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Field-evolved resistance to Bt maize by western corn rootworm: Predictions from the laboratory and effects in the field
Abstract
Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) provide an effective management tool for many key insect pests. However, pest species have repeatedly demonstrated their ability to adapt to management practices. Results from laboratory selection experiments illustrate the capacity of pest species to evolve Bt resistance. Furthermore, resistance has been documented to Bt sprays in the field and greenhouse, and more recently, by some pests to Bt crops in the field. In 2009, fields were discovered in Iowa (USA) with populations of western corn rootworm, Diabrotica virgifera virgifera LeConte, that had evolved resistance to maize that produces the Bt toxin Cry3Bb1. Fields with resistant insects in 2009 had been planted to Cry3Bb1 maize for at least three consecutive years and as many as 6years. Computer simulation models predicted that the western corn rootworm might evolve resistance to Bt maize in as few as 3years. Laboratory and field data for interactions between western corn rootworm and Bt maize indicate that currently commercialized products are not high-dose events, which increases the risk of resistance evolution because non-recessive resistance traits may enhance survival on Bt maize. Furthermore, genetic analysis of laboratory strains of western corn rootworm has found non-recessive inheritance of resistance. Field studies conducted in two fields identified as harboring Cry3Bb1-resistant western corn rootworm found that survival of western corn rootworm did not differ between Cry3Bb1 maize and non-Bt maize and that root injury to Cry3Bb1 maize was higher than injury to other types of Bt maize or to maize roots protected with a soil insecticide. These first cases of field-evolved resistance to Bt maize by western corn rootworm provide an early warning and point to the need to apply better integrated pest management practices when using Bt maize to manage western corn rootworm.
... Beginning in 2009, field-evolved Bt resistance by western corn rootworm to Cry3Bb1 maize was found in northeastern Iowa [12]. Subsequently, resistance to Cry3Bb1 was detected in fields distributed throughout Iowa [13,17,18]. Populations of Cry3Bb1-resistant western corn rootworm also have been identified in Illinois, Nebraska and Minnesota [19][20][21]. ...
... The large-scale adoption of Cry3Bb1 maize and the limited dispersal of rootworm adults has caused intense local selection in fields that are planted to Cry3Bb1 maize continuously [24,25]. Past research has shown rapid evolution of Cry3Bb1 resistance by western corn rootworm in some fields, with resistance developing after three or more years of continuous cultivation of Cry3Bb1 maize, which translates to three generations for western corn rootworm [12,13,17]. The rate of evolution of resistance to a Bt crop depends on several factors such as the intensity of selection for Bt resistance, initial frequency of resistant alleles, degree of dominance for a resistance trait and whether or not fitness costs accompany Bt resistance [26][27][28][29]. ...
... Where we were able to evaluate Cry3Bb1 resistance with both diet-based and plant-based bioassays (Tables 3 and 4; Fig 3), we found a significant positive correlation between. Additionally, LC 50 values for Cry3Bb1-susceptible control populations were similar to past research [44,56] as were levels of survival on Cry3Bb1 maize in plant-based bioassays [13,17,18]. This suggests that both plant-based and diet-based bioassays can be useful for quantifying Bt resistance. ...
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.
... These cases of field-evolved resistance include western corn rootworm resistance to mCry3A and Cry3Bb1 maize in the United States (Gassmann et al., 2011(Gassmann et al., , 2014, fall armyworm resistance to Cry1F maize in Puerto Rico and Brazil (Storer et al., 2010;Farias et al., 2014aFarias et al., , 2014b, and maize stem borer B. fusca resistance to Cry1Ab maize in South Africa (van Rensburg, 2007). Strategies to mitigate the effects of Bt resistance have included discontinuing the use of some types of Bt maize in certain geographies (Storer et al., 2010), increasing the use of conventional insecticides (Farias et al., 2014a), and pyramiding multiple Bt toxins to compensate for the loss of efficacy of a Bt trait (Gassmann, 2012). ...
... These included Cry1Ab and Cry1F toxins for the management of European corn borer (Huang et al., 2011). However, in other cases, such as Bt maize targeting western corn rootworm, the assumption of high dose was not satisfied (Gassmann, 2012). It is notable that after over a decade and a half of commercial use, and extensive cultivation within the United States, available data indicate a lack of field-evolved resistance by European corn borer to Bt maize (Siegfried and Hellmich, 2012). ...
... Factors contributing to resistance likely included the lack of a high dose for Bt maize targeting western corn rootworm, nonrecessive inheritance of resistance, and few accompanying fitness costs (Gassmann, 2012;Petzold-Maxwell et al., 2012b;Ingber and Gassmann, 2015). This suggests that for Bt crops that achieve less than a high dose, resistance may develop rapidly, and due to a lack of fitness costs, may persist once selected. ...
Maize is attacked by a range of lepidopteran and coleopteran pests, and these pests feed on plants at every phenological stage. Historically, the management of maize pests has focused on conventional insecticides. However, beginning in the 1990s, genetically engineered maize that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) was made commercially available for the management of pest insects. The first Bt maize produced single Bt toxins that targeted lepidopteran pests, but later Bt toxins targeted coleopteran pests, specifically corn rootworms Diabrotica spp., and multiple Bt toxins were placed into the same maize hybrids. The planting of Bt maize has provided an effective management option for many key pests of maize, but recent cases of pest resistance illustrate the potential limitations of this technology. More diversified management will increase the sustainability of pest management in maize. Past research has demonstrated the potential efficacy of a range of microbial control agents, including bacteria, fungi, nematodes, and viruses. Of particular interest are entomopathogenic fungi for the management of aboveground pests, and entomopathogenic fungi and nematodes for the management of belowground pests. One challenge to the implementation of microbial control agents is the need for pest management options that are cost-effective and may be applied easily and rapidly over a large area. Potential future applications of microbial control agents in maize may include bolstering populations of entomopathogens in the soil through conservation biological control, developing entomopathogenic fungi for application to maize seeds, and novel Bt toxins for genetically engineered maize.
... The high-dose-refuge approach has been successful for some lepidopteran pests (Huang et al. 2011). However, Bt corn hybrids developed for western corn rootworm are not high dose cultivar, which reduces the effectiveness of refuges at delaying resistance and may alter the effective dominance of resistance (Gassmann 2012(Gassmann , 2016Tabashnik and Gould 2012). Corn hybrids producing Bt toxin Cry3Bb1 were first planted commercially in 2003; after 6 yr of commercialization, the first case of field-evolved Cry3Bb1 resistance was found in Iowa in 2009 (Gassmann et al. 2011). ...
... Resistance ratios of field populations tested in this study ranged from 11 to 19 (Table 2) and plant-based bioassays of field populations with Cry3Bb1 resistance have reported similar resistance ratios (Gassmann et al. 2011. Because Bt corn targeting corn rootworm lacks a high dose, and susceptible western corn rootworm can survive on Bt corn, the modest resistance ratios observed here and elsewhere are sufficient for pest to achieve equivalent survival on Bt corn compared with non-Bt corn, and to impose substantial injury to Bt corn in the field (Gassmann 2012(Gassmann , 2016. This is in contrast to high-dose Bt crops where survival of susceptible individuals is absent or rare (Tabashnik and Carriere 2017). ...
Field-evolved resistance to Cry3Bb1 corn by western corn rootworm, Diabrotica virgifera virgifera LeConte (Colleoptera: Chrysomellidae), has been reported in field populations in Iowa, Illinois, Nebraska, and Minnesota. Inheritance and fitness costs associated with Cry3Bb1 resistance have been determined for non-diapausing laboratory strains of western corn rootworm with either laboratory-selected resistance or field-derived resistance. However, information on inheritance and fitness costs of Cry3Bb1 resistance in the diapausing field populations is lacking. In this study, we determined the inheritance of Cry3Bb1 resistance for four diapausing field strains of western corn rootworm using plant-based bioassays. We also determined the fitness costs for eight diapausing field populations in a greenhouse experiment. We found that Cry3Bb1 resistance was an autosomal trait and that the inheritance of resistance was mostly non-recessive; however, there was some variation in the dominance of Cry3Bb1 resistance. We did not find evidence of fitness costs affecting survival to adulthood, developmental rate, or adult dry mass. However, we did detect a fitness cost affecting adult size. The results of this study will add to the current understanding of field-evolved resistance to Cry3Bb1 corn by western corn rootworm and help in developing better strategies to manage resistance.
... Incomplete resistance to Cry3 toxins means that resistant individuals are significantly less susceptible to Cry3 toxins than susceptible individuals, yet they have lower fitness on Cry3 corn than on corn without rootworm-active Bt toxins (46). In fields with previously reported D. v. virgifera problems in Cry3Bb corn in Illinois and Iowa, root damage ratings were significantly lower for pyramided than Cry34/35Ab corn (23,47), and a consistent trend for lower adult emergence in pyramided than Cry34/35Ab corn occurred in three studies (23,45,47). In 2012 and 2013, Shrestha et al., (23) sampled 28 D. v. virgifera populations in Iowa, primarily from corn fields with a history of rootworm damage to Cry3Bb corn. ...
... Incomplete resistance to Cry3 toxins means that resistant individuals are significantly less susceptible to Cry3 toxins than susceptible individuals, yet they have lower fitness on Cry3 corn than on corn without rootworm-active Bt toxins (46). In fields with previously reported D. v. virgifera problems in Cry3Bb corn in Illinois and Iowa, root damage ratings were significantly lower for pyramided than Cry34/35Ab corn (23,47), and a consistent trend for lower adult emergence in pyramided than Cry34/35Ab corn occurred in three studies (23,45,47). In 2012 and 2013, Shrestha et al., (23) sampled 28 D. v. virgifera populations in Iowa, primarily from corn fields with a history of rootworm damage to Cry3Bb corn. ...
Significance
The western corn rootworm, a major insect pest in the Midwestern United States, has evolved resistance to genetically engineered corn that produces insecticidal proteins derived from the bacterium Bacillus thuringiensis (Bt). To evaluate tactics for reducing the damage caused by resistant rootworms, we analyzed field data for 2011 to 2016 from Illinois, Iowa, and Minnesota. The frequency of corn fields with severe rootworm damage was reduced by rotating corn with other crops and by not planting the same type of Bt corn year after year in the same field. These results support the EPA’s recommendations to decrease the negative impacts of rootworm resistance to Bt corn by rotating corn with other crops and diversifying the type of Bt corn planted.
... In addition, incomplete field-evolved resistance to Cry34/35Ab1 has also previously been documented for some D. v. virgifera populations , Ludwick et al. 2017. Most data suggest that cases of field-evolved D. v. virgifera resistance to Cry proteins are evolving independently under similar conditions (i.e., continuous deployment of Bt corn in the same fields) in Iowa, Illinois, Minnesota, Nebraska, and South Dakota (Gassmann et al. 2011(Gassmann et al. , 2014Gassmann 2012;Gray 2012;Porter et al. 2012;Wangila et al. 2015;Zukoff et al. 2016;Ludwick et al. 2017;Schrader et al. 2017). The potential for D. v. virgifera to develop resistance to Cry3Bb1, mCry3A, Cry34/35Ab1, and eCry3.1Ab ...
... Moreover, corrected larval survival on Cry3Bb1 was 94.2% lower in the laboratory control population than the Ransom population. Numerous authors have reported fieldevolved Cry3Bb1 resistance in D. v. virgifera, and most cases involved fields with a history of continuous Bt corn plantings (Gassmann et al. 2011(Gassmann et al. , 2014Gassmann 2012;Gray 2012;Porter et al. 2012;Wangila et al. 2015;Zukoff et al. 2016;Ludwick et al. 2017;Schrader et al. 2017). Thus, finding resistance to Cry3Bb1 in D. v. virgifera in North Dakota corn fields is not surprising, although the field history of the Ransom site is unknown. ...
Northern, Diabrotica barberi Smith & Lawrence, and western, D. virgifera virgifera LeConte, corn rootworms (Coleoptera: Chrysomelidae) are major economic pests of corn, Zea mays L., in North America. Corn hybrids expressing Bacillus thuringiensis Berliner (Bt) toxins are commonly used by growers to manage these pests. Several cases of field-evolved resistance to insecticidal proteins expressed by Bt corn hybrids have been documented in many corn-producing areas of North America, but only for D. v. virgifera. In 2016, beetles of both species were collected from five eastern North Dakota corn fields and reared in a growth chamber. In 2017, larvae reared from those populations were subjected to single-plant bioassays to screen for potential resistance to Cry3Bb1, Cry34/35Ab1, and pyramided Cry3Bb1 + Cry34/35Ab1 Bt toxins. Our results provide the first documented report of field-evolved resistance in D. barberi to corn hybrids expressing Cry3Bb1 (Arthur problem population) and Cry34/35Ab1 (Arthur and Page problem populations, and the Ransom and Sargent populations) proteins in North America. Resistance to Cry3Bb1 was also observed in the Ransom population of D. v. virgifera. Increased larval survival on the pyramided Cry3Bb1 + Cry34/35Ab1 hybrid was observed in both species. No cross-resistance was evident between Cry3Bb1 and Cry34/35Ab1 in any of the D. barberi populations tested. Our experiments identified field-evolved resistance to Bt toxins in some North Dakota populations of D. barberi and D. v. virgifera. Thus, more effective control tools and improved resistance management strategies are needed to prolong the durability of this technology for managing these important pests.
... Corn hybrids expressing Bacillus thuringiensis Berliner (Bt) insecticidal proteins have been widely used by U.S. corn producers to manage corn rootworms since 2003. Thus far, the rootworm-active Bt toxins incorporated into corn are expressed at 'less than highdose' concentrations (U.S. Environmental Protection Agency [USEPA 2002], Siegfried et al. 2005, Storer et al. 2006, Gassmann 2012, which is believed to increase the risk of resistance development in target pests (Gould 1998, Tabashnik et al. 2013, Andow et al. 2016). The first field-evolved resistance in D. v. virgifera to Bt insect-protected corn involved Iowa populations and hybrids expressing the Cry3Bb1 protein in 2009 (Gassmann et al. 2011). ...
