High-dose Refuge Research Articles

Performance of Sesamia nonagrioides on cultivated and wild host plants: Implications for Bt maize resistance management.

BACKGROUND Sesamia nonagrioides is an important maize pest in the Mediterranean basin that is effectively controlled by Cry1Ab‐expressing maize (Bt maize). The continued cultivation of Bt maize in Spain exerts high selection pressure on the target pests, which could lead to the development of resistance. Provision of refuges of non‐Bt plants is an essential component in the high‐dose/refuge (HDR) strategy to delay resistance evolution. Here we analyze the suitability of cultivated (rice and sorghum) and wild (Johnsongrass, cattail, common reed and giant reed) plants, reported as hosts of S. nonagrioides, for larval development and oviposition of this pest compared to maize, and we evaluate their potential role in delaying resistance development to Bt maize.RESULTSBioassays conducted with plant pieces or whole plants showed that the larval cycle could only be completed in the three cultivated plants and in Johnsongrass. Females showed a strong preference for ovipositing on maize in comparison with sorghum or rice. Although young larvae consumed more sorghum than maize in two‐choice bioassays, both larvae and adults had a better performance (shorter larval period and higher pupal weight, fecundity and fertility) when larvae fed on maize throughout their larval stage than when they fed on sorghum or rice.CONCLUSIONNone of the alternative hosts of S. nonagrioides tested here should be considered as natural unstructured refuges within the HDR strategy for Bt maize and this pest in Spain, as some of the necessary requirements to fulfill this strategy would not be met. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Efficacy of a cry1Ab Gene for Control of Maruca vitrata (Lepidoptera: Crambidae) in Cowpea (Fabales: Fabaceae).

Cowpea [Vigna unguiculata (L) Walp.] is an important staple legume in the diet of many households in sub-Saharan Africa. Its production, however, is negatively impacted by many insect pests including bean pod borer, Maruca vitrata F., which can cause 20-80% yield loss. Several genetically engineered cowpea events that contain a cry1Ab gene from Bacillus thuringiensis (Bt) for resistance against M. vitrata were evaluated in Nigeria, Burkina Faso, and Ghana (West Africa), where cowpea is commonly grown. As part of the regulatory safety package, these efficacy data were developed and evaluated by in-country scientists. The Bt-cowpea lines were planted in confined field trials under Insect-proof netting and artificially infested with up to 500 M. vitrata larvae per plant during bud formation and flowering periods. Bt-cowpea lines provided nearly complete pod and seed protection and in most cases resulted in significantly increased seed yield over non-Bt control lines. An integrated pest management strategy that includes use of Bt-cowpea augmented with minimal insecticide treatment for protection against other insects is recommended to control pod borer to enhance cowpea production. The insect resistance management plan is based on the high-dose refuge strategy where non-Bt-cowpea and natural refuges are expected to provide M. vitrata susceptible to Cry1Ab protein. In addition, there will be a limited release of this product until a two-toxin cowpea pyramid is released. Other than South African genetically engineered crops, Bt-cowpea is the first genetically engineered food crop developed by the public sector and approved for release in sub-Saharan Africa.

First detection of a Sesamia nonagrioides resistance allele to Bt maize in Europe

The Ebro Valley (Spain) is the only hotspot area in Europe where resistance evolution of target pests to Cry1Ab protein is most likely, owing to the high and regular adoption of Bt maize (>60%). The high-dose/refuge (HDR) strategy was implemented to delay resistance evolution, and to be effective it requires the frequency of resistance alleles to be very low (<0.001). An F2 screen was performed in 2016 to estimate the frequency of resistance alleles in Sesamia nonagrioides from this area and to evaluate if the HDR strategy is still working effectively. Out of the 137 isofemale lines screened on Cry1Ab maize leaf tissue, molted larvae and extensive feeding were observed for two consecutive generations in one line, indicating this line carried a resistance allele. The frequency of resistance alleles in 2016 was 0.0036 (CI 95% 0.0004–0.0100), higher but not statistically different from the value obtained in 2004–2005. Resistance does not seem to be evolving faster than predicted by a S. nonagrioides resistance evolution model, but the frequency of resistance is now triple the value recommended for an effective implementation of the HDR strategy. Owing to this, complementary measures should be considered to further delay resistance evolution in the Ebro Valley.

Impact of violated high-dose refuge assumptions on evolution of Bt resistance.

Transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been widely and successfully deployed for the control of target pests, while allowing a substantial reduction in insecticide use. The evolution of resistance (a heritable decrease in susceptibility to Bt toxins) can pose a threat to sustained control of target pests, but a high‐dose refuge (HDR) management strategy has been key to delaying countervailing evolution of Bt resistance. The HDR strategy relies on the mating frequency between susceptible and resistant individuals, so either partial dominance of resistant alleles or nonrandom mating in the pest population itself could elevate the pace of resistance evolution. Using classic Wright‐Fisher genetic models, we investigated the impact of deviations from standard refuge model assumptions on resistance evolution in the pest populations. We show that when Bt selection is strong, even deviations from random mating and/or strictly recessive resistance that are below the threshold of detection can yield dramatic increases in the pace of resistance evolution. Resistance evolution is hastened whenever the order of magnitude of model violations exceeds the initial frequency of resistant alleles. We also show that the existence of a fitness cost for resistant individuals on the refuge crop cannot easily overcome the effect of violated HDR assumptions. We propose a parametrically explicit framework that enables both comparison of various field situations and model inference. Using this model, we propose novel empiric estimators of the pace of resistance evolution (and time to loss of control), whose simple calculation relies on the observed change in resistance allele frequency.

The effect of nitrogen rate on transgenic corn Cry3Bb1 protein expression

Combining herbicide-resistant and Bacillus thuringiensis (Bt) traits in corn (Zea mays L.) hybrids may affect insect resistance management owing to volunteer corn. Some Bt toxins may be expressed at lower levels by nitrogen-deficient corn roots. Corn plants with sublethal levels of Bt expression could accelerate the evolution of Bt resistance in target insects. The present objective was to quantify the concentration of Bt (Cry3Bb1) in corn root tissue with varying tissue nitrogen concentrations. Expression of Cry3Bb1 toxin in root tissue was highly variable, but there were no differences in the overall concentration of Cry3Bb1 expressed between roots taken from Cry3Bb1-positive volunteer and hybrid corn plants. The nitrogen rate did affect Cry3Bb1 expression in the greenhouse, less nitrogen resulted in decreased Cry3Bb1 expression, yet this result was not documented in the field. A positive linear relationship of plant nitrogen status on Cry3Bb1 toxin expression was documented. Also, high variability in Cry3Bb1 expression is potentially problematic from an insect resistance management perspective. This variability could create a mosaic of toxin doses in the field, which does not fit into the high-dose refuge strategy and could alter predictions about the speed of evolution of resistance to Cry3Bb1 in western corn rootworm Diabrotica virgifera virgifera LeConte.

Resistance evolution to the first generation of genetically modified Diabrotica-active Bt-maize events by western corn rootworm: management and monitoring considerations

Western corn rootworm (Diabrotica virgifera virgifera; WCR) is a major coleopteran maize pest in North America and the EU, and has traditionally been managed through crop rotation and broad-spectrum soil insecticides. Genetically modified Bt-maize offers an additional management tool for WCR and has been valuable in reducing insecticide use and increasing farm income. A concern is that the widespread, repeated, and exclusive deployment of the same Bt-maize transformation event will result in the rapid evolution of resistance in WCR. This publication explores the potential of WCR to evolve resistance to plant-produced Bt-toxins from the first generation of Diabrotica-active Bt-maize events (MON 863 and MON 88017, DAS-59122-7 and MIR604), and whether currently implemented risk management strategies to delay and monitor resistance evolution are appropriate. In twelve of the twelve artificial selection experiments reported, resistant WCR populations were yielded rapidly. Field-selected resistance of WCR to Cry3Bb1 is documented in some US maize growing areas, where an increasing number of cases of unexpected damage of WCR larvae to Bt-maize MON 88017 has been reported. Currently implemented insect resistance management measures for Bt-crops usually rely on the high dose/refuge (HDR) strategy. Evidence (including laboratory, greenhouse and field data) indicates that several conditions contributing to the success of the HDR strategy may not be met for the first generation of Bt-maize events and WCR: (1) the Bt-toxins are expressed heterogeneously at a low-to-moderate dose in roots; (2) resistance alleles may be present at a higher frequency than initially assumed; (3) WCR may mate in a non-random manner; (4) resistance traits could have non-recessive inheritance; and (5) fitness costs may not necessarily be associated with resistance evolution. However, caution must be exercised when extrapolating laboratory and greenhouse results to field conditions. Model predictions suggest that a 20% refuge of non-Diabrotica-active Bt-maize can delay resistance evolution in WCR under certain conditions. This publication concludes that further research is needed to resolve the remaining scientific uncertainty related to the appropriateness of the HDR in delaying resistance evolution in WCR, resistance monitoring is essential to detect early warning signs indicating resistance evolution in the field, and that integrated pest management reliant on multiple tactics should be deployed to ensure effective long-term corn rootworm management and sustainable use of Bt-maize.

