A Single P450 Allele Associated with Insecticide Resistance in Drosophila

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Science  27 Sep 2002:
Vol. 297, Issue 5590, pp. 2253-2256
DOI: 10.1126/science.1074170


Insecticide resistance is one of the most widespread genetic changes caused by human activity, but we still understand little about the origins and spread of resistant alleles in global populations of insects. Here, via microarray analysis of all P450s in Drosophila melanogaster, we show that DDT-R, a gene conferring resistance to DDT, is associated with overtranscription of a single cytochrome P450 gene, Cyp6g1. Transgenic analysis ofCyp6g1 shows that overtranscription of this gene alone is both necessary and sufficient for resistance. Resistance and up-regulation in Drosophila populations are associated with a single Cyp6g1 allele that has spread globally. This allele is characterized by the insertion of an Accord transposable element into the 5′ end of the Cyp6g1 gene.

Insecticide resistance represents an important example of natural selection. Resistance can be mediated either by changes in the sensitivity of insecticide targets in the nervous system or by metabolism of insecticides before they reach these targets (1). Insecticide resistance associated with target site insensitivity is well documented within thepara-encoded voltage-gated sodium channel, theRdl-encoded ligand-gated chloride channel, and theAce-encoded acetylcholinesterase (1). However, the up-regulation of metabolic enzymes associated with resistance, such as the cytochrome P450s and glutathione S-transferases, remains less well understood (2–7). We are using Drosophila melanogaster as a model insect in which to dissect the genetic basis of metabolic insecticide resistance (8, 9), particularly at the DDT-Rlocus. This locus not only represents insect resistance to DDT (10), a compound largely withdrawn but still used in the control of disease vectors (11), but also confers cross-resistance to a wide range of other existing and novel insecticides (12–15). DDT-R is a dominant gene that maps to the right arm of chromosome II at 64.5 cM (16, 17). Recently, we have shown thatDDT-R is associated with overtranscription of the P450 geneCyp6g1 in three D. melanogaster strains (18). In this study, we were interested in answering three questions. First, is Cyp6g1 overtranscribed in all P450-mediated DDT-resistant D. melanogaster strains? Second, if so, is resistance globally associated with a single resistance allele? Third, is overtranscription of Cyp6g1 alone both necessary and sufficient for resistance?

The genes for the cytochrome P450s are a large family involved in a wide variety of metabolic functions. In insects, these enzymes play roles in key processes ranging from host plant utilization to xenobiotic resistance (3). Within the complete genome sequence of D. melanogaster, some 90 individual P450 genes have been identified (19). To determine the breadth of the correlation between Cyp6g1 overtranscription and DDT resistance in D. melanogaster, we challenged a microarray carrying polymerase chain reaction (PCR) products from all identified P450 open reading frames in the genome. Array analysis of Hikone-R (20), a resistant strain established from field collections in the early 1960s (16, 21), shows that onlyCyp6g1 is overtranscribed relative to Canton-S, a susceptible reference strain (Fig. 1A). Similar array analysis of a second DDT-resistant strain, WC2, which was recently collected in the field (U.S.A.), revealed the same result, with only Cyp6g1 being overtranscribed relative to P450 gene expression in the susceptible standard (Fig. 1B). To measure the level of overtranscription associated with resistance, we performed quantitative reverse transcriptase (RT)–PCR on mRNA from a range of resistant and susceptible strains (22), relative to the standard RP49 (23). This analysis confirms the relative overtranscription of Cyp6g1 in a range of strains and shows 10 to 100 times as much mRNA in resistant strains as in a range of susceptible strains (Fig. 1C). To examine a range of insecticides to which DDT-R confers cross-resistance, we mapped resistance to a chlorinated hydrocarbon (DDT), two neonicotinoid nicotinic acetylcholine receptor agonists (imidacloprid and nitenpyram), and a novel insect growth regulator (lufenuron). We measured recombination rates against visible genetic markers,P-element insertions of known location, and restriction fragment length polymorphisms (RFLPs). Resistance to all three different insecticides maps within the same genetic region encompassing the Cyp6g1 locus (Fig. 2A), which suggests that a single P450 may be capable of metabolizing a wide range of insecticide structures.

Figure 1

A single P450 gene, Cyp6g1, is overtranscribed in insecticide-resistant Drosophila. (A and B) Microarray analysis of D. melanogaster cytochrome P450 genes in two different DDT-resistant strains, Hikone-R (A) and WC2 (B). The relative hybridization ratio of resistant (Hikone-R or WC2) versus susceptible (Canton-S) mRNA to an array containing PCR-derived products of P450 gene open reading frames is shown against absolute intensity (relative transcript abundance) for each gene. Note that Cyp6g1 is overtranscribed in both resistant strains. (C) Measurement of Cyp6g1transcription, relative to the standard RP49.