... Therefore, it is reasonable to expect that tolerance to Cry3Bb1 could be increasing in some eastern North Dakota corn rootworm populations. Factors contributing to Bt resistance development include the following: rootworm-active Bt corn hybrids are typically considered low-dose-expressing products (USEPA 2002, Siegfried et al. 2005, Storer et al. 2006, Meihls et al. 2008, Gassmann 2012; continuous corn plantings (i.e., lack of crop rotation); and repeated use of Bt corn hybrids employing the same mode of action. As such, adherence to integrated pest management principles will be essential to extending the utility of Bt toxins to control corn rootworms, as well as insect pests targeted with this technology in other cropping systems. ...
Larval Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) were exposed to seven different entomopathogenic nematode species to test their potential infectivity in a laboratory setting. Known D. virgifera-infecting nematode species Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev, and Steinernema carpocapsae Weiser were tested in a concerted experiment alongside Steinernema diaprepesi Nguyen & Duncan, Steinernema riobrave Cabanillas, Poinar & Raulston, and a Missouri wild-type H. bacteriophora which have not been previously tested on D. virgifera. The species S. rarum Doucet was tested separately for D. virgifera infectivity. Third-instar D. virgifera were exposed to either 60 or 120 nematodes per larva for 6 d. Following exposure, mortality was recorded and larvae were examined to determine the presence of active nematode infections. Results indicated a significantly higher proportion of larvae with active infections from the Heterorhabditidae species and S. diaprepesi than the other Steinernematidae species for both exposure rates; mortality data indicated a similar trend. Steinernema rarum showed almost no infectivity in laboratory experiments.
... Corn hybrids expressing Bacillus thuringiensis Berliner (Bt) insecticidal proteins have been widely used by U.S. corn producers to manage corn rootworms since 2003. Thus far, the rootworm-active Bt toxins incorporated into corn are expressed at 'less than highdose' concentrations (U.S. Environmental Protection Agency [USEPA 2002], Siegfried et al. 2005, Storer et al. 2006, Gassmann 2012, which is believed to increase the risk of resistance development in target pests (Gould 1998, Tabashnik et al. 2013, Andow et al. 2016). The first field-evolved resistance in D. v. virgifera to Bt insect-protected corn involved Iowa populations and hybrids expressing the Cry3Bb1 protein in 2009 (Gassmann et al. 2011). ...
... Therefore, it is reasonable to expect that tolerance to Cry3Bb1 could be increasing in some eastern North Dakota corn rootworm populations. Factors contributing to Bt resistance development include the following: rootworm-active Bt corn hybrids are typically considered low-dose-expressing products (USEPA 2002, Siegfried et al. 2005, Storer et al. 2006, Meihls et al. 2008, Gassmann 2012; continuous corn plantings (i.e., lack of crop rotation); and repeated use of Bt corn hybrids employing the same mode of action. As such, adherence to integrated pest management principles will be essential to extending the utility of Bt toxins to control corn rootworms, as well as insect pests targeted with this technology in other cropping systems. ...
Northern, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), and western, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), corn rootworms are economic pests of corn, Zea mays L. in North America. We measured the impacts of corn hybrids incorporated with Cry3Bb1, Cry34/35Ab1, and pyramided (Cry3Bb1 + Cry34/35Ab1) Bacillus thuringiensis Berliner (Bt) proteins, tefluthrin soil insecticide, and clothianidin insecticidal seed treatment on beetle emergence, larval feeding injury, and corn yield at five locations from 2013 to 2015 in eastern North Dakota. In most cases, emergence was significantly lower in Bt-protected corn than in non-Bt corn hybrids. Exceptions included Wyndmere, ND (2013), where D. barberi emergence from Cry34/35Ab1 plots was not different from that in the non-Bt hybrid, and Arthur, ND (2013), where D. v. virgifera emergence from Cry3Bb1 plots did not differ from that in the non-Bt hybrid. Bt hybrids generally produced increased grain yield compared with non-Bt corn where rootworm densities were high, and larval root-feeding injury was consistently lower in Bt-protected plots than in non-Bt corn. The lowest overall feeding injury and emergence levels occurred in plots planted with the Cry3Bb1 + Cry34/35Ab1 hybrid. Time to 50% cumulative emergence of both species was 5-7 d later in Bt-protected than in non-Bt hybrids. Tefluthrin and clothianidin were mostly inconsequential in relation to beetle emergence and larval root injury. Our findings could suggest that some North Dakota populations could be in early stages of increased tolerance to some Bt toxins; however, Bt corn hybrids currently provide effective protection against rootworm injury in eastern North Dakota.
... The non-Bt portion of the field, or refuge, serves as a source of susceptible individuals that may mate with resistant insects, thereby producing heterozygous offspring and reducing the number of homozygous, resistant individuals [22]. The delay in resistance to a Bt crop achieved by the refuge strategy may be affected by both the dominance of resistance and fitness costs of resistance [23,24]. ...
... A high-dose Bt crop must either produce a concentration of Bt toxin that is 25 times greater than the concentration required to kill a susceptible individual, or kill 99.99% of susceptible individuals [41]. Maize hybrids currently available for management of western corn rootworm do not produce a high dose of Bt toxin [23,42,43], thus, resistance is expected to be inherited as a non-recessive trait. ...
The western corn rootworm, Diabrotica virgifera virgifera LeConte, is among the most serious insect pests of maize in North America. One strategy used to manage this pest is transgenic maize that produces one or more crystalline (Cry) toxins derived from the bacterium Bacillus thuringiensis (Bt). To delay Bt resistance by insect pests, refuges of non-Bt maize are grown in conjunction with Bt maize. Two factors influencing the success of the refuge strategy to delay resistance are the inheritance of resistance and fitness costs, with greater delays in resistance expected when inheritance of resistance is recessive and fitness costs are present. We measured inheritance and fitness costs of resistance for two strains of western corn rootworm with field-evolved resistance to Cry3Bb1 maize. Plant-based and diet-based bioassays revealed that the inheritance of resistance was non-recessive. In a greenhouse experiment, in which larvae were reared on whole maize plants in field soil, no fitness costs of resistance were detected. In a laboratory experiment, in which larvae experienced intraspecific and interspecific competition for food, a fitness cost of delayed larval development was identified, however, no other fitness costs were found. These findings of non-recessive inheritance of resistance and minimal fitness costs, highlight the potential for the rapid evolution of resistance to Cry3Bb1 maize by western corn rootworm, and may help to improve resistance management strategies for this pest.
... And it is used to eliminate weeds which grow near railway bars, gardens, parks, lakes and ponds. Human uses herbicides in their gardens to eliminate weeds such as dandelion and other weeds (12). ...
... However, in laboratory studies, Bt resistance had been acquired in WCR colonies, with one study noting the rise of resistance within several generations (Lefko et al. 2008;Meihls et al. 2008). The first report of field resistance evolved in WCR came in 2009 (Gassmann 2012). To combat Bt resistance in WCR, multi-toxin crops were adopted. ...
The western corn rootworm (Diabrotica virgifera virgifera LeConte) is the most important insect pest of corn in the United States Corn Belt, costing the agricultural industry over two billion dollars per year. As current management practices have been losing their effectiveness, new targets for corn rootworm control need to be explored. In this thesis, we focused on identifying rootworm chemoreceptors, which can be considered for potential targets for management. Transcriptomes from seven life stages were assembled for the western corn rootworm as well as two other related corn rootworm species, the northern corn rootworm (D. barberi) and the southern corn rootworm (D. undecimpunctata howardii). The three gustatory receptor genes (Gr1, Gr2, and Gr3) encoding the carbon dioxide receptors were identified from the three corn rootworm species. Unexpectedly, a duplication of Gr2 was identified. Many other candidate chemoreceptors, including a total of 200 gustatory receptors, 315 odorant receptors, and 80 ionotropic receptors, were also identified from the three rootworm species. Phylogenetic analysis among the chemoreceptors identified from corn rootworms and other insects (including both coleopteran and non-coleopteran species) revealed many coleopteran, especially rootworm-specific, expansions of some chemoreceptors. Gene expression analysus showed some of these chemoreceptors that have elevated expression in the neonate stage, indicating that these genes may have roles in host-seeking behavior of rootworm larvae. Potential ligand specificity of some rootworm chemoreceptors was also inferred based on the similarity to other insect chemoreceptors whose functions are known. Finally, using co-evolution analysis, protein structures of rootworm carbon dioxide receptors were predicted. The chemoreceptors identified in this study can serve as a valuable resource for developing new corn rootworm management strategies. Advisor: Etsuko N. Moriyama
... Our scouting data related to root injury did not show a clear difference between corn with or without a soilapplied insecticide. In the previous study conducted by Petzold-Maxwell et al. (2013) there was no significant difference in root injury between Bt corn with or without a soil-applied insecticide while Gassmann (2012) found that root injury was significantly lower for Bt corn with soil-applied insecticide compared to the control. ...
An on-farm research network is an organization of farmers that conducts agronomic experiments under local conditions. There is growing interest in on-farm research networks because they provide the infrastructure needed to test new products and management practices in farmers’ fields. Often, the results are usually presented as individual reports (i.e., a report summarizing the outcome for one trial), but this provides limited information difficult to generalize and does not allow presenting, in a synthetic way, all the results collected from the different trials. Moreover, there is an unexplored potential in detecting yield response variability patterns for better decision making. The overall objective of this thesis is to demonstrate the importance of identifying appropriate statistical methods for analyzing and visualizing on-farm research network data. Specifically, I focused on analyzing the on-farm research networks managed by the Iowa Soybean Association, and an adaptation was made with a French case-study. A data-analytics framework was developed to analyze multiple trials that use a common protocol and identify the conditions where an imposed treatment may or may not be effective. This framework used a random-effect model through a Bayesian approach and returned yield response estimates at the network and trial levels. The framework was implemented through a web-application for 51 different management practices on corn and soybean. The web-application includes dynamic data visualization features to enhance communication and information sharing, and is accessible to a broad audience to improve accessibility to on-farm research insights. A random-effects statistical model was used to compute prediction intervals describing a range of plausible yield response for a new (out-of-sample) trial, and compute the probability that the tested management practice will be ineffective in a new field. Depending on the level of between-trial variability, the prediction intervals were 2.2–12.1 times larger than confidence intervals for the estimated mean yield responses (i.e., at the network level) for all tested management practices. Using prediction intervals and the probability of ineffective treatment will prevent farmers from over-optimistic expectations that a significant effect at the network level will lead with high certainty to a yield gain on their farms. The data-analytic framework was adapted to a French on-farm research network focusing on the efficacy of biocontrol agent products against Botrytis cinerea, potassium bicarbonate and Aureobasidium pullulans, on organic vine. The results favored potassium bicarbonate as its efficacy on incidence at the network level is higher for diseased intensities between 0% and 10% than for Aureobasidium pullulans. For both biocontrol agents, the efficacy on incidence for a new trial is highly uncertain for intensity levels higher than 15%. Finally, this research investigated the impact of experimental plot scale (i.e., small-plot scale and field scale) on the effect of management practice on crop yield and identified the cause of potential discrepancies to inform on-farm decision-making better and adapt the extrapolation of the results. Taken together, this research represents the first major effort in consolidating results from on-farm research network and provides insight to make better farming management decisions.
... Europske populacije kukuruzne zlatice pokazale su rezistentnost na aldrin (Ciosi et al., 2009). Rezistentnosti na Bacillus thuringiensis (Bt) toksin Cry3Bb1 prvi puta je zabilježena kod populacija zlatica iz Iowe (Gassmann et al., 2011), a s vremenom se proširila i u ostale države SAD-a (Gassmann, 2012;Wangila et al., 2015). Pojava unakrsne rezistentnosti između toksina Cry3Bb1 i mCry3A stvorila je dodatne probleme u suzbijanju kukuruzne zlatice (Gassmann et al., 2014;Wangila et al., 2015). ...
Pojava rezistentnosti na insekticide kod kukaca ozbiljan je i rastući problem u poljoprivrednoj proizvodnji. Razvijanjem učinkovitih programa praćenja rezistentnosti omogućava se rano otkrivanje rezistentnosti te razvoj i pravovremena implementacija antirezistentnih strategija. Polimorfizam pojedinačnog nukleotida, engl. Syngle Nucleotide Polymorphisms (SNPs), novija je metoda analize cijelog genoma utvrđivanjem polimorfizma pojedinih nukleotida. Primjena SNPs-a na nemodelnim organizmima postala je pristupačno i lako dostupno sredstvo za generiranje važnih podataka o brojnim vrstama što bi inače bilo nemoguće zbog visokih troškova i često nedostatka stručnosti laboratorijskog osoblja. Obzirom na ogroman mogući broj pojedinačnih nukleotidnih polimorfizama koji se javljaju kod kukaca (od tisuće do milijuna), a koji se lako generiraju u jednom slijedu, pri izboru alata za utvrđivanje populacijske genetike neke vrste, SNPs tehnika je po svojim performansama daleko nadmašila primjenu mikrosatelita. S obzirom na velik broj SNPs-a (od tisuće do milijuna) koji se lako generiraju u jednom slijedu, nadmašili su mikrosatelite u izboru istraživačkog alata kada je u pitanju utvrđivanje. Kukuruzna zlatica (Diabrotica virgifera virgifera LeConte), krumpirova zlatica (Leptinotarsa decemlineata Say) i jabukin savijač (Cydia pomonella L.) najvažniji su štetnici koji ugrožavaju proizvodnju važnih ratarskih i voćarskih kultura u svijetu, ali i u Hrvatskoj. Sve navedene vrste razvile su rezistentnost na insekticide ili na strategije suzbijanja. U radu je prikazan pregled problema rezistentnosti kod navedenih štetnika, pregled genetskih istraživanja te način i mogućnost korištenja SNPs-ova za istraživanje rezistentnosti
... Europske populacije kukuruzne zlatice pokazale su rezistentnost na aldrin (Ciosi et al., 2009). Rezistentnosti na Bacillus thuringiensis (Bt) toksin Cry3Bb1 prvi puta je zabilježena kod populacija zlatica iz Iowe (Gassmann et al., 2011), a s vremenom se proširila i u ostale države SAD-a (Gassmann, 2012;Wangila et al., 2015). Pojava unakrsne rezistentnosti između toksina Cry3Bb1 i mCry3A stvorila je dodatne probleme u suzbijanju kukuruzne zlatice (Gassmann et al., 2014;Wangila et al., 2015). ...
