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Western corn rootworm, Diabrotica virgifera virgifera LeConte, is a serious pest of corn and is often managed with transgenic corn producing insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). This pest has developed field-evolved resistance to all commercially available Bt traits, beginning with Cry3Bb1 in 2009. Fitness costs may accompany Bt resistance, where individuals with alleles for Bt resistance have reduced fitness on non-Bt corn compared to Bt-susceptible individuals. In conjunction with non-Bt refuges, fitness costs can delay the evolution of Bt resistance. Importantly, ecological factors may affect the presence and magnitude of fitness costs. For western corn rootworm, available data suggest that fitness costs of Bt resistance may be present in some cases. Using two Cry3Bb1-resistant western corn rootworm strains (Hopkinton and Cresco), a fitness-cost experiment was performed by rearing rootworm in the absence of Bt for six generations to test for fitness costs of Cry3Bb1 resistance and the effect of larval rearing density on fitness costs. Fitness costs were detected for both strains; however, strains were still resistant to Cry3Bb1 corn at the end of the experiment. Cresco experienced a greater loss of resistance at low versus high density, but no effect of density was detected in Hopkinton. Our study shows that fitness costs can accompany Bt resistance in western corn rootworm and may be more pronounced under low larval density. Even though fitness costs were present, it appears that rootworm populations may remain resistant to Cry3Bb1 corn for years after resistance has evolved.

Background In the absence of vaccines or drugs, insecticides are the mainstay of Aedes-borne disease control. Their utility is challenged by the slow deployment of resources, poor community compliance and inadequate household coverage. Novel application methods are required. Methodology and principal findings A 10% w/w metofluthrin “emanator” that passively disseminates insecticide from an impregnated net was evaluated in a randomized trial of 200 houses in Mexico. The devices were introduced at a rate of 1 per room and replaced at 3-week intervals. During each of 7 consecutive deployment cycles, indoor resting mosquitoes were sampled using aspirator collections. Assessments of mosquito landing behaviours were made in a subset of houses. Pre-treatment, there were no differences in Aedes aegypti indices between houses recruited to the control and treatment arms. Immediately after metofluthrin deployment, the entomological indices between the trial arms diverged. Averaged across the trial, there were significant reductions in Abundance Rate Ratios for total Ae. aegypti, female abundance and females that contained blood meals (2.5, 2.4 and 2.3-times fewer mosquitoes respectively; P, Author summary Insecticidal control tools are heavily relied on for the control of mosquito-borne viruses such as dengue, chikungunya and Zika, but the logistics associated with conventional insecticide use (e.g. space sprays and residual formulations) are challenging. Considerable time and resources are required to treat household interiors; an impediment exacerbated by the difficulty in gaining entrance to households, and sometimes by limited compliance in the community. Another constraint to effective insecticide use is that many mosquito populations are resistant to the chemicals used. Volatile pyrethroids, exhibiting both lethal and behavioural effects on mosquitoes are available in formulations that release insecticides passively to the air, at room temperature. These may be suitable for deployment in houses with the aim of creating “bite-free” spaces. By removing the need for conventional application methods, these devices might be rapidly deployed with minimum disruption to households. This is the first large-scale, randomized control trial to evaluate the entomological impacts of volatile pyrethroids in an urban environment. Using metofluthrin as an example, we confirm that some formulations have a significant impact on Aedes aegypti densities and landing behaviour indoors. These effects occur despite the presence of pyrethroid-resistance alleles associated with conventional insecticide resistance.

