spatially-explicit test of the refuge strategy for delaying insecticide resistance

The refuge strategy is used worldwide to delay the evolution of pest resistance to insecticides that are either sprayed or produced by transgenic Bacillus thuringiensis (Bt) crops. This strategy is based on the idea that refuges of host plants where pests are not exposed to an insecticide promote survival of susceptible pests. Despite widespread adoption of this approach, large-scale tests of the refuge strategy have been problematic. Here we tested the refuge strategy with 8 y of data on refuges and resistance to the insecticide pyriproxyfen in 84 populations of the sweetpotato whitefly (Bemisia tabaci) from cotton fields in central Arizona. We found that spatial variation in resistance to pyriproxyfen within each year was not affected by refuges of melons or alfalfa near cotton fields. However, resistance was negatively associated with the area of cotton refuges and positively associated with the area of cotton treated with pyriproxyfen. A statistical model based on the first 4 y of data, incorporating the spatial distribution of cotton treated and not treated with pyriproxyfen, adequately predicted the spatial variation in resistance observed in the last 4 y of the study, confirming that cotton refuges delayed resistance and treated cotton fields accelerated resistance. By providing a systematic assessment of the effectiveness of refuges and the scale of their effects, the spatially explicit approach applied here could be useful for testing and improving the refuge strategy in other crop-pest systems. pesticide resistance | predictive evolutionary models | pest management | resistance management P opulation growth will continue to favor agricultural intensification for decades. Because agricultural intensification is associated with increased pest pressure, pesticides generally help to increase yield (1-3). Although significant progress has been made to reduce reliance on pesticides (4, 5), an increasing number of insects and mites exhibit field-evolved resistance to synthetic pesticides, Bacillus thuringiensis (Bt) sprays, and transgenic Bt crops (6, 7). Negative consequences of resistance include increased pesticide use, disruption of food webs and ecosystem services, increased risk to human health, and loss of profits for farmers and industry (1, 3). One of the main strategies for delaying resistance promotes survival of susceptible pests by providing refuges, which are areas of host plants where pests are not exposed to an insecticide. Theory predicts that refuges will slow the evolution of resistance by reducing the fitness advantage of resistant individuals (7-9). Refuges can also reduce the heritability of resistance when susceptible individuals mate with resistant individuals surviving exposure to an insecticide (7). Empirical support for the refuge strategy was provided by short-term laboratory and greenhouse experiments (10, 11). Although these experiments test the hypothesis that mating between susceptible and resistant individuals delays the evolution of resistance, they do not consider several factors that affect resistance in the field (7-9), and thus only provide partial support for effectiveness of the refuge strategy in the field. Retrospective analyses of variation in resistance evolution in the field also suggest that refuges have been effective, but these previous tests have been based primarily on comparisons among species, or qualitative comparisons within species based on a limited number of widely separated geographic areas (12, 13). In such tests, factors that vary among species or geographic areas can confound the effects of refuges. Accordingly, large-scale field tests of the refuge strategy for a single species within a geographic area where factors affecting resistance are similar are needed to test the refuge strategy more rigorously. Moreover, tests of predictive refuge strategy models are required to determine if the refuge strategy can delay resistance (14). Furthermore, to improve our ability to develop efficient refuge strategies, empirical approaches are necessary to characterize effects of refuges on resistance evolution (7, 15). Here we tested the refuge strategy using 8 y of data on refuges and resistance to the insecticide pyriproxyfen in 84 populations of the sweetpotato whitefly (Bemisia tabaci) sampled in cotton fields of central Arizona. We studied the B biotype of B. tabaci, also known as the Asia Minor-Middle East 1 species, which is a key pest of cotton and other crops in Arizona and worldwide (16). The insect growth regulators pyriproxyfen (a juvenile hormone analog) and buprofezin (a chitin synthesis inhibitor) are selective insecticides that have been used for whitefly control in Arizona cotton (Gossypium spp.) since 1996 (17, 18). A single application of either insecticide on cotton when B. tabaci populations start to increase has substantially reduced sprays of broad-spectrum insecticides, helped to conserve natural enemies, and restored farmers ' profits (18, 19). To deter rapid evolution of resistance, farmers in Arizona generally have not used pyriproxyfen to control B. tabaci on crops other than cotton Although B. tabaci is polyphagous, few whitefly crops other than cotton are available in central Arizona from June to September, when pyriproxyfen is sprayed on cotton. In principle, crops that could act as refuges include spring melons (Citrullus lanatus and Cucumis melo), alfalfa (Medicago sativa) and cotton not treated with pyriproxyfen (referred to hereafter as untreated cotton). B. tabac

