V-ATPase E mediates Cry2Ab binding and toxicity in Helicoverpa armigera.

Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H+-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.

Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility.

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

Stability of expression of Cry1Ac and Cry2Ab2 proteins in Bollgard-II hybrids at different stages of crop growth in different genotypes across cropping seasons and multiple geographies.

Bollgard-II cotton expressing Cry1Ac and Cry2Ab2 insecticidal proteins has been commercially cultivated in India since 2006 to control bollworms. These genes were introgressed into parental germplasm of numerous hybrids. Therefore, it is imperative that these insecticidal proteins are expressed in sufficient quantities in different tissues, throughout the season irrespective of genetic background or environmental conditions for effective performance. Here, we document results of a comprehensive study on pattern of expression of Bt proteins across different stages of crop growth in > 2000 cotton hybrids (Gossypium hirsutum), across 12 cropping seasons tested in the Northern, Southern or Central zones in India, in terminal leaf, pre-candle square and boll epicarp tissues. Statistical analysis of variability using Linear mixed effect model was used to estimate factors contributing to variability in expression of Bt proteins. For Cry1Ac, variability was maximally contributed by genotype × season × plant growth stage effect in terminal leaves and boll epicarp, while season effect drove variability in pre-candle square. In Cry2Ab2, season effect drove variability in three tissue types. Pre-candle square tissue had most variability in expression of both proteins followed by terminal leaf and boll epicarp. Further, expression of Bt proteins in 234 G. hirsutum × G. barbadense hybrids showed similar expression patterns as intra specific hybrids though there was a significant difference in expression levels. Cry2Ab2 was expressed in significantly higher amounts when genes were in homozygous state. Bt proteins were also found to be expressed in varied amounts in different tissues and were expressed even when hybrids were grown at sub-optimal temperatures.

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure.

Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.

Field-evolved resistance of Helicoverpa zea (Boddie) to transgenic maize expressing pyramided Cry1A.105/Cry2Ab2 proteins in northeast Louisiana, the United States.

The corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), is a major target pest of pyramided Bt maize and cotton in the U.S. In 2017 and 2018, notable ear damage and larval survival of H. zea were observed on pyramided Cry1A.105/Cry2Ab2 maize in some fields in northeast Louisiana, U.S. The objective of this study was to determine if the field control problem was due to resistance development to the Bt proteins in plants. A total of 15 H. zea populations were collected from Bt and non-Bt maize plants in 2017 and 2018 in multiple locations in Louisiana, Florida, and Georgia. Diet-overlay bioassays showed that LC50s of Cry1A.105 and Cry2Ab2 for populations collected from the areas with control problems in northeast Louisiana were as much as >1623- and 88-fold greater than that of a susceptible strain, respectively. In addition, two field trials in 2018 validated that Cry1A.105/Cry2Ab2 maize failed in managing natural H. zea populations, while Bt maize containing Vip3A was effective in northeast Louisiana. Results of the study documented that the observed field control problems of Cry1A.105/Cry2Ab2 maize against H. zea in northeast Louisiana were due to resistance development of the insect to the Bt proteins in plants. This is the first documentation of field-evolved resistance to pyramided Bt maize in a target insect species in southern U.S. However, susceptibility levels to Cry1A.105 and Cry2Ab2 varied greatly among populations collected from the three states, suggesting uneven distributions of the resistance in the region.

Fitness of Bt-resistant cabbage loopers on Bt cotton plants.