Arbovirus infection in Aedes aegypti has historically been quantified from a sample of the adult population by pooling collected mosquitoes to increase detectability. However, there is a significant knowledge gap about the magnitude of natural arbovirus infection within areas of active transmission, as well as the sensitivity of detection of such an approach. We used indoor Ae. aegypti sequential sampling with Prokopack aspirators to collect all mosquitoes inside 200 houses with suspected active ABV transmission from the city of Mérida, Mexico, and tested all collected specimens by RT-PCR to quantify: a) the absolute arbovirus infection rate in individually tested Ae. aegypti females; b) the sensitivity of using Prokopack aspirators in detecting ABV-infected mosquitoes; and c) the sensitivity of entomological inoculation rate (EIR) and vectorial capacity (VC), two measures ABV transmission potential, to different estimates of indoor Ae. aegypti abundance. The total number of Ae. aegypti (total catch, the sum of all Ae. aegypti across all collection intervals) as well as the number on the first 10-min of collection (sample, equivalent to a routine adult aspiration session) were calculated. We individually tested by RT-PCR 2,161 Aedes aegypti females and found that 7.7% of them were positive to any ABV. Most infections were CHIKV (77.7%), followed by DENV (11.4%) and ZIKV (9.0%). The distribution of infected Aedes aegypti was overdispersed; 33% houses contributed 81% of the infected mosquitoes. A significant association between ABV infection and Ae. aegypti total catch indoors was found (binomial GLMM, Odds Ratio > 1). A 10-min indoor Prokopack collection led to a low sensitivity of detecting ABV infection (16.3% for detecting infected mosquitoes and 23.4% for detecting infected houses). When averaged across all infested houses, mean EIR ranged between 0.04 and 0.06 infective bites per person per day, and mean VC was 0.6 infectious vectors generated from a population feeding on a single infected host per house/day. Both measures were significantly and positively associated with Ae. aegypti total catch indoors. Our findings provide evidence that the accurate estimation and quantification of arbovirus infection rate and transmission risk is a function of the sampling effort, the local abundance of Aedes aegypti and the intensity of arbovirus circulation., Author summary Aedes-borne diseases comprise a serious public health burden in many parts of the world, usually affecting low income areas. The ability to detect virus circulation within a population may be key in responding to the threat of outbreaks, providing a cost-effective approach for triggering vector control. Unfortunately, gaps in the knowledge of natural Aedes-borne virus (ABV) infection in Aedes aegypti have led to uncertainties in the consideration of arbovirus surveillance in mosquitoes. Here, we show that the natural infection rate in a mosquito population may not be a function of where Aedes aegypti are, but rather where key human-mosquito contacts occur. Sampling 200 houses with suspected ABV active transmission led us to quantify high virus infection rates in all Aedes aegypti present in the house and use such information to estimate the sensitivity of indoor aspiration with Prokopack devices and two measures of ABV transmission potential. Our findings provide evidence that the accurate quantification of arbovirus infection rate and transmission risk is a function of the sampling effort, the local abundance of Aedes aegypti and the intensity of arbovirus circulation. Results from this study are relevant to understand the value of virus testing of vector populations, and for the design of entomological endpoints relevant for epidemiological trials quantifying the impact of vector control on ABVs.

Cover crops are beneficial to agroecosystems because they decrease soil erosion and nutrient loss while increasing within-field plant diversity. Greater plant diversity within cropping systems can positively affect beneficial arthropod communities. We hypothesized that increasing plant diversity within annually rotated corn and soybean with the addition of a rye cover crop would positively affect the beneficial ground and canopy-dwelling communities compared with rotated corn and soybean grown without a cover crop. From 2011 through 2013, arthropod communities were measured at two locations in Iowa four times throughout each growing season. Pitfall traps were used to sample ground-dwelling arthropods within the corn and soybean plots and sweep nets were used to measure the beneficial arthropods in soybean canopies. Beneficial arthropods captured were identified to either class, order, or family. In both corn and soybean, community composition and total community activity density and abundance did not differ between plots that included the rye cover crop and plots without the rye cover crop. Most taxa did not significantly respond to the presence of the rye cover crop when analyzed individually, with the exceptions of Carabidae and Gryllidae sampled from soybean pitfall traps. Activity density of Carabidae was significantly greater in soybean plots that included a rye cover crop, while activity density of Gryllidae was significantly reduced in plots with the rye cover crop. Although a rye cover crop may be agronomically beneficial, there may be only limited effects on beneficial arthropods when added within an annual rotation of corn and soybean.

