Early detection of field-evolved resistance to Bt cotton in China: cotton bollworm and pink bollworm

Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some major insect pests, but pests can evolve resistance and thereby reduce the effectiveness of such Bt crops. The main approach for slowing pest adaptation to Bt crops uses non-Bt host plants as "refuges" to increase survival of susceptible pests. To delay evolution of pest resistance to cotton producing Bt toxin Cry1Ac, several countries have required refuges of non-Bt cotton, while farmers in China have relied on "natural" refuges of non-Bt host plants other than cotton. This strategy is designed for cotton bollworm (Helicoverpa armigera), which attacks many crops and is the primary target of Bt cotton in China, but it does not apply to pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we review evidence of field-evolved resistance to Cry1Ac by cotton bollworm in northern China and by pink bollworm in the Yangtze River Valley of China. For both pests, results of laboratory diet bioassays reveal significantly decreased susceptibility of field populations to Cry1Ac, yet field control failures of Bt cotton have not been reported. The early detection of resistance summarized here may spur countermeasures such as planting Bt cotton that produces two or more distinct toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

棉花无蜜腺性状抗棉铃虫及红铃虫效果研究

棉花无蜜腺性状抗棉铃虫及红铃虫效果研究郑冬官,方其英（安徽农业大学合肥２３００３３６）郑厚今（安徽省农业厅,合肥２３０００１）Ｒｅｓｉｓｔａｎｃｅｏｆｃｏｔｔｏｎｎｅｃｔａｒｉｌｅｓｓｃｈａｒａｃｔｅｒｔｏｂｏｌｌｗｏｒｍａｎｄｐｉｎｋｂｏｌｌｗｏｒ...     

Evidence that mass trapping suppresses pink bollworm populations in cotton fields

Both thelytokous and arrhenotokous Trichogramma minutum were collected from eggs of the spruce budmoth, Zeiraphera canadensis in New Brunswick, Canada and their phenotypic traits compared for use in biological control. The lower threshold temperature for development of thelytokous and arrhenotokous parasitoids was 9.7 and 10.1 °C, respectively; the former required significantly higher degree-days for development from egg to adult (–x±SE=165.1±5.8 °D) than the latter (128.1±4.9 °D). Thelytokous and arrhenotokous parasitoids had similar forewing length (0.49±0.01 vs. 0.49±0.01 mm) and adult lifespan (13.3±0.7 vs. 14.0±1.1 days), but significantly different fecundity and sex ratios. Thelytokous females produced fewer offspring (89.5±6.6 vs. 173.9±6.4) and fewer female progeny (77.2±5.4 vs. 109.8±3.3) despite an overall higher proportion of females (91.6±1.1% vs. 65.4±2.8%) than their arrhenotokous counterparts. Ovarian dissections showed that the number of eggs increased with parasitoid age in arrhenotokous parasitoids but remained steady in thelytokous parasitoids. The variation in ovarian development of the two forms was the major factor contributing to the differences in fecundity. Thelytokous parasitoids were more host-specific than arrhenotokous ones; when offered eight host species, the former rejected three whereas the latter rejected only one. Thelytokous parasitoids survived better than arrhenotokous ones when stored from 30 to 150 days at 4 °C. Thelytokous females were slower at initiating flight after emergence than arrhenotokous females but maintained flight activity longer (6 h). These results indicate that thelytokous T. minutum are different from their arrhenotokous counterparts physiologically, biologically and ecologically and that they may play different roles in the field. The potential for using thelytokous parasitoids in biological control programs is discussed.     

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.     

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.     

Effects of gossypol on fitness costs associated with resistance to Bt cotton in pink bollworm

Fitness costs associated with insect resistance to Bacillus thuringiensis (Bt) crops may help to delay or prevent the spread of resistance alleles, especially when refuges of non-Bt host plants are present. The potential for such delays increases as the magnitude and dominance of fitness costs increase. Here, we examined the idea that plant secondary chemicals affect expression of fitness costs associated with resistance to Bt cotton in Pectinophora gossypiella (Saunders). Specifically, we tested the hypotheses that gossypol affects the magnitude or dominance of fitness costs, by measuring performance of three independent sets of pink bollworm populations fed artificial diet with and without gossypol. Each set had an unselected susceptible population, a resistant population derived by selection from the susceptible population, and the F1 progeny of the susceptible and resistant populations. No individuals completed development on diets with gossypol in one set, suggesting that these individuals partially lost the ability to detoxify this chemical. In the other two sets, costs affecting survival did not support the hypotheses, but costs affecting pupal weight did. Adding gossypol to diet increased the magnitude and dominance of costs affecting pupal weight. In one of the two sets with survivors on diet with gossypol, costs affecting development time were less recessive when gossypol was present in diet. These results indicate that gossypol increased the magnitude and dominance of some fitness costs. Better understanding of the effects of natural plant defenses on fitness costs could improve our ability to design refuges for managing insect resistance to Bt crops.     