Development of resistance to the insecticidal toxins from Bacillus thuringiensis (Bt) in insects is the major threat to the continued success of transgenic Bt crops in agriculture. The fitness of Bt-resistant insects on Bt and non-Bt plants is a key parameter that determines the development of Bt resistance in insect populations. In this study, a comprehensive analysis of the fitness of Bt-resistant Trichoplusia ni strains on Bt cotton leaves was conducted. The Bt-resistant T. ni strains carried two genetically independent mechanisms of resistance to Bt toxins Cry1Ac and Cry2Ab. The effects of the two resistance mechanisms, individually and in combination, on the fitness of the T. ni strains on conventional non-Bt cotton and on transgenic Bt cotton leaves expressing a single-toxin Cry1Ac (Bollgard I) or two Bt toxins Cry1Ac and Cry2Ab (Bollgard II) were examined. The presence of Bt toxins in plants reduced the fitness of resistant insects, indicated by decreased net reproductive rate (R0 ) and intrinsic rate of increase (r). The reduction in fitness in resistant T. ni on Bollgard II leaves was greater than that on Bollgard I leaves. A 12.4-day asynchrony of adult emergence between the susceptible T. ni grown on non-Bt cotton leaves and the dual-toxin-resistant T. ni on Bollgard II leaves was observed. Therefore, multitoxin Bt plants not only reduce the probability for T. ni to develop resistance but also strongly reduce the fitness of resistant insects feeding on the plants.

Fitness costs and inheritance of Cry2Ab2 resistance in Spodoptera frugiperda (J.E. Smith).

Cry2Ab2 is a Bacillus thuringiensis (Bt) protein expressed in transgenic corn and cotton targeting above-ground lepidopteran pests including the fall armyworm, Spodoptera frugiperda (J.E. Smith). The objective of this study was to characterize fitness costs and inheritance of Cry2Ab2 resistance in S. frugiperda. To determine if fitness costs were associated with the resistance, life history parameters (larval survival, growth, development and egg production) of Cry2Ab2-resistant, -susceptible, and two reciprocal F1 colonies of S. frugiperda were assayed on non-toxic diet and non-Bt corn leaf tissue. The results showed that there were no significant differences among the four insect colonies for all the biological parameters measured with few exceptions, suggesting that the resistance in the colony was not associated with significant fitness costs in the test conditions. To examine the inheritance of resistance, susceptibilities of the resistant and susceptible parents, as well as eight additional colonies generated from various genetic crosses, were assayed using Cry2Ab2-treated diet and Cry2Ab2 corn leaf tissue. The Cry2Ab2 resistance in S. frugiperda in the colony was inherited as a single autosomal recessive or incompletely recessive gene. The results of the study suggest a potential risk of resistance development in S. frugiperda to the Cry2Ab2 protein and thus effective management strategies should be implemented for the sustainable use of the Bt corn technology for pest management.

The interaction of two-spotted spider mites, Tetranychus urticae Koch, with Cry protein production and predation by Amblyseius andersoni (Chant) in Cry1Ac/Cry2Ab cotton and Cry1F maize.

Crops producing insecticidal crystal (Cry) proteins from the bacterium, Bacillus thuringiensis (Bt), are an important tool for managing lepidopteran pests on cotton and maize. However, the effects of these Bt crops on non-target organisms, especially natural enemies that provide biological control services, are required to be addressed in an environmental risk assessment. Amblyseius andersoni (Acari: Phytoseiidae) is a cosmopolitan predator of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), a significant pest of cotton and maize. Tri-trophic studies were conducted to assess the potential effects of Cry1Ac/Cry2Ab cotton and Cry1F maize on life history parameters (survival rate, development time, fecundity and egg hatching rate) of A. andersoni. We confirmed that these Bt crops have no effects on the biology of T. urticae and, in turn, that there were no differences in any of the life history parameters of A. andersoni when it fed on T. urticae feeding on Cry1Ac/Cry2Ab or non-Bt cotton and Cry1F or non-Bt maize. Use of a susceptible insect assay demonstrated that T. urticae contained biologically active Cry proteins. Cry proteins concentrations declined greatly as they moved from plants to herbivores to predators and protein concentration did not appear to be related to mite density. Free-choice experiments revealed that A. andersoni had no preference for Cry1Ac/Cry2Ab cotton or Cry1F maize-reared T. urticae compared with those reared on non-Bt cotton or maize. Collectively these results provide strong evidence that these crops can complement other integrated pest management tactics including biological control.