The World Health Organization (WHO) has recently recommended indoor residual spraying (IRS) as part of a vector control strategy to combat Aedes-borne diseases, including dengue, chikungunya, and Zika viruses. Hand compression sprayers have been used in malaria prevention and control programs worldwide since the 1950s and are a standard for IRS application. However, there are technological advances that should be considered to improve IRS application (e.g., flow-control valves, rechargeable-battery equipment, reduced-drift nozzles, etc.), particularly if interventions are performed in urban areas to target Aedes aegypti. Using WHO guidelines, we contrasted technical characteristics of potential IRS equipment including hand compression sprayers (Hudson X-pert, Goizper IK Vector Control Super), rechargeable-battery sprayers (Solo 416, Birchmeier REC 15ABZ, Hudson NeverPump), and motorized sprayers (Honda WJR 2525, Kawashima AK35GX). Measurements included flow rate, droplet size, battery/fuel life, and technical/physical characteristics. Flow rate, the most important parameter, of the Hudson X-pert was stabilized at 550 ml/min by the use of a control flow valve (CFV). The IK Vector Control Super had integrated CFVs and produced a similar flow as the Hudson X-pert. Rechargeable-battery equipment provided consistent flow as well as negligible noise. Motorized sprayers also produced consistent flow, but their weight, high noise pollution when used indoors, and high engine temperature made them highly unpleasant for technicians. We identify alternatives to the more traditional hand compression Hudson X-pert sprayer with technical and operational considerations for performing IRS.

Background Quantification of adult Aedes aegypti abundance indoors has relied on estimates of relative density (e.g. number of adults per unit of sampling or time), most commonly using traps or timed collections using aspirators. The lack of estimates of the sensitivity of collections and lack of a numerical association between relative and the absolute density of adult Ae. aegypti represent a significant gap in vector surveillance. Here, we describe the use of sequential removal sampling to estimate absolute numbers of indoor resting Ae. aegypti and to calculate calibration coefficients for timed Prokopack aspirator collections in the city of Merida, Yucatan State, Mexico. The study was performed in 200 houses that were selected based on recent occurrence of Aedes-borne viral illness in residents. Removal sampling occurred in 10-minute sampling rounds performed sequentially until no Ae. aegypti adult was collected for 3 hours or over 2 consecutive 10-minute periods. Results A total of 3439 Ae. aegypti were collected. The sensitivity of detection of positive houses in the first sampling round was 82.5% for any adult Ae. aegypti, 78.5% for females, 75.5% for males and 73.3% for blood-fed females. The total number of Ae. aegypti per house was on average ~5 times higher than numbers collected for the first sampling round. There was a positive linear relationship between the relative density of Ae. aegypti collected during the first 10-min round and the absolute density for all adult metrics. Coefficients from the linear regression were used to calibrate numbers from 10-min collections into estimates of absolute indoor Ae. aegypti density for all adults, females and males. Conclusions Exhaustive removal sampling represents a promising method for quantification of absolute indoor Ae. aegypti density, leading to improved entomological estimates of mosquito distribution, a key measure in the assessments of the risk pathogen transmission, disease modeling and the evaluation of vector control interventions.

Crop rotation alters agroecosystem diversity temporally, and increasing the number of crops in rotation schemes can increase crop yields and reduce reliance on pesticides. We hypothesized that increasing the number of crops in annual rotation schemes would positively affect ground-dwelling beneficial arthropod communities. During 2012 and 2013, pitfall traps were used to measure activity-density and diversity of ground-dwelling communities within three previously established, long-term crop rotation studies located in Wisconsin and Illinois. Rotation schemes sampled included continuous corn, a 2-yr annual rotation of corn and soybean, and a 3-yr annual rotation of corn, soybean, and wheat. Insects captured were identified to family, and non-insect arthropods were identified to class, order, or family, depending upon the taxa. Beneficial arthropods captured included natural enemies, granivores, and detritivores. The beneficial community from continuous corn plots was significantly more diverse compared with the community in the 2-yr rotation, whereas the community in the 3-yr rotation did not differ from either rotation scheme. The activity-density of the total community and any individual taxa did not differ among rotation schemes in either corn or soybean. Crop species within all three rotation schemes were annual crops, and are associated with agricultural practices that make infield habitat subject to anthropogenic disturbances and temporally unstable. Habitat instability and disturbance can limit the effectiveness and retention of beneficial arthropods, including natural enemies, granivores, and detritivores. Increasing non-crop and perennial species within landscapes in conjunction with more diverse rotation schemes may increase the effect of biological control of pests by natural enemies.

