Nicholas P. Storer

Discovery and Characterization of Field Resistance to Bt Maize: Spodoptera frugiperda (Lepidoptera: Noctuidae) in Puerto Rico

ABSTRACT Transgenic maize, Zea mays L., event TC1507 produces the Cry1F protein to provide protection from feeding by several important lepidopteran pests, including Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). Reports of reduced field performance against this species in Puerto Rico were investigated, and laboratory bioassays showed that S. frugiperda collected from the affected area exhibited lower sensitivity to the Cry1F protein compared with typical colonies from other regions. The resistance was shown to be autosomally inherited and highly recessive. The Puerto Rico colony was shown to be moderately less sensitive than susceptible laboratory strains to Cry1Ab and Cry1Ac, but the differences in sensitivity were dramatically smaller than for Cry1F. Potential contributory factors to the emergence of resistance to Cry1F in Puerto Rico populations of S. frugiperda include the tropical island geography, unusually large population sizes in 2006, and drought conditions reducing the availability of alternative hosts. In response to this resistance incident, the technology providers have stopped commercial sales of TC1507 maize in Puerto Rico pending potential reversion to susceptibility.

Status of resistance to Bt maize in Spodoptera frugiperda: Lessons from Puerto Rico

In 2006, reports of potential Spodoptera frugiperda resistance to TC1507 maize in Puerto Rico were received. Subsequent investigation confirmed that pest populations collected from several sites in Puerto Rico were largely unaffected by the Cry1F protein in bioassays, with resistance ratios likely in excess of 1000. Since then, we have continued monitoring populations in Puerto Rico and in southern areas of the mainland US. The majority of the collections from Puerto Rico continue to show high levels of Cry1F resistance whereas populations collected from the southern US mainland continue to show full susceptibility to Cry1F and TC1507 maize. It does not appear that resistant populations have spread to any measurable extent from Puerto Rico to mainland US, nor that local selection pressure from Cry1F-expressing maize or cotton production in the southern US has caused a measurable change in population susceptibility. Lessons learned from Puerto Rico are being applied in other parts of the Americas where TC1507 maize is grown and additional steps being taken to protect the long-term durability of Cry1F in maize in areas where similar selection pressure may be expected. Tactics include using locally-adapted germplasm that contain native Spodoptera resistance, a robust education program to teach end-users about the potential for resistance to develop appropriate crop stewardship, resistance monitoring, and the use of insecticides under high S. frugiperda pressure. Perhaps most importantly, pyramided trait products that produce two or more different Bt proteins are being introduced to further delay resistance development to Cry1F.

Selection for Cry1F resistance in the European corn borer and cross‐resistance to other Cry toxins

Evolution of resistance by insect pests is the greatest threat to the continued success of Bacillus thuringiensis (Bt) toxins used in insecticide formulations or expressed by transgenic crop plants such as Cry1F‐expressing maize [(Zea mays L.) (Poaceae)]. A strain of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), obtained from field collections throughout the central US Corn Belt in 1996 was selected in the laboratory for resistance to Cry1F by exposure to the toxin incorporated into artificial diet. The selected strain developed more than 3000‐fold resistance to Cry1F after 35 generations of selection and readily consumed Cry1F expressing maize tissue; yet, it was as susceptible to Cry1Ab and Cry9C as the unselected control strain. Only a low level of cross‐resistance (seven‐fold) to Cry1Ac was observed. These lacks of cross‐resistance between Cry1F and Cry1Ab suggest that maize hybrids expressing these two toxins are likely to be compatible for resistance management of O. nubilalis.

