Inheritance and fitness cost of laboratory-selected resistance to Vip3Aa in Helicoverpa zea (Lepidoptera: Noctuidae).

The polyphagous pest Helicoverpa zea (Lepidoptera: Noctuidae) has evolved practical resistance to transgenic corn and cotton producing Cry1 and Cry2 crystal proteins from Bacillus thuringiensis (Bt) in several regions of the United States. However, the Bt vegetative insecticidal protein Vip3Aa produced by Bt corn and cotton remains effective against this pest. To advance knowledge of resistance to Vip3Aa, we selected a strain of H. zea for resistance to Vip3Aa in the laboratory. After 28 generations of continuous selection, the resistance ratio was 267 for the selected strain (GA-R3) relative to a strain not selected with Vip3Aa (GA). Resistance was autosomal and almost completely recessive at a concentration killing all individuals from GA. Declines in resistance in heterogeneous strains containing a mixture of susceptible and resistant individuals reared in the absence of Vip3Aa indicate a fitness cost was associated with resistance. Previously reported cases of laboratory-selected resistance to Vip3Aa in lepidopteran pests often show partially or completely recessive resistance at high concentrations and fitness costs. Abundant refuges of non-Bt host plants can maximize the benefits of such costs for sustaining the efficacy of Vip3Aa against target pests.

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea.

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab.

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world's most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.

Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States.

Invasive organisms pose a global threat and are exceptionally difficult to eradicate after they become abundant in their new habitats. We report a successful multitactic strategy for combating the pink bollworm (Pectinophora gossypiella), one of the world's most invasive pests. A coordinated program in the southwestern United States and northern Mexico included releases of billions of sterile pink bollworm moths from airplanes and planting of cotton engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). An analysis of computer simulations and 21 y of field data from Arizona demonstrate that the transgenic Bt cotton and sterile insect releases interacted synergistically to reduce the pest's population size. In Arizona, the program started in 2006 and decreased the pest's estimated statewide population size from over 2 billion in 2005 to zero in 2013. Complementary regional efforts eradicated this pest throughout the cotton-growing areas of the continental United States and northern Mexico a century after it had invaded both countries. The removal of this pest saved farmers in the United States $192 million from 2014 to 2019. It also eliminated the environmental and safety hazards associated with insecticide sprays that had previously targeted the pink bollworm and facilitated an 82% reduction in insecticides used against all cotton pests in Arizona. The economic and social benefits achieved demonstrate the advantages of using agricultural biotechnology in concert with classical pest control tactics.

Gene Flow Between Bt and Non-Bt Plants in a Seed Mixture Increases Dominance of Resistance to Pyramided Bt Corn in Helicoverpa zea (Lepidoptera: Noctuidae).

For delaying evolution of pest resistance to transgenic corn producing Bacillus thuringiensis (Bt) toxins, limited data are available to compare the effectiveness of refuges of non-Bt corn planted in seed mixtures versus blocks. Here we addressed this issue in the ear-feeding pest Helicoverpa zea Boddie by measuring its survival and development in the laboratory on ears from field plots with 90% Cry1A.105 + Cry2Ab corn and 10% non-Bt corn planted in a seed mixture or blocks. We compared a strain of H. zea selected for resistance to Cry1Ac in the laboratory, its parent strain not selected in the laboratory, and their F1 progeny. The relative survival of the F1 progeny and dominance of resistance were higher on ears from Bt plants in the seed mixture than the block. Half of the kernels in ears from non-Bt plants in the seed mixture produced both Cry1A.105 and Cry2Ab. However, survival on ears from non-Bt plants did not differ between the block and seed mixture. In simulations based on the observed survival, resistance to Cry1A.105 + Cry2Ab corn evolved faster with the seed mixture than the blocks, because of the higher dominance of resistance in the seed mixture. Increasing the refuge percentage improved durability of Cry1A.105 + Cry2Ab corn more for the blocks than the seed mixture. These findings imply that, for a given percentage of non-Bt corn, resistance of H. zea and other ear-feeding pests to multi-toxin Bt corn is likely to evolve faster for seed mixtures than blocks.

