Richard T. Roush

Mate finding, dispersal, number released, and the success of biological control introductions

Abstract. 1 An analysis of published data and a mathematical model of the population dynamics of introduced parasitoids were used to explore the possibility that biological control introductions fail because an Allee effect drives small, introduced populations extinct. Such an Allee effect would arise because low densities, resulting from dispersal into a new environment, lead to failure to mate, which leads to a male‐biased sex ratio, which, if extreme enough, could cause population extinction. 2 For chalcidoids, ichneumonoids and tachinids, the proportion of parasitoid populations that established when introduced for control of lepidopteran pests increased with the number of parasitoids per release, the total number released, and the number collected when each variable was analysed separately. For chalcidoids alone, establishment increased with the number of releases for this variable analysed separately. However, stepwise logistic regression of establishment on these variables included only the total number released for chalcidoids and the number per release for ichneumonoids and tachinids. This suggests that an Allee effect may limit the establishment of introduced parasitoids more than stochastic environmental variation or lack of genetic variation. 3 A reaction‐diffusion model of parasitoid introductions was developed, which included mate finding, dispersal, reproduction and survival. Sensitivity analysis showed that the critical number of females needed to establish a population decreased hyperbolically as mate detection distance and net reproductive rate were increased, but the critical number increased linearly as mean‐square displacement was increased. The critical number of females did not change when the gross distance traversed per generation was varied. This was because increased area searched by males compensated for increased displacement. Changing from virgin females producing all males (arrhenotoky) to virgin females producing no progeny increased the critical number of females by over 30%. 4 The analysis of past introductions and the sensitivity analysis of the reaction‐diffusion model both suggested a threshold of about 1000 insects per release to ensure establishment of introduced parasitoids. The implications of our results for the design of biological control introductions are discussed. Limitations in retrospective analyses and current knowledge indicate the need for an experimental approach to introductions.

Field tests on managing resistance to Bt-engineered plants.

Several important crops have been engineered to express toxins of Bacillus thuringiensis (Bt) for insect control. In 1999, US farmers planted nearly 8 million hectares (nearly 20 million acres) of transgenic Bt crops approved by the EPA. Bt-transgenic plants can greatly reduce the use of broader spectrum insecticides, but insect resistance may hinder this technology. Present resistance management strategies rely on a “refuge” composed of non-Bt plants to conserve susceptible alleles. We have used Bt-transgenic broccoli plants and the diamondback moth as a model system to examine resistance management strategies. The higher number of larvae on refuge plants in our field tests indicate that a “separate refuge” will be more effective at conserving susceptible larvae than a “mixed refuge” and would thereby reduce the number of homozygous resistant (RR) offspring. Our field tests also examined the strategy of spraying the refuge to prevent economic loss to the crop while maintaining susceptible alleles in the population. Results indicate that great care must be taken to ensure that refuges, particularly those sprayed with efficacious insecticides, produce adequate numbers of susceptible alleles. Each insect/Bt crop system may have unique management requirements because of the biology of the insect, but our studies validate the need for a refuge. As we learn more about how to refine our present resistance management strategies, it is important to also develop the next generation of technology and implementation strategies.

The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants

A Bacillus thuringiensis (B.t.) cryIIIA δ-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicct (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250 000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of δ-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of δ-endotoxin RNA and protein.

Managing pests and their resistance to Bacillus thuringiensis: Can transgenic crops be better than sprays?

Insecticidal toxins from Bacillus thuringiensis (Bt) can now be deployed either in sprays or transgenic plants. Some entomologists and environmentalists have argued that the sprays are preferable to plants because they are less likely to cause resistance. However Bt sprays are not generally competitive with chemical insecticides and seem unlikely to displace them. In contrast, transgenic plants appear to be sufficiently effective to displace chemicals, making such plants attractive from the standpoint of environmental protection. Further, simulation models using data from the diamondback moth and a laboratory experiment using the Indianmeal moth suggest that wider at least some circumstances, transgenic plants bearing only one Bt gene may be more effective than sprays for delaying resistance to Bt Resistance in a laboratory‐selected strain of the Colorado potato beetle is especially interesting because a strain that can survive Bt sprays and develop to maturity cannot develop successfully on transgenic pl...