Invazivna alohtona vrsta europski mračnjak (Abutilon theophrasti Medik.) danas je jedna od najagresivnijih i najštetnijih korovnih vrsta u Hrvatskoj. Jakim habitusom s naglašeno velikim listovima vrlo brzo može zasjeniti uzgajanu kulturu uzrokujući značajne gubitke prinosa. Relativno krupno sjeme omogućuje mu nicanje i iz dubljih slojeva tla što zbog kontinuiranog nicanja s različitih dubina iz tla predstavlja problem u suzbijanju. Uz spomenuto, dormantnost sjemena ograničava prognozu nicanja i uspješno suzbijanje ove vrste. Kompeticija mračnjaka, osim morfološkim značajkama, izražena je i alelopatskim sposobnostima. Europski mračnjak eksudatima svih biljnih dijelova može izazvati inhibiciju rasta i razvoja biljaka u svojoj blizini. Za pouzdano i učinkovito suzbijanje europskog mračnjaka, osobito u kulturama slabijih kompetitivnih sposobnosti, nužno je integrirati sve raspoložive mjere suzbijanja
... However, resistance to maize expressing Cry3Bb1 was reported in Iowa in 2009 [19]. Afterwards, resistance to Cry3Bb1 was detected in fields throughout Iowa [20,21] but also in WCR populations found in Illinois, Nebraska, and Minnesota [22][23][24]. Selected rootworm populations developed resistance to the toxins Cry34/Cry35Ab1, Cry3Bb1, and mCry3A under laboratory and greenhouse conditions [25][26][27][28]. ...
The western corn rootworm (WCR), is one of the most serious pests of maize in the United States. In this study, we aimed to find a reliable pattern of difference related to resistance type using population genetic and geometric morphometric approaches. To perform a detailed population genetic analysis of the whole genome, we used single nucleotide polymorphisms (SNPs) markers. For the morphometric analyses, hindwings of the resistant and non-resistant WCR populations from a laboratory in the US were used. Genetic results showed that there were some differences among the resistant US populations. The low value of pairwise FST = 0.0181 estimated suggests a lack of genetic differentiation and structuring among the putative populations genotyped. However, STRUCTURE analysis revealed three genetic clusters. Heterozygosity estimates (HO and HE) over all loci and populations were very similar. There was no exact pattern, and resistance could be found throughout the whole genome. The geometric morphometric results confirmed the genetic results, with the different genetic populations showing similar wing shape. Our results also confirmed that the hindwings of WCR carry valuable genetic information. This study highlights the ability of geometric morphometrics to capture genetic patterns and provides a reliable and low-cost alternative for preliminary estimation of population structure. The combined use of SNPs and geometric morphometrics to detect resistant variants is a novel approach where morphological traits can provide additional information about underlying population genetics, and morphology can retain useful information about genetic structure. Additionally, it offers new insights into an important and ongoing area of pest management on how to prevent or delay pest evolution towards resistant populations, minimizing the negative impacts of resistance.
... Insect pest species which have developed field-evolved resistance to a single or more than one Bt proteins in the transgenic crops and reduced crop efficacy have reached 7 in 2019 [5]. Some examples are Busseola fusca (Corn) (Cry1Ab) [6]; Spodoptera frugiperda (Corn) (Cry1F) [7]; Diabrotica virgifera virgifera (Corn) (Cry3Bb) [8]; Helicoverpa zea (Corn) (Cry1Ab) [9]; Pectinophora gossypiella (cotton) (Cry1Ac) [10]. The incidence of pink bollworm, P. gossypiella, has been also observed in Bollgard II versions of Bt cotton in India [11]. ...
In this study, we report a novel indigenous Bacillus thuringiensis (Bt) isolate, T26, which showed spores and crystals under scanning electron microscope and pathogenicity against the pink bollworm (Pectinophora gossypiella Saunders) in artificial diet based bioassay. SDS-PAGE analysis of the spore-crystal mixture of the Bt isolate, T26 revealed presence of three major protein bands of approximate molecular weights of 80, 55 and 40 kDa. The draft genome assembly consists of 56 scaffolds with an entire draft genome size of 5,054,095 bp. NCBI blast analysis revealed that assembled draft genome is spread over in a chromosome (4,818,543 bp) and one plasmid (235,552 bp). NCBI Prokaryotic Genome Annotation Pipeline (PGAP) showed presence of 5033 coding gene sequences and 159 RNAs genes. None of the known lepidopteran active genes (cry1, cry2 and cry9) could be detected with PCR or with whole genome sequence analysis using Bt toxin scanner tool or CryProcessor tool. Thus, presence of protein crystals and toxicity towards cotton pink bollworm and absence of any known cry/vip/cyt type of genes in draft genome indicates it is a novel type of Bt isolate. Further investigation of this genome sequence along with protein sequencing will lead to understand the novel factors responsible for its virulence and could be a useful tool for the insect resistance management in pink bollworm.
... Indeed, the commercially used proteinaceous insecticidal Cry toxins from Bacillus thuringiensis have toxic effects through binding to protein receptors in the insect midgut [21,44]. These protein receptors were shown to be frequently modified, thus conferring resistance against these biopesticides to the insect [44][45][46]. ...
Oyster mushrooms (Pleurotus spp.) have recently been shown to produce insecticidal bi-component protein complexes based on the aegerolysin proteins. A role for these proteins is thus indicated for defence and protection of the mushroom, and we propose their use as new environmentally friendly bioinsecticides. These aegerolysin-based protein complexes permeabilise artificial lipid vesicles through aegerolysin binding to an insect-specific sphingolipid, ceramide phosphoethanolamine (CPE), and they are cytotoxic for the Spodoptera frugiferda (Sf9) insect cell line. Tandem mass spectrometry analysis of the Sf9 lipidome uncovered lipids not previously reported in the literature, including in particular C14 sphingosine-based CPE molecular species, which comprised ~4 mol% of the whole lipidome. Further analysis of the lipid binding specificity of an aegerolysin from P. ostreatus, ostreolysin A6 (OlyA6), to lipid vesicles composed of commercial lipids, to lipid vesicles composed of the total lipid extract from Sf9 cells, and to HPLC-separated Sf9 cell lipid fractions containing ceramides, confirmed CPE as the main OlyA6 receptor, but also highlighted the importance of membrane cholesterol for formation of strong and stable interactions of OlyA6 with artificial and natural lipid membranes. Binding assays performed with glycan arrays and surface plasmon resonance, which included invertebrate-specific glycans, excluded these saccharides as potential additional OlyA6 receptors.
... Consequently, WCR has developed resistance to conventional insecticides, 5-10 crop rotation, 11 and commercial maize hybrids expressing the Bt insecticidal proteins Cry3Bb1, mCry3A, Cry3A.1Ab. [12][13][14] More recently there are indications of WCR resistance maize hybrids that express the Bt toxin Cry34/35Ab1. 15 Over the past decade, RNA interference (RNAi) 16 has been explored as an additional control measure for WCR. ...
Background:
RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins.
Results:
Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays.
Conclusion:
We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. This article is protected by copyright. All rights reserved.
... Our scouting data related to root injury did not show a clear difference between corn with or without a soil-applied insecticide. In the previous study conducted by Petzold-Maxwell et al. (2013) there was no significant difference in root injury between Bt-corn with or without a soil-applied insecticide while Gassmann (2012) found that root injury was significantly lower for Bt-corn with soil-applied insecticide compared to the control. ...
An on‐farm research network is an organization of farmers that conducts agronomic experiments under local conditions. It is common that an elementary statistical analysis be conducted for individual studies. However, there is unexplored potential in detecting yield response variability patterns for better decision making. We developed a data‐analytics framework and web‐application program that allows users to analyze multiple studies that use a common protocol and can identify the conditions where an imposed treatment may or may not be effective. The development of this data‐analytics framework is needed to improve predictions at the farm level that can lead to more cost‐effective, sustainable and environmentally sound agricultural production. Data visualization is an important part of data‐analytics. In this paper, we have developed and tested a Bayesian hierarchical model that can be used to assess the general agronomic performance of different management practices. Decision making related to new management practices should be based on the complete evidence, local conditions and economic considerations. The web‐application includes dynamic data visualization features to enhance communication and sharing of information with the goal to reach a broader audience.
Core Ideas We develop a data‐analytics framework and web‐application for on‐farm research trials.
A Bayesian hierarchical model quantifies the uncertainty in yield response.
The model helps assess alternative practices, products, and technologies among trial locations.
The framework provides a reactive break‐even economic analysis of alternative management practices.
... However, insects develop resistance against such transgenic plants in due course for their survival. A number of major pest species have developed resistance (field-evolved resistance) to one or more than one Bt toxins in crops and reduced transgenic crop efficacy viz., Busseola fusca (Corn) (Cry1Ab) [4]; Spodoptera frugiperda (Corn) (Cry1F) [5]; Diabrotica virgifera virgifera (Corn) (Cry3Bb) [6]; Helicoverpa zea (Corn) (Cry1Ab) [7]; Pectinophora gossypiella (cotton) (Cry1Ac) [8] and (Cry1Ac + Cry2Ab) [9]. Hence, isolation, identification and characterization of new Bt isolates with novel proteins are of significance for providing alternatives to counteract these problems. ...
Bacillus thuringiensis is a spore-forming bacterium that is pathogenic towards a range of insect and nematode species and had been widely used as a biopesticide. In this study, we present the morphological, molecular and genetic characteristics of an indigenous Bt isolate T414 which displayed an effective toxicity against Pectinophora gossypiella. Scanning electron microscopy revealed the presence of bipyramidal, spherical and cubic shaped protein crystals in its spore-crystal suspension. SDS-PAGE analysis of its spore-crystal mixture showed the presence of two major protein bands viz.130 and 65 kDa. Whole genome sequencing with MiSeq divulged that it contains a chromosome and many plasmids. The assembled genome finally contained 6493494bp. Automated annotation of this genome draft predicted 6877 coding sequences and 152 RNAs (rRNAs, tRNAs and ncRNAs). NCBI blast analysis showed that assembled genome was distributed in a chromosome and 15 different types of plasmids. Further analysis of draft sequence revealed it harbors parasporal crystal protein genes (cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab and cyt1), vegetative insecticidal protein gene (vip3Aa), all plasmid borne and various additional virulence factors such as chitinases, proteases, bacteriocins and hemolysins. From the analysis it is evident that all the Cry, Cyt or Vip toxins are plasmid borne and are present on two types of plasmids named as p414A and p414E in the present study. A cry2A type gene was cloned and sequenced. It was named as cry2Aa21 by Bt nomenclature committee.
... Crop rotation was found to be less effective in portions of the eastern Corn Belt due to the selection of D. virgifera with a lower fidelity to corn (Levine et al. 2002). Resistance to transgenic plants expressing endotoxins of the bacterium Bacillus thuringiensis Berliner (Bt) in the field was discovered in Iowa, with populations having reduced susceptibility to Cry3Bb1 (Gassmann et al. 2011, Gassmann 2012). More cases of Bt resistance were reported across the Corn Belt to products expressing the Cry3Bb1 (Gray 2012, Wangila et al. 2015, Zukoff et al. 2016, Ludwick et al. 2017, Schrader et al. 2017, as well as resistance to mCry3A with apparent cross-resistance to Cry3Bb1 and eCry.1Ab ...
Larval Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) were exposed to seven different entomopathogenic nematode species to test their potential infectivity in a laboratory setting. Known D. virgifera-infecting nematode species Heterorhabditis bacteriophora Poinar, Heterorhabditis megidis Poinar, Jackson & Klein, Steinernema feltiae Filipjev, and Steinernema carpocapsae Weiser were tested in a concerted experiment alongside Steinernema diaprepesi Nguyen & Duncan, Steinernema riobrave Cabanillas, Poinar & Raulston, and a Missouri wild-type H. bacteriophora which have not been previously tested on D. virgifera. The species S. rarum Doucet was tested separately for D. virgifera infectivity. Third-instar D. virgifera were exposed to either 60 or 120 nematodes per larva for 6 d. Following exposure, mortality was recorded and larvae were examined to determine the presence of active nematode infections. Results indicated a significantly higher proportion of larvae with active infections from the Heterorhabditidae species and S. diaprepesi than the other Steinernematidae species for both exposure rates; mortality data indicated a similar trend. Steinernema rarum showed almost no infectivity in laboratory experiments.
... We herein showed that Bt resistance is not necessarily beneficial to the insect on Bt plants in a multi-trophic environment. Bt plants suffer more damage when fed on by Bt-resistant WCR larvae (Gassmann 2012, Meihls et al. 2012, Frank et al. 2013. As a result from this extended damage, the induction of indirect defenses was stronger in Bt plants damaged by Bt-resistant WCR than in Bt hybrids attacked by susceptible larvae (Fig. 2). ...