Effects of cotton cultivar on fitness costs associated with resistance of pink bollworm (Lepidoptera: Gelechiidae) to Bt cotton

Fitness costs associated with insect resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) reduce the fitness on non-Bt refuge plants of resistant individuals relative to susceptible individuals. Because costs may vary among host plants, choosing refuge cultivars that increase the dominance or magnitude of costs could help to delay resistance. Specifically, cultivars with high concentrations of toxic phytochemicals could magnify costs. To test this hypothesis, we compared life history traits of three independent sets of pink bollworm, Pectinophora gossypiella (Saunders), populations on two cotton cultivars that differed in antibiosis against this cotton pest. Each set had an unselected susceptible population, a resistant population derived by selection from the susceptible population, and the F1 progeny of the susceptible and resistant populations. Confirming previous findings with pink bollworm feeding on cotton, costs primarily affected survival and were recessive on both cultivars. The magnitude of the survival cost did not differ between cultivars. Although the experimental results did not reveal differences between cultivars in the magnitude or dominance of costs, modeling results suggest that differences between cultivars in pink bollworm survival could affect resistance evolution. Thus, knowledge of the interaction between host plants and fitness costs associated with resistance to Bt crops could be helpful in guiding the choice of refuge cultivars.     

Oviposition on and mining in bolls of Bt and non-Bt cotton by resistant and susceptible pink bollworm (Lepidoptera: Gelechiidae)

Transgenic cotton that produces insecticidal crystal protein Cry1Ac of Bacillus thuringiensis (Bt) has been effective in controlling pink bollworm, Pectinophora gossypiella (Saunders). We compared responses to bolls of Bt cotton and non-Bt cotton by adult females and neonates from susceptible and Cry1Ac-resistant strains of pink bollworm. In choice tests on caged cotton plants in the greenhouse, neither susceptible nor resistant females laid fewer eggs on Bt cotton bolls than on non-Bt cotton bolls, indicating that the Bt toxin did not deter oviposition. Multiple regression revealed that the number of eggs laid per boll was negatively associated with boll age and positively associated with boll diameter. Females also laid more eggs per boll on plants with more bolls. The distribution of eggs among bolls of Bt cotton and non-Bt cotton was clumped, indicating that boll quality rather than avoidance of previously laid eggs was a primary factor in oviposition preference. Parallel to the results from oviposition experiments, in laboratory no-choice tests with 10 neonates per boll, the number of entrance holes per boll did not differ between Bt cotton and non-Bt cotton for susceptible and resistant neonates. Also, like females, neonates preferred younger bolls and larger bolls. Thus, acceptance of bolls by females for oviposition and by neonates for mining was affected by boll age and diameter, but not by Bt toxin in bolls. The lack of discrimination between Bt and non-Bt cotton bolls by pink bollworm from susceptible and resistant strains indicates that oviposition and mining initiation are independent of susceptibility to Cry1Ac.     

Overwintering cost associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelechiidae)

Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) may have important effects on the evolution of resistance. We investigated overwintering costs in pink bollworm, Pectinophora gosypiella (Saunders), strains with different degrees of resistance to Bt cotton. Frequency of resistant individuals in a strain was not associated with induction of diapause or emergence from diapause in early winter. Emergence from diapause in the spring was 71% lower in three highly resistant strains than in two heterogeneous strains from which the resistant strains were derived. This underestimates the overwintering cost because the frequency of the resistance allele was relatively high in the heterogeneous strains. Emergence in the spring in hybrid progeny from crosses between the resistant and heterogeneous strains was greater than in resistant strains but did not differ from susceptible strains, showing that the overwintering cost was recessive to some extent.     

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.     

Effects of Bt cotton and crylac toxin on survival and development of pink bollworm (Lepidoptera: Gelechiidae).

We evaluated the effects of Bacillus thuringiensis (Bt) toxin CrylAc on survival and development of a susceptible strain and laboratory-selected resistant strains of pink bollworm, Pectinophora gossypiella (Saunders). For susceptible and resistant strains tested on artificial diet, increases in CrylAc concentration reduced developmental rate and pupal weight. In greenhouse tests, survival of resistant larvae on transgenic cotton that produces CrylAc (Bt cotton) was 46% relative to their survival on non-Bt cotton. In contrast, Bt cotton killed all susceptible larvae tested. F1 hybrid progeny of resistant and susceptible adults did not survive on Bt cotton, which indicates recessive inheritance of resistance. Compared with resistant or susceptible larvae reared on non-Bt cotton, resistant larvae reared on Bt cotton had lower survival and slower development, and achieved lower pupal weight and fecundity. Recessive resistance to Bt cotton is consistent with one of the basic assumptions of the refuge strategy for delaying resistance to Bt cotton. Whereas slower development of resistant insects on Bt cotton could increase the probability of mating between resistant adults and accelerate resistance, negative effects of Bt cotton on the survival and development of resistant larvae could delay evolution of resistance.     

