Insertion of an intact CR1 retrotransposon in a cadherin gene linked with Bt resistance in the pink bollworm, Pectinophora gossypiella.

Three mutations in the Pectinophora gossypiella cadherin gene PgCad1 are linked with resistance to Bacillus thuringiensis (Bt) toxin Cry1Ac. Here we show that the r3 mutation entails recent insertion into PgCad1 of an active chicken repeat (CR1) retrotransposon, designated CR1-1_Pg. Unlike most other CR1 elements, CR1-1_Pg is intact, transcribed by a flanking promoter, contains target site duplications and has a relatively low number of copies. Examination of transcripts from the PgCad1 locus revealed that CR1-1_Pg disrupts both the cadherin protein and a long noncoding RNA of unknown function. Together with previously reported data, these findings show that transposable elements disrupt eight of 12 cadherin alleles linked with resistance to Cry1Ac in three lepidopteran species, indicating that the cadherin locus is a common target for disruption by transposable elements.

piggyBac-like elements in the pink bollworm, Pectinophora gossypiella.

A transgenic line of the pink bollworm, Pectinophora gossypiella, a key lepidopteran cotton pest, was generated previously using the piggyBac transposon IFP2 from Trichoplusia ni. Here we identified an endogenous piggyBac-like element (PLE), designated as PgPLE1, in the pink bollworm. A putatively intact copy of PgPLE1 (PgPLE1.1) presents the canonical features of PLE: inverted terminal repeats with three C/G residues at the extreme ends, inverted subterminal repeats, TTAA target site and an open reading frame encoding transposase with 68% similarity to IFP2. Vectorette PCR revealed large variation in the insertion sites of PgPLE1 amongst worldwide populations, indicating the potential mobility of PgPLE1. The PgPLE1 was undetectable in the genome of Pectinophora endema, implying the recent invasion of PgPLE1 after the divergence of these two closely related species.

Analyzing haplodiploid inheritance of insecticide resistance in whitefly biotypes.

We developed new methods for analyzing inheritance of insecticide resistance in haplodiploid arthropods and applied them to elucidate resistance of the whitefly Bemisia tabaci (Gennadius) to an insect growth regulator, pyriproxyfen. Two invasive biotypes of this devastating crop pest, the B biotype in Arizona and the Q biotype in Israel, have evolved resistance to pyriproxyfen. Here, we incorporated data from laboratory bioassays and crossing procedures exploiting haplodiploidy into statistical and analytical models to estimate the number of loci affecting pyriproxyfen resistance in strains of both biotypes. In tests with models of one to ten loci, the best fit between expected and observed mortality occurred with a two-locus model for the B biotype strain (QC-02) and for one- and two-locus models for the Q biotype strain (Pyri-R). The estimated minimum number of loci affecting resistance was 1.6 for the B biotype strain and 1.0 for the Q biotype strain. The methods used here can be applied to insecticide resistance and other traits in haplodiploid arthropods.

Binding of Bacillus thuringiensis toxin Cry1Ac to multiple sites of cadherin in pink bollworm.

Toxins from Bacillus thuringiensis (Bt) are widely used for pest control. In particular, Bt toxin Cry1Ac produced by transgenic cotton kills some key lepidopteran pests. We found that Cry1Ac binds to recombinant peptides corresponding to extracellular regions of a cadherin protein (BtR) in a major cotton pest, pink bollworm (Pectinophora gossypiella) (PBW). In conjunction with previous results showing that PBW resistance to Cry1Ac is linked with mutations in the BtR gene, the results reported here support the hypothesis that BtR is a receptor for Cry1Ac in PBW. Similar to other lepidopteran cadherins that bind Bt toxins, BtR has at least two Cry1Ac-binding domains in cadherin-repeat regions 10 and 11, which are immediately adjacent to the membrane proximal region. However, unlike cadherins from Manduca sexta and Bombyx mori, toxin binding was not seen in regions more distal from the membrane proximal region. We also found that both the protoxin and activated toxin forms of Cry1Ac bound to recombinant BtR fragments, suggesting that Cry1Ac activation may occur either before or after receptor binding.

Inheritance of resistance to the Cry1Ab Bacillus thuringiensis toxin in Ostrinia nubilalis (Lepidoptera: Crambidae).

