John A. McKenzie

The genetic, molecular and phenotypic consequences of selection for insecticide resistance

Studies of insecticide resistance allow theories of the adaptive process to be tested where the selective agent, the insecticide, is unambiguously defined. Thus, the consequences of selection of phenotypic variation can be investigated in genetic, biochemical, molecular, population biological and, most recently, developmental contexts. Are the options limited biochemically and molecularly? Is the genetic mechanism monogenic or polygenic, general or population/species specific? Are fitness and developmental patterns associated? These questions of general evolutionary significance can be considered with experimental approaches to determine how insecticide resistance evolves.

The effect of genetic background on the fitness of diazinon resistance genotypes of the Australian sheep blowfly, Lucilia cuprina

SummaryLaboratory and field experimentation has shown that resistant and susceptible diazinon genotypes of flies collected from the field may have similar fitness in an environment free of diazinon. If the genetic background of resistant genotypes from the field is disrupted, the fitness of the resistant genotype declines. These results, in conjunction with previous data, indicate a modification of the genetic background in field populations following the spread of the resistance allele some ten years earlier. It is suggested that this outcome is dependent on the availability of genetic variability, the intensity of selection and the duration of insecticide usage after resistance develops.

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.

Modification of developmental instability and fitness: Malathion-resistance in the Australian sheep blowfly,Lucilia cuprina

The evolution of resistance to malathion byLucilia cuprina initially results in an increase in fluctuating asymmetry. Resistant flies are at a selective disadvantage, relative to susceptibles, in the absence of the insecticide. A fitness/asymmetry modifier of diazinon-resistant phenotypes ameliorates these effects resulting in malathion-resistant phenotypes of relative fitness and asymmetry similar to susceptibles. For the nine genotypic combinations of the modifier and malathion-resistance alleles, developmental time increases linearly with increasing asymmetry. Percentage egg hatch decreases linearly with increasing asymmetry. The initially disruptive effect of the malathion-resistant allele was partially dominant, the effect of the modifier dominant. The results are discussed in terms of developmental perturbation, asymmetry estimation and relative fitness to consider whether it is adequate to use changes in fluctuating asymmetry alone as measures of developmental instability. It is suggested that in some circumstances antisymmetry may indicate developmental instability and that the diazinon/malathion-resistance systems inL. cuprina may allow the relative importance of genetical and/or environmental developmental perturbations to be ascertained.

Mutations in Dα1 or Dβ2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster

Resistance to insecticides by modification of their molecular targets is a serious problem in chemical control of many arthropod pests. Neonicotinoids target the nicotinic acetylcholine receptor (nAChR) of arthropods. The spectrum of possible resistance-conferring mutations of this receptor is poorly understood. Prediction of resistance is complicated by the existence of multiple genes encoding the different subunits of this essential component of neurotransmission. We focused on the cluster of three Drosophila melanogaster nAChR subunit genes at cytological region 96A. EMS mutagenesis and selection for resistance to nitenpyram was performed on hybrids carrying a deficiency for this chromosomal region. Two complementation groups were defined for the four strains isolated. Molecular characterisation of the mutations found lesions in two nAChR subunit genes, Dalpha1 (encoding an alpha-type subunit) and Dbeta2 (beta-type). Mutations conferring resistance in beta-type receptors have not previously been reported, but we found several lesions in the Dbeta2 sequence, including locations distant from the predicted neonicotinoid-binding site. This study illustrates that mutations in a single-receptor subunit can confer nitenpyram resistance. Moreover, some of the mutations may protect the insect against nitenpyram by interfering with subunit assembly or channel activation, rather than affecting binding affinities of neonicotinoids to the channel.

Selection at the diazinon resistance locus in overwintering populations of Lucilia cuprina (the Australian sheep blowfly).