Effect of resistance to Bacillus thuringiensis cotton on pink bollworm (Lepidoptera: Gelechiidae) response to sex pheromone

Fitness costs associated with resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) could reduce male response to pheromone traps. Such costs would cause underestimation of resistance frequency if monitoring was based on analysis of males caught in pheromone traps. To develop a DNA-based resistance monitoring program for pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), we compared the response to pheromone traps of males with and without cadherin alleles associated with resistance to Bt cotton (Gossypium hirsutum L.). When irradiated males from two hybrid laboratory strains with an intermediate frequency of resistance alleles were released in large field cages, the probability of capture in pheromone traps was not lower for males with resistance alleles than for males without resistance alleles. These results suggest that analysis of trapped males would not underestimate the frequency of resistance. As the time males spent in traps in the field increased from 3 to 15 d, the success of DNA amplification declined from 100 to 30%. Thus, the efficiency of a DNA-based resistance monitoring program would be improved by analyzing males remaining in traps for 3 d or less.     

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.     

Effects of kaolin particle film on oviposition,larval mining,and infestation of cotton by pink bollworm (Lepidoptera: Gelechiidae)

We tested effects of kaolin particle film on oviposition, larval mining, and infestation of cotton by pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), in the laboratory, greenhouse, and field. In laboratory choice tests, females laid seven times more eggs on untreated bolls than on bolls treated with kaolin. When neonates were put on bolls in the laboratory, each boll with a treated and untreated half, larvae and mines were found 24 h later on the untreated half but not on the treated half. In oviposition choice tests with whole plants in the greenhouse, females laid four times more eggs on untreated plants than on treated plants and the number of eggs on bolls was five times higher for untreated plants than for treated plants. Kaolin treatments altered the distribution of eggs among plant parts, with untreated bolls receiving a higher percentage than treated bolls, whereas the opposite occurred for petioles. In field tests, treatment with kaolin alone reduced the proportion of bolls infested with pink bollworm, but a mixture of kaolin and the pyrethroid lambda-cyhalothrin was most effective. The results suggest that kaolin particle film may be useful against pink bollworm, particularly in conjunction with other control tactics.     

The effects of the infestation ratio of spiny bollworm (Earias insulana) and pink bollworm (Pectinophora gossypiella) on cotton yield grown in semi-arid region of Turkey

Abstract:  Field studies were conducted in Harran Plain, Turkey, to investigate the infestation of spiny bollworm (SBW) and pink bollworm (PBW) in cotton blind bolls during the period 1999–2001. Blind bolls left on the cotton plants after the harvest were collected, and the PBW and SBW larvae, which are the causes of infection were counted in the laboratory by splitting the bolls. The statistical methods reveal that the infestation ratio variable plays an important role in reducing cotton yield. The infestation ratio variable was found to be statistically significant in all models used. Different soil types may have little effects on yield. We also estimate elasticity of yield with respect to infestation ratio variable. A 1% increase in infestation ratio would reduce about 2.5–6% of cotton yield. Although the infestation ratio of cotton yield is inelastic, which means that the yield is less responsive to the infestation ratio, the elasticity is however an economically important indication because additional cost spent on pests will increase total cost thereby increasing the level where marginal revenue equals the marginal cost.     

DNA screening reveals pink bollworm resistance to Bt cotton remains rare after a decade of exposure

Transgenic crops producing toxins from the bacterium Bacillus thuringiensis (Bt) kill insect pests and can reduce reliance on insecticide sprays. Although Bt cotton (Gossypium hirsutum L.) and Bt corn (Zea mays L.) covered 26 million ha worldwide in 2005, their success could be cut short by evolution of pest resistance. Monitoring the early phases of pest resistance to Bt crops is crucial, but it has been extremely difficult because bioassays usually cannot detect heterozygotes harboring one allele for resistance. We report here monitoring of resistance to Bt cotton with DNA-based screening, which detects single resistance alleles in heterozygotes. We used polymerase chain reaction primers that specifically amplify three mutant alleles of a cadherin gene linked with resistance to Bt cotton in pink bollworm, Pectinophora gossypiella (Saunders), a major pest. We screened DNA of 5,571 insects derived from 59 cotton fields in Arizona, California, and Texas during 2001-2005. No resistance alleles were detected despite a decade of exposure to Bt cotton. In conjunction with data from bioassays and field efficacy tests, the results reported here contradict predictions of rapid pest resistance to Bt crops.     

DNA-based detection of Bt resistance alleles in pink bollworm

Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins. We previously identified three mutant alleles (r1, r2, r3) of a cadherin gene in pink bollworm (Pectinophora gossypiella) linked with recessive resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton. Here we describe a polymerase chain reaction (PCR)-based method that detects the mutation in genomic DNA of each of the three resistant alleles. Using primers that distinguish between resistant and susceptible (s) alleles, this method enables identification of 10 genotypes (r1r1, r1r2, r1r3, r2r2, r2r3, r3r3, r1s, r2s, r3s, and ss) at the cadherin locus. For each of the three resistant alleles, the method detected the resistance allele in a single heterozygote (r1s, r2s, or r3s) pooled with DNA from the equivalent of 19 susceptible (ss) individuals. The results suggest that the DNA-based detection method described here could greatly increase the efficiency of monitoring for resistance to Cry1Ac compared to bioassays that detect rare individuals with homozygous resistance.     

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.