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.     

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.     

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...     

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.     

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.     

Helicoverpa zea and Bt cotton in the United States

Helicoverpa zea (Boddie), the bollworm or corn earworm, is the most important lepidopteran pest of Bt cotton in the United States. Corn is the preferred host, but the insect feeds on most flowering crops and wild host plants. As a cotton pest, bollworm has been closely linked to the insecticide-resistance prone Heliothis virescens (F.), tobacco budworm. Immature stages of the two species are difficult to separate in field environments. Tobacco budworm is very susceptible to most Bt toxins, and Bt cotton is considered to be "high dose." Bollworm is less susceptible to Bt toxins, and Bt cotton is not "high dose" for this pest. Bt cotton is routinely sprayed with traditional insecticides for bollworm control. Assays of bollworm field populations for susceptibility to Bt toxins expressed in Bt cotton have produced variable results since pre-deployment of Bt cottons in 1988 and 1992. Analyses of assay response trends have been used by others to suggest that field resistance has evolved to Bt toxins in bollworm, but disagreement exists on definitions of field resistance and confidence of variable assay results to project changes in susceptibility of field populations. Given historical variability in bollworm response to Bt toxins, erratic field control requiring supplemental insecticides since early field testing of Bt cottons, and dramatic increases in corn acreage in cotton growing areas of the Southern US, continued vigilance and concern for resistance evolution are warranted.     

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.     

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.     

Increased frequency of pink bollworm resistance to Bt toxin Cry1Ac in China

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The main approach for delaying 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 transgenic cotton producing Bt toxin Cry1Ac, the United States and some other 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. The "natural" refuge strategy focuses on cotton bollworm (Helicoverpa armigera), the primary target of Bt cotton in China that attacks many crops, but it does not apply to another major pest, pink bollworm (Pectinophora gossypiella), which feeds almost entirely on cotton in China. Here we report data showing field-evolved resistance to Cry1Ac by pink bollworm in the Yangtze River Valley of China. Laboratory bioassay data from 51 field-derived strains show that the susceptibility to Cry1Ac was significantly lower during 2008 to 2010 than 2005 to 2007. The percentage of field populations yielding one or more survivors at a diagnostic concentration of Cry1Ac increased from 0% in 2005-2007 to 56% in 2008-2010. However, the median survival at the diagnostic concentration was only 1.6% from 2008 to 2010 and failure of Bt cotton to control pink bollworm has not been reported in China. The early detection of resistance reported here may promote proactive countermeasures, such as a switch to transgenic cotton producing toxins distinct from Cry1A toxins, increased planting of non-Bt cotton, and integration of other management tactics together with Bt cotton.     

转基因抗虫棉种植面积变化对花生田棉铃虫种群影响

【目的】棉铃虫Helicoverpa armigera(Hübner)是花生田主要害虫之一。转基因抗虫棉全面推广后,棉铃虫种群(包括棉田、花生田、玉米田)得到有效控制。近年,随着转基因抗虫棉种植面积逐年下降,花生田棉铃虫种群呈现逐年上升趋势。【方法】本文通过调查山东省花生主产区(非棉区)龙口、招远、莱阳、文登四个县级市花生田棉铃虫成虫、卵量、幼虫量等的发生情况,分析了棉铃虫发生面积及种群数量15年(2000—2014年)的变化规律,并重点分析了主要影响因素。【结果】结果表明:花生棉铃虫发生面积受棉花种植面积影响较大,二者呈显著负相关性;花生田棉铃虫累计诱蛾量总体呈下降趋势,但与棉花种植面积相关性不显著;棉铃虫卵量、虫量与抗虫棉种植面积呈显著负相关性。【结论】所以,转基因抗虫棉种植面积减少是导致花生田棉铃虫种群数量上升的主要因素。另外,近年玉米田棉铃虫发生面积也呈逐年上升趋势,与抗虫棉种植面积呈显著负相关。所以,随转基因抗虫棉种植面积的下降,若不采取有效措施,棉铃虫种群可能会出现大发生趋势。     

Antennal response of cotton bollworm (Heliocoverpa armigera) to volatiles in transgenic Bt cotton

Abstract:  Volatile compositions in transgenic Bt cotton variety 'GK-97' and its parental variety 'Simian No. 3' were analysed, and antennal response of cotton bollworm ( Heliocoverpa armigera ) to the volatiles were investigated. Volatile components in Bt cotton and regular cotton were almost same, except for absence of a sesquiterpene and a minor unknown compound in regular cotton. The results of gas chromatography-electroantennographic detection (GC-EAD) experiments showed that cotton bollworm antennae responded to seven compounds and two minor unknown compounds in Bt volatiles, among which concentrations of α -pinene and β -pinene in Bt cotton were relatively higher than those in regular cotton, and one minor unknown compound was absent in regular cotton.     

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.     

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.     

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.     

Comparing the predatory performance of green lacewing on cotton bollworm on conventional and Bt cotton

We compared the survival of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) eggs and larvae on Bt and conventional cotton, in the presence or absence of the generalist predator, green lacewing larvae, Mallada signatus, (Schneider) (Neuroptera: Chrysopidae). In small arenas, green lacewings consumed a similar number of H. armigera eggs (ave. 15.8 ± 1.3 on conventional, 12.6 ± 1.4 on Bt cotton per predator over 24 h) and larvae (ave. 6.8 ± 0.7 conventional, 6.5 ± 0.8 Bt per predator over 24 h) whether on Bt or conventional cotton leaves. Likewise, similar numbers of eggs were consumed by each lacewing larva searching whole plants of either Bt (ave. 15.5 ± 0.6 of 49 over 24 h) or conventional (ave. 13.6 ± 1.1 of 49 over 24 h). On conventional plants over 72 h, survival of H. armigera larvae was 72.8% and decreased to 37.7% when lacewings were present, giving a net consumption rate of 35.1% (8.6 prey per predator over 72 h). On Bt cotton plants, 13.6% of the H. armigera larvae survived after 72 h and this decreased to 1.7% when lacewings were present. This combination of mortality factors operated synergistically. Helicoverpa armigera larvae moved to fruiting structures on conventional or Bt cotton but failed to survive in the squares (young flower buds) when the impacts of Bt and lacewings were combined. The removal of first to second instar H. armigera larvae from squares of Bt cotton by predators has the potential to reduce immediate pest damage and, perhaps more importantly, remove potentially Bt‐resistant genotypes.     

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.