Graham P. Head

Cry1Ab protein levels in phytophagous insects feeding on transgenic corn: implications for secondary exposure risk assessment

Enzyme‐linked immunosorbent assays (ELISA) and bioassays were used to estimate levels of Cry1Ab protein in four species of phytophagous insects after feeding on transgenic Bt‐corn plants expressing Cry1Ab protein or artificial diets containing Cry1Ab protein. The level of Cry1Ab in insects feeding on sources containing the Cry1Ab protein was uniformly low but varied with insect species as well as food source. For the corn leaf aphid, Rhopalosiphum maidis (Fitch), feeding on diet solutions containing Cry1Ab protein, the level of the protein in the aphid was 250–500 times less than the original levels in the diet, whereas no Cry1Ab was detected by ELISA in aphids feeding on transgenic Bt‐Corn plants. For the lepidopteran insects, Ostrinia nubilalis (Hübner), Helicoverpa zea (Boddie), and Agrotis ipsilon (Hufnagel), levels of Cry1Ab in larvae varied significantly with feeding treatment. When feeding for 24 h on artificial diets containing 20 and 100 ppm of Cry1Ab, the level of Cry1Ab in the larvae was about 57 and 142 times lower, respectively, than the original protein level in the diet for O. nubilalis, 20 and 34 times lower for H. zea, and 10 to 14 times lower for A. ipsilon. Diet incorporation bioassays with a susceptible insect (first instar O. nubilalis) showed significant Cry1Ab bioactivity present within whole body tissues of R. maidis and O. nubilalis that had fed on diet containing a minimum of 20 ppm or higher concentrations (100 or 200 ppm) of Cry1Ab, but no significant bioactivity within the tissues of these insects after feeding on transgenic Bt‐corn plants. The relevance of these findings to secondary exposure risk assessment for transgenic Bt crops is discussed.

No Detection of Cry1Ac Protein in Soil After Multiple Years of Transgenic Bt Cotton (Bollgard) Use

Abstract Soil samples were collected from within and outside six fields where insect-resistant transgenic cotton (Bollgard) encoding the Bacillus thuringiensis Berliner (Bt) subsp. kurstaki cry1Ac gene had been grown and subsequently incorporated into soil by postharvest tillage for 3–6 consecutive years. The level of Cry1Ac protein in these samples (collected 3 mo after the last season’s tillage) was evaluated using both enzyme-linked immunosorbent assays (ELISA) and bioassays with a susceptible insect species, Heliothis virescens (F.), the tobacco budworm. Both methods revealed that no detectable Cry1Ac protein was present in any of the soil samples collected from within or outside the Bollgard fields. Based on the results from reference standards, the limit of detection for the ELISA was 3.68 ng of extractable protein per gram of soil, and that of the bioassay (measured by EC50) was 8 ng of biologically active protein per gram of soil. Together, these findings demonstrate that the amount of Cry1Ac protein accumulated as a result of continuous use of transgenic Bt cotton, and subsequent incorporation of plant residues into the soil by postharvest tillage, is extremely low and does not result in detectable biological activity.

Transgenic Bt potato and conventional insecticides for Colorado potato beetle management: comparative efficacy and non-target impacts

Field studies were conducted in 1992 and 1993 in Hermiston, Oregon, to evaluate the efficacy of transgenic Bt potato (Newleaf®, which expresses the insecticidal protein Cry3Aa) and conventional insecticide spray programs against the important potato pest, Leptinotarsa decemlineata (Say), Colorado potato beetle (CPB), and their relative impact on non‐target arthropods in potato ecosystems. Results from the two years of field trials demonstrated that Newleaf potato plants were highly effective in suppressing populations of CPB, and provided better CPB control than weekly sprays of a microbial Bt‐based formulation containing Cry3Aa, bi‐weekly applications of permethrin, or early‐ and mid‐season applications of systemic insecticides (phorate and disulfoton). When compared with conventional potato plants not treated with any insecticides, the effective control of CPB by Newleaf potato plants or weekly sprays of a Bt‐based formulation did not significantly impact the abundance of beneficial predators or secondary potato pests. In contrast to Newleaf potato plants or microbial Bt formulations, however, bi‐weekly applications of permethrin significantly reduced the abundance of several major generalist predators such as spiders (Araneae), big‐eyed bugs (Geocorus sp.), damsel bugs (Nabid sp.), and minute pirate bugs (Orius sp.), and resulted in significant increases in the abundance of green peach aphid (GPA), Myzus persicae (Sulzer) – vector of viral diseases, on the treated potato plots. While systemic insecticides appeared to have reduced the abundance of some plant sap‐feeding insects such as GPA, lygus bugs, and leafhoppers, early and mid‐season applications of these insecticides had no significant impact on populations of the major beneficial predators. Thus, transgenic Bt potato, Bt‐based microbial formulations and systemic insecticides appeared to be compatible with the development of integrated pest management (IPM) against other potato pests such as GPA because these CPB control measures have little impact on major natural enemies. In contrast, the broad‐spectrum pyrethroid insecticide (permethrin) is less compatible with IPM programs against GPA and the potato leafroll viral disease.

