of the Cry protein concentration in prey. For the nontarget risk assessment, this indicates that Bt protein concentrations decrease considerably from one trophic level to the next in the food web, except for spider mites that contain Bt protein concentrations higher than those measured in the leaves. Exposure of phloem sucking hemipterans is negligible.     

Use of transgenic plants to measure insect herbivore movement

Use of ingested transgenic corn tissue as a marker for measuring movement of adult Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae; western corn rootworm) was investigated. Laboratory observations of beetles feeding on corn foliage, pollen, silks, or soybean foliage provided background on feeding patterns. The interval between food consumption and its appearance in feces (gut passage time) ranged from 102.7 +/- 11 min for soybean foliage to 56.7 +/- 2.9 min for corn silks. In a laboratory assay, protein expression tests identified the presence of Cry3Bb1 protein inside 50% of adult D. virgifera for up to 16 h after they had last consumed Cry3Bb1 protein-expressing corn silks from 'YieldGard Rootworm' corn plants (Monsanto Co.). Cry3Bb1 protein could not be detected by 32 h postfeeding. The proportion of Cry3Bb1 protein-positive beetles declined linearly with increasing time since feeding on 'YieldGard Rootworm' tissue. Approximately 20% of adult D. virgifera collected near 'YieldGard Rootworm' corn plots tested positive for Cry3Bb1 protein, indicating 'YieldGard Rootworm' tissue consumption within the last 16-32 h. Based on a 16- to 32-h postfeeding detection interval for Cry3Bb1 protein and the distance between 'YieldGard Rootworm' sources and sites where Cry3Bb1-positive insects were collected, 85.3% of males and females moved < or = 4.6-9.1 m/d through R2-R3 stage corn. Among Cry3Bb1-positive adults that left corn and were captured in an adjacent soybean field, 86.4% of males and 93.1% of females moved < or = 4.6-9.1 m/d through soybean. Detection of transgenic plant tissues in mobile insect herbivores is a novel application of biotechnology to the study of insect movement.     

Determination of baseline susceptibility to Cry1Ab protein for Asian corn borer (Lep., Crambidae)

Abstract: Although transgenic Bacillus thuringiensis (Bt) corn can provide a new tool for control of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), concern has been raised regarding the possibility of the target insect evolving resistance to the Bt protein under intensive selection pressure from Bt corn. Therefore, it is necessary to establish baseline data to enable detection of changes in susceptibility in field populations after prolonged exposure to Bt corn. Susceptibility to purified Cry1Ab protein from Bt was determined for 10 populations of ACB from the major corn‐growing regions of China, ranging geographically from Heilongjiang Province in the northeast to Shaanxi Province in the east‐central part. Neonate ACB were exposed to semi‐artificial diet incorporated with increasing Cry1Ab protein concentrations, and mortality and growth inhibition were evaluated after 7 days. The range of LC₅₀ (50% lethal concentration) among the populations was 0.10 to 0.81 μg/g (Cry1Ab protein/diet). Differences (P < 0.05) in susceptibility among the populations were significant. LC₅₀s generated from the Huanghuaihai Summer Corn Region were higher than those from the Spring Corn Regions. Bt was one of the significant natural biomortality factors of overwintering generation ACB. There was a significant correlation between percentage of the larvae infected with Bt and their LC₅₀ values to Cry1Ab protein in geographic distinct populations (r = 0.7350*, d.f. = 8, _r0.05 = 0.632). Based on the background of Bt formulations used for corn insect pests control in these areas, these differences were not caused by prior exposure to Bt insecticides. Instead, the small differences likely reflect natural Bt selection pressure. Because the variation in susceptibility to Cry1Ab was small (<10‐fold), the ACB apparently is susceptible to Cry1Ab across its range within China.