Inheritance of Cry1F resistance in laboratory-selected European corn borer and its survival on transgenic corn expressing the Cry1F toxin

A major assumption of the high-dose/refuge strategy proposed for insect resistance management strategies for transgenic crop plants that express toxins from Bacillus thuringiensis is that resistance traits that evolve in pest species will be recessive. The inheritance of Cry1F resistance and larval survival on commercially available Cry1F corn hybrids were determined in a laboratory-selected strain of European corn borer, Ostrinia nubilalis (Hübner), displaying more than 3000-fold resistance to Cry1F. Concentration-response bioassays of reciprocal parental crosses indicated that the resistance is autosomal and recessive. Bioassays of the backcross of the F1 generation with the selected strain were consistent with the hypothesis that a single locus, or a set of tightly linked loci, is responsible for the resistance. Greenhouse experiments with Cry1F-expressing corn hybrids indicated that some resistant larvae survived the high dose of toxin delivered by Cry1F-expressing plants although F1 progeny of susceptible by resistant crosses had fitness close to zero. These results provide the first direct evidence that the high dose/refuge strategy currently in place to manage resistance in Cry1F-expressing corn is appropriate.

Stability of a set of allergens and non-allergens in simulated gastric fluid

Stability in simulated gastric fluid has been suggested as a parameter for consideration in the allergenicity assessment of transgenic proteins. However, the relationship between the stability of proteins in simulated gastric fluid and allergenicity has been inconsistent among studies conducted with reference allergens and non-allergens. Differences in laboratory methods and data interpretation have been implicated as possible causes for conflicting study results. We attempted to mitigate some of the methodological inconsistencies among laboratory methods by applying a kinetic interpretation to results of digestion experiments conducted with a set of known allergens and putative non-allergens. We found that pepsinolysis in simulated gastric fluid generally followed an exponential (pseudo-first-order) pattern of decay, at least during the terminal (slower) phase of digestion, allowing the calculation of digestion half-lives. While digestibility estimates were reproducible and robust, results for the proteins evaluated in this study did not support a significant association between stability in simulated gastric fluid and allergenicity.

Digestion assays in allergenicity assessment of transgenic proteins

The food-allergy risk assessment for transgenic proteins expressed in crops is currently based on a weight-of-evidence approach that holistically considers multiple lines of evidence. This approach recognizes that no single test or property is known to distinguish allergens from nonallergens. The stability of a protein to digestion, as predicted by an in vitro simulated gastric fluid assay, currently is used as one element in the risk assessment process. A review of the literature on the use of the simulated gastric fluid assay to predict the allergenic status of proteins suggests that more extensive kinetic studies with well-characterized reference proteins are required before the predictive value of this assay can be adequately judged.

Estimating the frequency of Cry1F resistance in field populations of the European corn borer (Lepidoptera: Crambidae)

BACKGROUND Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) have suppressed European corn borer populations and reduced the pest status of this insect throughout much of the US corn belt. A major assumption of the high-dose/refuge strategy proposed for insect resistance management and Bt corn is that the frequency of resistance alleles is low so that resistant pests surviving exposure to Bt corn will be rare. RESULTS The frequency of resistance to the Cry1F Bt toxin was estimated using two different screening tools and compared with annual susceptibility monitoring based on diagnostic bioassays and LC50 and EC50 determinations. An F1 screening approach where field-collected individuals were mated to a resistant laboratory strain and progeny were assayed to determine genotype revealed that resistance alleles could be recovered even during the first year of commercially available Cry1F corn (2003). Estimates of frequency from 2003-2005 and 2006-2008 indicated that, although allele frequency was higher than theoretical assumptions (0.0286 and 0.0253 respectively), there was no indication that the frequency was increasing. Similar estimates in 2008 and 2009 using an F2 screening approach confirmed the presence of non-rare resistance alleles (frequency ≈ 0.0093 and 0.0142 for 2008 and 2009, respectively). The results of both screening methods were in general agreement with the observed mortality in diagnostic bioassays and LC50 and EC50 determinations. CONCLUSIONS These results are consistent with previous modeling results, suggesting that the high-dose/refuge strategy that is in place for Bt corn may be effective in delaying resistance evolution even when a relatively high frequency of resistance alleles exists.

Measurements of Cry1F binding and activity of luminal gut proteases in susceptible and Cry1F resistant Ostrinia nubilalis larvae (Lepidoptera: Crambidae).

