Molecular Mechanisms of Insecticide Resistance - American Chemical

Chapter 7 Cloning of a Locus Associated with Cyclodiene Resistance in Drosophila A Model System in a Model Insect 1

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Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0505.ch007

R. H. ffrench-Constant and R. T. Roush

1Department of Entomology, 237 Russell Laboratories, 1630 Linden Drive, University of Wisconsin, Madison, WI 53706 Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY 14853 A locus associated with cyclodiene resistance has been cloned from Drosophila. A strain showing high levels of resistance to cyclodienes was isolated from the field and the gene mapped to the polytene subregion 66F on the left arm of chromosome three. The gene was cloned following a cosmid walk across the region and identification of several inversion breakpoints uncovering resistance. A number of cDNAs have been isolated from the locus. Sequencing of one of these showed high homology to vertebrate GABAA subunits. The susceptible phenotype has been rescued following P-element mediated germline transformation of a cosmid containing the cloned susceptible gene. The use of the cloned gene to study gene dosage, protein expression, and identification of the resistance associated mutation is discussed. Functional expression studies are described to determine the precise nature of the receptor. 2

In order to overcome the genetic intractability of most pest insects in which resistance is found, Drosophila has been proposed as a particularly efficient model insect for the study and cloning of insecticide resistance genes (7,2). The aim of this chapter is to illustrate the application of this approach to die cloning of cyclodiene resistance and to show how cloned genes can be used to elucidate the basis of resistance following their isolation from Drosophila. Drosophila as a Model Insect for Cloning Insensitive Target Sites Drosophila, alongside the housefly, has been used extensively to study metabolic resistance based on mixed function oxidases (Scott, Feyereisen, Waters, in this volume) and glutathione-S-transferases (Cochrane, in this volume). Enzymes from these metabolic systems have been purified and the genes coding for them are now becoming accessible to molecular genetics via the screening of expression libraries with antibodies raised against purified proteins. In contrast, the gene products of most target based resistance mechanisms, such as knock down resistance (kdr) to pyrethroids (Osborne, in this volume) or cyclodiene resistance, remain inaccessible or uncertain. The purpose of the present chapter is therefore to illustrate how Drosophila can be used to clone resistance genes with no previous knowledge of their product and to show, through the example of cyclodiene resistance, how this approach could be used to clone other insensitive target sites. 0097-6156/92/0505-0090$06.00/0 © 1992 American Chemical Society

In Molecular Mechanisms of Insecticide Resistance; Mullin, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