Shared and Independent Genetic Basis of Resistance to Bt Toxin Cry2Ab in Two Strains of Pink Bollworm.

Evolution of pest resistance threatens the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Field populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton, have evolved practical resistance to transgenic cotton producing Bt toxin Cry2Ab in India, but not in the United States. Previous results show that recessive mutations disrupting an autosomal ATP-binding cassette gene (PgABCA2) are associated with pink bollworm resistance to Cry2Ab in field-selected populations from India and in one lab-selected strain from the United States (Bt4-R2). Here we discovered that an independently derived, lab-selected Cry2Ab-resistant pink bollworm strain from the United States (BX-R) also harbors mutations that disrupt PgABCA2. Premature stop codons introduced by mis-splicing of PgABCA2 pre-mRNA were prevalent in field-selected larvae from India and in both lab-selected strains. The most common mutation in field-selected larvae from India was also detected in both lab-selected strains. Results from interstrain crosses indicate BX-R has at least one additional mechanism of resistance to Cry2Ab that does not involve PgABCA2 and is not completely recessive or autosomal. We conclude that recessive mutations disrupting PgABCA2 are the primary, but not the only, mechanism of resistance to Cry2Ab in pink bollworm.

Reduced cadherin expression associated with resistance to Bt toxin Cry1Ac in pink bollworm.

BACKGROUND: Better understanding of the molecular basis of resistance is needed to improve management of pest resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Here we analyzed resistance of the pink bollworm (Pectinophora gossypiella) to Bt toxin Cry1Ac, which is used widely in transgenic Bt cotton. Field-evolved practical resistance of pink bollworm to Cry1Ac is widespread in India, but not in China or the United States. Previous work with laboratory- and field-selected pink bollworm indicated that resistance to Cry1Ac is caused by changes in the amino acid sequence of a midgut cadherin protein (PgCad1) that binds Cry1Ac in susceptible larvae. RESULTS: Relative to a susceptible strain, the laboratory-selected APHIS-R strain had 530-fold resistance to Cry1Ac with autosomal recessive inheritance. Unlike previous results, resistance in this strain was not consistently associated with insertions or deletions in the expected amino acid sequence of PgCad1. However, this resistance was associated with 79- to 190-fold reduced transcription of the PgCad1 gene and markedly lower abundance of PgCad1 protein. CONCLUSION: The ability of pink bollworm and other major pests to evolve resistance to Bt toxins via both qualitative and quantitative changes in receptor proteins demonstrates their remarkable adaptability and presents challenges for monitoring and managing resistance to Bt crops. © 2019 Society of Chemical Industry.

Seasonal Declines in Cry1Ac and Cry2Ab Concentration in Maturing Cotton Favor Faster Evolution of Resistance to Pyramided Bt Cotton in Helicoverpa zea (Lepidoptera: Noctuidae).

Under ideal conditions, widely adopted transgenic crop pyramids producing two or more distinct insecticidal proteins from Bacillus thuringiensis (Bt) that kill the same pest can substantially delay evolution of resistance by pests. However, deviations from ideal conditions diminish the advantages of such pyramids. Here, we tested the hypothesis that changes in maturing cotton producing Cry1Ac and Cry2Ab affect evolution of resistance in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), a pest with low inherent susceptibility to both toxins. In terminal leaves of field-grown Bt cotton, the concentration of both toxins was significantly higher for young, squaring plants than for old, fruiting plants. We used laboratory bioassays with plant material from field-grown cotton to test H. zea larvae from a strain selected for resistance to Cry1Ac in the laboratory, its more susceptible parent strain, and their F1 progeny. On young Bt cotton, no individuals survived to pupation. On old Bt cotton, survival to pupation was significantly higher for the lab-selected strain and the F1 progeny relative to the unselected parent strain, indicating dominant inheritance of resistance. Redundant killing, the extent to which insects resistant to one toxin are killed by another toxin in a pyramid, was complete on young Bt cotton, but not on old Bt cotton. No significant fitness costs associated with resistance were detected on young or old non-Bt cotton. Incorporation of empirical data into simulations indicates the observed increased selection for resistance on old Bt cotton could accelerate evolution of resistance to cotton producing Cry1Ac and Cry2Ab in H. zea.