Sweetpotato Whitely Control on Cotton by Treating Only the Field Edges

The edges of one of each of five pairs of long staple cotton fields were treated for sweetpotato whiteflies. Treated fields had 61% fewer eggs and 53% fewer nymphs than untreated fields. Adult populations were reduced 64% in the treated fields at the edges. In the center of treated fields adult populations remained low and unchanged but in untreated fields there was a 70% increase. According to minicard tests, cotton from treated fields was not sticky but cotton from untreated fields was sticky. Thus, populations of whiteflies and their damage can be significantly reduced by treating only the periphery of cotton fields at the onset of infestation. The treating of only 12 to 15% of the acreage greatly reduces costs and preserves the untreated center for beneficial insects

Sweetpotato Whitefly Parasites Abundant in Some Cotton Fields During October

Surveys of whitefly parasites in cotton showed few or none were present during July and October in some areas, preliminary observations of sticky traps show that large numbers of parasites were present in some fields during October

Preliminary Investigation of Sweet Potato Whitefly Population Dynamics Across Arizona

The sweet potato whitefly can have an impact on cotton by reducing yields through direct feeding damage, by transmitting the cotton crumple leaf virus during feeding and by the production of large amounts of sticky honeydew that interferes with the harvesting and ginning process. Data on whitefly populations collected weekly from 938 yellow sticky traps near cotton fields have been entered into a geographic information system (GIS) database. In general, whitefly populations were high near cotton fields in the Yuma area before July 6th. They rose rapidly in central Arizona between July 6th and July 20th. During the month of August, counts continued to rise in central Arizona, particularly in western Pinal County. Populations began to fall during October. Whitefly populations in eastern La Paz County were slower to develop than in other areas in western Arizona. Whitefly populations in Graham and Cochise County were not significant throughout the growing season. Cotton crumple leaf virus was observed in parts of central and western Arizona

Susceptibility of Southwestern Pink Bollworm to Bt toxins Cry1Ac and Cry2Ab2 in 2005