How Governmental Regulation Can Help or Hinder the Integration of Bt Crops within IPM Programs

Regulatory risk assessments are an important part of the introduction of insect-resistant genetically modified (GM) crops (e.g., Bacillus thuringiensis [Bt] crops) into the environment to ensure the safe use of such products. In doing so, the regulatory assessment process can be clearly beneficial to integrated pest management (IPM) programs. In general, the regulatory framework for insect-resistant GM crops includes an assessment of the following: effects of the insecticidal trait on non-target organisms, other potential adverse environmental impacts, evolution of resistance to target pests, and environmental and agronomic benefits of the insecticidal trait. Each country’s regulatory system is dependent on the overall environmental risk management goals, relevant and available risk information, scientific capacity, and the available financial resources. A number of regulatory activities can help to ensure that new products such as Bt crops fit well within IPM programs: (1) evaluation of the environmental safety of new products, and their ability to enhance IPM; (2) encouragement of the adoption of new technologies with improved environmental safety profiles; (3) adoption of an expedited regulatory review system; and (4) encouragement and appropriate oversight of sustainable use of such products. Governmental regulation of insect-resistant GM crops can also hinder IPM programs by creating significant barriers to the adoption of such technologies. Such barriers include: (1) absence of functioning regulatory systems in many developing countries; (2) meeting the obligations and understanding the various interpretations of international treaties, e.g., Cartagena Protocol on Biosafety; (3) lack of public sector research to generate data supporting the safety of these crops; and (4) regulatory costs involved in the development and commercialization of novel products for small market sectors. Ways in which regulatory data requirements can be globally harmonized need to be considered to decrease the regulatory barriers for insect-resistant GM crops and comparable technologies. International organizations can play a key role in rationalizing regulatory systems; however, public sector research will also be needed to make sure that the risk assessment process is scientifically sound and transparent.

A Multiyear, Large-Scale Comparison of Arthropod Populations on Commercially Managed Bt and Non-Bt Cotton Fields

Abstract Field studies were conducted in 2000–2002 to compare foliage-dwelling arthropod populations on Bacillus thuringiensis Berliner (Bt) (Bollgard) cotton and non-Bt (conventional) cotton season-long in South Carolina, Georgia, northern Alabama, and southern Alabama. For each of these four regions, three or four paired fields were sampled weekly in each of the 3 yr. Each pair of fields consisted of a Bt and a non-Bt cotton field, both at least 5 ha in size. The dominant arthropod taxa collected included target pests (heliothine moths and Spodoptera spp.), nontarget pests (stink bugs and plant bugs), and generalist natural enemies [Geocoris spp., Orius spp., Solenopsis invicta (Buren), ladybeetles, and spiders]. Where target pests were present, particularly Helicoverpa zea (Boddie), their numbers were consistently significantly lower in the Bt cotton fields. Natural enemy populations generally were not significantly different between the Bt and the non-Bt cotton fields (50% of all comparisons) and, where significant differences were present, natural enemy abundance usually was higher in the Bt than the non-Bt cotton fields. These differences were correlated with lower insecticide use on the Bt than the non-Bt cotton fields, particularly in South Carolina, where target pest pressure was heaviest. When presented with insect eggs or larvae as prey items, the larger natural enemy populations in Bt cotton fields exhibited significantly higher predation rates. These results show that Bt cotton has no significant adverse impacts on the nontarget arthropod populations studied and, compared with insecticide-treated non-Bt cotton, Bt cotton supports higher natural enemy populations with significant positive impacts on biological control.