Assessing the potential for interaction between the insecticidal activity of two genetically engineered cotton events combined by conventional breeding: An example with COT102 × MON 15985.

Bollgard(®) III was developed by combining cotton events COT102 and MON 15985 through conventional breeding to improve efficacy against lepidopteran feeding damage. COT102 produces the Vip3Aa19 protein and MON 15985 produces the Cry1Ac and Cry2Ab2 proteins. COT102 × MON 15985 has also been bred with Roundup Ready Flex(®) cotton (MON 88913) that confers glyphosate tolerance. This study evaluated the activity of COT102 and MON 15985 and the combined activity of COT102 and MON 15985 against the cotton bollworm (CBW, Helicoverpa zea). COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 have comparable Vip3Aa19 and/or Cry1Ac, Cry2Ab2 protein expression levels as determined by enzyme-linked immunosorbent assay. CBW demonstrated concentration-dependent growth inhibition after 7-days of feeding on lyophilized leaf tissue derived from COT102, MON 15985, COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 incorporated into an artificial diet. Observed EC50 values for COT102 × MON 15985 and COT102 × MON 15985 × MON 88913 were comparable (≤4% deviation) with the predicted EC50 value under the assumption of additivity using the combined activity of COT102 and MON 15985. No interaction in biological activity between COT102 and MON 15985 is consistent with results from competition and ligand blotting assays that demonstrated that Vip3Aa does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. The results from this study demonstrate that the activity of COT102 × MON 15985 against CBW is consistent with predictions of additivity.

F2 screen for resistance to Bacillus thuringiensis Cry2Ab2-maize in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) from the southern United States.

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target pest of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established using single-pair mating of field individuals collected from seven locations in four states of the southern U.S.: Texas, Louisiana, Georgia, and Florida. The objective of the investigation was to detect resistance alleles in field populations to Cry2Ab2, a common Bt protein produced in transgenic maize and cotton. For each F2 family, 128 F2 neonates were screened on leaf tissue of Cry2Ab2 maize plants in the laboratory. A conservative estimate of the frequency of major Cry2Ab2 resistance alleles in S. frugiperda from the four states was 0.0023 with a 95% credibility interval of 0.0003-0.0064. In addition, six families were considered to likely possess minor resistance alleles at a frequency of 0.0082 with a 95% credibility interval of 0.0033-0.0152. One F2 family from Georgia (GA-15) was confirmed to possess a major resistance allele to the Cry2Ab2 protein. Larvae from this family survived well on whole maize plants expressing Cry2Ab2 protein and demonstrated a significant level (>15-fold) of resistance when fed with the same protein incorporated in a meridic diet. The detection of the major resistance allele along with the relatively abundant minor resistance alleles revealed in this study may have important implications for resistance management.

Identification, inheritance, and fitness costs of Cry2Ab2 resistance in a field-derived population of sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae).

The sugarcane borer, Diatraea saccharalis (F.), is one of the major target pests of transgenic maize, Zea mays, expressing Bacillus thuringiensis (Bt) proteins in South America and mid-southern region of the U.S. The MON89034 maize expresses Cry2Ab2 and Cry1A.105 Bt proteins and it was developed to provide better control of key lepidopteran pests of maize including D. saccharalis. The objectives of this study were to select and characterize the resistance of D. saccharalis to Cry2Ab2 using a non-commercial Cry2Ab2 single gene Bt maize line. A Cry2Ab2-resistant strain (Cry2Ab2-RR) of D. saccharalis was established from 28 two-parent families collected from fields in northeast Louisiana, U.S. The Cry2Ab2-RR showed a high level of resistance to Cry2Ab2 in both diet-incorporated and whole maize plant bioassays. The Cry2Ab2 resistance in D. saccharalis was likely inherited as a single or a few tightly linked autosomal genes. The resistance was non-recessive and not associated with fitness costs. The results should provide valuable information in resistance monitoring, assessing resistance risk, and developing effective management strategies for the sustainable use of Bt maize technology for managing maize stalk borers.