Laboratory selection with Cry1Ab, the predominant Bacillus thuringiensis (Bt) toxin in transgenic corn, Zea mays L., produced >1000-fold resistance in two laboratory strains of European corn borer, Ostrinia nubilalis (Hübner). We tested the offspring of various crosses to determine the mode of inheritance of resistance to Cry1Ab. Patterns of inheritance of resistance were similar in the two resistant strains. The progeny of reciprocal F1 crosses (resistant male x susceptible female and vice versa) responded alike in bioassays, indicating autosomal inheritance. The median lethal concentrations (LC50 values) of F1 were intermediate between the resistant and susceptible parents, indicating approximately additive inheritance. However, the dominance of resistance increased as the concentration of Cry1Ab decreased. Analysis of progeny from backcrosses (F1 x susceptible strain) suggests that resistance was controlled by more than one locus. In particular, the fit of observed to expected mortality improved as the number of putative loci increased from 1 to 10. The polygenic nature of resistance in these two laboratory strains suggests that major genes for resistance to Cry1Ab were not common in the founding populations of O. nubilalis. A low initial frequency of major genes for Cry1Ab resistance might be an important factor in delaying evolution of resistance to Bt corn in this pest.

Reversing insect adaptation to transgenic insecticidal plants.

The refuge-high-dose strategy for delaying insect adaptation to transgenic plants produces non-transgenic plants that enable survival of susceptible individuals. Previous theoretical work has suggested three requirements for success of the refuge-high-dose strategy: a low initial frequency of the resistance allele, extensive mating between resistant and susceptible adults and recessive inheritance of resistance. In order to understand an observed decrease in resistance frequency and improve the potential for managing resistance better, we used analytical and simulation models for exploring the conditions that prevent or reverse the evolution of resistance, even when resistance is not rare initially. Assuming random mating and recessive or nearly recessive inheritance of resistance, the factors favouring reversal of resistance are non-recessive costs of resistance, low initial resistance allele frequency, large refuges, incomplete resistance and density-independent population growth in refuges.

Mutations in the Bemisia tabaci para sodium channel gene associated with resistance to a pyrethroid plus organophosphate mixture.

The voltage-gated sodium channel is the primary target site of pyrethroid insecticides. In some insects, super knockdown resistance (super-kdr) to pyrethroids is caused by point mutations in the linker fragment between transmembrane segments 4 and 5 of the para-type sodium channel protein domain II (IIS4-5). Here, we identify two mutations in the IIS4-5 linker of the para-type sodium channel of the whitefly, BEMISIA TABACI: methionine to valine at position 918 (M918V) and leucine to isoleucine at position 925 (L925I). Although each mutation was isolated independently from strains >100-fold resistant to a pyrethroid (fenpropathrin) plus organophosphate (acephate) mixture, only L925I was associated with resistance in strains derived from the field in 2000 and 2001. The L925I mutation occurred in all individuals from nine different field collections that survived exposure to a discriminating concentration of fenpropathrin plus acephate. Linkage analysis of hemizygous male progeny of unmated heterozygous F1 females (L925Ixwild-type) shows that the observed resistance is tightly linked to the voltage-gated sodium channel locus. The results provide a molecular tool for better understanding, monitoring and managing pyrethroid resistance in B. tabaci.

Determining the mode of inheritance of pesticide resistance with backcross experiments.

The most widely used method for evaluating the mode of inheritance of pesticide resistance is based on bioassays of individuals from a backcross between F1 (hybrid of resistant and susceptible strains) and parental resistant or susceptible strains. Monte Carlo simulations of the standard backcross method showed that the probability of incorrectly rejecting the null hypothesis of monogenic inheritance (Type I error) was generally more than double the conventional value of P = 0.05. Conversely, the null hypothesis of monogenic inheritance was likely to be accepted in a relatively large proportion of cases in which resistance is controlled by two or more loci. Expected differences in mortality of backcross offspring between monogenic and additive polygenic models approached zero as dose approached extremely low values, extremely high values, and the LD50 of the backcross generation. Thus, the effectiveness of the backcross method depended strongly on dose. The power of the standard backcross method to correctly reject the null hypothesis of monogenic inheritance increased as number of loci, slope of parental dose-mortality lines, magnitude of resistance, and sample size increased. Guidelines for improving the design and interpretation of backcross experiments are presented.

Implications of gene amplification for evolution and management of insecticide resistance.

Simulations were used to compare evolution of insecticide resistance predicted by a conventional two-allele model with predictions from three- and four-allele models that assume resistance is based on gene amplification. Results were similar between models when insecticide concentration was low or moderate. In contrast, when 10% of the population was not exposed to insecticide each generation, high insecticide concentrations slowed resistance development in the two-allele model, but caused rapid development of high levels of resistance in the three- and four-allele models. The presence of a third allele at an initial frequency as low as 10-7 doubled or tripled the rate of resistance development in some cases. Attempts to slow evolution of resistance by overwhelming it with high concentrations of insecticides are not likely to succeed if gene amplification or other mechanisms generate alleles that confer high levels of resistance.

Managing resistance with multiple pesticide tactics: theory, evidence, and recommendations.