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), and northern corn rootworm, Diabrotica barberi Smith & Lawrence, are major pests of corn (Zea mays L.). Corn producing Bacillus thuringiensis (Bt) toxins are widely used to manage Diabrotica spp.; however, Bt resistance by D. v. virgifera has led to high levels of feeding injury in the field. We tested whether field history affected root injury and abundance of adult Diabrotica spp. In 2013 and 2014, four types of cornfields were sampled: 1) recently rotated fields, 2) continuous cornfields, 3) fields with a history of injury to Bt corn (past problem fields), and 4) fields with greater than one node of injury to Bt corn at the time of sampling (current problem fields). Data were collected on field history, root injury, and the abundance of adult Diabrotica spp. from each field. Root injury and the abundance of D. v. virgifera were significantly greater in current problem fields compared to the other field types, while D. barberi were significantly more abundant in recently rotated fields. Root injury and the abundance of D. v. virgifera did not differ among recently rotated fields, continuous cornfields, and past problem fields. Analysis of field history showed that recently rotated fields were characterized by significantly less Bt corn, soil-applied insecticides, and years planted to corn continuously. These results suggest that greater cropping practice diversity can reduce management inputs for Diabrotica spp.; however, its effects on resistance evolution remain undetermined.

Decreased pest pressure is sometimes associated with more diverse agroecosystems, including the addition of a rye cover crop (Secale cereale L.). However, not all pests respond similarly to greater vegetational diversity. Polyphagous pests, such as true armyworm (Mythimna unipuncta Haworth), black cutworm (Agrotis ipsilon Hufnagel), and common stalk borer (Papaipema nebris Guenee), whose host range includes rye have the potential to cause injury to crops following a rye cover crop. The objectives of this study were to compare the abundance of early-season insect pests and injury to corn (Zea mays L.) from fields with and without a rye cover crop on commercial farms. Fields were sampled weekly to quantify adult and larval pests and feeding injury to corn plants from mid-April until corn reached V8 stage, during 2014 and 2015. Measurements within fields were collected along transects that extended perpendicularly from field edges into the interior of cornfields. Adult true armyworm and adult black cutworm were captured around all cornfields, but most lepidopteran larvae captured within cornfields were true armyworm and common stalk borer. Cornfields with a rye cover crop had significantly greater abundance of true armyworm and greater proportion of injured corn. Both true armyworm abundance and feeding injury were significantly greater in the interior of cornfields with rye. Common stalk borer abundance did not differ between cornfields with or without rye cover. Farmers planting corn following a rye cover crop should be aware of the potential for increased presence of true armyworm and for greater injury to corn.