Control of Resistant Pink Bollworm (Pectinophora gossypiella) by Transgenic Cotton That Produces Bacillus thuringiensis Toxin Cry2Ab

ABSTRACT Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 μg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 μg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 μg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.

Cry1F Resistance in Fall Armyworm Spodoptera frugiperda: Single Gene versus Pyramided Bt Maize

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012–2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda.

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.

Field evaluation of the impact of corn rootworm (Coleoptera: Chrysomelidae) -protected Bt corn on foliage-dwelling arthropods

Abstract A 3-yr field study was conducted in Monmouth, IL, to evaluate the effect of transgenic Bt corn expressing a Cry3Bb1 protein (MON 863) on foliage-dwelling arthropods. The study employed a split-plot design with MON 863 corn and a conventional non-Bt near isoline (RX670) as the main plots and insecticide regimens (no insecticide, imidacloprid applied as seed treatment, tefluthrin applied as soil treatment, and permethrin applied as foliar treatment) as subplots. Foliage-dwelling arthropods were sampled with sticky traps during each of the 3 yr (2000–2002). The most abundant taxa collected included corn pests, such as Diabrotica virgifera virgifera LeConte and Chaetocnema pulicaria Melsheimer (Coleoptera:Chrysomelidae), and Rhopalosiphum maidis Fitch (Homoptera:Aphididae). The most abundant generalist predators captured by sticky traps were Coccinellidae (ladybird beetles), Nabidae (damsel bugs), Orius insidiosus Say (minute pirate bugs), Syrphidae (flower flies), Chrysoperla carnea Stephens (green lacewings), Macrocentrus cingulum Rienhardt (a braconid parasitoid), and Araneae (spiders). MON 863 corn had no consistent adverse impact on the relative abundance of any nontarget foliage-dwelling arthropod taxon, including predators and parasitoids. However, insecticide applications of foliar insecticide (permethrin) significantly and consistently decreased the abundance of ladybird beetles, green lacewings, and damsel bugs compared with the insecticidal seed treatment or no insecticide application. The abundance of the pest R. maidis also was observed to increase in the foliar-applied insecticide treatment. Therefore, adoption of MON 863 and the concurrent reduction in broad-spectrum foliar-applied insecticide use for control of adult Diabrotica spp. have the potential to enhance biological control within corn agro-ecosystems.

Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins.

Sugarcane borer, Diatraea saccharalis (F.), is a primary corn stalk borer pest targeted by transgenic corn expressing Bacillus thuringiensis (Bt) proteins in many areas of the mid-southern region of the United States. Recently, genes encoding for Cry1A.105 and Cry2Ab2 Bt proteins were transferred into corn plants (event MON 89034) for controlling lepidopteran pests. This new generation of Bt corn with stacked-genes of Cry1A.105 and Cry2Ab2 will become commercially available in 2009. Susceptibility of Cry1Ab-susceptible and -resistant strains of D. saccharalis were evaluated on four selected Bt proteins including Cry1Aa, Cry1Ac, Cry1A.105, and Cry2Ab2. The Cry1Ab-resistant strain is capable of completing its larval development on commercial Cry1Ab-expressing corn plants. Neonates of D. saccharalis were assayed on a meridic diet containing one of the four Cry proteins. Larval mortality, body weight, and number of surviving larvae that did not gain significant weight (<0.1mg per larva) were recorded after 7 days. Cry1Aa was the most toxic protein against both insect strains, followed in decreasing potency by Cry1A.105, Cry1Ac, and Cry2Ab2. Using practical mortality (larvae either died or no significant weight gain after 7 days), the median lethal concentration (LC(50)) of the Cry1Ab-resistant strain was estimated to be >80-, 45-, 4.1-, and -0.5-fold greater than that of the susceptible strain to Cry1Aa, Cry1Ac, Cry1A.105 and Cry2Ab2 proteins, respectively. This information should be useful to support the commercialization of the new Bt corn event MON 89034 for managing D. saccharalis in the mid-southern region of the United States.