Sequences, mixtures, rotations, and mosaics are potential strategies for using more than one pesticide to manage pest populations and for slowing the evolution of pesticide resistance. Results from theoretical models suggest that, under certain conditions, mixtures might be especially effective for resistance management. The assumptions of such models, however, are probably not widely applicable. Potential disadvantages associated with mixtures that are usually not considered in modeling studies include disruption of biological control, promotion of resistance in secondary pests, and intense selection for cross-resistance. Results from limited experimental work suggest that pesticide combinations do not consistently suppress resistance development. More thorough evaluation of tactics that seek to optimize benefits of more than one insecticide will require rigorous experiments with the particular pest and pesticide combinations. Because of the difficulty in generalizing results across systems and the potential negative effects of multiple insecticide use, emphasis on minimizing insecticide use is recommended.

Evolution of pesticide resistance: interactions between generation time and genetic, ecological, and operational factors.

We used computer simulation modeling to clarify the relationship between generation time and the rate of evolution of pesticide resistance. We examined the influence of generation time under various assumptions about genetics, population dynamics and selection pressures. The simplest model demonstrated that the time required for resistance to evolve can be independent of generation time. However, interactions of generation time with genetic, biological and operational factors resulted in positive, negative, and U-shaped relationships between the number of generations per year and the time required for resistance to evolve. These results preclude any generalizations concerning the influence of generation time on resistance evolution. Some ability to predict the influence of generation time may still exist on a case-by-case basis if the context of the resistance episode can be specified.

Roles of selection intensity, major genes, and minor genes in evolution of insecticide resistance.

A prominent hypothesis about insecticide resistance is that genes of major effect play a key role in field-evolved resistance because the intensity of selection is extremely high in the field. A corollary hypothesis is that the lower intensity of selection in laboratory selection experiments favors polygenic control of insecticide resistance. Contrary to these hypotheses, a literature review revealed that the intensity of selection for insecticide resistance in the field varies widely and overlaps broadly with selection intensities in the laboratory. Also contrary to these hypotheses, results from simulations of population genetic models suggest that selection intensities typical of laboratory selection experiments favor resistance that is conferred by major genes. Major genes dominated responses to selection for resistance across a wide range of simulated selection intensities, with and without fitness costs and refuges. The simulation results also suggest that the intensity of selection, rather than the number of loci conferring resistance, is central in determining rates of resistance evolution and effectiveness of refuges.

Efficacy of a dehydrated steinernematid nematode against black cutworm (Lepidoptera: Noctuidae) and diamondback moth (Lepidoptera: Plutellidae).

We compared the ability of in vitro-produced, commercially formulated with in vivo-produced, nonformulated Steinernema carpocapsae (Weiser) Poinar. All strain to infect and kill larvae of black cutworm, Agrotis ipsilon (Hufnagel), and diamondback moth, Plutella xylostella (L.). In vitro-produced nematodes formulated in wettable dispersible granules, which were stored dry, were rehydrated in water for 0-72 h before application. Against black cutworms, the efficacy of nematodes was (from most to least effective): in vivo > in vitro rehydrated for 72 h > in vitro rehydrated for 48 h > in vitro rehydrated for 24 h > dehydrated (0 h). Nematodes rehydrated for 72 h in water or moist soil were equally effective against black cutworm larvae, and both were significantly more effective than nematodes without rehydration. These results indicated that nematodes in the wettable dispersible granule formulation required time to rehydrate in the soil before infecting black cutworm larvae. Nematode treatments described above were applied to radish plants held at 100 or 75% RH and tested against diamondback moth larvae. At 100% RH, nematode efficacy was (from most to least effective): in vitro rehydrated for 72 h > in vivo > in vitro rehydrated 48 h > in vitro rehydrated 24 h > dehydrated (0 h). The efficacy of all treatments was lower at 75% than at 100% RH, and the ranking of in vivo and in vitro nematodes rehydrated for 72 h was reversed. The nematodes in the wettable dispersible granule formulation were effective for foliar treatments when humidity was high and nematodes were rehydrated for at least 48 h before application. The data show that nematode infectivity was reduced unless nematodes were rehydrated.

Variation in tolerance to Bacillus thuringiensis among and within populations of the spruce budworm (Lepidoptera: Tortricidae) in Ontario.