Plants genetically modified to produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bt) have been extensively used to manage the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) in the United States. Evolution of WCR resistance to Bt toxins has forced the consideration of alternative pest management and improved insect resistance management. Entomopathogenic nematodes (EPNs), obligate insect parasites, are attracted toward volatile organic compounds (VOCs) emitted by maize roots after WCR herbivory. The production of VOCs of two types of Bt maize (MON88017 and MIR604) and their near-isolines was evaluated after induction with Bt-susceptible and resistant WCR. The attraction of EPNs toward the Bt hybrids was tested in the laboratory and the field. Bt hybrids emitted VOCs when induced by Bt-resistant insects whereas induction by Bt-susceptible WCR did not elicit a plant response. Survival of Bt-resistant WCR was lower on the hybrid attracting EPNs and similar to the survival of Bt-susceptible WCR without EPNs. This trade-off of Bt-resistance is defined here as an induced fitness cost, and offers a viable tool to management of Bt-resistant WCR.
... Current approaches for controlling WCR include crop rotation, broad-spectrum soil insecticides, and transgenic crops expressing protein toxins from Bacillus thuringiensis. However, WCR has developed resistance to many of these practices (Gray et al., 2009;Gassmann, 2012;Gassmann et al., 2011;Siegfried and Spencer, 2012), making it necessary to develop novel and more sustainable measures to control this pest. ...
RNA interference (RNAi) has proven effective for controlling pest insects such as western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). Previous studies have shown that WCR adults display a robust RNAi response to orally-administered double-stranded RNA (dsRNA). However, it is unclear how quickly the response occurs after ingestion or how long RNAi effect lasts after WCR stop ingesting diet containing dsRNA. In the current study, WCR adult females were provided with diet treated with dsRNAs of Laccase 2 and Argonaute 2, two nonessential genes, for 8 days. RNAi response in WCR females commenced as early as 10 hours after exposure to dsRNA and lasted up to 40 days after exposure to dsRNA ended. Our results show that dsRNA-mediated RNAi response in WCR females is rapid and long-lasting. These findings suggest that even a short-term ingestion of transgenic plants expressing dsRNA by WCR may have a sustained impact on this insect.
... This variety of findings owes to the diversity of mechanism, timing, and form (i.e., strength of competition) that density dependence can take. In general, negative density dependence may extend durability when it acts prior to selection for resistance (Heimpel et al. 2005) or incurs a fitness cost (Gassmann et al. 2012). Conversely, density dependence shortens durability when it acts after selection (Comins 1977, Tabashnik and Croft 1982, Glaum et al. 2012. ...
It has long been recognized that pest population dynamics can affect the durability of a pesticide, but dose remains the primary component of insect resistance management (IRM). For transgenic pesticidal traits such as Bt (Bacillus thuringiensis Berliner (Bacillales: Bacillaceae)), dose (measured as the mortality of susceptibles caused by a toxin) is a relatively fixed characteristic and often falls below the standard definition of high dose. Hence, it is important to understand how pest population dynamics modify durability and what targets they present for IRM. We used a deterministic model of a generic arthropod pest to examine how timing and strength of density dependence interacted with population growth rate and Bt mortality to affect time to resistance. As in previous studies, durability typically reached a minimum at intermediate doses. However, high population growth rates could eliminate benefits of high dose. The timing of density dependence had a more subtle effect. If density dependence operated simultaneously with Bt mortality, durability was insensitive to its strengths. However, if density dependence was driven by postselection densities, decreasing its strength could increase durability. The strength of density dependence could affect durability of both single traits and pyramids, but its influence depended on the timing of density dependence and size of the refuge. Our findings suggest the utility of a broader definition of high dose, one that incorporates population-dynamic context. That maximum growth rates and timing and strength of interactions causing density dependent mortality can all affect durability, also highlights the need for ecologically integrated approaches to IRM research.
... Gene silencing pathways involving siRNA and miRNA can potentially be exploited to control agricultural insect pests such as western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, a major insect pest of corn in the United States. Western corn rootworm presents a particular challenge to manage due to its ability to evolve resistance to insecticides, including Bacillus thuringiensis toxins expressed by transgenic corn plants [34][35][36][37]. Alternative pest management strategies have been proposed to treat insect pests that are prone to develop resistance to conventional control measures, including the use of transgenic plants expressing dsRNA or artificial miRNA that are specific to essential genes of insect pests. ...
RNA interference (RNAi) based approaches can potentially be used to control insect pests. These approaches may depend on the usage of microRNA (miRNA) or double stranded RNA (dsRNA) mediated gene knockdown, which likely involves proteins that regulate these pathways, such as Argonaute 1 (Ago1), Argonaute 2 (Ago2), Dicer 1 (Dcr1), Dicer 2 (Dcr2), and Drosha in insects. We previously performed functional characterization of Ago2 and Dcr2 of western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) and observed that knockdown of Ago2 and Dcr2 ameliorated the lethal effect induced by the dsRNA-mediated knockdown of an essential gene in WCR, thereby confirming the involvement of Ago2 and Dcr2 in the dsRNA pathway. In the current study, we identified and characterized additional members of the Argonaute and Dicer gene families, namely Ago1, Ago3, Aubergine, and Dcr1, in a previously developed WCR transcriptome. We also identified a Drosha homolog in the same transcriptome. We evaluated the impacts on WCR adult fitness associated with the dsRNA-mediated knockdown of Ago1, Ago2, Dcr1, Dcr2, and Drosha genes. Among these putative RNAi pathway genes, only the knockdown of Ago1 incurred significant fitness costs such as reduced survival and oviposition rate, as well as decreased egg viability. The present study, to our knowledge, represents the first report showing that Ago1 is critical to the survival of insect adults. Our findings suggest that Ago1 plays an essential role in broader life stages of an insect than previously thought. Importantly, since fitness costs were not observed, downregulation or loss of function of RNAi pathway genes such as Ago2 or Dcr2 may confer resistance to pest control measures that rely on the normal functions of these genes. However, the precise roles of these genes under field conditions (i.e., in the presence of possible viral pathogens) requires further investigation.
... Hence, Mp708 displays a suite of resistance traits that encompass both constitutive and inducible defense responses to three types of insect pests: a whorl feeder (fall armyworm), phloem feeder (corn leaf aphid) and root feeder (WCR). Because populations of WCR Are developing resistance to Bt-transgenes (Flagel et al. 2015;Gassmann 2012), soil-applied insecticide and persist despite crop rotation with soybean (Bigger 1932;Gray et al. 2009), the availability of a non-transgenic genotype with this remarkable range of native host plant resistance will be especially useful for discovering new resistance traits that can be implemented in plant breeding and pest management programs against a highly adaptable insect like WCR. Fig. 8 Time course of mir1 transcript in roots of Mp708 in response to WCR infestation. ...
Insect resistance against root herbivores like the western corn rootworm (WCR, Diabrotica virgifera virgifera) is not well understood in non-transgenic maize. We studied the responses of two American maize inbreds, Mp708 and Tx601, to WCR infestation using biomechanical, molecular, biochemical analyses, and laser ablation tomography. Previous studies performed on several inbreds indicated that these two maize genotypes differed in resistance to pests including fall armyworm (Spodoptera frugiperda) and WCR. Our data confirmed that Mp708 shows resistance against WCR, and demonstrates that the resistance mechanism is based in a multi-trait phenotype that includes increased resistance to cutting in nodal roots, stable root growth during insect infestation, constitutive and induced expression of known herbivore-defense genes, including ribosomal inhibitor protein 2 (rip2), terpene synthase 23 (tps23) and maize insect resistance cysteine protease-1 (mir1), as well high constitutive levels of jasmonic acid and production of (E)-β-caryophyllene. In contrast, Tx601 is susceptible to WCR. These findings will facilitate the use of Mp708 as a model to explore the wide variety of mechanisms and traits involved in plant defense responses and resistance to herbivory by insects with several different feeding habits.
... a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 gossypiella (Saunders) for Cry1Ac in India [26]; Western corn rootworm, Diabrotica v. virgifera LeConte for Cry3Bb and mCry3A in U.S. [27][28][29]; and corn earworm, H. zea (Boddie) for Cry1Ac in U.S [16,30]. Reports of field-evolved resistance by H. zea to Cry1Ac was refuted by Moar et al. [31], to which Tabashnik et al. [32] responded with further confirmations on the initial report. ...
[This corrects the article DOI: 10.1371/journal.pone.0169115.].
... One of two critical assumptions of the high-dose/refuge strategy documented to diminish evolution of resistance in target insects for Bt crops expressing Bt toxins is recessive or incompletely recessive inheritance of resistance, i.e., progenies from mating between homozygous susceptible and homozygous resistant adults are susceptible to the high-dose expression [14]. To some extent, the fitness of heterozygous individuals is lower on a Bt crop than the homozygous resistant parent, the maximum interruption in resistance can be acquired with resistance traits that are functionally recessive [15,16]. ...
A strain of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), has evolved >800-fold resistance to Cry1Ie (ACB-IeR) after 49 generations of selection. The inheritance pattern of resistance to Cry1Ie in ACB-IeR strain and its cross-resistance to other Bt toxins were determined through bioassay by exposing neonates from genetic-crosses to toxins incorporated into the diet. The response of progenies from reciprocal F1 crosses were similar (LC50s: 76.07 vs. 74.32 μg/g), which suggested the resistance was autosomal. The effective dominance (h) decreased as concentration of Cry1Ie increased. h was nearly recessive or incompletely recessive on Cry1Ie maize leaf tissue (h = 0.02), but nearly dominant or incompletely dominant (h = 0.98) on Cry1Ie maize silk. Bioassay of the backcross suggested that the resistance was controlled by more than one locus. In addition, the resistant strain did not perform cross-resistance to Cry1Ab (0.8-fold), Cry1Ac (0.8-fold), Cry1F (0.9-fold), and Cry1Ah (1.0-fold). The present study not only offers the manifestation for resistance management, but also recommends that Cry1Ie will be an appropriate candidate for expression with Cry1Ab, Cry1Ac, Cry1F, or Cry1Ah for the development of Bt maize.
... Similar to earlier results obtained by Prasifka et al. 3 and Head et al. 4 for SmartStax, the results here show that the pyramid products SmartStax and SmartStax PRO can provide protection from larval feeding and are both capable of significantly reducing adult CRW emergence over a range of relevant conditions. As resistance evolves to single-Bt traits that have been deployed for nearly a decade, 6,7,26,27 the durability of pyramid products containing these Bt traits is reduced, emphasizing the need for novel modes of action against CRW. Here we provide evidence that on grower fields where WCR densities are high and resistance to Cry3Bb1may be present, the addition of DvSnf7 in SmartStax PRO can reduce root damage compared to SmartStax and prolong the durability of Cry3Bb1 and Cry34Ab1/Cry35Ab1. ...
Background:
Cases of western corn rootworm (WCR) field-evolved resistance to Cry3Bb1 and other corn rootworm (CRW) control traits have been reported. Pyramid products expressing multiple CRW traits can delay resistance compared to single trait products. We used field studies to assess the pyramid CRW corn products, SmartStax (expressing Cry3Bb1 and Cry34Ab1/Cry35Ab1) and SmartStax PRO (expressing Cry3Bb1, Cry34Ab1/Cry35Ab1 and DvSnf7), at locations with high WCR densities and possible Cry3Bb1 resistance, and to assess the reduction in adult emergence attributable to DvSnf7 and other traits. Insect resistance models were used to assess durability of SmartStax and SmartStax PRO to WCR resistance.
Results:
SmartStax significantly reduced root injury compared to non-CRW-trait controls at all but one location with measurable WCR pressure, while SmartStax PRO significantly reduced root injury at all locations, despite evidence of Cry3Bb1 resistance at some locations. The advantage of SmartStax PRO over SmartStax in reducing root damage was positively correlated with root damage on non-CRW-trait controls. DvSnf7 was estimated to reduce WCR emergence by approximately 80-95%, which modelling indicated will improve durability of Cry3Bb1 and Cry34Ab1/Cry35Ab1 compared to SmartStax.
Conclusion:
The addition of DvSnf7 in SmartStax PRO can reduce root damage under high WCR densities and prolong Cry3Bb1 and Cry34Ab1/Cry35Ab1 durability.
... Bats have a lower pest-control impact on Bt than on conventional cotton [13]; however, over the past two years, mounting evidence from around the world suggests that insect pests are evolving resistance to Btmodified crops [18]. While not yet widespread, Bt-resistant pests have been found in the field in India, China, and the U.S., and in laboratory studies [19][20][21][22][23]. ...
... a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 gossypiella (Saunders) for Cry1Ac in India [26]; Western corn rootworm, Diabrotica v. virgifera LeConte for Cry3Bb and mCry3A in U.S. [27][28][29]; and corn earworm, H. zea (Boddie) for Cry1Ac in U.S [16,30]. Reports of field-evolved resistance by H. zea to Cry1Ac was refuted by Moar et al. [31], to which Tabashnik et al. [32] responded with further confirmations on the initial report. ...
Background
Transgenic corn engineered with genes expressing insecticidal toxins from the bacterium Bacillus thuringiensis (Berliner) (Bt) are now a major tool in insect pest management. With its widespread use, insect resistance is a major threat to the sustainability of the Bt transgenic technology. For all Bt corn expressing Cry toxins, the high dose requirement for resistance management is not achieved for corn earworm, Helicoverpa zea (Boddie), which is more tolerant to the Bt toxins.