A long non-coding RNA regulates cadherin transcription and susceptibility to Bt toxin Cry1Ac in pink bollworm, Pectinophora gossypiella.

Extensive planting of transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has spurred increasingly rapid evolution of resistance in pests. In the pink bollworm, Pectinophora gossypiella, a devastating global pest, resistance to Bt toxin Cry1Ac produced by transgenic cotton is linked with mutations in a gene (PgCad1) encoding a cadherin protein that binds Cry1Ac in the larval midgut. We previously reported a long non-coding RNA (lncRNA) in intron 20 of cadherin alleles associated with both resistance and susceptibility to Cry1Ac. Here we tested the hypothesis that reducing expression of this lncRNA decreases transcription of PgCad1 and susceptibility to Cry1Ac. Quantitative RT-PCR showed that feeding susceptible neonates small interfering RNAs (siRNAs) targeting this lncRNA but not PgCad1 decreased the abundance of transcripts of both the lncRNA and PgCad1. Moreover, neonates fed the siRNAs had lower susceptibility to Cry1Ac. The results imply that the lncRNA increases transcription of PgCad1 and susceptibility of pink bollworm to Cry1Ac. The results suggest that disruption of lncRNA expression could be a novel mechanism of pest resistance to Bt toxins.

Effects of seasonal changes in cotton plants on the evolution of resistance to pyramided cotton producing the Bt toxins Cry1Ac and Cry1F in Helicoverpa zea.

BACKGROUND: In pests with inherently low susceptibility to Bacillus thuringiensis (Bt) toxins, seasonal declines in the concentration of Bt toxins in transgenic crops could accelerate evolution of resistance by increasing the dominance of resistance. Here, we evaluated Helicoverpa zea survival on young and old cotton plants that produced the Bt toxins Cry1Ac and Cry1F or did not produce Bt toxins. RESULTS: Using a strain selected for resistance to Cry1Ac in the laboratory, its parent strain that was not selected in the laboratory, and their F1 progeny, we showed that resistance to Cry1Ac + Cry1F cotton was partially dominant on young and old plants. On Cry1Ac + Cry1F cotton, redundant killing was incomplete on young plants but nearly complete on old plants. No significant fitness costs on non-Bt cotton occurred on young plants, but large recessive costs affected survival on old plants. Simulation models incorporating the empirical data showed that the seasonal changes in fitness could delay resistance to Cry1Ac + Cry1F cotton by inducing low equilibrium frequencies of resistance alleles when refuges are sufficiently large. CONCLUSION: Our results suggest that including effects of seasonal changes in fitness of pests on Bt crops and refuge plants can enhance resistance risk assessment and resistance management. © 2017 Society of Chemical Industry.

Binding and Oligomerization of Modified and Native Bt Toxins in Resistant and Susceptible Pink Bollworm.

Insecticidal proteins from Bacillus thuringiensis (Bt) are used extensively in sprays and transgenic crops for pest control, but their efficacy is reduced when pests evolve resistance. Better understanding of the mode of action of Bt toxins and the mechanisms of insect resistance is needed to enhance the durability of these important alternatives to conventional insecticides. Mode of action models agree that binding of Bt toxins to midgut proteins such as cadherin is essential for toxicity, but some details remain unresolved, such as the role of toxin oligomers. In this study, we evaluated how Bt toxin Cry1Ac and its genetically engineered counterpart Cry1AcMod interact with brush border membrane vesicles (BBMV) from resistant and susceptible larvae of Pectinophora gossypiella (pink bollworm), a global pest of cotton. Compared with Cry1Ac, Cry1AcMod lacks 56 amino acids at the amino-terminus including helix α-1; previous work showed that Cry1AcMod formed oligomers in vitro without cadherin and killed P. gossypiella larvae harboring cadherin mutations linked with >1000-fold resistance to Cry1Ac. Here we found that resistance to Cry1Ac was associated with reduced oligomer formation and insertion. In contrast, Cry1AcMod formed oligomers in BBMV from resistant larvae. These results confirm the role of cadherin in oligomerization of Cry1Ac in susceptible larvae and imply that forming oligomers without cadherin promotes toxicity of Cry1AcMod against resistant P. gossypiella larvae that have cadherin mutations.

