Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in conventional sprayable formulations and in transgenic crops, and it is the most promising alternative to synthetic insecticides. However, evolution of resistance in insect populations is a serious threat to this technology. So far, only one insect species has evolved significant levels of resistance in the field, but laboratory selection experiments have shown the high potential of other species to evolve resistance against Bt. We have reviewed the current knowledge on the biochemical mechanisms and genetics of resistance to Bt products and insecticidal crystal proteins. The understanding of the biochemical and genetic basis of resistance to Bt can help design appropriate management tactics to delay or reduce the evolution of resistance in insect populations.

Biochemistry and Genetics of Insect Resistance to Bacillus thuringiensis

Transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) were first commercialized in 1996 amid concern from some scientists, regulators and environmentalists that the widespread use of Bt crops would inevitably lead to resistance and the loss of a 'public good,' specifically, the susceptibility of insect pests to Bt proteins. Eight years later, Bt corn and cotton have been grown on a cumulative area >80 million ha worldwide. Despite dire predictions to the contrary, resistance to a Bt crop has yet to be documented, suggesting that resistance management strategies have been effective thus far. However, current strategies to delay resistance remain far from ideal. Eight years without resistance provides a timely opportunity for researchers, regulators and industry to reassess the risk of resistance and the most effective strategies to preserve Bt and other novel insect-resistant crops in development.

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Insect resistance management in GM crops: past, present and future

Evolution of resistance by insect pests threatens the continued effectiveness of Bacillus thuringiensis (Bt) toxins in sprays and transgenic crops. Fitness costs of Bt resistance occur when, in the absence of Bt toxins, fitness is lower for resistant insects than for susceptible insects. Modeling results show that fitness costs can delay resistance by selecting against Bt-resistant genotypes in refuges where insects are not exposed to Bt toxins. In 77 studies including 18 species, fitness costs were detected in 62% of experiments testing for declines in resistance and in 34% of fitness component comparisons. Mean fitness costs were 15.5% for survival, 7.4% for development time, and 2.5% for mass. Although most fitness costs were recessive, nonrecessive costs can select more strongly against resistance. Because fitness costs vary with ecological conditions, refuges designed to increase the dominance or magnitude of fitness costs could be especially useful for delaying pest resistance.

Fitness Costs of Insect Resistance to Bacillus thuringiensis

The Colorado potato beetle, Leptinotarsa decemlineata (Say), is widely regarded as the most important insect defoliator of potatoes. Its current range covers about 16 million km2 in North America, Europe, and Asia and continues to expand. This insect has a complicated and diverse life history, which is well-suited to agricultural environments, and makes it a complex and challenging pest to control. Dispersal, closely connected with diapause, feeding, and reproduction, allow the Colorado potato beetle to employ “bet-hedging” reproductive strategies, distributing its offspring in both space (within and between fields) and time (within and between years). The Colorado potato beetle played a large role in creating the modern pesticide industry, with hundreds of chemicals tested against it. High selection pressure, together with natural propensity to adapt to toxic substances, eventually resulted in a large number of insecticide-resistant Colorado potato beetle populations. Since the middle of the last century, the beetle has developed resistance to 52 different compounds belonging to all major insecticide classes. Resistance levels vary greatly among different populations and between beetle life stages, but in some cases can be very high (up to 2,000-fold). Known mechanisms of Colorado potato beetle resistance to insecticides include enhanced metabolism involving esterases, carboxylesterases and monooxygenases, and target site insensitivity, as well as reduced insecticide penetration and increased excretion. There is also some evidence of behavioral resistance. Resistance mechanisms are sometimes highly diverse even within a relatively narrow geographical area. Resistance is usually inherited as an incompletely dominant or incompletely recessive trait, with one or several genes involved in its determination. Because of pleiotropic effects of resistant alleles, insecticide-resistant beetles often have reduced relative fitness in the absence of insecticides. Rotating different classes of insecticides and reducing insecticidal pressure on pest populations by provision of temporal and spatial refuges from exposure to toxins have been proposed to delay evolution of resistance. However, insecticide resistance in this insect will likely remain a major challenge to the pest control practitioners. Still limited understanding of beetle biology, its flexible life history, and grower reluctance to adopt some of the resistance management techniques create impediments to successful resistance management. Overcoming these obstacles is not an easy task, but it will be crucial for sustainable potato production.

Colorado Potato Beetle Resistance to Insecticides

Insects are exposed to a variety of stress factors in their environment, and, in many cases for insect pests to agriculture, those factors include toxic chemical insecticides. Coping with the toxicity of insecticides can be costly and requires energy and resource allocation for adaptation and survival. Several behavioural, physiological and genetic mechanisms are used by insects to handle toxic insecticides, sometimes leading to resistance by constitutive overexpression of detoxification enzymes or inducing mutations in the target sites. Such actions are costly and may affect reproduction, impair dispersal ability and have several other effects on the insect's fitness. Fitness costs resulting from resistance to insecticides has been reported in many insects from different orders, and several examples are given in this mini-review.

Fitness costs associated with insecticide resistance.