7. FFRENCH-CONSTANT & ROUSH

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One strategy for the cloning of genes in Drosophila relies upon the localization of the gene coding for a mutant phenotype on the detailed map of the salivary gland polytene chromosomes. Following such a localization a number of techniques exist to facilitate cloning of the gene of interest, due to the high density of cloned DNA at known positions along these chromosomes. This chapter will illustrate the isolation of a cyclodiene resistant mutant in Drosophila, the mapping of the gene responsible, its cloning based upon this chromosomal location, and its putative identification. Although the discussion will be based around the cloning of cyclodiene resistance, this approach would be applicable for other resistance mechanisms for which homologs could be found in Drosophila. Cyclodiene Resistance and the GABA Receptor. Cyclodienes are thought to act at the picrotoxinin (PTX) receptor within the GAB A A receptor/chloride ionophore complex (3). Consistent with this theory, cyclodiene resistant insects also show resistance to PTX (4). Ligand binding studies in strains of the German cockroach Blatella germanica (L.) have shown that PTX binding sites on the GAB A A receptor of resistant strains possess only one tenth of the affinity for PTX of those of susceptibles, and that resistant strains may also show a reduction in the number of receptors (5). Recent studies with a new ligand, ethynylbicycloorthobenzoate (EBOB), have demonstrated that the binding affinity for this radioligand is reduced fourfold in a cyclodiene resistant strain of houseflies (6). Resistance is thus postulated to be associated with insensitivity of the cyclodiene/PTX binding site on the GAB A A receptor. Our present knowledge of GABA receptor structure and function comes from a number of cDNA's cloned from vertebrates (7) and one from invertebrates (8). These receptors are composed of several different polypeptide types that assemble to form the chloride ionophore. These polypeptide types (a, p, y and 8) show 20-40% amino acid identity with one another. Further, each type is represented by a family of genes (e.g. pi, p2, p3) showing 60-80% identity. The composition of the different subunits forming the ionophore has been shown to affect the pharmacology of expressed vertebrate receptors. However, current understanding is insufficient to predict the location of the cyclodiene/PTX binding site and thus the possible nature of the resistance associated mutation(s). Recently, an invertebrate G A B A A receptor has been isolated from the snail Lymnaea stagnalis (8) by homology with vertebrate receptors. Genomic clones were isolated using a vertebrate GAB A A Pi clone. Following the identification of several exons encoding a polypeptide with strong similarity to vertebrate P subunits, RACE (a PCR variant, rapid amplification of £DNA ends) was used to isolate a cDNA. Insect G A B A A receptors have remained uncloned due to the difficulty of obtaining suitable ligands for protein purification and the failure of vertebrate genes as heterologous probes. Thus, cloning of the cyclodiene resistance gene from Drosophila represented a method of not only understanding the basis of resistance but also of cloning a putative invertebrate GABA receptor. It should be stressed at the outset however that the cloning of the gene described here relies in no way upon previous knowledge or any assumption about the nature of the gene product. Thus, although current evidence suggests that cyclodiene resistance is associated with an insensitive G A B A A receptor, we are still conducting functional expression assays to confirm that cDNAs isolated from the locus, with high amino acid similarity to vertebrate G A B A A receptors, actually form GABA receptors. Throughout this text, the gene product will thus be referred to as a putative G A B A A receptor or susceptible allele.


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Cloning Cyclodiene Resistance Isolation of Cyclodiene Resistant Mutant. High levels of insensitivity (about 4,000 fold) to dieldrin, a cyclodiene, were isolated by screening field-collected Drosophila melanogaster. The strain was made homozygous by 2-4 generations of selection. The single locus (Rdl) conferring resistance was mapped to the left arm of chromosome III. The mutant shows a semi-dominant phenotype following contact exposure to dieldrin, in common with the phenotype displayed in cyclodiene resistant insects and vertebrates. A dose of 30 \ig of dieldrin applied to the inside of a glass vial discriminated between resistant homozygotes Rdfi/Rdfi and heterozygotes Rdl /Rdl (hereafter R/R and R/S respectively), whereas a dose of 0.5 u,g distinguished between R/S and Rdl /Rdl (hereafter S/S) flies (9). R

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Cross-resistance, Homology and Nervous System Insensitivity. Following repeated backcrossing to a susceptible strain, lacking elevated mixed function oxidase activity, the mutants still showed resistance to a range of cyclodienes (dieldrin, aldrin and endrin), lindane and PTX. Similar levels of resistance to these compounds were displayed as those found in other insects (10). Further, Rdl (on the left arm of chromosome three) occupies a chromosomal location homologous to that for dieldrin resistance in Musca domestica (chromosome IV) and Lucilia cuprina (chromosome V)(77). In order to prove that resistance was associated with the nervous system, suction electrode recordings were taken from the peripheral nerves of transected larval central nervous systems (72). Treatment of nerve preparations with GABA reduced the spontaneous firing of peripheral nerves. This inhibition could be effectively reversed by the addition of dieldrin or PTX to susceptible preparations, but neither compound had an effect on resistant individuals. Thus cyclodiene resistance in Drosophila is present at the level of the nervous system and extends to PTX. In summary, dieldrin resistance in Drosophila extends to other cyclodienes and PTX, is present at the level of the nervous system, and appears to be fully representative of cyclodiene resistance in other insects. Cyclodiene resistance is a common kind of pesticide resistance, found in at least 276 species (7 J). Recombinational and Deficiency Mapping. Following recombinational mapping of the gene to approximately map unit 26 cM on the left arm of chromosome III, a number of deficiencies from this region were screened to see if they uncovered resistance. When uncovered by a deficiency, and therefore in the absence of any susceptible gene product (sensitive receptors), the resistant allele was expected to show full levels of insensitivity. Thus, for a deficiency (Df) uncovering resistance, RIDf flies will survive a dose of 30 jig dieldrin in a fashion similar to R/R flies. Only one deficiency within the region Df(3L)29A6 was found to uncover resistance, whereas the overlapping deficiency Df(3L)ACl did not. This localized the gene to the polytene subregion 66F which is the only region of Df(3L)29A6 not common to Df(3L)ACl as indicated in previous cytological mapping (10). Generation and Characterization of New Rearrangements. In order to further localize the gene within the 66F subregion, new rearrangements uncovering the gene were generated by y-irradiation. As flies heterozygous for resistance and any new rearrangement uncovering resistance can survive 30 u.g dieldrin, new rearrangements were screened for by irradiating male SIS flies at 4,000 rads, crossing them to R/R females and screening their progeny at 30 u.g dieldrin.



