Association between resistance to Bt cotton and cadherin genotype in pink bollworm

Two strains of pink bollworm, Pectinophora gossypiella (Saunders), each derived in 1997 from a different field population, were selected for resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac in the laboratory. One strain (MOV97-R) originated from Mohave Valley in western Arizona; the other strain (SAF97-R) was from Safford in eastern Arizona. Relative to a susceptible laboratory strain, Cry1Ac resistance ratios were 1700 for MOV97-R and 520 for SAF97-R. For the two resistant strains, larval survival did not differ between non-Bt cotton and transgenic cotton producing CrylAc. In contrast, larval survival on Bt cotton was 0% for the two unselected parent strains from which the resistant strains were derived. Previously identified resistance (r) alleles of a cadherin gene (BtR) occurred in both resistant strains: r1 and r3 in MOV97-R, and r1 and r2 in SAF97-R. The frequency of individuals carrying two r alleles (rr) was 1.0 in the two resistant strains and 0.02 in each of the two unselected parent strains. Furthermore, in two hybrid strains with a mixture of susceptible (s) and r alleles at the BtR locus, all survivors on Bt cotton had two r alleles. The results show that resistance to Cry1Ac-producing Bt cotton is associated with recessive r alleles at the BtR locus in the strains of pink bollworm tested here. In conjunction with previous results from two other Bt-resistant strains of pink bollworm (APHIS-98R and AZP-R), results reported here identify the cadherin locus as the leading candidate for molecular monitoring of pink bollworm resistance to Bt cotton.     

Similar genetic basis of resistance to Bt toxin Cry1Ac in Boll-selected and diet-selected strains of pink bollworm

Genetically engineered cotton and corn plants producing insecticidal Bacillus thuringiensis (Bt) toxins kill some key insect pests. Yet, evolution of resistance by pests threatens long-term insect control by these transgenic Bt crops. We compared the genetic basis of resistance to Bt toxin Cry1Ac in two independently derived, laboratory-selected strains of a major cotton pest, the pink bollworm (Pectinophora gossypiella [Saunders]). The Arizona pooled resistant strain (AZP-R) was started with pink bollworm from 10 field populations and selected with Cry1Ac in diet. The Bt4R resistant strain was started with a long-term susceptible laboratory strain and selected first with Bt cotton bolls and later with Cry1Ac in diet. Previous work showed that AZP-R had three recessive mutations (r1, r2, and r3) in the pink bollworm cadherin gene (PgCad1) linked with resistance to Cry1Ac and Bt cotton producing Cry1Ac. Here we report that inheritance of resistance to a diagnostic concentration of Cry1Ac was recessive in Bt4R. In interstrain complementation tests for allelism, F(1) progeny from crosses between AZP-R and Bt4R were resistant to Cry1Ac, indicating a shared resistance locus in the two strains. Molecular analysis of the Bt4R cadherin gene identified a novel 15-bp deletion (r4) predicted to cause the loss of five amino acids upstream of the Cry1Ac-binding region of the cadherin protein. Four recessive mutations in PgCad1 are now implicated in resistance in five different strains, showing that mutations in cadherin are the primary mechanism of resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona.     

Shared genetic basis of resistance to Bt toxin Cry1ac in independent strains of pink bollworm

Classical and molecular genetic analyses show that two independently derived resistant strains of pink bollworm, Pectinophora gossypiella (Saunders), share a genetic locus at which three mutant alleles confer resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac. One laboratory-selected resistant strain (AZP-R) was derived from individuals collected in 1997 from 10 Arizona cotton fields, whereas the other (APHIS-98R) was derived from a long-term susceptible laboratory strain. Both strains were previously reported to show traits of "mode 1" resistance, the most common type of lepidopteran resistance to Cry1A toxins. Inheritance of resistance to a diagnostic concentration of Cry1Ac (10 microg per gram of diet) was recessive in both strains. In interstrain complementation tests for allelism, F1 progeny from crosses between the two strains were resistant to the diagnostic concentration of Cry1Ac. These results indicate that a major resistance locus is shared by the two strains. Analysis of DNA from the pink bollworm cadherin gene (BtR) using allele-specific polymerase chain reaction (PCR) tests showed that the previously identified resistance alleles (r1, r2, and r3) occurred in both strains, but their frequencies differed between strains. In conjunction with previous findings, the results reported here suggest that PCR-based detection of the three known cadherin resistance alleles might be useful for monitoring resistance to Cry1Ac-producing Bt cotton in field populations of pink bollworm.     