La pyrale se disperse-t-elle suffisamment pour limiter durablement la résistance au maïs Bt via la stratégie « haute dose/refug

The use of transgenic crops producing toxins from the bacterium Bacillus thuringiensis - or Bt crops - is associated with the risk that the targeted pests become resistant to these toxins. To reduce this risk, the US government required the implementation of a strategy named High Dose/Refuge (HDR). This strategy is based on maintaining Bt toxin-free plants or crops - referred to as « refuges » - to preserve a pool of susceptible insects in the vicinity of Bt fields. Among other factors, its efficiency relies on a high gene flow between these susceptible individuals and any resistant individuals selected in Bt fields. For several pests targeted by these toxins, this strategy was nevertheless implemented when little was in fact know, as to the life history traits likely to influence the intensity of the gene flow. Part of this gap has been filled since then: we summarize here the recent advances on the European corn borer (ECB), one of the main targets of insecticidal Bt maize. Although this moth pest is highly polyphagous, its other host plants - whether wild or cultivated - do not provide a sufficient source of susceptible individuals to efficiently prevent toxin resistance from developing. Moreover, a fraction of the ECB reproduce in close vicinity of their place of emergence, so that refuges situated a few hundred meters from Bt maize fields - the maximal distance currently required is 800 meters - may not warrant a sufficient intermixing between susceptible and resistant individuals. In crop rotation situations, this intermixing could however be facilitated through a contrasted management of herbaceous maize field borders. Although 10 years after the beginning of Bt maize cultivation no resistance has yet broken out, our data suggests that it is illusory to aim at a universally suitable strategy, and that the HDR strategy - as currently implemented - may not necessarily be optimal.

Dispersal propensity and settling preferences of European corn borers in maize field borders

Summary Bacillus thuringiensis (Bt) crops kill pest larvae but have led to resistance evolution in several target pests. The high dose‐refuge (HDR) strategy aimed at delaying Bt resistance evolution depends on dispersal patterns of target pests. Examination of adult dispersal of the European corn borer Ostrinia nubilalis (ECB), the main target of Bt maize, can help to improve resistance management. Estimated recapture rates over 20 mark–release–recapture sessions in herbaceous field borders, where ECB adults rest during the day and mate at night, were used to examine the influence of sex, release period and site on ECB dispersal. Data from an additional 30 sessions were used to test the influence of night temperature, humidity, dew index and wind speed. Average recaptures within 50 m of release were lower 12 h after night (7·7%) than 12 h after day (34·5%) releases, did not differ between sexes, and decreased during nights with higher temperatures and lower wind speed. Local habitat had a major influence on dispersal. The number of unmarked adults caught initially in a given section of field border was strongly correlated with those subsequently captured in the same section, suggesting that moths flying in from the surroundings consistently settle in the same preferred spots. Moreover, recapture rates of marked adults were positively correlated with the prior density of unmarked adults in the release section. The spatial distribution of recaptured moths around the release point suggests that they moved on a very local scale, while those not recaptured probably left the area by a different, long‐range type of dispersal. Synthesis and applications. A proportion of European corn borer adults typically remained within a few metres of their initial location for at least 12 h. This should favour non‐random mating early in the flight season when nights are cold, population mixture is low and most individuals are unmated. Non‐random mating can accelerate the evolution of resistance, but this effect may be offset by non‐random oviposition. Our findings suggest that the intensity and direction of dispersal could be manipulated by field border management. Our data on the range and prevalence of short‐range dispersal and the factors influencing this process, support the view that resistance evolution is multifactorial. Our results can be used to parameterize multifactorial models from which specific management recommendations can be formulated.

Insecticide Use in Bt and Non-Bt Field Corn in the Western Corn Belt: As Reported by Crop Consultants in a Mail Survey

The use of foliar insecticides to control arthropod pests in corn is reported to be more extensive in the western high plains (western Kansas and Nebraska and bordering areas) than in the Midwest corn belt. Recently, the extent of insecticide use has become an important matter because the U.S. Environmental Protection Agency (EPA) has implemented the high-dose–refuge strategy for insect resistance management (IRM) to delay the development of resistance to transgenic corn in the European corn borer, Ostrinia nubilalis (Hubner). The use of foliar insecticide applications could reduce the effectiveness of the refuge for the IRM strategy. To get better data on this matter, we developed a crop consultant survey to collect information on insecticide use on Bt and non-Bt corn in Kansas and Nebraska. Data were compiled according to three agronomic regions within these two states based on different agricultural practices and insect complexes (southwest, west, and east). The consultants reported that during the early-whorl stage, relatively few corn acres were treated with foliar insecticides (≤14.2%) in any of the three regions, and the trends suggested that non-Bt corn received slightly more insecticide than did Bt corn. The consultants also reported that during late-whorl stage, the dryland corn acres also received fewer foliar insecticide treatments (≤12.4%) and again, non-Bt corn received slightly more treatment than did Bt corn. However, the consultants reported that during the late-whorl stage, irrigated corn received considerable foliar insecticide treatment, particularly in the southwest region: 66% in the non-Bt corn and 40% in the Bt corn. They also reported that 34% and 31% of these treated acreages received two treatments for pests, such as southwestern corn borer, Diatraea grandiosella Dyar, and spider mites, Tetranychidae (which are not as important in the other regions), in addition to treatments for European corn borers and western corn rootworm, Diabrotica virgifera virgifera LeConte (which are present in all three regions). The difference in insecticide use between non-Bt and Bt corn was 26, 6, and 19% in the southwest, west, and east regions (respectively) during 2002, and 20, 12, and 12% in the respective regions during the previous 5 yr. This level of treatment differential between Bt and non-Bt could potentially reduce the time to resistance in the European corn borer from 30 yr to 18 yr based on modeling projections with similar assumptions. These data also document that the use of insecticides is lower in Bt corn than in non-Bt corn, making this one of the first surveys to document a reduction in the use of foliar insecticides in Bt corn as compared with non-Bt corn.