Fall Armyworm (Lepidoptera: Noctuidae) Development, Survivorship, and Damage on Cotton Plants Expressing Insecticidal Plant-Incorporated Protectants.

Cotton, Gossypium hirsutum (L.), plants expressing insecticidal crystal (Cry) proteins of Bacillus thuringiensis (Bt) Berliner are planted on significant acreage across the southern region of the United States. Fall armyworm, Spodoptera frugiperda (J. E. Smith), can be a significant cotton pest in some years, but this species has not been a primary target of Bt cotton technologies. The objective of this study was to quantify fall armyworm larval survivorship and fruiting form injury on transgenic cotton lines expressing Cry1Ac (Bollgard), Cry1Ac+Cry2Ab (Bollgard II), and Cry1Ac+Cry1F (WideStrike) Bt proteins. Larval survivorship and fruiting form damage of fall armyworm on Bollgard, Bollgard II, WideStrike, and non-Bt (control) cotton lines were evaluated in no-choice field studies. Fall armyworm (third instars) were placed on flower buds (squares), white flowers, and bolls, enclosed within a nylon mesh exclusion cage, and evaluated at selected intervals after infestation. Exposure of fall armyworm larvae to Bollgard cotton lines generally resulted in no significant effects on survivorship compared with larvae exposed to the non-Bt cotton line. Survivorship and plant injury by fall armyworm on Bollgard II cotton lines was variable compared with that on non-Bt cotton lines, and significant differences between treatments were inconsistent. Fall armyworm had significantly lower survivorship and caused less plant injury on WideStrike cotton lines than on non-Bt cotton lines across all plant structures. Development and survivorship of fall armyworm larvae on these cotton lines also were evaluated in no-choice laboratory assays by offering the previously described fruiting forms to third instars. Bollgard II and WideStrike cotton lines significantly reduced fall armyworm development and survivorship compared with those larvae offered non-Bt tissue. These results suggest that differences exist among selected Bt cotton technologies in their performance against fall armyworm.

Coexpression of the silent cry2Ab27 together with cry1 genes in Bacillus thuringiensis subsp. aizawai SP41 leads to formation of amorphous crystal toxin and enhanced toxicity against Helicoverpa armigera.

The unexpressed cry2Ab27 gene of Bacillus thuringiensis subsp. aizawai SP41 (SP41) consists of a single open reading frame (ORF) of 1902bp encoding for 634 amino acid residues. The cry2Ab27 gene appears to be silent due to the lack of promoter and terminator sequences. In this study we fused the cry2Ab27 ORF with the cry1Ab promoter (500bp) and the terminator (300bp) in vector pHT304-18Z in order to drive the expression of cry2Ab27 in both SP41 and an acrystaliferous, B. thuringiensis subsp. thuringiensis 407 (407). A protein with a molecular mass of 65kDa, consistent with the Cry2Ab protein, was detected in both transformants using SDS-PAGE and Western blot analysis. Bipyramidal crystals were observed in SP41 and its transformant containing the pHT304-18Z vector (SPHT) in contrast, cells expressing cry2Ab27 (SPC2) exhibited crystal proteins with irregular shapes. No inclusion protein was detected in the 407 transformant expressing the cry2Ab27 gene. Cry2Ab27 was found in the purified crystal toxin from strain SPC2. The solubilized crystal toxin proteins from SPC2 were 6.9-fold more toxic toward the larvae of Helicoverpa armigera compared to toxin proteins from SPHT. However SPC2 crystal toxin displayed only slightly higher toxicity against the larvae of Spodoptera litura and S. exigua compared to SPHT produced toxin. Our data support the use of Cry2Ab in combination with the Cry1 toxin for enhanced control of heliothine insect pests.