Non-Recessive Bt Toxin Resistance Conferred by an Intracellular Cadherin Mutation in Field-Selected Populations of Cotton Bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.     

High-level resistance to Bacillus thuringiensis toxin crylac and cadherin genotype in pink bollworm

Resistance to transgenic cotton, Gossypium hirsutum L., producing Bacillus thuringiensis (Bt) toxin Cry1Ac is linked with three recessive alleles of a cadherin gene in laboratory-selected strains of pink bollworm, Pectinophora gossypiella (Saunders), a major cotton pest. Here, we analyzed a strain (MOV97-R) with a high frequency of cadherin resistance alleles, a high frequency of resistance to 10 microg of Cry1Ac per milliliter of diet, and an intermediate frequency of resistance to 1000 microg of Cry1Ac per ml of diet. We selected two strains for increased resistance by exposing larvae from MOV97-R to diet with 1000 microg of Cry1Ac per ml of diet. In both selected strains, two to three rounds of selection increased survival at 1000 microg of CrylAc per ml of diet to at least 76%, indicating genetic variation in survival at this high concentration and yielding >4300-fold resistance relative to a susceptible strain. Variation in cadherin genotype did not explain variation in survival at 1000 microg of Cry1Ac per ml of diet, implying that one or more other loci affected survival at this concentration. This conclusion was confirmed with results showing that when exposure to Cry1Ac stopped, survival at 1000 microg of Cry1Ac per ml of diet dropped substantially, but survival at 10 microg Cry1Ac per ml of diet remained close to 100% and all survivors had two cadherin resistance alleles. Although survival at 1000 microg of Cry1Ac per ml of diet is not required for resistance to Bt cotton, understanding how genes other than cadherin confer increased survival at this high concentration may reveal novel mechanisms of resistance.     

Sequence variation and differential splicing of the midgut cadherin gene in Trichoplusia ni

The insect midgut cadherin serves as an important receptor for the Cry toxins from Bacillus thuringiensis (Bt). Variation of the cadherin in insect populations provides a genetic potential for development of cadherin-based Bt resistance in insect populations. Sequence analysis of the cadherin from the cabbage looper, Trichoplusia ni, together with cadherins from 18 other lepidopterans showed a similar phylogenetic relationship of the cadherins to the phylogeny of Lepidoptera. The midgut cadherin in three laboratory populations of T. ni exhibited high variability, although the resistance to Bt toxin Cry1Ac in the T. ni strain is not genetically associated with cadherin gene mutations. A total of 142 single nucleotide polymorphisms (SNPs) were identified in the cadherin cDNAs from the T. ni strains, including 20 missense mutations. In addition, insertion and deletion polymorphisms (indels) were also identified in the cadherin alleles in T. ni. More interestingly, the results from this study reveal that differential splicing of mRNA also occurs in the cadherin gene expression. Therefore, variation of the midgut cadherin in insects may not only be caused by cadherin gene mutations, but could also result from alternative splicing of its mRNA regulated by factors acting in trans. Analysis of cadherin gene alleles in F2, F3 and F4 progenies from the cross between the Cry1Ac resistant and the susceptible strain after consecutive selections with Cry1Ac for three generations showed that selection with Cry1Ac did not result in an increase of frequencies of the cadherin alleles originated from the resistant strain.     