The biochemical mechanism of resistance to the Bacillus thuringiensis Cry1F toxin was studied in a laboratory-selected strain of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) showing more than 3000-fold resistance to Cry1F and limited cross resistance to other Cry toxins. Analyses of Cry1F binding to brush border membrane vesicles of midgut epithelia from susceptible and resistant larvae using ligand immunoblotting and Surface Plasmon Resonance (SPR) suggested that reduced binding of Cry1F to insect receptors was not associated with resistance. Additionally, no differences in activity of luminal gut proteases or altered proteolytic processing of the toxin were observed in the resistant strain. Considering these results along with previous evidence of relatively narrow spectrum of cross resistance and monogenic inheritance, the resistance mechanism in this Cry1F selected strain of O. nubilalis appears to be specific and may be distinct from previously identified resistance mechanisms reported in other Lepidoptera.

Assessing the ecological risks from the persistence and spread of feral populations of insect-resistant transgenic maize.

One source of potential harm from the cultivation of transgenic crops is their dispersal, persistence and spread in non-agricultural land. Ecological damage may result from such spread if the abundance of valued species is reduced. The ability of a plant to spread in non-agricultural habitats is called its invasiveness potential. The risks posed by the invasiveness potential of transgenic crops are assessed by comparing in agronomic field trials the phenotypes of the crops with the phenotypes of genetically similar non-transgenic crops known to have low invasiveness potential. If the transgenic and non-transgenic crops are similar in traits believed to control invasiveness potential, it may be concluded that the transgenic crop has low invasiveness potential and poses negligible ecological risk via persistence and spread in non-agricultural habitats. If the phenotype of the transgenic crop is outside the range of the non-transgenic comparators for the traits controlling invasiveness potential, or if the comparative approach is regarded as inadequate for reasons of risk perception or risk communication, experiments that simulate the dispersal of the crop into non-agricultural habitats may be necessary. We describe such an experiment for several commercial insect-resistant transgenic maize events in conditions similar to those found in maize-growing regions of Mexico. As expected from comparative risk assessments, the transgenic maize was found to behave similarly to non-transgenic maize and to be non-invasive. The value of this experiment in assessing and communicating the negligible ecological risk posed by the low invasiveness potential of insect-resistant transgenic maize in Mexico is discussed.

Evaluation of Corn Hybrids Expressing Cry34Ab1/Cry35Ab1 and Cry3Bb1 Against the Western Corn Rootworm (Coleoptera: Chrysomelidae)

ABSTRACT Studies were conducted across nine U.S. states, over 5 yr, to characterize the efficacy of transgenic corn (Zea mays L.) hybrids producing insecticidal proteins derived from Bacillus thuringiensis (Bt) for control of western corn rootworm, Diabrotica virgifera virgifera Le Conte. Hybrids tested had the same genetic background, contained one of two single events (DAS-59122-7 expressing Cry34Ab1/Cry35Ab1 or MON 88017 expressing Cry3Bb1) or a pyramid consisting of both rootworm-active events (SmartStax traits) and were compared with a non-Bt near isoline. Frequency analyses of root feeding data showed that hybrids containing both events sustained less root damage (0–3 node injury scale) than hybrids containing either event alone. The levels of root protection provided by MON 88017 and DAS-59122-7 were not different from each other. Efficacy was also evaluated based on consistency of protection, based on the proportion of plants with root ratings of either ≤0.25 or <1.00 on the node injury scale. The combination of two modes of action in SmartStax provided greater product consistency over a single mode of action at the 0.25 level and all hybrids producing Bt proteins provided equally high consistency at the 1.00 level. Overall these data show single and multiple mode of action hybrids provided high, consistent protection over the past 5 yr across the trial geography; however, pyramiding the rootworm Bt events provided greater and more consistent root protection. These findings also support that pyramided traits like SmartStax (Cry3Bb1 + Cry34Ab1/Cry35Ab1) remain a viable strategy for delaying resistance to either trait.