Engineering of Bacillus thuringiensis Cry2Ab toxin for improved insecticidal activity.

Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC50 (0.129 µg/cm2) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm2). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin's future application.

Impact of Bt corn expressing Bacillus thuringiensis Berliner insecticidal proteins on the growth and survival of Spodoptera frugiperda larvae in Colombia.

Bioassays were conducted under controlled conditions to determine the response of Spodoptera frugiperda (J. E. Smith) larvae fed with corn materials expressing Bacillus thuringiensis (Bt) insecticidal endotoxins: (1) VT Double Pro® (VT2P) expressing Cry1A.105-Cry2Ab2 proteins and (2) VT Triple Pro® (VT3P) expressing Cry1A.105-Cry2Ab2-Cry3Bb1 proteins. The parameters assessed were: (i) mortality rate, and (ii) growth inhibition (GI) with respect to the control. To conduct this study, larvae were collected from commercial non-Bt corn fields, in four agricultural sub-regions in Colombia, between 2018 and 2020. Fifty-two populations were assessed from the field and neonate larvae from each of the populations were used for the bioassays. The study found that mortality rates in the regions for larvae fed with VT2P corn ranged from 95.1 to 100.0%, with a growth inhibition (%GI) higher than 76.0%. Similarly, mortality rate for larvae fed with VT3P corn were between 91.4 and 100.0%, with a %GI above 74.0%. The population collected in Agua Blanca (Espinal, Tolima; Colombia) in 2020, showed the lowest mortality rate of 53.2% and a %GI of 73.5%, with respect to the control. The population that exhibited the lowest %GI was collected in 2018 in Agua Blanca (Espinal, Tolima, Colombia) with a 30.2%, growth inhibition, with respect to the control. In recent years, the use of plant tissue to monitor susceptibility to fall armyworm has proven to be useful in the resistance management program for corn in Colombia determining that the FAW populations are still susceptible to Bt proteins contained in VT2P and VT3P.

A Modified F2 Screen for Estimating Cry1Ac and Cry2Ab Resistance Allele Frequencies in Helicoverpa zea (Lepidoptera: Noctuidae).

Evaluating the frequency of resistance alleles is important for resistance management and sustainable use of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis. Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) is a major crop pest in the United States that has evolved practical resistance to the crystalline (Cry) proteins in Bt corn and cotton. The standard F2 screen for estimating resistance allele frequency does not work well for H. zea because successful single-pair matings are rare. In this study, we developed and implemented a modified F2 screen for H. zea that generates F1 progeny by crossing three laboratory susceptible female moths with one feral male moth instead of single-pair crosses. During 2019-2020, we used this modified method to establish 192 F2 families from 623 matings between susceptible females and feral males from Arkansas, Louisiana, Mississippi, and Tennessee. From each F2 family, we screened 128 neonates against discriminating concentrations of Cry1Ac and Cry2Ab in diet overlay bioassays. Based on these discriminating concentration bioassays, families were considered positive for resistance if at least five larvae survived to second instar, including at least one to third instar. The percentage of positive families was 92.7% for Cry1Ac and 38.5% for Cry2Ab, which yields an estimated resistance allele frequency (with 95% confidence interval) of 0.722 (0.688-0.764) for Cry1Ac and 0.217 (0.179-0.261) for Cry2Ab. The modified F2 screen developed and implemented here may be useful for future resistance monitoring studies of H. zea and other pests.

Reduced expression of the P-glycoprotein gene HaABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin but not Cry2Ab toxin in Helicoverpa armigera.