Methodology/Major Findings
We present field monitoring data using Cry1Ab (1996–2016) and Cry1A.105+Cry2Ab2 (2010–2016) expressing sweet corn hybrids as in-field screens to measure changes in field efficacy and Cry toxin susceptibility to H. zea. Larvae successfully damaged an increasing proportion of ears, consumed more kernel area, and reached later developmental stages (4th - 6th instars) in both types of Bt hybrids (Cry1Ab—event Bt11, and Cry1A.105+Cry2Ab2—event MON89034) since their commercial introduction. Yearly patterns of H. zea population abundance were unrelated to reductions in control efficacy. There was no evidence of field efficacy or tissue toxicity differences among different Cry1Ab hybrids that could contribute to the decline in control efficacy. Supportive data from laboratory bioassays demonstrate significant differences in weight gain and fitness characteristics between the Maryland H. zea strain and a susceptible strain. In bioassays with Cry1Ab expressing green leaf tissue, Maryland H. zea strain gained more weight than the susceptible strain at all concentrations tested. Fitness of the Maryland H. zea strain was significantly lower than that of the susceptible strain as indicated by lower hatch rate, longer time to adult eclosion, lower pupal weight, and reduced survival to adulthood.
Conclusions/Significance
After ruling out possible contributing factors, the rapid change in field efficacy in recent years and decreased susceptibility of H. zea to Bt sweet corn provide strong evidence of field-evolved resistance in H. zea populations to multiple Cry toxins. The high adoption rate of Bt field corn and cotton, along with the moderate dose expression of Cry1Ab and related Cry toxins in these crops, and decreasing refuge compliance probably contributed to the evolution of resistance. Our results have important implications for resistance monitoring, refuge requirements and other regulatory policies, cross-resistance issues, and the sustainability of the pyramided Bt technology.
Cotton crop yields are largely affected by infestations of Anthonomus grandis , which is its main pest. Although Bacillus thuringiensis (Bt) derived proteins can limit insect pest infestations, the diverse use of control methods becomes a viable alternative in order to prolong the use of technology in the field. One of the alternative methods to Bt technology has been the utilization of certain Pseudomonas species highly efficient in controlling coleopteran insects have been used to produce highly toxic insecticidal proteins. This study aimed to evaluate the toxicity of IPD072Aa and PIP-47Aa proteins, isolated from Pseudomonas spp., in interaction with Cry1Ia10, Cry3Aa, and Cry8B proteins isolated from B . thuringiensis , to control A . grandis in cotton crops. The genes IPD072Aa and PIP-47Aa were synthesized and cloned into a pET-SUMO expression vector. Moreover, Cry1Ia10, Cry3Aa, and Cry8B proteins were obtained by inducing recombinant E . coli clones, which were previously acquired by our research group from the Laboratory of Bacteria Genetics and Applied Biotechnology (LGBBA). These proteins were visualized in SDS-PAGE, quantified, and incorporated into an artificial diet to estimate their lethal concentrations (LC) through individual or combined bioassays. The results of individual toxicity revealed that IPD072Aa, PIP-47Aa, Cry1Ia10, Cry3Aa, and Cry8B were efficient in controlling A . grandis , with the latter being the most toxic. Regarding interaction assays, a high synergistic interaction was observed between Cry1Ia10 and Cry3Aa. All interactions involving Cry3Aa and PIP-47Aa, when combined with other proteins, showed a clear synergistic effect. Our findings highlighted that the tested proteins in combination, for the most part, increase toxicity against A . grandis neonate larvae, suggesting possible constructions for pyramiding cotton plants to the manage and the control boll weevils.
Nearly all maize seed sold in the United States includes a neonicotinoid seed treatment (NST), meant to protect seedlings against early-season insect pests. For key pests, including western corn rootworm (Diabrotica virgifera virgifera LeConte) (D.v.v), insecticidal proteins derived from Bacillus thuringiensis (Bt) are expressed in plant tissues as alternatives to soil-applied insecticides. Insect resistance management (IRM) plans use non-Bt “refuges” to encourage survival of Bt-susceptible D.v.v., which maintains susceptible alleles in the population. In non-cotton producing regions, IRM guidelines require a minimum 5% blended refuge for maize expressing more than 1 trait targeting D.v.v. Prior work has shown that 5% blends yield insufficient proportions of refuge beetles to contribute reliably to IRM. Whether NSTs interfere with survivorship of refuge beetles is unknown. Our objective was to determine whether NSTs affect proportions of refuge beetles, and secondarily, to determine whether NSTs provide agronomic advantages over Bt seed alone. To reveal host plant type (i.e., Bt or refuge), we used a stable isotope (15N) to mark refuge plants in plots with 5% seed blends. To assess refuge performance between treatments, we compared proportions of beetles from respective natal hosts. In all site-years, NSTs showed inconsistent effects on proportions of refuge beetles. Treatment comparisons showed inconsistent agronomic benefits of NSTs when combined with Bt traits. Our results demonstrate that NSTs have a negligible impact on refuge performance and reinforces the assertion that 5% blends are serving little benefit for IRM. Plant stand and yield were not improved by NSTs.
Two species of Diabrotica have evolved resistance to crop rotation that involves planting the same primary crop every second year in the same location. Diabrotica barberi has been adapted by extending egg diapause for more than one winter. Diabrotica virgifera virgifera has lost its fidelity for corn, Zea mays, during the ovipositional period; eggs are laid in most fields of vegetation in the crop landscape. Both adaptations permit eggs to hatch in cornfields. D. v. virgifera has also evolved resistance to most other integrated pest management (IPM) tactics over the past 60 years, including all currently commercialized insecticidal corn hybrids. This chapter explores the diverse approaches to IPM and the role of insect behavior in resistance management.
The concepts of population genetics and evolution seem simple from an abstract, academic perspective, but in reality, nature is too heterogeneous and dynamic to allow science to be so easy. To provide a reference scenario, we describe evolution by natural selection in patchy landscapes without consideration of pest density. Complications involving a variety of ecological processes are discussed. We describe random genetic drift and demographic Allee effects on evolution. We explore the complexities of genetic architecture for arthropods that evolve resistance. The roles of mutation rate, gene interaction, fitness costs, and dominance (expression of resistance) are presented. Even though most research has been on diploid arthropods, many species can be haplodiploid (males are haploid, females are diploid). The relationship between generation time and resistance evolution is evaluated. We emphasize the importance of temporal and spatial scales for hypotheses and recommendations regarding evolution of resistance.
The insecticidal crystalline proteins (Crys) are a family of insect endotoxin functioning in crop protection. As insects keep evolving into tolerance to the existing Crys, it is necessary to discover new Cry proteins to overcome potential threatens. Crys possess three functional domains at their N-termini, and the most active region throughout evolution was found at the domain-III. We swapped domain-IIIs from various Cry proteins and generated seven chimeric proteins. All recombinants were expressed in Escherichia coli and their toxicity was assessed by dietary exposure assays. Three of the seven Crys exhibited a high toxicity to Asian corn borer over the controls. One of them, Cry1Ab-Gc, a chimeric Cry1Ab being replaced with the domain-III of Cry1Gc, showed the highest toxicity to rice stem borer when it was over-expressed in Oryza sativa. Furthermore, it was also transformed into maize, backcrossed into commercial maize inbred lines and then produced hybrid to evaluate their commercial value. Transgenic maize performed significant resistance to the Asian corn borer without affecting the yield. We further showed that this new protein did not have adverse effects on the environment. Our results indicated that domain III swapped of Crys could be used as an efficient method for developing new engineered insecticidal protein.
Though cry gene transformed crops have successfully revolutionized modern agriculture, it is still necessary to discover new Cry proteins to overcome potential threatens from the development of resistant insect populations. We swapped domain-IIIs with various Cry proteins and engineered seven chimeric proteins, aiming to produce new engineered hybrid insecticidal proteins. Seven recombinant proteins were expressed in Escherichia coli . Three proteins exhibited high toxicity against Asian corn borer in dietary exposure assays. Three hybrid proteins were further transformed to rice (cv. Jijing88) to determine their insecticidal activity. Cry1Ab/Gc hybrid proteins, Cry1Ab being replaced by the domain-III of Cry1Gc, showed significantly more toxic against rice stem borer than others. Furthermore, Cry1Ab/Gc gene was transformed into maize (cv. HiII), then backcrossed into commercial maize inbred lines (cv. Ji853 and Y822), and formulated into Xiangyu 998 hybrid to evaluate their commercial value. Transgenic maize performed significant resistance improvement to the Asian corn borer without affecting the yield, and this new protein did not have adverse effects on the environment. Our result proved domain-swapped could be used as an efficient method for exploring new cry genes and engineered hybrid insecticidal protein. Cry1Ab/Gc provides a new tool for Lepidopteran insects resistant management in rice and maize.
Insect pests are a threat to agriculture as they cause a loss of 15–22% to economically important crops every year. Bacillus thuringiensis produces parasporal crystal inclusions that have insecticidal ‘Cry’ proteins which are toxic to insect larvae of the order Lepidoptera, Coleoptera and Diptera, etc. In the present study, 40 different soil samples from Amritsar and its surrounding areas were selected for isolation of B. thuringiensis. The rod shaped, gram-positive bacterial isolates were further analyzed for characteristic crystal formation using phase contrast and scanning electron microscopy. 6 Bacillus samples containing cry genes were identified using the universal primers for cry genes, of which one isolate exhibited a protein band of ~95 kDa. This protein was purified using a Sephadex G-75 column. The insecticidal assays conducted with purified Cry protein on insect larvae of lepidopteran and dipteran orders viz. Spodoptera litura, Galleria malonella, Bactrocera cucurbitae and Culex pipens revealed considerable detrimental effects. A significant increase in larval mortality was observed for the larvae of all insects in a concentration dependent manner when treated with Cry protein purified from B. thuringenisis VIID1. The purified Cry protein did not have any significant effect on honey bee larvae.
BACKGROUND
The corn earworm, Helicoverpa zea (Boddie), is a major target pest of pyramided Bt corn and cotton in the U.S. Field‐evolved practical resistance to Cry1 and Cry2 proteins in H. zea has been documented in multiple locations in the U.S. Understanding the genetic basis of Bt resistance is essential in developing insect resistance management (IRM) strategies for the sustainable use of the Bt crop technology. In this study, we characterized the genetic bases of Cry2Ab2 resistance in H. zea using diet‐overlay bioassays with two different forms of Cry2Ab2 protein.
RESULTS
Laboratory bioassays using a Cry2Ab2‐resistant (RR) strain, a susceptible (SS) strain, as well as cross and backcross strains, revealed that resistance to Cry2Ab2 was autosomally inherited and controlled by more than one locus. In diet bioassays, the dominance of Cry2Ab2 resistance in H. zea varied from incompletely recessive to incompletely dominant across all tested Cry2Ab2 concentrations of either Bt corn leaf powder or solubilized protein. On leaf tissue of TwinLink cotton (expressing Cry1Ab and Cry2Ae), Cry2Ab2 resistance in H. zea was completely dominant.
CONCLUSION
These results have significant implications for understanding the widespread field‐evolved resistance of H. zea against Cry1 and Cry2 proteins in Bt corn and cotton and should be useful in developing effective IRM strategies for H. zea.
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The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is the most damaging corn pest in the U.S. Corn Belt, costing producers over $1 billion annually in control and damage costs. Currently, corn producers rely on three control strategies for WCR management: crop rotation, chemical insecticides, and transgenic corn expressing Bacillus thuringiensis Berliner (Bt) proteins. Populations of WCR have evolved resistance to all of these tactics, limiting effective control strategies for producers. RNA interference (RNAi), is the newest mode of action developed for WCR management. In July 2017, the first RNAi plant-incorporated protectant (PIP) corn product was approved for production in the United States. This product, marketed under the trade name SmartStax PRO®, will express two Bt proteins and DvSnf7 double-stranded RNA for WCR control. Similar to current PIPs, resistance monitoring protocols must be established before adoption of this technology to delay resistance evolution. This study characterized the variability of adult susceptibility due to age and sex. Male beetles were most tolerant to dsRNA at 2-days post-emergence, but responded uniformly to dsRNA at 10-, 20-, and 30-days post-emergence and were significantly more susceptible than their female counterparts at 10- and 20-days post-emergence. Female adults responded uniformly for 2-, 10-, and 20-days post-emergence, but were significantly more susceptible at 30-days post-emergence. Baseline susceptibilities for U.S. Corn Belt populations of WCR were established and the potential for use of adult WCR for DvSnf7 dsRNA susceptibility monitoring in field populations was evaluated. Overall, most field populations were uniform in their larval susceptibility to DvSnf7 dsRNA. Adult male susceptibility was more variable compared to larvae and correlation ratios between adult males and larvae were not always consistent. Therefore, it may not be possible to use adult WCR to monitor changes in DvSnf7 dsRNA susceptibility, especially if small shifts in susceptibility impact product performance. Advisors: Ana M. Vélez Arango and Lance J. Meinke
Recent studies suggest that resistance in Helicoverpa zea (Boddie) (Lepidoptera, Noctuidae) to Cry1A(b/c) and Cry2Ab2 toxins from the bacterium Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) has increased and field efficacy is impacted in transgenic corn and cotton expressing these toxins. A third toxin, Vip3A, is available in pyramids expressing two or more Bt toxins in corn hybrids and cotton varieties, but uncertainty exists regarding deployment strategies. During a growing season, H. zea infests corn and cotton, and debate arises over use of Vip3A toxin in corn where H. zea is not an economic pest. We used a three-locus, spatially explicit simulation model to evaluate when using Vip3A in corn might hasten evolution of resistance to Vip3A, with implications in cotton where H. zea is a key pest. When using a conventional refuge in corn and initial resistance allele frequencies of Cry1A and Cry2A were 10%, transforming corn with Vip3A slowed resistance to these toxins and delayed resistance evolution to the three-toxin pyramid as a whole. When Cry resistance allele frequencies exceeded 30%, transforming corn with Vip3A hastened the evolution of resistance to the three-toxin pyramid in cotton. When using a seed blend refuge strategy, resistance was delayed longest when Vip3A was not incorporated into corn and used only in cotton. Simulations of conventional refuges were generally more durable than seed blends, even when 75% of the required refuge was not planted. Extended durability of conventional refuges compared to other models of resistance evolution are discussed as well as causes for unusual survivorship in seed blends.