Bt cotton is an extremely important tool for integrated pest management in the Southwest. It has been a major factor in the current historic low levels of conventional insecticide use in cotton of this region. This is due to Bt cotton’s unprecedented efficacy against the pink bollworm, Pectinophora gossypiella, and its selectivity in favor of key natural enemies of arthropod pests. Due to the pivotal importance of Bt cotton and widespread concerns about the development of pest resistance to transgenic crops, a multi-agency resistance management program was established to monitor and pro-actively manage resistance development in the pink bollworm. This report constitutes results from the ninth year of this monitoring program. Larvae were obtained from bolls collected in cotton fields located throughout the Southwest, cultured in the laboratory, and offspring tested using diet-incorporation bioassays that discriminate between susceptible and resistant pink bollworm. A total of 11 Arizona and four California collections were successfully reared and tested for susceptibility to Cry1Ac using a discriminating concentration of 10 μg Cry1Ac/ml of diet. Susceptibility to Cry2Ab2 was estimated similarly for 12 strains from Arizona and four from California using diagnostic concentrations of 1.0 and 10 μg Cry2Ab2/ml of diet. Success of pink bollworm eradication in suppressing pink bollworm populations in New Mexico and Texas precluded successful collection of samples in those states. No survivors of 10 μg Cry1Ac/ml were detected in any bioassays of 2005 strains (n=5358). The grand mean frequency of PBW survival of 10 μg Cry1Ac/ml in 2005 was 0.000%. A susceptible culture, APHIS-S, used each year as an internal control, yielded 99.3% corrected mortality in tests of 10μg/ml Cry1Ac (n=490). All twelve pink bollworm strains collected in 2005 were highly susceptible to Cry2Ab2, based on contrasts with baseline data collected from 2001-2003. There were no survivors of bioassays of either 1.0 μg Cry2Ab2/ml (n=1,000) or 10 μg Cry2Ab2/ml (n=3425). The susceptible APHIS-S culture had 82.5% corrected mortality in tests of 10 μg/ml Cry2Ab2 (n=200) and 100% mortality in tests of 10 μg/ml Cry2Ab2 (n=120). Field evaluations of efficacy of Bt cotton were conducted by the Arizona Cotton Research and Protection Council in adjacent pairs of Bt and non-Bt fields at 44 Arizona locations. Statewide, large pink bollworm larvae were found in an average of 15% of non-Bt bolls sampled from borders of refuge fields. This was on the low end of the range of infestation levels observed in refuges during the past decade. Bolls from adjacent Bt cotton (Bollgard™) fields yielded an average of 0.28% infested bolls. This value was down slightly from the previous year. Over 70% of the pink bollworm recovered from collections in Bt fields were from bolls that did not express Bt toxin. We conclude that there was no indication of problems with pink bollworm resistance to Cry1Ac or Cry2Ab2 at the locations sampled in 2005. Moreover, Bt cotton continued to exhibit exceptional field performance in Arizona

Susceptibility of Arizona Pink Bollworm to Cry1Ac

Genetically modified cotton expressing the Cry1Ac toxin has been used in Arizona since 1996 with exceptionally positive results in terms of economic returns to growers and reductions in insecticide use in cotton. Since 1995, average insecticide use in Arizona cotton has declined from greater than six applications per acre to less than two in 2000. Bt cotton has contributed greatly to these savings to growers, as have insect growth regulators used for whitefly control. Collections of pink bollworm, Pectinophora gossypiella, made in 1997 and subsequently exposed to Cry1Ac in the laboratory from 1998 to 2000, yielded a laboratory strain with susceptibility to Cry1Ac reduced 1,000 to 3,000- fold, relative to highly susceptible field populations. Unparalleled measures have been taken to detect and manage this resistance. In this report we summarize results of statewide monitoring of pink bollworm susceptibility to Cry1Ac conducted from 1997 to 2000, and results of field evaluations of the effectiveness of Bt cotton from 1995 to 2001. Susceptibility of Arizona pink bollworm to Cry1Ac, increased from 1997 to 2000. Mean corrected mortality in 1μg/ml Cry1Ac assays was 57.4% in 1997, 90.6% in 1998, 97.9% in 1999 and 97.4% in 2000. Mean corrected mortality in bioassays of 10 μg/ml also increased: it was 94.1% in 1997, 99.9% in 1998, 100% in 1999 and 100 % in 2000. Field performance of Bt cotton in 2000 continued to be excellent at 39 locations throughout Arizona cotton at which paired Bt and non-Bt fields were evaluated. Whereas non-Bt cotton fields had mean infestations of over 15% infested bolls, Bt cotton fields averaged less than 0.15% infested bolls. Thus, after six years of intensive use of Bt cotton in Arizona, pink bollworm populations show no signs of being resistant to Bollgard cotton. Indeed, for reasons that are not understood at this time, they have been found to be significantly more susceptible to the Bt toxin in Bollgard cotton at the end of the 2000 season than they were in 1997