Rapid evolution of pest resistance to Bt insecticidal proteins presents a serious threat to the sustainable use of Bt crops. The cotton bollworm has been extensively exposed to Bt cotton worldwide and has evolved resistance in laboratory and field. Previous studies have highlighted the significant roles played by the ABC transporter proteins in Bt resistance. In this study, the ORF of HaABCB1 was cloned and analyzed. The expression of HaABCB1 was detected in all developmental stages and tissues, with the highest expression in third instar larvae stage and hindgut tissue. Compared with susceptible strain, a remarkable decrease of HaABCB1 expression in Cry1Ac resistant strain while no significant change in Cry2Ab resistant strain were found. The HaABCB1 expression reduced after susceptible larvae induced by Cry1Ac, but no obvious expression changes after Cry2Ab exposure. RNAi-mediated down-regulation of HaABCB1 could lead to a significant reduction in larval susceptibility to Cry1Ac, but not to Cry2Ab, in susceptible strain. Genetic linkage analysis confirmed that decreased expression of the HaABCB1 mediates resistance to Cry1Ac, but not Cry2Ab resistance. This knowledge contributes to better understanding of the complex molecular mechanisms underlying Bt resistance and provide theoretical foundation for the development of new strategies for pest resistance management.

Baseline Susceptibility of the Field Populations of Ostrinia furnacalis in Indonesia to the Proteins Cry1A.105 and Cry2Ab2 of Bacillus thuringiensis.

Genetically modified MON 89034 corn (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins, viz. Cry1A.105 and Cry2Ab2, is a biotechnological option being considered for the management of the major corn pest in Indonesia, the Asian corn borer (Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae)). As a part of a proactive resistance-management program for MON 89034 corn in Indonesia, we assessed the baseline susceptibility of field-collected populations of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins. Dose-response bioassays using the diet-dipping method indicated that the lethal concentration (LC50) values of Cry1A.105 and Cry2Ab2 in 24 different field populations of O. furnacalis ranged from 0.006 to 0.401 µg/mL and from 0.044 to 4.490 µg/mL, respectively, while the LC95 values ranged from 0.069 to 15.233 µg/mL for Cry1A.105 and from 3.320 to 277.584 µg/mL for Cry2Ab2. The relative resistance ratios comparing the most tolerant field populations and an unselected laboratory population were 6.0 for Cry1A.105 and 2.0 for Cry2Ab2 based on their LC50 values. Some field populations were more susceptible to both proteins than the unselected laboratory population. The LC99 and its 95% fiducial limits across the field populations were calculated and proposed as candidate diagnostic concentrations. These data provide a basis for resistance monitoring in Bt Corn and further support building resistance-management strategies in Indonesia.

Relative fitness of susceptible and Cry1A.105/Cry2Ab2-single-/dual-protein-resistant Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) on non-Bt diet and a diet containing a low concentration of two proteins.

Helicoverpa zea (Boddie) is a destructive agricultural pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1A.105 and Cry2Ab2 are two Bt proteins expressed in a widely planted maize event MON 89034. In this study, two tests (Test-I and Test-II) were conducted to evaluate the relative fitness of Bt-susceptible and -resistant H. zea on non-Bt diet (Test-I and Test-II) and a diet containing a mix of Cry1A.105 and Cry2Ab2 at a low concentration (Test-II only). Insect populations evaluated in Test-I were two Bt-susceptible strains and three Bt-resistant strains (a single-protein Cry1A.105-, a single-protein Cry2Ab2-, and a dual-protein Cry1A.105/Cry2Ab2-resistant strains). Test-II analyzed the same two susceptible strains, three backcrossed-and-reselected Cry1A.105/Cry2Ab2-single-/dual-protein-resistant strains, and three F1 heterozygous strains. Measurements of life table parameters showed that neither the single- nor dual-protein Cry1A.105/Cry2Ab2 resistance in H. zea was associated with fitness costs under the test conditions. The single Cry protein resistances at a concentration of a mix of Cry1A.105 and Cry2Ab2 that resulted in a zero net reproductive rate for the two susceptible strains were functionally incomplete recessive or codominant, and the dual-protein resistance was completely dominant. The lack of fitness costs could be a factor contributing to the rapid revolution of resistance to the Cry proteins in this species. Data generated from this study should aid our understanding of Cry protein resistance evolution and help in refining IRM programs for H. zea.