Examination of the F2 screen for rare resistance alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae)

A synthetic laboratory population of the diamondback moth, Plutella xylostella (L.), was used to test the F2 screen developed for detecting the frequency of rare resistance alleles to Cry1Ac and Cry1C toxins of Bacillus thuringiensis (Bt). Of the 120 single-pair matings set up, 106 produced enough F2 families for screening of Cry1Ac or Cry1C resistance alleles using both transgenic broccoli and an artificial diet overlay assay with a diagnostic dose. When using Bt broccoli plants as the F2 screen method, only one F2 family was detected for Cry1Ac resistance and no family was detected for Cry1C resistance. Six families were detected for either Cry1Ac or Cry1C resistance using the diet assay. The survivors in the diagnostic diet assay were crossed with the resistant individuals to confirm their resistance genotypes. Four F2 families were confirmed to contain one copy of an allele resistant to Cry1Ac in the original single-pairs and four other F2 families contained an allele resistant to Cry1C. Our results suggest that using transgenic plants expressing a high level of a Bt toxin in an F2 screen may underestimate the frequency of resistance alleles with high false negatives, or fail to detect true resistance alleles. The diagnostic diet assay was a better F2 screen method to detect alleles, especially for the Cry1Ac resistance with monogenic inheritance in the diamondback moth. The estimated probabilities of false positives and false negatives were 33 and 1%, respectively, for detecting Cry1Ac resistance at the allele frequency of 0.012 using the diagnostic diet assay. Careful validation of the screening method for each insect-crop system is necessary before the F2 screen can be used to detect rare Bt resistance alleles in field populations.     

Mutated cadherin alleles from a field population of Helicoverpa armigera confer resistance to Bacillus thuringiensis toxin Cry1Ac

The cotton bollworm Helicoverpa armigera is the major insect pest targeted by cotton genetically engineered to produce the Bacillus thuringiensis toxin (transgenic Bt cotton) in the Old World. The evolution of this pest's resistance to B. thuringiensis toxins is the main threat to the long-term effectiveness of transgenic Bt cotton. A deletion mutation allele (r(1)) of a cadherin gene (Ha_BtR) was previously identified as genetically linked with Cry1Ac resistance in a laboratory-selected strain of H. armigera. Using a biphasic screen strategy, we successfully trapped two new cadherin alleles (r(2) and r(3)) associated with Cry1Ac resistance from a field population of H. armigera collected from the Yellow River cotton area of China in 2005. The r(2) and r(3) alleles, respectively, were created by inserting the long terminal repeat of a retrotransposon (designated HaRT1) and the intact HaRT1 retrotransposon at the same position in exon 8 of Ha_BtR, which results in a truncated cadherin containing only two ectodomain repeats in the N terminus of Ha_BtR. This is the first time that the B. thuringiensis resistance alleles of a target insect of Bt crops have been successfully detected in the open field. This study also demonstrated that bollworm larvae carrying two resistance alleles can complete development on Bt cotton. The cadherin locus should be an important target for intensive DNA-based screening of field populations of H. armigera.     

Effects of refuge contamination by transgenes on Bt resistance in pink bollworm (Lepidoptera: Gelechiidae)

Refuges of non-Bacillus thuringiensis (Bt) cotton, Gossypium hirsutum L., are used to delay Bt resistance in pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), a pest that eats cotton seeds. Contamination of refuges by Bt transgenes could reduce the efficacy of this strategy. Previously, three types of contamination were identified in refuges: 1) homozygous Bt cotton plants, with 100% of their seeds producing the Bt toxin Cry1Ac; 2) hemizygous Bt plants with 70-80% of their seeds producing Cry1Ac; and 3) non-Bt plants that outcrossed with Bt plants, resulting in bolls with Cry1Ac in 12-17% of their seeds. Here, we used laboratory bioassays to examine the effects of Bt contamination on feeding behavior and survival of pink bollworm that were resistant (rr), susceptible (ss), or heterozygous for resistance (rs) to Cry1Ac. In choice tests, rr and rs larvae did not differ from ss in preference for non-Bt versus Bt seeds. Survival of rr and rs also did not differ from ss on artificial outcrossed bolls (a mixture of 20% Bt and 80% non-Bt cotton seeds). On artificial hemizygous Bt bolls (70% Bt seeds) and homozygous Bt bolls (100% Bt seeds), rr had higher survival than ss, although rs and ss did not differ. In a simulation model, levels of refuge contamination observed in the field had negligible effects on resistance evolution in pink bollworm. However, in hypothetical simulations where contamination conferred a selective advantage to rs over ss individuals in refuges, resistance evolution was accelerated.     