Laboratory experiments were conducted to compare relative fitness of strains of Colorado potato beetle resistant and susceptible to Bacillus thuringiensis subsp. tenebrionis Cry3A toxin. Net replacement rates and intrinsic rates of population increase were calculated for resistant and susceptible populations. During the experiment, susceptible males on average copulated 13.3 6 1.5 times, whereas resistant males copulated only 8.0 6 1.0 times. Susceptible females produced an average of 824.2 6 68.1 eggs and 590.9 6 58.5 larvae, which was significantly .484.6 6 48.0 eggs and 334.9 6 39.7 larvae produced by an average resistant female. As a result, both net replacement rate and intrinsic rate of increase were reduced for the resistant population. Furthermore, twice as many susceptible beetles as resistant beetles survived overwintering diapause. Our results clearly indicate that relative fitness of resistant individuals is reduced in the absence of B. thuringiensis in the environment. Therefore, relaxation of selection pressure through refugia and insecticide rotation will favor decrease in the frequency of resistant alleles in beetle populations.

http://www.potatobeetle.org/Alyokhin/Reprints/relfitn_reprint.pdf

Relative Fitness of Colorado Potato Beetle (Coleoptera: Chrysomelidae) Resistant and Susceptible to the Bacillus thuringiensis Cry3A Toxin

Colorado potato beetle dispersal and reproduction was investigated under field and laboratory conditions. Movement and mating of newly emerged summer-generation Colorado potato beetle adults was monitored in the field using a mark-recapture technique, and beetle mating within experimental plots was recorded. The number of degree-days (DD) required for the beetles to become reproductive was tested in an environmental chamber using 10°C as a developmental threshold. A computer-linked flight mill system was used to quantify the influence of mating on the flight behavior of male and female beetles. Adult dispersal started within the first 24 h after eclosion from the pupae, but a significant proportion of newly emerged beetles stayed close to the place of their larval development until reaching reproductive maturity. The beetles required at least 34 DD before mating produced viable offspring, and females did not start laying eggs until a minimum of 51 DD after eclosion. Mating had a pronounced effect on beetle flight, decreasing flight activity of the females and increasing flight activity of the males. Enhancing gene flow between beetles surviving on transgenic plants and susceptible beetles in refugia should be advantageous in managing beetle resistance to transgenic plants.

http://www.potatobeetle.org/Alyokhin/Reprints/sumgen_reprint.pdf

Reproduction and Dispersal of Summer-Generation Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Laboratory strains of Colorado potato beetle, Leptinotarsa decemlineata (Say), physiologically resistant and susceptible to Bacillus thuringiensis (Berliner) subsp. tenebrionis Cry3A toxin were reared to adults on caged potato plants. Influence of three different diets (transgenic potatoes, regular potatoes, and regular potatoes followed by the transgenic potatoes) on beetle mortality, fecundity, and flight behavior were tested under laboratory conditions. A computer-linked flight mill system was used to quantify beetle flight, and dissections were performed to determine the level of flight muscle development. Susceptible beetles continuously fed on transgenic foliage suffered heavy mortality, did not develop flight muscles, and did not produce any eggs. Resistant beetles continuously fed on transgenic foliage were capable of flight and reproduction; however, it took them longer to initiate flight behavior, and their fecundity was lower than fecundity of other treatments. In both strains, detrimental effects became significantly less severe when the beetles were allowed to feed on regular foliage prior to toxin ingestion. In the resistant strain, ingestion of Cry3A toxin significantly increased flight activity, indicating that physiological resistance was probably reinforced by the behavioral escape from toxic environments. No such response was observed for susceptible beetles. When fed on regular foliage, resistant Colorado potato beetles engaged in significantly fewer flights than susceptible beetles. Behavioral differences between resistant and susceptible beetles observed in the present study are likely to affect gene flow between transgenic crops and adjacent refugia, and should be taken in consideration when designing resistance management plans for transgenic potato crops.

Modifications in dispersal and oviposition of Bt-resistant and Bt-susceptible Colorado potato beetles as a result of exposure to Bacillus thuringiensis subsp. tenebrionis Cry3A toxin

Mating behavior of post-diapause Colorado potato beetles, Leptinotarsa decemlineata (Say), was observed within an overwintering site, a rotated potato field, newly colonized potato plants, and under laboratory conditions. The influence of spring mating on beetle flight in the presence and in the absence of host plants was investigated using a computer-linked flight mill system. Diapause was terminated simultaneously in male and female beetles, and the first matings were observed as early as within the first 24 h after the beetles emerged from the soil (60–90 DD accumulated). The beetles mated within the overwintering site, the potato field, and the fields rotated out of potatoes. Mating status did not affect flight behavior of overwintered beetles; however, unfed beetles displayed higher flight activity than fed beetles. Most flight activity took place soon after flight muscle regeneration, and then declined sharply by the 5th day after flight initiation. Mating in or near overwintering sites soon after diapause termination might be an important factor in providing gene flow between insecticide-resistant and insecticide-susceptible Colorado potato beetle populations, and should be considered in designing resistance management plans.

Reproductive status and flight activity of the overwintered Colorado potato beetle.

Sperm precedence in the Colorado potato beetle, Leptinotarsa decemlineata (Say), was investigated using phosphoglucomutase (Pgm) allozymes as genetic markers. Females were mated to 2 males carrying different Pgm alleles, and the proportion of offspring sired by each male was determined by cellulose acetate gel electrophoresis. Our results indicate that Pgm alleles in the Colorado potato beetle are inherited in the Mendelian fashion, and there is no change in sperm utilization over time. We also were able to confirm that sperm precedence in this insect is incomplete, with approximately equal to 72% of the larvae fathered by males at the 2nd mating. Possible implications of incomplete sperm precedence for insecticide resistance management are discussed.

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Andrei V. Alyokhin

Papers

Relative Fitness of Colorado Potato Beetle (Coleoptera: Chrysomelidae) Resistant and Susceptible to the Bacillus thuringiensis Cry3A Toxin

Reproduction and Dispersal of Summer-Generation Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Modifications in dispersal and oviposition of Bt-resistant and Bt-susceptible Colorado potato beetles as a result of exposure to Bacillus thuringiensis subsp. tenebrionis Cry3A toxin

Reproductive status and flight activity of the overwintered Colorado potato beetle.

Electrophoretic confirmation of sperm mixing in mated Colorado potato beetles (Coleoptera : Chrysomelidae)