This review follows progress in the analysis of cyclodiene insecticide resistance from the initial isolation of the mutant, through cloning of the resistance gene, to an examination of the distribution of resistance alleles in natural populations. Emphasis is given to the use of a resistant Drosophila mutant as an entry point to cloning the associated gamma-aminobutyric acid (GABA) receptor subunit gene, Resistance to dieldrin. Resistance is associated with replacements of a single amino acid (alanine302) in the chloride ion channel pore of the protein. Replacements of alanine302 not only directly affect the drug binding site but also allosterically destabilize the drug preferred conformation of the receptor. Resistance is thus conferred by a unique dual mechanism associated with alanine302, which is the only residue replaced in a wide range of different resistant insects. The underlying mutations appear either to have arisen once, or multiply, depending on the population biology of the pest insect. Although resistance frequencies decline in the absence of selection, resistance alleles can persist at relatively high frequency and may cause problems for compounds to which cross-resistance is observed, such as the novel fipronils.

The eastern tiger swallowtail butterfly Papilio glaucus shows a striking example of Batesian mimicry. In this species, females are either wild type (yellow and black) or melanic (where most of the yellow colour is replaced by black). In order to understand how these different colour patterns are regulated, we examined the temporal order of wing pigment synthesis via precursor incorporation studies, enzyme assays, and in situ hybridisation to mRNA encoding a key enzyme, dopa decarboxylase. We show that dopa decarboxylase provides dopamine to both of the two major colour pigments, papiliochrome (yellow) and melanin (black). Interestingly, however, dopa decarboxylase activity is spatially and temporally regulated, being utilised early in presumptive yellow tissues and later in black. Further, in melanic females, both dopa decarboxylase activity and early papiliochrome synthesis are suppressed in the central forewing and this normally yellow area is later melanised. These results show that the regulation of enzyme synthesis observed in the yellow/black pattern of a single wing, is similar to that involved in melanism. We infer that dopa decarboxylase activity must be regulated in concert with downstream enzymes of either the melanin and/or the papiliochrome specific pathways, forming part of a developmental switch between yellow or black. This modification of multiple enzyme activities in concert is consistent with a model of melanisation involving coordinate regulation of the underlying synthetic pathways by a single Y-linked (female) factor.

Antibodies against the Drosophila gamma-aminobutyric acid (GABA) receptor subunit RDL were used to investigate the significance of inhibitory inputs to the mushroom bodies in the blowfly (Calliphora erythrocephala) brain. The pedunculus and the lobes of the mushroom body, which mainly consist of Kenyon cell fibers, revealed strong immunoreactivity against RDL. Pedunculi, alpha- and beta-lobe show characteristic unstained core structures with concentric labeling along the neuropile axis. The gamma-lobes in contrast exhibit a compartmentalized RDL-immunoreactive pattern. These data suggest an important role of GABAergic inhibition in the pedunculus and the lobes of insect mushroom bodies. It is most likely that the RDL-immunoreactivity in the mushroom bodies is closely related to Kenyon cell fibers suggesting that Kenyon cells are an inhomogeneous class of neurons, only part of which receive inhibitory GABAergic input from extrinsic elements. GABAergic inhibition, therefore, may play a substantial role in the process of learning and memory formation in the insect mushroom bodies. (C) 1997 Wiley-Liss, Inc.

The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction.

Previously we have described the distribution of the Rdl GABA receptor subunit in the Drosophila CNS. Knowing that Rdl can coassemble with LCCH3 (a Drosophila GABA receptor-like subunit showing sequence similarity to vertebrate beta subunit GABAA receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast, Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.

The coffee berry borer, Hypothenemus hampei, is the most important insect pest of coffee worldwide and has an unusual life history that ensures a high degree of inbreeding. Individual females lay a predominantly female brood within individual coffee berries and because males are flightless there is almost entirely full sib mating. We investigated the genetics associated with this interesting life history after the important discovery of resistance to the cyclodiene type insecticide endosulfan. Both the inheritance of the resistance phenotype and the resistance-associated point mutation in the gamma-aminobutyric acid receptor gene Rdl were examined. Consistent with haplodiploidy, males failed to express and transmit paternally derived resistance alleles. Furthermore, while cytological examination revealed that males are diploid, one set of chromosomes was condensed, and probably nonfunctional, in the somatic cells of all males examined. Moreover, although two sets of chromosomes were present in primary spermatocytes, the chromosomes failed to pair before the single meiotic division, and only one set was packaged in sperm. Thus, the coffee berry borer is "functionally" haplodiploid. Its genetics and life history may therefore represent an interesting intermediate step in the evolution of true haplodiploidy. The influence of this breeding system on the spread of insecticide resistance is discussed.