Creation of genetically modified plants with an incorporated system protecting them from pests is one of the main goals in modern agricultural biotechnology. From the mid-1990s to the present, the majority of such transgenic crops is represented by carriers of the insecticidal Cry and Vip genes of the bacterium Bacillus thuringiensis. However, a tendency to change this strategy in favor of the incorporated defense systems based on RNA interference has become evident during the recent decade. Evolutional paths of phytophages' responses to insecticidal GM plants that are armed with these genetic constructions are discussed in the review.
Western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), has developed resistance to transgenic corn that produces the insecticidal toxin Cry3Bb1 derived from the bacterium Bacillus thuringiensis (Bacillales: Bacillaceae) (Bt), with cross-resistance extending to corn with Bt toxins mCry3A and eCry3.1Ab. Additionally, some populations of western corn rootworm have evolved resistance to Cry34/35Ab1 corn. We conducted a 2-yr field and laboratory study that included three field locations: 1) Bt-susceptible population, 2) field with a recent history of Cry3Bb1 resistance, and 3) field with a long-term history of Cry3Bb1 resistance. The population with recently evolved Cry3Bb1 resistance showed resistance to Cry3Bb1 corn in both laboratory bioassays and field evaluations; by contrast, the population with a long-term history of Cry3Bb1 resistance showed resistance, in both laboratory and field experiments to Cry3Bb1 corn and corn with a pyramid of mCry3A plus eCry3.1Ab corn. Field-based evaluations also showed that the field population with a long-term history of Cry3Bb1 resistance imposed higher root injury to Cry3Bb1 corn and the pyramid of mCry3A plus eCry3.1Ab compared with the susceptible control. The results of this study are discussed in the context of developing strategies to manage western corn rootworm in areas where populations have evolved resistance to Cry3Bb1 corn.
Aegerolysins ostreolysin A (OlyA) and pleurotolysin A (PlyA), and pleurotolysin B (PlyB) with the membrane-attack-complex/perforin domain are proteins from the mushroom genus Pleurotus. Upon binding to sphingomyelin/cholesterol-enriched membranes, OlyA and PlyA can recruit PlyB to form multimeric bi-component transmembrane pores. Recently, Pleurotus aegerolysins OlyA, PlyA2 and erylysin A (EryA) were demonstrated to preferentially bind to artificial lipid membranes containing 50 mol% ceramide phosphoethanolamine (CPE), the main sphingolipid in invertebrate cell membranes. In this study, we demonstrate that OlyA6, PlyA2 and EryA bind to insect cells and to artificial lipid membranes with physiologically relevant CPE concentrations. Moreover, these aegerolysins permeabilize these membranes when combined with PlyB. These aegerolysin/PlyB complexes show selective toxicity toward western corn rootworm larvae and adults and Colorado potato beetle larvae. These data strongly suggest that these aegerolysin/PlyB complexes recognize CPE as their receptor molecule in the insect midgut. This mode of binding is different from those described for similar aegerolysin-based bacterial complexes, or other Bacillus thuringiensis Cry toxins, which have protein receptors. Targeting of Pleurotus aegerolysins to CPE and formation of transmembrane pores in concert with PlyB suggest the use of aegerolysin/PlyB complexes as novel biopesticides for the control of western corn rootworm and Colorado potato beetle.
High adoption rates of single-gene Bacillus thuringiensis (Bt) Cry1Ac soybean impose selection pressure for resistance in the soybean looper, Chrysodeixis includens, a major defoliator in soybean and cotton crops. To anticipate and characterize resistance profiles that can evolve, soybean looper larvae collected from field crops in Brazil in 2013 were selected for resistance to Cry1Ac. Using two methods of selection viz., chronic exposure to Cry1Ac cotton leaves and the seven-day larval exposure to purified Cry1Ac on the artificial diet, 31 and 127-fold resistance was obtained in 11 and 6 generations of selection, respectively. The resistance trait had realized heritability of 0.66 and 0.72, respectively, indicating that most of the phenotypic variation in Cry1Ac susceptibility of the soybean looper larvae was due to additive genetic variation. The Cry1Ac-selected populations showed positive cross-resistance to Cry1Ab (6.7–8.7 fold), likely because these Bt toxins have a very similar molecular structure. Importantly, the Cry1Ac-selected populations became more susceptible to Cry2Aa and Cry1Fa, showing negative crossresistance (up to 6-fold, P < 0.05). These results indicate that Cry1Ac, Cry1Fa, and Cry2A are compatible
in a multi-toxin approach to minimize the risk of rapid adaptation of the soybean looper to Bt toxins.
The western corn rootworm, Diabrotica virgifera virgifera LeConte, is the most serious pest of maize across the US Corn Belt and now Europe. The beetle has repeatedly demonstrated its ability to adapt to pest management strategies through evolution of resistance to conventional insecticides, the cultural practice of crop rotation and now to the Bacillus thuringiensis (Bt) toxin which is produced by genetically modified maize. In this study, the wing morphology of 358 western corn rootworm adults from Iowa, Indiana and Illinois, USA, was investigated using geometric morphometric procedures. The populations investigated comprised resistant (i.e. soybean–maize rotation and Bt-maize variants) and non-resistant beetle populations. Data analysis was divided into two groups: (1) resistant versus non-resistant and (2) rotation-resistant versus Bt-maize rootworm populations. Results showed that morphological differences exist in the hind-wing shape of both rotation and Bt-maize-resistant versus non-resistant populations and rotation-resistant versus Bt-maize-resistant variants. Across all three types of rootworm variants investigated, the movement of landmarks 8, 9 and 14 drove the wing shape differences found. These landmarks relate to the basal radial vein and are a key anatomical character used to distinguish different wing morphotypes in rootworm. This study demonstrates the utility of hind-wing morphology/shape as an inexpensive and accessible population biomarker for rootworm. With simple equipment (camera mounted microscopes or flatbed scanners) and readily available free software to capture and analyse landmark (shape and size) data, it is possible to effectively monitor pest resistance development and associated field-based population-level differences. The biological implications of the differences in wing shape found and how this relates to rootworm flight and consequently its dispersal and invasion capabilities are also explored.
Phytophagous insect pests strongly affect the productivity and profitability of agriculture and forestry. Despite the well-known sensitivity of insects to abiotic effects such as temperature, their potential responses to ongoing climate change remain unclear. Here we compile and review documented climate change responses of 31 of the globally most severe insect pests of agriculture and forestry, focussing on species for which long-term, high-quality data are available. Most of the selected species show at least one response affecting their severity as pests, including changes in geographic range, population dynamics, life-history traits, and/or trophic interactions. The agricultural pests show strikingly more diverse and generally weaker responses to climate change than the forestry pests. However, the agricultural pests seem to increase more in detrimental ecological impact than do the forestry pests. Unexpectedly, 59% of the species show responses of reduced potential impacts as pests under ongoing climate change. This reduction in impact is further supported by a thermal sensitivity analysis showing little benefit of climate warming in relation to the optimal developmental temperatures for the majority of these pests under both current climate and future projections. The documented variation in responses indicates that efforts to mitigate undesirable climate change effects must target individual species, taking into account the complex ecological and evolutionary mechanisms underlying their responses.
RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.
Plant-mediated RNA interference (RNAi) shows great potential in crop protection. It relies on plants stably expressing double-stranded RNAs (dsRNAs) that target essential genes in pest insects. Practical application of this strategy is challenging because producing sufficient amounts of stable dsRNA in plants has proven to be difficult to achieve with conventional transgenesis. In addition, many insects do not respond to exogenously applied dsRNAs, either degrading them or failing to import them into the cytoplasm. We summarize recent progress in RNAi-mediated insect pest control and discuss factors determining its efficacy. Expressing dsRNA in chloroplasts overcomes many of the difficulties previously encountered. We also highlight remaining challenges and discuss the environmental and biosafety issues involved in the use of this technology in agriculture.
Understanding how soil ecosystem responds to transgenic Bacillus thuringiensis (Bt) rice is necessary for environmental risk assessment. While the influences of short-term cultivation of Bt rice on soil properties have been reported previously, little is known about the long-term effects of Bt rice on soil ecosystems. In this study, soil samples were taken from a long-term rice cultivation site in Fujian Province, China, where transgenic Bt rice (Kefeng-6) and its non-Bt parent breed (Minghui-86) had been continuously cultivated for 8 years. Soil Bt protein concentration and a total of 16 variables were analyzed to assess potential risks of soil health under Bt rice cultivation. The results revealed that soil Bt protein is unlikely to accumulate after Bt rice cultivated in the field, and no consistently significant changes were observed in soil enzymatic activities (catalase, dehydrogenase, acid phosphatase, and urease), microbial biomass (microbial carbon and nitrogen), total organic carbon, decomposition (soil respiration, Q10, and qCO2), soil nitrogen and phosphorus contents. Due to a local tradition that aboveground biomass was removed after harvest, the increased net primary productivity by Bt rice cultivation did not significantly change soil C cycling. Results of this study suggested that on the aspects of soil microbial functioning and C cycling, long-term cultivation of Bt rice is unlikely to result in significant effects on soil health.
Foliar applications of commercial formulations of the insecticidal spore-crystal protein complex of Bacillus thuringiensis subsp. kUTstaki caused development of resistance in field populations of a major lepidopteran pest of vegetables, diamondback moth, Plutella xylostella (L.). Laboratory bioassays of larvae showed that the LC"o" and LC•• for a field population of diamondback moth treated repeatedly with B. thuTingiensis were 25 to 33 times greater than the respective LC"o"sand LC••'s for two susceptible laboratory colonies. Mortality at the field rate of B. thuTingiensis was 34-35% in two resistant populations compared with 90-100% in two susceptible laboratory colonies. The results suggest that the potential for resistance development in pest populations is an important consideration for deployment of B. thuTingiensis toxin genes in genetically-engineered crop plants and use of B. thuringiensis in related tactics.
Topical bioassays were conducted in 1995 to estimate the susceptibility of adult western corn rootworm, Diabrotica virgifera virgifera LeConte, populations from Nebraska to technical grade methyl parathion, carbaryl, and bifenthrin. Significant differences in susceptibility occurred among populations for each insecticide. The largest relative differences in LD50 values between the most tolerant and susceptible field populations were 16.4- and 9.4-fold for methyl parathion and carbaryl, respectively. The F1 colonies also exhibited significant differences in susceptibility to methyl parathion and carbaryl indicating that susceptibility traits are heritable. The response to bifenthrin (up to 4-fold difference in LD50 values) was more homogeneous across populations than the response to the other 2 compounds. Populations with the largest LD50 values were located in 2 areas where adult management programs using carbamate and organophosphate insecticides have been extensively applied and control failures have been commonly reported. Results strongly suggest that corn rootworm management practices have selected for significant levels of resistance to methyl parathion, carbaryl, or both in certain areas of Nebraska.
Transgenic crops producing Bacillus thuringiensis (Bt) toxins are used worldwide to control major pests of corn and cotton. Development of strategies to delay the evolution of pest resistance to Bt crops requires an understanding of factors affecting responses to natural selection, which include variation in survival on Bt crops, heritability of resistance, and fitness advantages associated with resistance mutations. The two main strategies adopted for delaying resistance are the refuge and pyramid strategies. Both can reduce heritability of resistance, but pyramids can also delay resistance by reducing genetic variation for resistance. Seasonal declines in the concentration of Bt toxins in transgenic cultivars, however, can increase the heritability of resistance. The fitness advantages associated with resistance mutations can be reduced by agronomic practices, including increasing refuge size, manipulating refuges to increase fitness costs, and manipulating Bt cultivars to reduce fitness of resistant individuals. Manipulating costs and fitness of resistant individuals on transgenic insecticidal crops may be especially important for thwarting evolution of resistance in haplodiploid and parthenogenetic pests. Field-evolved resistance to Bt crops in only five pests during the last 14 years suggests that the refuge strategy has successfully delayed resistance, but the accumulation of resistant pests could accelerate.
A simulation model of the temporal and spatial dynamics and population genetics of western corn rootworm, Diabrotica virgifera virgifera LeConte, was created to evaluate the use of block refuges and seed blends in the management of resistance to transgenic insecticidal corn (Zea mays L.). This Bt corn expresses one transgenic corn event, DAS-59122-7, that produces a binary insecticidal protein toxin (Cry34Ab1/Cry35Ab1) and provides host-plant resistance. The model incorporates the latest information about larval and adult behavior. Results of this modeling effort indicate that the seed-blend scenarios in many cases produced equal or greater durability than block refuges that were relocated each year. Resistance evolved in the most likely scenarios in 10-16 yr. Our standard analysis presumed complete adoption of 59122 corn by all farmers in our hypothetical region, no crop rotation, and 100% compliance with Insect Resistant Management (IRM) regulations. As compliance levels declined, resistance evolved faster when block refuges were deployed. Seed treatments that killed the pest when applied to all seeds in a seed blend or just to seeds in Bt corn blocks delayed evolution of resistance. Greater control of the pest population by the seed treatment facilitated longer durability of the transgenic trait. Therefore, data support the concept that pyramiding a transgenic insecticidal trait with a highly efficacious insecticidal seed treatment can delay evolution of resistance.