Cadherin-based resistance to Bacillus thuringiensis cotton in hybrid strains of pink bollworm: fitness costs and incomplete resistance

Recessive resistance to Bacillus thuringiensis (Bt) cotton, Gossypium hirsutum L., in laboratory-selected strains of pink bollworm, Pectinophora gossypiella (Saunders), is associated with three resistance alleles (r1, r2, and r3) of a cadherin gene. Previous experiments based on measurement of fitness components in Bt-resistant and Bt-susceptible strains revealed that fitness costs and incomplete resistance are associated with resistance. Here, we used two hybrid strains of pink bollworm, each containing a mixture of susceptible and resistant individuals, and polymerase chain reaction (PCR) amplifications to test the association between cadherin genotype and fitness components for individuals sharing a common genetic background. All survivors on Bt cotton had two r alleles, confirming that recessive cadherin alleles are tightly linked with resistance to Bt cotton. On non-Bt cotton, significantly greater developmental time for rr than ss larvae indicated a recessive fitness cost, but costs did not affect survival or pupal weight. Incomplete resistance was manifested as longer developmental time, lower survival, and smaller pupal weight in rr individuals developing on Bt cotton compared with non-Bt cotton. As in previous experiments, no significant variation in performance on Bt cotton was detected among rr genotypes. However, a meta-analysis of data from seven experiments revealed that survival on Bt cotton relative to non-Bt cotton was lower in r2r3 and higher in r1r2 compared with the other rr genotypes. Assessment of fitness components associated with cadherin genotypes in hybrid strains of pink bollworm confirms that recessive resistance to Bt cotton is associated with recessive fitness costs and incomplete resistance.     

Fitness cost of resistance to Bt cotton linked with increased gossypol content in pink bollworm larvae

Fitness costs of resistance to Bacillus thuringiensis (Bt) crops occur in the absence of Bt toxins, when individuals with resistance alleles are less fit than individuals without resistance alleles. As costs of Bt resistance are common, refuges of non-Bt host plants can delay resistance not only by providing susceptible individuals to mate with resistant individuals, but also by selecting against resistance. Because costs typically vary across host plants, refuges with host plants that magnify costs or make them less recessive could enhance resistance management. Limited understanding of the physiological mechanisms causing fitness costs, however, hampers attempts to increase costs. In several major cotton pests including pink bollworm (Pectinophora gossypiella), resistance to Cry1Ac cotton is associated with mutations altering cadherin proteins that bind this toxin in susceptible larvae. Here we report that the concentration of gossypol, a cotton defensive chemical, was higher in pink bollworm larvae with cadherin resistance alleles than in larvae lacking such alleles. Adding gossypol to the larval diet decreased larval weight and survival, and increased the fitness cost affecting larval growth, but not survival. Across cadherin genotypes, the cost affecting larval growth increased as the gossypol concentration of larvae increased. These results suggest that increased accumulation of plant defensive chemicals may contribute to fitness costs associated with resistance to Bt toxins.     

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.     