The design of three PCR-based monitoring techniques for the genotyping of cyclodiene-resistant insects are described: (1) PCR followed by a diagnostic restriction enzyme digest or PCR/REN, (2) PCR amplification of specific alleles or PASA and (3) single-stranded conformational polymorphism analysis of PCR-amplified DNA or SSCP. The relative disadvantages and potential applications of each of these techniques are discussed and compared to the use of insecticide bioassays. It is concluded that, although such techniques will probably never replace bioassays for routine monitoring, they can more readily address several fundamental questions relating to the evolution and spread of specific resistance alleles in insect populations.

Cyclodiene insecticide resistance persists in field populations of Drosophila spp. at a frequency of approximately 1% (0.01), despite the withdrawal of most cyclodiene type insecticides except endosulfan. However, we have previously documented that resistance-associated amino acid replacements in the gene Rdl, a gamma-aminobutyric acid receptor, can significantly affect several channel functions of the integral chloride ionophore. We were therefore interested in investigating if different resistance-associated replacements confer significant fitness disadvantages and whether the use of endosulfan could be maintaining selection for cyclodiene resistance in the field. Using PCR amplification of specific alleles (PASA) within 3000 individual flies, we report that neither the alanine302 > serine (allele I) replacement in Drosophila melanogaster Meigen nor the alanine302 > serine (allele I) or alanine302 > glycine (allele 2) replacements in D. simulans Sturtevant showed any reduction in frequency in cage experiments nm for one year in the laboratory in the absence of selection. Further, repeated applications of endosulfan selected significantly for cyclodiene resistance in the field. Thus the apparent absence of fitness cost, combined with the continued use of endosulfan, may maintain cyclodiene resistance at this relatively high frequency in field populations.

The efficiency of monitoring for cyclodiene resistance in Drosophila populations via insecticide bioassay was compared directly with results from PCR-based genotyping of individual insects (PCR/REN). Insecticide bioassays were performed on several hundred insects collected from each of 10 sites and PCR/REN on only 100 insects per site. The mean resistance frequency estimate by bioassay (0.02) was twice as high as that observed by PCR/REN (0.01) and was associated with a greater variance. We were also able to document a high frequency of expected RS survivors in the bioassay that were later genotyped as susceptible (SS). If similar trends hold at higher frequencies of resistance, this could lead to serious overestimation of resistance frequencies. This study highlights two advantages of PCR/REN over insecticide bioassay (i). Estimations of resistance frequency via PCR/REN are more precise that those derived by bioassay, and (ii) resistance frequencies can be determined by PCR/REN with correspondingly smaller sample sizes. The implications of these results for resistance monitoring and for the persistence of resistance genes in untreated populations are discussed.

Resistance to cyclodiene insecticides, documented in at least 277 species, is perhaps the most common kind of resistance to any pesticide. By using cyclodiene resistance to localize the responsible gene, a gamma-aminobutyric acid type A receptor/chloride ion-channel gene was previously cloned and sequenced from an insecticide-susceptible Drosophila melanogaster strain. We now describe the molecular genetics of the resistance allele. A single-base-pair mutation, causing a single-amino acid substitution (Ala-->Ser) within the second membrane-spanning region of the channel, was found to be the only consistent difference between resistant and susceptible strains of D. melanogaster. Some resistant strains of Drosophila simulans show the same mutation, whereas others show an alternative single-base-pair mutation in the same codon, resulting in the substitution of a different amino acid (glycine). These constitute single-box-pair mutations in insects that confer high levels of resistance to insecticides. The presence of the resistance mutations was then tested in a much larger set of strains by the PCR and subsequent digestion with a diagnostic restriction endonuclease. Both resistance-associated mutations cause the loss of a Hae II site. This site was invariably present in 122 susceptible strains but absent in 58 resistant lines of the two species sampled from five continents. PCR/restriction endonuclease treatment was also used to examine linkage of an EcoRI polymorphism in a neighboring intron in D. melanogaster, which was found associated with resistance in all but 3 of 48 strains examined. These PCR-based techniques are widely applicable to examination of the uniqueness of different resistance alleles in widespread populations, the identification of resistance mechanisms in different species, and the determination of resistance frequencies in monitoring.

Susceptibility to cyclodiene insecticides in Drosophila melanogaster (Meigen) has been rescued (restored) by P-element mediated germline transformation of a 40-kilobase cosmid carrying a functional copy of the susceptible gene. The inserted cosmid is carried on chromosome two and the native gene is on chromosome three; thus, up to four alleles can now be crossed into one insect. In this study, we examined the effect of varying the number and ratio of susceptible and resistant alleles of this gene to examine whether resistance was conferred by alteration of the number or affinity of cyclodiene receptors. These experiments show that the heterozygous phenotype is restored in individuals with an even proportion of resistant and susceptible alleles and that susceptibility is independent of allele number. These results are consistent with susceptibility being related to the ratio of sensitive and insensitive receptors, rather than variation in total receptor number. The significance of altered receptor number or affinity in insecticide resistance is discussed.