Mortality of the western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae due to feeding on maize, Zea mays L., expressing Bacillus thuringiensis Berliner (Bt) was evaluated in five Missouri sites in 2007, 2008, and 2009. Specifically, eCry3.1Ab (5307), mCry3A (MIR604), and eCry3.1Ab plus mCry3A proteins relative to survivorship on maize with the same genetic background without these genes (isoline maize) was evaluated. An average of 890.8 +/- 152.3 beetles emerged from isoline plots, whereas average beetle emergence from 5307, MIR604, and 5307 x MIR604 was 1.9 +/- 0.6, 19.3 +/- 6.3, and 0.8 +/- 0.3, respectively, when averaged across 22 replications in five environments. Overall, 66, 50, 61, and 51% of beetles recovered from 5307, MIR604, 5307 x MIR604, and isoline maize, respectively, were female, and there was no significant difference between the number of male and female beetles that emerged from any of these treatments. Mortality due to 5307, MIR604, and 5307 x MIR604 was 99.79, 97.83, and 99.91%, respectively. There was an 8.0-d delay in time to 50% beetle emergence from 5307 compared with isoline maize, which was significantly later than to the other three maize lines. The average delay to 50% emergence from MIR604 and 5307 x MIR604 averaged 4.1 and 4.6 d, respectively later than 50% emergence from isoline maize. Female beetles had a significant delay in time to 50% emergence compared with male beetles from all treatments with the exception of 5307 x MIR604. Data are discussed in terms of insect resistance management in relation to other control measures for western corn rootworm.
Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to promote survival of susceptible pests. To delay pest resistance to transgenic cotton producing Bt toxin Cry1Ac, farmers in the United States and Australia planted refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. Here we report data from a 2010 survey showing field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera), in northern China. Laboratory bioassay results show that susceptibility to Cry1Ac was significantly lower in 13 field populations from northern China, where Bt cotton has been planted intensively, than in two populations from sites in northwestern China where exposure to Bt cotton has been limited. Susceptibility to Bt toxin Cry2Ab did not differ between northern and northwestern China, demonstrating that resistance to Cry1Ac did not cause cross-resistance to Cry2Ab, and implying that resistance to Cry1Ac in northern China is a specific adaptation caused by exposure to this toxin in Bt cotton. Despite the resistance detected in laboratory bioassays, control failures of Bt cotton have not been reported in China. This early warning may spur proactive countermeasures, including a switch to transgenic cotton producing two or more toxins distinct from Cry1A toxins.
Crops engineered to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt) are planted on millions of hectares annually, reducing the use of conventional insecticides and suppressing pests. However, the evolution of resistance could cut short these benefits. A primary pest targeted by Bt maize in the United States is the western corn rootworm Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae).
We report that fields identified by farmers as having severe rootworm feeding injury to Bt maize contained populations of western corn rootworm that displayed significantly higher survival on Cry3Bb1 maize in laboratory bioassays than did western corn rootworm from fields not associated with such feeding injury. In all cases, fields experiencing severe rootworm feeding contained Cry3Bb1 maize. Interviews with farmers indicated that Cry3Bb1 maize had been grown in those fields for at least three consecutive years. There was a significant positive correlation between the number of years Cry3Bb1 maize had been grown in a field and the survival of rootworm populations on Cry3Bb1 maize in bioassays. However, there was no significant correlation among populations for survival on Cry34/35Ab1 maize and Cry3Bb1 maize, suggesting a lack of cross resistance between these Bt toxins.
This is the first report of field-evolved resistance to a Bt toxin by the western corn rootworm and by any species of Coleoptera. Insufficient planting of refuges and non-recessive inheritance of resistance may have contributed to resistance. These results suggest that improvements in resistance management and a more integrated approach to the use of Bt crops may be necessary.
To investigate the development of resistance to mCry3A, a laboratory colony of the western corn rootworm, Diabrotica virgifera virgifera LeConte, was established from field survivors of mCry3A-expressing (MIR604) corn, Zea mays L. Feral adults emerging from MIR604 (selected) and isoline (control) field plots were collected and returned to the laboratory. Progeny of each colony was reared one generation on isoline corn and then crossed reciprocally with a nondiapausing colony. The resulting nondiapausing progeny were then reared on greenhouse corn in accordance with the wild type parent's origin (on MIR604 or isoline corn). After four, seven, and 10 total generations of selection, the resistance ratio of the selected colony was 0.5, 4.3, and 15.4 in terms of lethal concentration (LC)50 values in toxicity assays, with the latter two LC50 values being significant. After seven generations of selection in total, selected and control colonies were screened on MIR604 and isoline corn under field conditions. There was a significant colony x corn pedigree interaction in terms of plant damage. There was no significant difference in damage between MIR604 and isoline corn, whereas this difference was significant for the control colony. After 14 generations of selection, a seedling bioassay was performed. Again, there was a significant colony x corn pedigree interaction, this time in terms of the number of larvae recovered. There was no significant difference in the number of larvae recovered from MIR604 and isoline corn for the selected colony, whereas this difference was significant for the control colony, although larval size was greater on isoline corn for both colonies. Resistance has developed in western corn rootworm laboratory colonies to all Bt proteins currently registered for corn rootworm management, which emphasizes the importance of adhering to resistance management plans for maintaining product efficacy.
Five short-diapause laboratory lines of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), were selected for resistance to MON863, a variety of corn genetically modified with the Bacillus thuringiensis Berliner (Bt) transgene that expresses the Cry3Bb1 delta-endotoxin. Three of the selected lines were developed by incremental increase in the duration of exposure to MON863 over 11 generations (moderate selected lines). Two selected lines were developed from a control group by constant exposure to MON863 for at least 14 d posthatch over seven generations (intense selected lines). At the end of the experiment, survivorship, as measured by adult emergence, was approximately 4 times higher in each of the selected lines reared on MON863 compared with control lines. Estimates of realized heritabilities (h2) were 0.16 and 0.15 for the moderate and intense selected lines, respectively, and are consistent with h2 estimates reported previously from a variety of pest insects. These lines provide data necessary for evaluating the potential for Bt resistance within diabroticite beetles and will be useful for developing improved insect resistance management strategies.
Mortality of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae due to MIR604 transgenic corn, Zea mays L., expressing the modified Cry3A (mCry3A) protein relative to survivorship on corn with the same genetic background without the gene (isoline corn) was evaluated at three Missouri sites in both 2005 and 2006. We made these comparisons by using wild-type western corn rootworm at three different egg densities (6,000, 3,000, and 1,500 eggs per m) so that the role of density-dependent mortality would be known. The mortality due to the mCry3A protein was 94.88% when averaged across all environments and both years. Fifty percent emergence of beetles was delayed approximately 5.5 d. Beetles were kept alive and their progeny evaluated on MIR604 and isoline corn in the greenhouse to determine whether survivorship on MIR604 in the field for one generation increased survivorship on MIR604 in the greenhouse in the subsequent generation. There was no significant difference in survivorship on MIR604 in greenhouse assays between larvae whose parents survived isoline and larvae whose parents survived MIR604 in the field the previous generation, indicating that many susceptible beetles survived MIR604 in the field the previous season along with any potentially resistant beetles. The data are discussed in terms of rootworm insect resistance management.
Combinations of dissimilar insecticidal proteins ("pyramids") within transgenic plants are predicted to delay the evolution of pest resistance for significantly longer than crops expressing a single transgene. Field-evolved resistance to Bacillus thuringiensis (Bt) transgenic crops has been reported for first generation, single-toxin varieties and the Cry1 class of proteins. Our five year data set shows a significant exponential increase in the frequency of alleles conferring Cry2Ab resistance in Australian field populations of Helicoverpa punctigera since the adoption of a second generation, two-toxin Bt cotton expressing this insecticidal protein. Furthermore, the frequency of cry2Ab resistance alleles in populations from cropping areas is 8-fold higher than that found for populations from non-cropping regions. This report of field evolved resistance to a protein in a dual-toxin Bt-crop has precisely fulfilled the intended function of monitoring for resistance; namely, to provide an early warning of increases in frequencies that may lead to potential failures of the transgenic technology. Furthermore, it demonstrates that pyramids are not 'bullet proof' and that rapid evolution to Bt toxins in the Cry2 class is possible.
A simulation model of the population dynamics and genetics of western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), was created to evaluate the use of refuges in the management of resistance to transgenic insecticidal corn, Zea mays L., expressing one or two toxin traits. Hypothetical scenarios and a case study of a corn hybrid pyramided with existing toxins are simulated. In the hypothetical situations, results demonstrated that evolution is generally delayed by pyramids compared with deployment of a single-toxin corn hybrid. However, soil insecticide use in the refuge reduced this delay and quickened the evolution of resistance. Results were sensitive to the degree of male beetle dispersal before mating and to the effectiveness of both toxins in the pyramid. Resistance evolved faster as fecundity increased for survivors of insecticidal corn. Thus, effects on fecundity must be measured to predict which resistance management plans will work well. Evolution of resistance also occurred faster if the survival rate due to exposure to the two toxins was not calculated by multiplication of two independent survival rates (one for each insect gene) but was equivalent to the minimum of the two. Furthermore, when single-trait and pyramided corn hybrids were planted within rootworm-dispersal distance of each other, the toxin traits lost efficacy more quickly than they did in scenarios without single-trait corn. For the case study involving transgenic corn expressing Cry34/35Ab1 and Cry3Bb1, the pyramid delayed evolution longer than a single trait corn hybrid and longer than a sequence of toxins based on at least one resistance-allele frequency remaining below 50%. Results are discussed within the context of a changing transgenic corn marketplace and the landscape dynamics of resistance management.
The percentage of viable eggs of the western corn rootworm, Diabrotica virgifera virgifera LeConte, which survived to the adult stage was evaluated for the effect of egg density in 2005 and 2007 in central Missouri. In 2005, each plot was 2.44 by 3.05 m and contained 64 maize (corn), Zea mays L., plants. In 2007, plots were 3.05 by 3.05 m and again contained 64 corn plants. Seven egg densities (2,400, 1,200, 600, 300, 100, 50, and 25 viable eggs per 30.5 cm) were evaluated with four to six replications in each year in a completely randomized design. In 2007 only, an additional row was infested near each plot to evaluate plant damage. In both years, there was no correlation of infestation level and percentage of emergence between infestation levels of 25-600 viable eggs per 30.5 cm, indicating that density-dependent mortality did not occur at these egg densities. In 2005, 8.04% of the viable eggs established on a corn plant and produced an adult at these lower infestation rates. In 2007, this value was 2.9%. Regardless of egg density, approximately 92-97% failed to establish and produce adults (density-independent mortality). In 2005 and in the combined analysis, as viable egg densities increased from 600 to 2400 per 30.5 cm there was a significant decrease in percentage of emergence. In a broken line analysis of the 2005 data, the point where density-dependent mortality began in the combined analysis was 851 eggs per 30.5 cm with a 95% confidence interval from 678 to 1024. That year density-dependent mortality was important at high infestations and killed 54.4% of those larvae that successfully established on a plant at the highest egg density. However, little or no density-dependent mortality occurred at infestation levels <850 viable eggs per 30.5 cm in either year of the study. Combining data from both years with all previously published data in a broken line analysis indicated that density-dependent mortality began at approximately 800 viable eggs per 30.5 cm. These data are discussed in terms of dose calculations for products targeting the western corn rootworm.
The western corn rootworm, Diabrotica virgifera virgifera LeConte, is an established insect pest of maize (Zea mays L.) in North America. The rotation of maize with another crop, principally soybeans, Glycine max (L.), was the primary management strategy utilized by North American producers and remained highly effective until the mid-1990s. In 1995, widespread and severe root injury occurred in east-central Illinois and northern Indiana maize fields that had been annually rotated with soybeans on a regular basis for several decades. The failure of this cultural tactic from a pest management perspective was attributed to a behavioral adaptation by a variant western corn rootworm that had lost fidelity to maize for egg laying. In 1992, an infestation of western corn rootworm was found within a small maize field near the Belgrade Airport. By 2007, the presence of this insect pest had been confirmed in 20 European countries. More recent molecular studies have confirmed that at least three separate invasions (until 2004) of western corn rootworms have occurred in Europe, increasing the risk that rotation-resistant western corn rootworms will be introduced into a new continent. Although biological control and use of conventional resistant maize hybrids have not achieved widespread success in the management of western corn rootworms in North America, these tactics are being evaluated in Europe.
In her seminal work, Silent Spring, Rachel Carson writes: “If Darwin were alive today the insect world would delight and astound him with its impressive verification of his theories of survival of the fittest. Under the stress of intensive chemical spraying the weaker members of the insect populations are being weeded out.” (1)
To delay evolution of insect resistance to transgenic crops producing Bacillus thuringiensis (Bt) toxins, nearby "refuges" of host plants not producing Bt toxins are required in many regions. Such refuges are expected to be most effective in slowing resistance when the toxin concentration in Bt crops is high enough to kill all or nearly all insects heterozygous for resistance. However, Bt corn, Zea mays, introduced recently does not meet this "high-dose" criterion for control of western corn rootworm (WCR), Diabrotica virgifera virgifera. A greenhouse method of rearing WCR on transgenic corn expressing the Cry3Bb1 protein was used in which approximately 25% of previously unexposed larvae survived relative to isoline survival (compared to 1-4% in the field). After three generations of full larval rearing on Bt corn (Constant-exposure colony), WCR larval survival was equivalent on Bt corn and isoline corn in greenhouse trials, and the LC(50) was 22-fold greater for the Constant-exposure colony than for the Control colony in diet bioassays with Cry3Bb1 protein on artificial diet. After six generations of greenhouse selection, the ratio of larval recovery on Bt corn to isoline corn in the field was 11.7-fold greater for the Constant-exposure colony than the Control colony. Removal from selection for six generations did not decrease survival on Bt corn in the greenhouse. The results suggest that rapid response to selection is possible in the absence of mating with unexposed beetles, emphasizing the importance of effective refuges for resistance management.