Challenges in maintaining high effectiveness of classic vector control in urban areas has renewed the interest in indoor residual spraying (IRS) as a promising approach for Aedes-borne disease prevention. While IRS has many benefits, application time and intrusive indoor applications make its scalability in urban areas difficult. Modifying IRS to account for Ae. aegypti resting behavior, named targeted IRS (TIRS, spraying walls below 1.5 m and under furniture) can reduce application time; however, an untested assumption is that modifications to IRS will not negatively impact entomological efficacy. We conducted a comparative experimental study evaluating the residual efficacy of classically-applied IRS (as developed for malaria control) compared to two TIRS application methods using a carbamate insecticide against a pyrethroid-resistant, field-derived Ae. aegypti strain. We performed our study within a novel experimental house setting (n = 9 houses) located in Merida (Mexico), with similar layouts and standardized contents. Classic IRS application (insecticide applied to full walls and under furniture) was compared to: a) TIRS: insecticide applied to walls below 1.5 m and under furniture, and b) Resting Site TIRS (RS-TIRS): insecticide applied only under furniture. Mosquito mortality was measured eight times post-application (out to six months post-application) by releasing 100 Ae. aegypti females /house and collecting live and dead individuals after 24 hrs exposure. Compared to Classic IRS, TIRS and RS-TIRS took less time to apply (31% and 82% reduction, respectively) and used less insecticide (38% and 85% reduction, respectively). Mortality of pyrethroid-resistant Ae. aegypti did not significantly differ among the three IRS application methods up to two months post application, and did not significantly differ between Classic IRS and TIRS up to four months post application. These data illustrate that optimizing IRS to more efficiently target Ae. aegypti can both reduce application time and insecticide volume with no apparent reduction in entomological efficacy., Author summary Vector control is the primary strategy for managing Aedes aegypti and reducing transmission of Aedes-borne diseases; however, the indoor resting behavior of Ae. aegypti and the evolution of insecticide resistance reduces the effectiveness of many vector control tactics. Indoor residual spraying (IRS) is effective against Ae. aegypti, but lengthy application time makes IRS difficult to scale within urban environments. We compared the application and entomological efficacy of Classic IRS against two novel Aedes-targeting IRS application methods (Targeted IRS [TIRS]- insecticide applied to walls below 1.5 m and under furniture and Resting Site TIRS [RS-TIRS]- insecticide applied only under furniture) within experimental houses using a carbamate insecticide. Both TIRS and RS-TIRS took less time to apply and used less insecticide compared to Classic IRS. Mortality of pyrethroid-resistant Ae. aegypti did not differ among treatments out to two months post-application, and there was no difference in mortality between Classic IRS and TIRS out to four months post-application. These data provide evidence that IRS application methods can be improved to take less time and insecticide yet not lose entomological efficacy, making TIRS more scalable within urban environments. However, larger field studies with epidemiologic endpoints are needed to further assess the efficacy of these modified TIRS techniques.

Mosquito abatement programs in Midwestern communities frequently exist within landscapes dominated by agriculture. Although separately managed, both agricultural pests and mosquitoes are targeted by similar classes of insecticides. As a result, there is the potential for unintended insecticide exposure to mosquito populations from agricultural pest management. To determine the impact that agricultural management practices have on mosquito insecticide susceptibility we compared the mortality of Aedes vexans (Meigen; Diptera: Culicidae) between populations sampled from locations with and without mosquito abatement in South Dakota, a region dominated by agricultural production. Collection locations were either within towns with mosquito abatement programs (n = 2; Brookings and Sioux Falls, SD) or located > 16 km from towns with mosquito abatement programs (n = 2; areas near Harrold and Willow Lake, SD). WHO bioassays were used to test susceptibly of adults to differing insecticide classes relative to their respective controls; 1) an organochlorine (dieldrin 4%), 2) an organophosphate (malathion 5%), and 3) a pyrethroid (lambda-cyhalothrin 0.05%). Corrected mortality did not significantly differ between locations with or without abatement; however, when locations were analized by proportion of developed land within the surrounding landscape pyrethroid mortality was significantly lower where crop production dominated the surrounding landscape and mosquito abatement was present. These data suggest that agricultural pest management may incidentally contribute to reduced mosquito susceptibility where overlap between agricultural pest management and mosquito abatement exists. Decoupling insecticide classes used by both agricultural and public health pest management programs may be necessary to ensure continued efficacy of pest management tools.

Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States, and recent management of western corn rootworm has included planting of Bt corn. Beginning in 2009, western corn rootworm populations with resistance to Cry3Bb1 corn and mCry3A corn were found in Iowa and elsewhere. To date, western corn rootworm populations have remained susceptible to corn producing Bt toxin Cry34/35Ab1. In this study, we used single-plant bioassays to test field populations of western corn rootworm for resistance to Cry34/35Ab1 corn, Cry3Bb1 corn, and mCry3A corn. Bioassays included nine rootworm populations collected from fields where severe injury to Bt corn had been observed and six control populations that had never been exposed to Bt corn. We found incomplete resistance to Cry34/35Ab1 corn among field populations collected from fields where severe injury to corn producing Cry34/35Ab1, either singly or as a pyramid, had been observed. Additionally, resistance to Cry3Bb1 corn and mCry3A corn was found among the majority of populations tested. These first cases of resistance to Cry34/35Ab1 corn, and the presence of resistance to multiple Bt toxins by western corn rootworm, highlight the potential vulnerability of Bt corn to the evolution of resistance by western corn rootworm. The use of more diversified management practices, in addition to insect resistance management, likely will be essential to sustain the viability of Bt corn for management of western corn rootworm.