Variation in tolerance to Bacillus thuringiensis Berliner subsp. kurstaki (strain HD-1-S-1980) among and within populations of the spruce budworm, Choristoneura fumiferana (Clemens), was assessed in the laboratory. Force-feeding assays using offspring of females collected as pupae from nine locations throughout Ontario and from a laboratory colony (DCF) demonstrated limited variation in tolerance among populations. Variation among populations was comparable with the variation observed among repeated assays with different batches of larvae from the DCF colony. Population LC50s were not significantly associated with age of the outbreak, host-plant species, incidence of the microsporidian Nosema fumiferanae (Thomson), or size of the female parent. Upper limits for genetic variation in tolerance were estimated by examining variation among full-sibling families within same populations. Mortality of individual families ranged from 6.5 to 70.9% within five field populations and from 2.7 to 93.3% within two laboratory colonies in response to a dose that caused a mean mortality of 40%. Familial factors accounted for 32.8% of the phenotypic variation in response across field populations, as compared with 3% for population factors. These data suggest that the phenotypic variation in tolerance to B. thuringiensis has a substantial genetic component and may provide a basis for evolution of resistance given sufficient selection pressure.

Binding and toxicity of Bacillus thuringiensis protein Cry1C to susceptible and resistant diamondback moth (Lepidoptera: Plutellidae).

We studied mechanisms of resistance to Bacillus thuringiensis insecticidal crystal protein Cry1C in the diamondback moth, Plutella xylostella (L.). Binding assays with midgut brush border membrane vesicles prepared from whole larvae showed no significant difference between resistant and susceptible strains in binding of radioactively-labeled Cry1C. These results indicate that reduced binding of Cry1C to midgut membrane target sites did not cause resistance to Cry1C. Thus, the mechanism of resistance to Cry1C differs from that observed in several previously reported cases of resistance to Cry1A toxins in diamondback moth. We tested Cry1C toxin and Cry1C crystalline protoxin against resistant and susceptible larvae using leaf disk bioassays. After adjusting for the size difference between Cry1C toxin and protoxin, we found that with resistant larvae, toxin was significantly more toxic than protoxin. In contrast, with susceptible larvae, no significant difference in toxicity occurred between Cry1C toxin and protoxin. The resistance ratios for Cry1C were 19 for toxin and 48 for protoxin. These results suggest that reduced conversion of Cry1C protoxin to toxin is a minor mechanism of resistance to Cry1C. Because neither reduced binding nor reduced conversion of protoxin to toxin appear to be major mechanisms, one or more other mechanisms are important in diamondback moth resistance to Cry1C.

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.

Suppression of diamondback moth (Lepidoptera: Plutellidae) with an entomopathogenic nematode (Rhabditida: Steinernematidae) and Bacillus thuringiensis Berliner.

We tested the efficacy of the All strain of Steinernema carpocapsae (Weiser) against larvae of the diamondback moth, Plutella xylostella (L.). In laboratory bioassays we found that (1) commercially formulated nematodes produced in vitro were as effective as nematodes produced in vivo, (2) resistance of P. xylostella to Bacillus thuringiensis Berliner subsp. kurstaki did not confer cross-resistance to nematodes, (3) mortality caused by nematodes was higher for early than late 3rd-instar P. xylostella larvae, and (4) no interaction occurred when B. thuringiensis and nematodes were combined against a susceptible strain of P. xylostella, but an antagonistic interaction occurred between the 2 pathogens against a strain of P. xylostella resistant to B. thuringiensis. In field trials conducted on 2 watercress [Rorippa Nasturtium-aquaticum (L.) Hayek] farms in Hawaii, nematodes provided 41% control, B. thuringiensis subsp. aizawai gave 44% control, and the combined treatment (B. thuringiensis plus nematodes both at half rate) resulted in 58% control. Using nemodes to control diamondback moth can theoretically reduce resistance development in diamondback moth populations to B. thuringiensis products, but repeated applications of nematodes will probably be ineffective in attaining control (suggested in simulation model). The results of this study demonstrate that nematodes may be a useful component of integrated pest management programs if efficacy can be increased, especially for populations of P. xylostella that are resistant to B. thuringiensis.

Genetics of pink bollworm resistance to Bacillus thuringiensis toxin Cry1Ac.

Laboratory selection increased resistance of pink bollworm (Pectinophora gossypiella) to the Bacillus thuringiensis toxin Cry1Ac. Three selections with Cry1Ac in artificial diet increased resistance from a low level to >100-fold relative to a susceptible strain. We used artificial diet bioassays to test F1 hybrid progeny from reciprocal crosses between resistant and susceptible strains. The similarity between F1 progeny from the two reciprocal crosses indicates autosomal inheritance of resistance. The dominance of resistance to Cry1Ac depended on the concentration. Resistance was codominant at a low concentration of Cry1Ac, partially recessive at an intermediate concentration, and completely recessive at a high concentration. Comparison of the artificial diet results with previously reported results from greenhouse bioassays shows that the high concentration of Cry1Ac in bolls of transgenic cotton is essential for achieving functionally recessive inheritance of resistance.