This book provides a comprehensive review of the current knowledge of Western corn rootworm, Diabrotica virgifera virgifera , and how it might be managed both in North America and in Europe. Comparisons are drawn between plant protection techniques currently applied in North America and their potential application in Europe. Cultural, biotechnical and biological control measures are also addressed, as are ecological baseline data, i.e. population dynamics of the pest in North America and Europe, economic thresholds and aspects of its behaviour.
Efficacy of Bollgard (DP50B) and Bollgard II (DP50BX) cottons that express either one or two Bacillus thuringiensis Berliner proteins, respectively, along with the conventional sister genotype (DP50), was determined for a feral strain of bollworm, Helicoverpa zea (Boddie), and a Cry1Ac-selected bollworm strain in 1999. In 2000, a greenhouse study was designed to compare the efficacy of three transgenic cottons expressing either the Cry1Ac endotoxin alone (DP50B), the Cry2Ab endotoxin alone (DP50X), or both the Cry1Ac and Cry2Ab endotoxins (DP50BX) against a feral and a Cry1Ac-selected bollworm strain. Results from the 1999 greenhouse study evaluating both a feral and a Cry1Ac-selected bollworm strain demonstrated that when averaged across bollworm strains, the Bollgard II genotype significantly reduced larval survival and fruit penetration by bollworm compared to the Bollgard variety. Also, the Cry1Ac-selected bollworm strain displayed increased larval survival, superficial fruit damage, and fruit penetration compared to the feral strain when averaged across genotypes. In the 2000 study, the Bollgard II genotype significantly reduced fruit penetration by bollworm below that of the Bollgard variety when averaged across strains; however, the single Cry2Ab-producing genotype performed similarly to both Bollgard and Bollgard II with respect to fruit penetration. The Cry1Ac-selected bollworm strain exhibited significantly greater larval survival and superficial fruit damage on the Bollgard variety compared to the feral strain, but no differences among larval strains were evident for other genotypes. Also, when averaged across genotypes, the Cry1Ac-selected bollworm strain penetrated a higher proportion of cotton fruit compared to the feral strain. These results suggest that commercialization of Bollgard II cottons would significantly reduce bollworm survival and damage compared to that experienced by current Bollgard varieties. Bollgard II plantings also should have a positive impact on Bt resistance management of bollworm.
Intensified management of agricultural land has produced dramatic yield increases over the last century. Intensification involves increased mechanization, irrigation and use of synthetic fertilizers and pesticides, in conjunction with use of crops bred to respond to high-input environments (Matson et al. 1997). Accordingly, the large monocultures currently epitomizing agricultural intensification differ in many ways from natural ecosystems. Compared to plants in natural environments, crops in monocultures may be easier to find for insect herbivores (Root 1973; Feeny 1976; Andow 1991), more suitable nutritionally (Scriber and Slansky 1981; Myers 1985), or less defended by natural enemies (Croft 1990; Andow 1991). Moreover, economically acceptable damage to crops can be low compared to damage occurring in natural environments. Such differences facilitate pest outbreaks in monocultures, which are suppressed most of the time with synthetic insecticides.
The remarkable ability of insects to adapt quickly to toxins used to control them threatens agriculture and human health worldwide. The quantity and variety of examples of pesticide resistance also offer opportunities for determining how response to selection is affected by various factors, including behavior, dominance, fitness trade-offs, founder events, gene flow, genetic constraints, haplodiploidy, life-history traits, major and minor genes, multitrophic interactions, and population dynamics. While study of resistance can provide fundamental insights about evolution, efforts to manage resistance enable application and testing of evolutionary theories. In particular, can strategies based on evolutionary principles delay evolution of pest resistance to insecticidal transgenic crops? This chapter deals with the evolution of insect resistance to transgenic plants, focusing on transgenic crops with Bacillus thuringiensis (Bt) toxins, Bt toxins and their mode of action, the genetic basis of resistance to Bt toxins, the refuge strategy for delaying pest resistance to Bt crops, insect gene flow between refuges and Bt crops, and pink bollworm resistance to Bt cotton in Arizona.
The remarkable ability of insects to adapt quickly to toxins used to control them threatens agriculture and human health worldwide. The quantity and variety of examples of pesticide resistance also offer opportunities for determining how response to selection is affected by various factors, including behavior, dominance, fitness trade-offs, founder events, gene flow, genetic constraints, haplodiploidy, life-history traits, major and minor genes, multitrophic interactions, and population dynamics. While study of resistance can provide fundamental insights about evolution, efforts to manage resistance enable application and testing of evolutionary theories. In particular, can strategies based on evolutionary principles delay evolution of pest resistance to insecticidal transgenic crops? This chapter deals with the evolution of insect resistance to transgenic plants, focusing on transgenic crops with Bacillus thuringiensis (Bt) toxins, Bt toxins and their mode of action, the genetic basis of resistance to Bt toxins, the refuge strategy for delaying pest resistance to Bt crops, insect gene flow between refuges and Bt crops, and pink bollworm resistance to Bt cotton in Arizona.
Transgenic Bt (Bacillus thuringiensis) corn (Zea mays L.) hybrids with resistance to corn rootworm (CRW; Diabrotica spp.) or European corn borer [ECB; Ostrinia nubilalis (Hubner)] can have greater tolerance to water and nutrient stress, and thus may have higher optimum plant densities. Experiments were conducted following soybean [Glycine max (L.) Merr.] over nine site-years in Illinois to determine whether the response to plant density for corn grain yield and net return to seed cost differ among near-isoline hybrids with no insect resistance, Bt resistance to CRW, or Bt resistance to CRW plus ECB. Similar experiments were conducted over three site-years in Iowa following both soybean and corn for near-isoline hybrids with Bt resistance to ECB or ECB plus CRW. Larval CRW injury was low in Iowa and stalk lodging was minimal in all experiments. Across site-years in Illinois and in both crop sequences in Iowa, grain yield and net return to seed cost were not affected by hybrid. Net return to seed cost within $2.50 ha(-1) of the maximum occurred with densities of 76,300 to >98,600 plants ha(-1) following soybean in Illinois, 87,100 to 93,400 plants ha(-1) following soybean in Iowa, and 87,400 to 95,700 plants ha(-1) following corn in Iowa. Yields within these optimum plant densities were 15.9, 16.1, and 15.4 Mg ha(-1) respectively. When CRW and ECB are managed or are at low levels, optimum plant density is similar between hybrids with or without resistance to these pests.
The western corn rootworm Diabrotica virgifera virgifera and northern corn rootworm D. barberi are the most serious insect pests of corn Zea mays in Canada and N-central USA. Management options are examined, with consideration of crop rotation, tillage and soil environment, planting and harvesting dates, host-plant resistance, and options for biological and insecticidal control. Particular attention is paid to integrated pest management which involves crop rotation, scouting fields to determine the need for control measures for silk clipping and root damage the following years, use of insecticides only when necessary, and consideration of environmental, biological, chemical and physical features that contribute to corn rootworm control. -P.J.Jarvis
A 3-yr study assessed the influence of three commonly used planting-time soil insecticides (carbofuran, chlorpyrifos, and terbufos) on emergence of western corn rootworm, Diabrotica virgifera virgifera LeConte, from different tillage systems located near Champaign (1983 and 1984) and Monmouth (1985), III. The potential interaction of insecticides and tillage systems was not significant in any of the 3 yr with regard to beetle emergence. In 1983, significantly fewer (P > F = 0.0107) western com rootworm beetles emerged from control plots than from insecticide-treated plots. Conversely, beetle emergence in 1984 was greater from the control plots than from the insecticide-treated plots (P > F = 0.0001). In 1985, the main effect of insecticide application on beetle emergence was not significant. Total precipitation during the peak larval feeding period in June was distinctly different each year of the study (1983, 23.2 em; 1984, 3.1 cm; 1985, 8.0 cm) and may have influenced the dynamics of soil insecticide and beetle emergence interactions. In 1983 and 1984, beetle emergence among the insecticide-treated plots was greatest in plots treated with carbofuran, the most water-soluble of the compounds examined. The effect of tillage on root damage was not significant; whereas, the insecticides provided satisfactory root protection compared with the control. Results suggest that producers who use planting-time soil insecticides to protect corn roots from damage are not actually managing rootworm populations (i.e., reducing the population level) in some seasons. Although applying an insecticide in a band is intended only to protect the root system, greater beetle emergence from insecticide-treated areas in some years implies that the long-term severity of western corn rootworm infestations may be exacerbated.
For the first time, resistance to the CRYIIIA coleopteran specific delta endotoxin of Bacillus thuringiensis var. san diego or tenebrionis is reported. Colorado potato beetles, Leptinotarsa decemlineata (Say), were selected in the laboratory after exposure in potato fields in Michigan. After 12 generations of selection, the selected strain was 59 times more resistant than the unselected strain and 24-35 times more resistant than other susceptible or insecticide-resistant strains. We observed no cross-resistance between organophosphate, carbamate, or pyrethroid resistance and B. thuringiensis resistance. No difference in movement induced by B. thuringiensis or initial feeding behavior was observed between selected and unselected strains, although a shift toward petiole feeding behavior was observed in adults (of both strains) that were stressed by B. thuringiensis. The selected strain oviposited at a reduced rate of 0.1 ±0.15 (mean ± SD) egg masses per day) on treated foliage; the unselected strain was oostatic. Based on these results, it is recommended that resistance management strategies be developed and deployed for both conventionally applied B. thuringiensis products and B. thuringiensis transgenic plants.
In late June 1987, severe western com rootworm, Diabrotica virgifera virgifera LeConte, larval injury to com grown for seed production (inbred com) was observed within a 3-km2 area near Piper City (Ford County), IL. The rootworm injury occurred in 6 fields that in the prior year had been planted to weed-free soybeans grown for seed production. The severe rootworm injury problem reoccurred in 1st-yr seed com in the same area in 1988 and to varying degrees in the years since (through 1994). Laboratory and field studies were initiated in 1987 to investigate several possible causes of the problem. Under simulated field soil temperature conditions, western com rootworm eggs from the Piper City population did not show evidence of the prolonged diapause trait. Although higher than expected oviposition took place in Piper City soybean fields, a large field study with staggered plantings of soybeans at Urbana, IL, <75 km away, confirmed earlier studies that few western com rootworm eggs are laid in weed-free soybean fields. Pyrethroid insecticides are routinely used in seed corn for com earworm, Helicoverpa zea (Baddie), control. In laboratory bioassays, the pyrethroid insecticide, permethrin, repelled western com rootworm female beetles from treated com to lay eggs in untreated soybeans and demonstrated that western com rootworm damage to 1st-yr seed com could have been caused by pyrethroid use the previous summer in adjacent cornfields.
Colonies of Indian meal moth, Plodia interpunctella (Hübner), and almond moth, Cadra cautella (Walker), reared in the laboratory on diet treated with Bacillus thuringiensis, became resistant to B. thuringiensis. However, resistance did not progress at the same rate or to the same extent in all of the colonies. Resistance in five Indian meal moth colonies increased from 2- to 29-fold within three generations, and from 15- to 1000fold in ca. 40 generations under relatively low selection pressure. With higher selection pressure, resistance in one colony increased >250-fold. Resistance in an almond moth colony increased only ca. 7-fold in 21 generations of intensive selection. Resistance was stable when selection was discontinued after the resistance levels reached a plateau, but declined if selection was discontinued earlier. The resistance was partially recessive in the five Indian meal moth colonies, but not to an equal extent. The resistance assorted independently of the recessive genetic markers copper, golden, and white-eye.
Heliothis virescens (F.), tobacco budworms eggs were collected from 3 adjacent counties in North Carolina. A laboratory strain (YDK) was established from these eggs using precautions to avoid loss of genetic diversity. A subset of this laboratory strain (YHD2) was selected on artificial diet containing the Bacillus thuringiensis (Berliner) toxin, CryIA(c). In the, first 12 episodes of selection only moderate resistance (≍7- to 8-fold) was found. However, after 19 episodes of selection the strain had developed >500-fold resistance to the CryIA(c) toxin. Further selection lead to higher levels of resistance with the greatest resistance ratio recorded being ≍10,000-fold. The YHD2 strain was cross-resistant to CryIA(a), CryIA(b) and CryIF. We also found some resistance to CryIB, CryIC and CryIA, but the level of resistance to these toxins was more moderate. Reciprocal genetic crosses between the resistant and control strains indicated that resistance to CryIA(c) and to CryIA(b) was partially recessive, but that the low level of resistance to CryIA was more dominant. Progeny from backcrosses of F1 larvae to the resistant parent were placed on artificial diet containing a concentration of CryIA(b) that had previously been found to slow the growth of F1 larvae. When these larvae were weighed after 10 d, two clearly demarcated size classes were found in ≍1:1 ratios as is expected in backcrosses when a single locus (or a set of tightly linked loci) is coding for a major component of a recessive trait. Adults that developed from the larger size class of larvae were mated and their offspring inherited the ability to grow well on CryIA(b). As expected from the single locus model, one quarter of the offspring from matings of the small backcross larvae grew well on CryIA(b). Results of this selection experiment indicate that the initial frequency of this resistance trait could be approximately 10-3, but field tests will be needed to confirm this rough estimate.
Adults of the western corn rootworm. Diabrotica virgifera LeConte, collected from two widely separated areas in Nebraska, were assayed for susceptibility to aldrin, Diazinon® (O,O-diethyl O-(2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate), and heptachlor. Dosage-mortality curves and LD50 values were determined for each insecticide. Results indicated that adults of D. virgifera collected from the central part of the State required about 100 times as much aldrin or heptachlor per insect to produce an LD50in 2 hours as adults collected in eastern Nebraska. LD50 values for Diazinon tested on beetles from the two areas were virtually the same. The reasons for such differential resistance are considered.