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.

Western corn rootworm, Diabrotica virgifera virgifera LeConte, has evolved resistance to transgenic maize, Zea maize L., that produces the insecticidal protein Cry3Bb1, which is derived from the bacterium Bacillus thuringiensis. We hypothesized that the level of Cry3Bb1 resistance in populations of western corn rootworm could be influenced by farming practices. To test this hypothesis, we evaluated the effect of field history on resistance to Cry3Bb1 maize by western corn rootworm. In 2013 and 2014, rootworm adults were collected from the four types of maize fields: 1) current problem fields, 2) past problem fields, 3) rotated maize fields, and 4) continuous maize fields. Those field populations along with seven Bt-susceptible control populations were tested for Cry3Bb1 resistance with both plant-based and diet-based bioassays. All field populations were resistant to Cry3Bb1 regardless of field history, however, some variation in the degree of resistance was found. For all categories of field populations, larval survivorship on Cry3Bb1 maize was significantly higher than control populations, and did not differ from survival on non-Bt maize. Evidence of resistance to Cry3Bb1 maize in plant-based bioassays was further supported by diet-based bioassays and we found a positive relationship between LC50 values from diet-based bioassays and the larval survivorship in plant-based bioassays. This study provides evidence of Cry3Bb1 resistance throughout the agricultural landscape studied, irrespective of the field history, and highlights the need for improved resistance management approaches, such as better use of integrated pest management to better delay pest resistance.

Diabrotica virgifera virgifera LeConte is a major pest of corn and causes over a billion dollars of economic loss annually through yield reductions and management costs. Corn producing toxins derived from Bacillus thuringiensis (Bt) has been developed to help manage D. v. virgifera. However, previous studies have demonstrated the ability of this species to evolve resistance to Bt toxins in both laboratory and field settings.We used an experimental evolution approach to test the refuge strategies for delaying resistance of D. v. virgifera to corn producing Bt toxin Cry34/35Ab1. In the absence of refuges, D. v. virgifera developed resistance to Bt corn after three generations of selection. In some cases, non-Bt refuges reduced the level of resistance compared with the strain selected in the absence of refuges, but refuge strains did show reduced susceptibility to Bt corn compared with the unselected strain.In this study, non-Bt refuges delayed resistance to Bt corn by D. v. virgifera in some cases but not others. Combining the refuge strategy with pyramids of multiple Bt toxins and applying other pest management strategies will likely be necessary to delay resistance of D. v. virgifera to Bt corn.

The widespread planting of crops genetically engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) places intense selective pressure on pest populations to evolve resistance. Western corn rootworm is a key pest of maize, and in continuous maize fields it is often managed through planting of Bt maize. During 2009 and 2010, fields were identified in Iowa in which western corn rootworm imposed severe injury to maize producing Bt toxin Cry3Bb1. Subsequent bioassays revealed Cry3Bb1 resistance in these populations. Here, we report that, during 2011, injury to Bt maize in the field expanded to include mCry3A maize in addition to Cry3Bb1 maize and that laboratory analysis of western corn rootworm from these fields found resistance to Cry3Bb1 and mCry3A and cross-resistance between these toxins. Resistance to Bt maize has persisted in Iowa, with both the number of Bt fields identified with severe root injury and the ability western corn rootworm populations to survive on Cry3Bb1 maize increasing between 2009 and 2011. Additionally, Bt maize targeting western corn rootworm does not produce a high dose of Bt toxin, and the magnitude of resistance associated with feeding injury was less than that seen in a high-dose Bt crop. These first cases of resistance by western corn rootworm highlight the vulnerability of Bt maize to further evolution of resistance from this pest and, more broadly, point to the potential of insects to develop resistance rapidly when Bt crops do not achieve a high dose of Bt toxin.