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All the expected R/S progeny will die at this dose except for any progeny where the S allele is broken or deleted by a rearrangement uncovering resistance. New rearrangements uncovering resistance were generated at a frequency of one per 35,000 progeny. As this number of progeny can be screened overnight this forms a very powerful screen. All rearrangements uncovering the resistance gene were found to be lethal when crossed between themselves. This suggests that the resistance gene product is essential for formation of a viable fly. Chromosomal Walk and Localization of Rearrangement Breakpoints. A chromosomal walk (14) was carried out in a cosmid library in order to clone the 66F subregion and identify any rearrangement breakpoints marking the location of the resistance gene. The walk was initiated from a X genomic clone (XI21) which hybridized in situ to 66F1,2. Six cosmids, containing approximately 200 kb, were isolated from across the 66F sub-region (Figure 1). The last cosmids in the walk extended into 67A1,2 but failed to enter the deficiency Df(3L)ACl. Single breakpoints for five of the new rearrangements generated have been located within the walk by probing Southern blots of genomic DNA from rearrangement strains with fragments from cosmid steps of the walk. One of these is from a new deficiency only visibly deleting only the cytological region 66F5. Four breakpoints are clustered within cosmid 6 (although only the first three identified are shown in Figure 1) and give two new recombinant bands on a Southern, indicating the breakpoints of either an inversion or an insertion. Cytological examination revealed that two of these breakpoints were from inversions, In(3L)Rdl~ll and In(3L)Rdl-20 with one breakpoint in 66F/67A and others outside the region (75). These independent inversion breakpoints in cosmid 6 must therefore mark the location of the resistance gene (Figure 1). 9

Isolation and sequencing of cDNA's. In order to establish what cDNA's were being produced at the locus, a 10 kb EcoRI fragment, spanning two of the three clustered breakpoints, was used to screen an embryonic cDNA library. A cDNA, designated NB14.1, was isolated which spanned the cluster of three rearrangement breakpoints (75). Sequence analysis of this cDNA revealed one long open readingframeof 606 amino acids. The sequence of this open readingframewas used to scan DNA and protein databases, revealing highest homology with several vertebrate G A B A A receptor subunits (75) and glycine receptors (76) but lacks the strychnine binding domain characteristic of the latter. Locus complexity. A number of different cDNA's ranging in size from 1.02.5kb have been identifiedfromthe cyclodiene resistance locus. These cDNA's have been aligned based on their restriction maps and their pattern of hybridization with genomic fragments (data not shown). Due to the similarity of these different cDNAs, probing of Northern blots (unpublished data) with whole cDNAs or fragments thereof reveals a number of small transcripts (2.0-2.5kb) and one larger transcript (9kb). The status of the large transcript is uncertain but it raises the possibility that the smaller cDNAs are not full length. The purpose of the large extent of message that is presumably not coding (open readingframeof NB14.1 only 2.0kb) is unclear, but large transcripts are also found in other Drosophila ion channel genes such as the sodium channel locus para. Many of the cDNAs we have sequenced have contained unspliced introns, thus limiting the number of candidate cDNAs. However, several cDNAs are of equal length to NB14.1 and possess similar yet different restriction maps. Experiments are therefore in progress to test the hypothesis that the locus codes for a number of different receptor subunit cDNAs by alternative splicing. This hypothesis is based on



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Figure 1. Chromosomal walk through the 66F polytene subregion of Drosophila melanogaster. The positions of the cosmids in the walk are shown below an EcoRI restriction map. The solid boxes represent the positions of rearrangements uncovering the resistance gene. The location of the gene is marked by a cluster of three inversion breakpoints in cosmid 6.