Multi-Toxin Resistance Enables Pink Bollworm Survival on Pyramided Bt Cotton.

Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant "pyramids" producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India.

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects.

Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.

Cross-resistance to toxins used in pyramided Bt crops and resistance to Bt sprays in Helicoverpa zea.

To delay evolution of resistance by insect pests, farmers are rapidly increasing their use of transgenic crops producing two or more Bacillus thuringiensis (Bt) toxins that kill the same pest. A key condition favoring durability of these "pyramided" crops is the absence of cross-resistance between toxins. Here we evaluated cross-resistance in the major lepidopteran pest Helicoverpa zea (Boddie) to Bt toxins used in pyramids. In the laboratory, we selected a strain of this pest with Bt toxin Cry1Ac followed by selection with MVP II, a formulation containing a hybrid protoxin that is identical to Cry1Ac in the active portion of the toxin and 98.5% identical overall. We calculated the resistance ratio as the EC50 (concentration causing mortality or failure to develop beyond the first instar of 50% of larvae) for the laboratory-selected strain divided by the EC50 for its field-derived parent strain that was not selected in the laboratory. The resistance ratio was 20.0-33.9 (mean=27.0) for MVP II, 57.0 for Cry1Ac, 51.3 for Cry1A.105, 22.4 for Cry1Ab, 3.3 for Cry2Ab, 1.8 for Cry1Fa, and 1.6 for Vip3Aa. Resistance ratios were 2.9 for DiPel ES and 2.0 for Agree VG, which are commercial Bt spray formulations containing Cry1Ac, other Bt toxins, and Bt spores. By the conservative criterion of non-overlap of 95% fiducial limits, the EC50 was significantly higher for the selected strain than its parent strain for MVP II, Cry1Ac, Cry1A.105, Cry1Ab, Cry2Ab and DiPel ES. For Cry1Fa, Vip3Aa, and Agree VG, significantly lower susceptibility to a high concentration indicated low cross-resistance. The resistance ratio for toxins other than Cry1Ac was associated with their amino acid sequence similarity to Cry1Ac in domain II. Resistance to Cry1Ac and the observed cross-resistance to other Bt toxins could accelerate evolution of H. zea resistance to currently registered Bt sprays and pyramided Bt crops.

Alternative splicing and highly variable cadherin transcripts associated with field-evolved resistance of pink bollworm to bt cotton in India.

Evolution of resistance by insect pests can reduce the benefits of insecticidal proteins from Bacillus thuringiensis (Bt) that are used extensively in sprays and transgenic crops. Despite considerable knowledge of the genes conferring insect resistance to Bt toxins in laboratory-selected strains and in field populations exposed to Bt sprays, understanding of the genetic basis of field-evolved resistance to Bt crops remains limited. In particular, previous work has not identified the genes conferring resistance in any cases where field-evolved resistance has reduced the efficacy of a Bt crop. Here we report that mutations in a gene encoding a cadherin protein that binds Bt toxin Cry1Ac are associated with field-evolved resistance of pink bollworm (Pectinophora gossypiella) in India to Cry1Ac produced by transgenic cotton. We conducted laboratory bioassays that confirmed previously reported resistance to Cry1Ac in pink bollworm from the state of Gujarat, where Bt cotton producing Cry1Ac has been grown extensively. Analysis of DNA from 436 pink bollworm from seven populations in India detected none of the four cadherin resistance alleles previously reported to be linked with resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona. However, DNA sequencing of pink bollworm derived from resistant and susceptible field populations in India revealed eight novel, severely disrupted cadherin alleles associated with resistance to Cry1Ac. For these eight alleles, analysis of complementary DNA (cDNA) revealed a total of 19 transcript isoforms, each containing a premature stop codon, a deletion of at least 99 base pairs, or both. Seven of the eight disrupted alleles each produced two or more different transcript isoforms, which implicates alternative splicing of messenger RNA (mRNA). This represents the first example of alternative splicing associated with field-evolved resistance that reduced the efficacy of a Bt crop.