In excess of 70 per cent mortality is observed during the overwintering stage of the life cycle of L. cuprina. The mortality is selective in the absence of a fitness modifier; phenotypes resistant to diazinon overwinter less successfully than susceptibles. In the presence of the modifier the overwintering success of all genotypes is similar. The effect is dominant. Laboratory and field experiments show that selection against resistant individuals increases with time in arrested development. The relevance of these results to the evolution of insecticide resistance is discussed.

Measuring fitness and intergenic interactions: The evolution of resistance to diazinon inLucilia cuprina

Resistance at the diazinon-resistance locus (Rop-1) is recessive with respect to fitness. Selection initially occurs at concentrations lower than those required to controlLucilia cuprina. The presence of theRop-1 allele initially disrupted development so that in the absence of diazinon, carriers were at a relative selective disadvantage. Continued use of the chemical, subsequent to resistance evolving, selected a modifier to ameliorate this effect. Modified resistant phenotypes show similar developmental stability and relative fitness to susceptible individuals. Frequency-dependent interactions are observed between resistant and susceptible phenotypes of theRop-1 locus. The interactions are determined by the concentration of diazinon and range from competitive to facilitative. The results are discussed in the context of the contribution insecticide resistance systems can make to the study of general evolutionary phenomena.

Diazinon resistance in Lucilia cuprina ; mapping of a fitness modifier

Modification of the fitness of diazinon resistance genotypes of the Australian sheep blowfly, Lucilia cuprina, in the absence of the insecticide from SS > RS > RR to SS = RS = RR (McKenzie et al., 1982) has been shown previously to be due to the segregation of a gene(s) on chromosome III (McKenzie and Purvis, 1984). In this study the gene (gene complex) is mapped to the w locus region of that chromosome by comparing changes in frequency of SS individuals in population cages initiated with RS genotypes segregating for field derived regions of chromosome III. Comparison of percentage egg hatch, the percentage of first instar larvae reaching adulthood and the time of development from egg to adult for combinations of modifier and resistance genotypes show that the modifier affects only the latter. Developmental time is decreased for RS and RR genotypes. The effect is dominant. The developmental time of SS genotypes is unaffected by modifier genotype.

Genotype, environment and the asymmetry phenotype. Dieldrin-resistance in Lucilia cuprina (the Australian sheep blowfly)

Dieldrin-resistant (Rdl/Rdl and Rdl/ +) and susceptible (+/+) phenotypes of Lucilia cuprina were scored for departures from bilateral symmetry for bristle characters after development at different temperatures, larval densities or concentrations of dieldrin. The asymmetry phenotype of resistant flies was dominant and independent of developmental temperature and larval density. The asymmetry of susceptibles increased for temperatures and larval densities above and below standard rearing conditions. A positive correlation was observed between asymmetry score and dieldrin concentration for all genotypes. The susceptible phenotype did not attain the asymmetry score of resistants in any environment. Resistant phenotypes showed an antisymmetric pattern in each environment; fluctuating asymmetry was observed for susceptibles. The relevance of the results of genetic and general or specific environmental stresses to estimates of developmental perturbation is discussed.

Chromosomal localisation of fitness modifiers of diazinon resistance genotypes of Lucilia cuprina

SummaryPrevious results demonstrated that modification of the genetic background produced changes in the fitness of genotypes at the diazinon resistance locus in Lucilia cuprina (McKenzie et al., 1982). Fitness sets were estimated from population cage studies following disruption of the field genome by generations of backcrossing of a resistant field strain to males of a susceptible laboratory strain.The present experiments report the results of a similar procedure where females of the laboratory strain have been used as the recurrent backcross parent. In this case recombination between the field and laboratory genomes is precluded during backcrossing. Population cage results were analogous to those of the previous experiments suggesting fitness modifiers were unlinked to the diazinon resistance locus on chromosome IV. The use of chromosome substitution lines provided confirmation. Major fitness modifiers were mapped to chromosome III.The relevance of changes in the fitness sets of insecticide resistance genotypes, because of genetic background modification, to models of the evolution of insecticide resistance is discussed.