Susceptibility of Arizona Pink Bollworm to Cry1Ac Following Six Years of Intensive Use of Transgenic Bt Cotton in Arizona

We summarize information on the performance of Bt cotton against pink bollworm (PBW), Pectinophora gossypiella, in Arizona following six years of use of this new technology. Monitoring of PBW susceptibility to Bt toxin Cry1Ac was conducted annually since 1997 by collecting insects from ten to 17 cotton fields, culturing strains in the laboratory, and measuring susceptibility to Cry1Ac in diet-incorporation bioassays. Based on survival in discriminating concentration bioassays of 10 μg Cry1Ac/ml of insect diet, resistant PBW were detected in low frequencies at 10 out of 17 Arizona locations sampled in 2001 and ranged from 0.0 to 4.0%. Though significantly more abundant than in the previous three seasons, resistant PBW were statistically less abundant in 2001 than they were in 1997. One collection from Paloma, AZ, had 4.0% survivors (uncorrected, actual survival) in bioassays of 10 μg/ml and samples from Coolidge, Maricopa, and Parker Arizona yielded • 1.0% survivors of this concentration. Susceptibility of a limited numbers of 2001 collections of PBW from California, New Mexico and Texas is also reported. Bioassays of 2002 collections are underway at the time of this writing. In a parallel effort, field efficacy of Bt cotton against PBW was documented at five to 39 Arizona locations per year since 1995 by collecting cotton bolls at seasons’ end and counting PBW and exit holes. In 39 pairs of adjacent Bt and non-Bt fields evaluated in 2001 by the Arizona Cotton Research and Protection Council, mean end-of-season pink bollworm infestation levels were > 15% for non-Bt fields and were < 0.15% in adjacent Bt fields. Thus, field observations indicated that performance of Bt cotton continued to be excellent throughout Arizona in the 2002 season

Update on Pink Bollworm Resistance to Bt Cotton in the Southwest

Monitoring of Arizona pink bollworm (PBW), Pectinophora gossypiella, susceptibility to the Bt toxin Cry1Ac has been conducted annually since 1997. PBW were collected from cotton fields located throughout the Southwest in 2002, cultured in the laboratory, and tested for susceptibility to Cry1Ac using diet-incorporation bioassays. A total of 13 Arizona collections were successfully reared and bioassayed. Collections from California (6), New Mexico (1), and Texas (1) were also tested. Laboratory selection of pink bollworms collected from Arizona in 1997 and exposed to Cry1Ac in diet produced a strain capable of surviving on Bollgard® cotton. Subsequent studies showed that 10 g Cry1Ac/ml of insect diet was a reliable diagnostic concentration for detection of pink bollworm that were homozygous for resistance to Cry1Ac. On this basis, resistant PBW were detected in 2002 in only 2 out of 13 Arizona strains. The overall frequency of resistant PBW in 2002 for Arizona was 0.17% and ranged from 0.0 to 1.7%. One of six California collections evaluated had a single resistant survivor. No resistant pink bollworms were detected in the single New Mexico and Texas collections evaluated. Resistant PBW were significantly more abundant in Arizona in 2001 and 2002 than they were in 1998, 1999 or 2000. However, the frequency of resistant survivors in bioassays was low for 2001 and 2002, and markedly lower than in 1997. The Arizona Cotton Research and Protection Council evaluated the efficacy of Bt cotton in 2002 using adjacent pairs of Bt and non-Bt fields at 43 locations across Arizona. Pink bollworms were found in an average of 23.3% of these non-Bt boll fields. Bolls from Bt cotton fields yielded an average of 0.144% (range 0 to 1.300%) infested bolls. Of these, all but three of the pink bollworm recovered from Bt cotton plantings came from bolls that tested negative for Cry1Ac. We conclude from these findings that there is no indication that pink bollworm resistance to Cry1Ac was a problem at the locations sampled in 2002. Bt cotton continued to exhibit exceptional field performance in Arizona