A 2 yr field study was conducted to determine how a blend of entomopathogens interacted with Bt maize to affect mortality of Diabrotica spp. (Coleoptera: Chrysomelidae), root injury to maize (Zea maize L.) and yield. The blend of entomopathogens included two entomopathogenic nematodes, Steinernema carpocapsae Weiser and Heterorhabditis bacteriophora Poinar, and one entomopathogenic fungus, Metarhizium brunneum (Metschnikoff) Sorokin. Bt maize (event DAS59122-7, which produces Bt toxin Cry34/35Ab1) decreased root injury and survival of western corn rootworm (Diabrotica virgifera virgifera LeConte) and northern corn rootworm (Diabrotica barberi Smith & Lawrence) but did not affect yield. During year 1 of the study, when rootworm abundance was high, entomopathogens in combination with Bt maize led to a significant reduction in root injury. In year 2 of the study, when rootworm abundance was lower, entomopathogens significantly decreased injury to non-Bt maize roots, but had no effect on Bt maize roots. Yield was significantly increased by the addition of entomopathogens to the soil. Entomopathogens did not decrease survival of corn rootworm species. The results suggest that soil-borne entomopathogens can complement Bt maize by protecting roots from feeding injury from corn rootworm when pest abundance is high, and can decrease root injury to non-Bt maize when rootworm abundance is low. In addition, this study also showed that the addition of entomopathogens to soil contributed to an overall increase in yield.

The western corn rootworm Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) and the northern corn rootworm Diabrotica barberi SmithLawrence (Coleoptera: Chrysomelidae) are major pests of corn (Zea mays L.). Historically, crop rotation has been an effective management strategy, but both species have adapted to crop rotation in the Midwest. For both species in eastern Iowa, we measured abundance and prevalence of rotation resistance using sticky traps and emergence cages in fields of corn and soybean (Glycine max L.). Based on currently available data, we calculated the economic thresholds for these pests at two Diabrotica spp. per trap per day in cornfields and 1.5 D. v. virgifera per trap per day in soybean fields. The economic injury level of rotation-resistant D. barberi was determined to be 3.5 adult insects per emergence cage per year. Peak abundance of rootworm adults in cornfields was below economic thresholds in the majority of fields sampled, suggesting that management of rootworm larvae in continuous cornfields may not always be necessary. Rotation-resistant D. barberi was found throughout eastern Iowa using emergence cages in first-year cornfields, however, the abundance was below levels expected to impose economic injury in 14 of 17 fields evaluated. The presence of rotation-resistant D. v. virgifera, as measured by the occurrence of this insect in soybean fields, occurred only in northeastern Iowa and was also below the economic threshold. These data suggests that crop rotation remains a viable pest management strategy in eastern Iowa.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a major pest of corn (Zea mays L.) in North America and has evolved resistance to crop rotation by ovipositing in alternate crops such as soybeans [Glycine max (L.) Merr.]. Through experiments with plants grown in the greenhouse and the field, we tested whether soybeans with resistance to the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), affected survival, fecundity, and consumption of soybean for D. v. virgifera. Soybean varieties tested included those types resistant to A. glycines (Rag1 and rag1/rag3) and a susceptible near isoline of the Rag1 variety. Females were provided with a diet of corn tissue for 4 d after which they were fed a diet of tissue from one of three soybean varieties for 4 d, starved for 4 d, or fed corn tissue. When fed greenhouse grown plants, strains differed significantly in survival and consumption, but consumption did not differ by variety of soybean. Diet treatment only affected fecundity; individuals fed corn continuously had greater fecundity than those individuals fed soybeans. In the experiment with plants grown in the field, leaf consumption differed among strains and individuals fed corn continuously had greater fecundity than the other treatments. Soybean varieties with Rag1 and rag1/rag3 resistance to A. glycines did not appear to affect the fitness of D. v. virgifera. Thus, planting of these A. glycines-resistant soybean varieties should not directly affect the spread of rotation-resistant D. v. virgifera.

BackgroundCrops 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 findingsWe 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/significanceThis 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.