Mating Patterns and Bt Resistance: The Case of the European Corn Borer

Mating Patterns and Bt Resistance: The Case of the European Corn Borer

Resistance Evolution to Bt Crops: Predispersal Mating of European Corn Borers

Over the past decade, the high-dose refuge (HDR) strategy, aimed at delaying the evolution of pest resistance to Bacillus thuringiensis (Bt) toxins produced by transgenic crops, became mandatory in the United States and is being discussed for Europe. However, precopulatory dispersal and the mating rate between resident and immigrant individuals, two features influencing the efficiency of this strategy, have seldom been quantified in pests targeted by these toxins. We combined mark-recapture and biogeochemical marking over three breeding seasons to quantify these features directly in natural populations of Ostrinia nubilalis, a major lepidopteran corn pest. At the local scale, resident females mated regardless of males having dispersed beforehand or not, as assumed in the HDR strategy. Accordingly, 0–67% of resident females mating before dispersal did so with resident males, this percentage depending on the local proportion of resident males (0% to 67.2%). However, resident males rarely mated with immigrant females (which mostly arrived mated), the fraction of females mating before dispersal was variable and sometimes substantial (4.8% to 56.8%), and there was no evidence for male premating dispersal being higher. Hence, O. nubilalis probably mates at a more restricted spatial scale than previously assumed, a feature that may decrease the efficiency of the HDR strategy under certain circumstances, depending for example on crop rotation practices.

Frequency of Resistance to Bacillus thuringiensis Toxin Cry1Ab in Southern United States Corn Belt Population of European Corn Borer (Lepidoptera: Crambidae)

The high-dose refuge resistance management strategy is the main approach used to delay resistance in targeted pests to Bacillus thuringiensis (Bt) toxins in transgenic crops. We used an F2 screen to test a critical assumption of the high-dose refuge strategy, which is that resistance allele (R) frequencies are initially rare (<10−3) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) from the southern Corn Belt. We expanded the methodological scope of the F2 screen so that both males and females may be used to initiate a screen and determined how the results from both sexes may be combined. In total, 62 female and 131 male O. nubilalis lines from Kansas and 39 female and four male lines from Texas were screened. No major resistance alleles were found and estimated R frequency for the southern Corn Belt was updated to between 0 and 0.0044 with 95% credibility. The experiment-wise detection probability was 98.7%. These results suggest the frequency of resistance alleles is low enough that the high-dose refuge resistance management strategy may be effective for delaying resistance evolution in O. nubilalis to Bt corn in the southern Corn Belt.

Frequency of Resistance to &lt;I&gt;Bacillus thuringiensis&lt;/I&gt; Toxin Cry1Ab in Southern United States Corn Belt Population of European Corn Borer (Lepidoptera: Crambidae)

The high-dose refuge resistance management strategy is the main approach used to delay resistance in targeted pests to Bacillus thuringiensis (Bt) toxins in transgenic crops. We used an F2 screen to test a critical assumption of the high-dose refuge strategy, which is that resistance allele (R) frequencies are initially rare (<10(-3)) in Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) from the southern Corn Belt. We expanded the methodological scope of the F2 screen so that both males and females may be used to initiate a screen and determined how the results from both sexes may be combined. In total, 62 female and 131 male O. nubilalis lines from Kansas and 39 female and four male lines from Texas were screened. No major resistance alleles were found and estimated R frequency for the southern Corn Belt was updated to between 0 and 0.0044 with 95% credibility. The experiment-wise detection probability was 98.7%. These results suggest the frequency of resistance alleles is low enough that the high-dose refuge resistance management strategy may be effective for delaying resistance evolution in O. nubilalis to Bt corn in the southern Corn Belt.

Genetics of Resistance to Transgenic Bacillus thuringiensis Poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae)

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.