Inheritance of resistance to Bt toxin crylac in a field-derived strain of pink bollworm (Lepidoptera: Gelechiidae)

Laboratory selection with Cry1Ac, the Bacillus thuringiensis (Bt) toxin in transgenic cotton, initially produced 300-fold resistance in a field-derived strain of pink bollworm, Pectinophora gossypiella (Saunders), a major cotton pest. After additional selection increased resistance to 3,100-fold, we tested the offspring of various crosses to determine the mode of inheritance of resistance to Cry1Ac. The progeny of reciprocal F1 crosses (resistant male x susceptible female and vice versa) responded alike in bioassays, indicating autosomal inheritance. Consistent with earlier findings, resistance was recessive at a high concentration of Cry1Ac. However, the dominance of resistance increased as the concentration of Cry1Ac decreased. Analysis of survival and growth of progeny from backcrosses (F1 x resistant strain) suggest that resistance was controlled primarily by one or a few major loci. The progression of resistance from 300- to 3,100-fold rules out the simplest model with one locus and two alleles. Overall the patterns observed can be explained by either a single resistance gene with three or more alleles or by more than one resistance gene. The pink bollworm resistance to Cry1Ac described here fits "mode 1" resistance, the most common type of resistance to Cry1A toxins in Lepidoptera.     

Resistance of Trichoplusia ni Populations Selected by Bacillus thuringiensis Sprays to Cotton Plants Expressing Pyramided Bacillus thuringiensis Toxins Cry1Ac and Cry2Ab

Two populations of Trichoplusia ni that had developed resistance to Bacillus thuringiensis sprays (Bt sprays) in commercial greenhouse vegetable production were tested for resistance to Bt cotton (BollGard II) plants expressing pyramided Cry1Ac and Cry2Ab. The T. ni colonies resistant to Bacillus thuringiensis serovar kurstaki formulations were not only resistant to the Bt toxin Cry1Ac, as previously reported, but also had a high frequency of Cry2Ab-resistant alleles, exhibiting ca. 20% survival on BollGard II foliage. BollGard II-resistant T. ni strains were established by selection with BollGard II foliage to further remove Cry2Ab-sensitive alleles in the T. ni populations. The BollGard II-resistant strains showed incomplete resistance to BollGard II, with adjusted survival values of 0.50 to 0.78 after 7 days. The resistance to the dual-toxin cotton plants was conferred by two genetically independent resistance mechanisms: one to Cry1Ac and one to Cry2Ab. The 50% lethal concentration of Cry2Ab for the resistant strain was at least 1,467-fold that for the susceptible T. ni strain. The resistance to Cry2Ab in resistant T. ni was an autosomally inherited, incompletely recessive monogenic trait. Results from this study indicate that insect populations under selection by Bt sprays in agriculture can be resistant to multiple Bt toxins and may potentially confer resistance to multitoxin Bt crops.     

Frequency of Cry1F Non-Recessive Resistance Alleles in North Carolina Field Populations of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a target species of transgenic corn (Zea mays L.) that expresses single and pyramided Bacillus thuringiensis (Bt) toxin. In 2014, S. frugiperda were collected from a light trap in North Carolina, and a total of 212 F₁/F₂ isofemale lines of S. frugiperda were screened for resistance to Bt and non-Bt corn. All of the 212 isolines were susceptible to corn tissue expressing Cry1A.105 + Cry2Ab, Cry1F + Cry1A.105 + Cry2Ab, and Cry1F + Cry1Ab + Vip3Aa20. Growth rate bioassays were performed to isolate non-recessive Bt resistance alleles. Seven individuals out of the 212 isofemale lines carried major non-recessive alleles conferring resistance to Cry1F. A pooled colony was created from the seven individuals. This colony was 151.21 times more resistant to Cry1F than a known-susceptible population and was also resistant to Cry1A.105, but was not resistant to Cry2Ab and Vip3Aa20. The results demonstrate that field populations of S. frugiperda collected from North Carolina are generally susceptible to Cry1F, but that some individuals carry resistant alleles. The data generated in this study can be used as baseline data for resistance monitoring.     

Efficacy of Genetically Modified Bt Toxins Alone and in Combinations Against Pink Bollworm Resistant to Cry1Ac and Cry2Ab

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.