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the difference in restriction maps of the cDNA's, presenting the intriguing possibility that a number of different receptor subunit variants are being produced by the same locus.


Use of the Cloned Gene to Investigate Insecticide Resistance Germline transformation to rescue susceptibility. P-element mediated germline transformation was used to test if cosmid 6, the cosmid spanning the three inversion breakpoints, contained a complete functional copy of the susceptible gene. Flies heterozygous for resistance and the deficiency Df(3L)29A6 (i.e. RIDf) display full levels of resistance (15% mortality after 24hrs exposure to 30 \ig dieldrin), whereas all R/S flies die after such exposure. Therefore, RIDf flies with and without an inserted copy of cosmid 6 (on chromosome II) were generated to see if the R/S phenotype could be rescued by the insert The resulting flies carrying the insert were susceptible (93% mortality at 24hrs) and those without were resistant (3% mortality), thus proving that cosmid 6 carries a susceptible copy of the resistance gene. The pattern of mortality of these flies over time when exposed to 30 \ig dieldrin shows that the inserted copy of the gene does not fully restore the RIS phenotype (Figure 2). This may be due to reduced expression of the inserted gene associated with its new position in the genome. Transformation with an overlapping cosmid (5B), displaced only 5kb distally from cosmid 6, failed to rescue susceptibility. As cosmid 5B lacks only 5kb from the proximal end of cosmid 6 and the resistance associated breakpoints occupy the opposite end of cosmid 6 (near its overlap with cosmid 5), it appears that the locus is spread across at least the 40kb of genomic DNA in cosmid 6 and that the proximal end of cosmid 6 may contain elements of the gene promoter. The full extent of the locus will be determined by mapping the 5' and 3' ends of the cDNAs to the genomic map. Effects of gene dosage on resistance. The insertion of a susceptible allele onto chromosome H, independent of the native allele on chromosome in, allows for the generation of flies with varying numbers of S and R alleles and an examination of the effects of gene dosage. The LTso's for flies with varying numbers of R and S alleles exposed to 30 \ig dieldrin are shown in Figure 3. This figure shows that, independent of the number of copies of S and R, whenever the proportions of S and R alleles are equal, flies are effectively susceptible to this dose. It has previously been uncertain whether resistance to cyclodienes is conferred either by a change in receptor sensitivity or in receptor density (5). These results show that altering die number of S and R alleles does not alter susceptibility, as would be expected if resistance was associated with a change in receptor density. However, the results are consistent with the resistant allele coding for an insensitive receptor whose effect can be countered by the addition of sensitive receptors. Identification of resistance associated mutation. The apparent size and complexity of the cyclodiene resistance locus complicates the identification of the resistance associated mutation. We will proceed to establish precisely which cDNA or cDNAs from the susceptible library is/are responsible for rescuing susceptibility by injecting each under control of its own promoter isolated from the genomic DNA of cosmid 6. Sequence differences between candidate cDNA's from resistant and susceptible flies will then be determined.


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MOLECULAR MECHANISMS OF INSECTICIDE RESISTANCE

Chromosome

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Figure 2. Time/mortality responses of SIS, R/S and R/Df sibs with and without an inserted copy of cosmid 6 on chromosome II. RIDf flies carrying an inserted copy of cosmid 6 show partial rescue of the R/S phenotype and have significandy greater and more rapid mortality than their RIDf sibs without the insert.

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Molecular Mechanisms of Insecticide Resistance - American Chemical

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