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.

The changing nutrition scenario.

The past seven decades have seen remarkable shifts in the nutritional scenario in India. Even up to the 1950s severe forms of malnutrition such as kwashiorkar and pellagra were endemic. As nutritionists were finding home-grown and common-sense solutions for these widespread problems, the population was burgeoning and food was scarce. The threat of widespread household food insecurity and chronic undernutrition was very real. Then came the Green Revolution. Shortages of food grains disappeared within less than a decade and India became self-sufficient in food grain production. But more insidious problems arising from this revolution were looming, and cropping patterns giving low priority to coarse grains and pulses, and monocropping led to depletion of soil nutrients and 'Green Revolution fatigue'. With improved household food security and better access to health care, clinical manifestations of severe malnutrition virtually disappeared. But the decline in chronic undernutrition and "hidden hunger" from micronutrient deficiencies was slow. On the cusp of the new century, an added factor appeared on the nutritional scene in India. With steady urban migration, upward mobility out of poverty, and an increasingly sedentary lifestyle because of improvements in technology and transport, obesity rates began to increase, resulting in a dual burden. Measured in terms of its performance in meeting its Millennium Development Goals, India has fallen short. Despite its continuing high levels of poverty and illiteracy, India has a huge demographic potential in the form of a young population. This advantage must be leveraged by investing in nutrition education, household access to nutritious diets, sanitary environment and a health-promoting lifestyle. This requires co-operation from all the stakeholders, including governments, non government organizations, scientists and the people at large.

Sustained susceptibility of pink bollworm to Bt cotton in the United States.

Evolution of resistance by pests can reduce the benefits of transgenic crops that produce toxins from Bacillus thuringiensis (Bt) for insect control. One of the world's most important cotton pests, pink bollworm (Pectinophora gossypiella), has been targeted for control by transgenic cotton producing Bt toxin Cry1Ac in several countries for more than a decade. In China, the frequency of resistance to Cry1Ac has increased, but control failures have not been reported. In western India, pink bollworm resistance to Cry1Ac has caused widespread control failures of Bt cotton. By contrast, in the state of Arizona in the southwestern United States, monitoring data from bioassays and DNA screening demonstrate sustained susceptibility to Cry1Ac for 16 y. From 1996-2005, the main factors that delayed resistance in Arizona appear to be abundant refuges of non-Bt cotton, recessive inheritance of resistance, fitness costs associated with resistance and incomplete resistance. From 2006-2011, refuge abundance was greatly reduced in Arizona, while mass releases of sterile pink bollworm moths were made to delay resistance as part of a multi-tactic eradication program. Sustained susceptibility of pink bollworm to Bt cotton in Arizona has provided a cornerstone for the pink bollworm eradication program and for integrated pest management in cotton. Reduced insecticide use against pink bollworm and other cotton pests has yielded economic benefits for growers, as well as broad environmental and health benefits. We encourage increased efforts to combine Bt crops with other tactics in integrated pest management programs.

Identification of salt responsive genes using comparative microarray analysis in Upland cotton (Gossypium hirsutum L.).

Salinity negatively impacts plant growth and productivity, and little is known about salt responsive genes in cotton. In this study, an intra-specific backcross population of cotton (Gossypium hirsutum L.) was treated with 200 mM NaCl after which differentially expressed genes were identified by comparison between salt tolerant and susceptible segregant bulks using comparative microarray analysis. Microarray analysis identified 720 salt-responsive genes, of which 695 were down-regulated and only 25 were up-regulated in the salt tolerant bulk. Gene ontology of annotated genes revealed that at least some of the identified salt responsive transcripts belong to pathways known to be associated with salt stress including osmolyte and lipid metabolism, cell wall structure, and membrane synthesis. About 48% of all salt-responsive genes were functionally unknown. Quantitative RT-PCR was used to validate 17 selected salt responsive genes. This work represents the first study in employing microarray to investigate the possible mechanisms of the salt response in cotton. Further analysis of salt-responsive genes associated with salt tolerance in cotton will assist in laying a foundation for molecular manipulation in development of new cultivars with improved salt tolerance.