Resistance Detection and Documentation: The Relative Roles of Pesticidal and Biochemical Assays

The last decade has witnessed significant changes in both the philosophy and methods used for the monitoring of insecticide and acaricide resistance. The traditional emphasis has been on the development of precise but artificial techniques that measure change only in the physiological resistance of a strain under laboratory conditions (Busvine 1957). These techniques commonly use topical application of technical-grade insecticide in a suitable solvent and the calculation of median lethal dose estimates (e.g., LD50 or LC50) on a per-body-weight basis.

Male production induced by antibiotic treatment inEncarsia formosa (Hymenoptera: Aphelinidae), an asexual species

The production of large numbers of males in the thelytokous speciesEncarsia formosa was induced by feeding antibiotics to their mothers. The males induced by antibiotic treatment produce sperm and sometimes mate with females, but insemination does not occur.

Transgenic broccoli expressing aBacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies

We usedAgrobacterium tumefaciens to transform flowering stalk explants of five genotypes of broccoli with a construct containing the neomycin phosphotransferase gene and aBacillus thuringiensis (Bt) gene [CryIA(c) type] optimized for plant expression. Overall transformation efficiency was 6.4%; 181 kanamycin-resistant plants were recovered. Of the 162 kanamycin-resistant plants tested, 112 (69%) caused 100% morality of 1st-instar larvae of aBt-susceptible diamondback moth strain. Southern blots of some resistant transformants confirmed presence of theBt gene. Selected plants that gave 100% mortality of susceptible larvae allowed survival of a strain of diamondback moth that had evolved resistance toBt in the field. F1 hybrids between resistant and susceptible insects did not survive. Analysis of progeny from 26 resistant transgenic lines showed 16 that gave segregation ratios consistent with a single T-DNA integration. Southern analysis was used to verify those plants possessing a single T-DNA integration. Because these transgenic plants kill susceptible larvae and F1 larvae, but serve as a suitable host for resistant ones, they provide an excellent model for tests ofBt resistance management strategies.

Greenhouse Tests on Resistance Management of Bt Transgenic Plants Using Refuge Strategies

Abstract Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops. Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth. In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P. xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants. Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage. Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants. In the second test, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges. P. xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth. Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants. Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays. Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.

Gene mapping and cross-resistance in cyclodiene insecticide-resistant Drosophila melanogaster (Mg.).

Resistance to the cyclodiene insecticide dieldrin maps to a single gene (Rdl) on the left arm of chromosome III in Drosophila melanogaster (Meigen). The gene was further mapped by the use of chromosomal deficiencies to a single letter sub-region, 66F, on the polytene chromosome. The cross-resistance spectrum of a backcrossed strain lacking elevated mixed function oxidase activity, a common resistance mechanism, was examined. Levels of resistance similar to those found in other insects were found to dieldrin, aldrin, endrin, lindane, and picrotoxinin. Strong similarity of this single major gene with that found in other cyclodiene resistant insects is suggested by its cross-resistance spectrum and chromosomal location, via homology with other Diptera. The significance of major genes in insecticide resistance is discussed.

Selection of high-level abamectin resistance from field-collected house flies,Musca domestica

Abamectin is a novel, highly promising insecticide with activity against many pests. To determine if resistance to abamectin could occur, we collected house flies from several New York dairies and selected them in the laboratory. Resistance developed repidly and to a high level (36 or>60,000-fold, depending upon test technique and/or adjuvant) that could not be overcome by the synergists piperonyl butoxide orS,S,S-tributylphosphorotrithioate. There was no increase in (cross)resistance to crotoxyphos, dichlorvos, dimethoate, tetrachlorvinphos, permethrin, dieldrin or lindane following abamectin selection. Our results suggest the potential for abamectin resistance is high, at least in house flies, and that the judicious use of abamectin will be needed to prolong its usefulness as an insecticide.