Figure 2

Resistance to DDT and several novel insecticides maps to the region encompassing the Cyp6g1locus. (A) Recombinational mapping against both visible mutants (cn, cinnabar eyes, and vg,vestigial wings), P-element insertions of known genomic location, and RFLPs show that resistance to DDT and the novel insecticides imidacloprid (IMI), nitenpyram (NIT), and lufenuron (LUF) maps to a region encompassing Cyp6g1. Map estimates for each compound are given (solid bars represent confidence intervals) alongside the predicted cytological regions encompassed (in parentheses). (B) Map of the genomic structure of theCyp6g1 locus showing the intron-exon organization of the gene and the location of the Accord element insertion in the resistant (Hikone-R) allele (GenBank AY131284). Note the location of the PCR primers used in the diagnostic for the presence of theAccord element (22).

To examine the population genetics of DDT-R in global populations of D. melanogaster, we completely sequenced two different resistant alleles, Hikone-R and WC2. These alleles have an identical nucleotide sequence, and both carry an insertion in the 5′ end of Cyp6g1 that shows homology to the terminal direct repeat of an Accord transposable element (Fig. 2B). To further examine the similarity of DDT-R resistance alleles, we screened with DDT field-collected strains available fromDrosophila stock centers. We did not find DDT resistance in strains established in the laboratory in the 1930s (Oregon-R-C, Swedish-C, and Canton-S) and collected before DDT usage, but did find resistance in 28 of 75 strains established from different locations across the globe in the 1960s and later (table S1). Although this high frequency (37%) of resistant strains does not strictly represent the past or current frequency of resistance in global populations, as resistance alleles may have been lost by chance in subculturing, it does support two hypotheses. First, resistance to DDT was widespread, as expected, and second, resistance can persist in laboratory strains in the absence of pesticide selection, which suggests that little or no fitness cost is associated with this mechanism. To examine the apparent correlation between the presence of the transposon and resistance, we surveyed resistant and susceptible strains for the presence or absence of the Accord element. We also sequenced the first intron of the Cyp6g1 gene in the same resistant and susceptible strains to determine the relatedness of the DDT-R alleles. A diagnostic, using PCR to detect the presence of the transposon and based on the length of the product generated, showed the insertion to be present in all 20 resistant alleles examined (Fig. 3A). The similarity of all the resistant alleles is also supported by a phylogeny of DNA sequences (fig. S1) derived from the first intron of the Cyp6g1 gene. This shows that the susceptible alleles belong to several different clades of diverse geographic origin. In contrast, the resistant alleles all belong to a single well-supported clade (Fig. 3B), also containing a single susceptible genotype. This genotype, which does not carry theAccord transposable element, is therefore the putative susceptible progenitor into which the transposable element inserted, thus forming the resistant allele. This allele has spread globally, under the combined influences of insecticide selection and migration (24). The observation that the nucleotide sequence around the first intron in Cyp6g1 (291 bp away from the site of the insertion) is identical in all the resistant alleles supports the concept of this global spread and suggests strong linkage disequilibrium or “hitchhiking” of nucleotide variation with the spread of DDT resistance.

Figure 3

All resistant Cyp6g1 alleles carry the Accord insertion and show identical nucleotide sequence within the first intron, supporting a single global origin ofDDT-R. (A) Survey of a range of susceptible (S1-20) and resistant (R1-20) fly strains (see table S1 for strain origins) with a PCR-based diagnostic to detect the presence or absence of the Accord element in the 5′ end of the Cyp6g1 gene. Note the perfect correlation between the presence of the element (larger, Accord associated 243-bp PCR product) and resistance. The relative positions of PCR primers used in the diagnostic are described in Fig. 2B. (B) Phylogeny of a global collection of susceptible (S1-20) and resistant (R1-20) DDT-R alleles. Note that the susceptible alleles form seven groups or “clades” and that the eighth clade contains all the resistant alleles and a single susceptible allele found in two different susceptible strains.

Finally, to verify that overtranscription of Cyp6g1alone is responsible for DDT resistance, we produced resistance in transgenic flies carrying an inserted copy of Cyp6g1 driven by the GAL4/UAS system (25). After germline transformation mediated by P-element transposition of constructs containing UAS-Cyp6g1, we were able to overtranscribe the inserted copy of the gene using heat shock–based GAL4 drivers. We again quantified the level of overtranscription from the Cyp6g1 transgene via quantitative RT-PCR relative toRP49 transcription (Fig. 4A). These results show that transgenic overtranscription leads to an overabundance of Cyp6g1 transcript of about 100 times that in susceptible nontransgenic fly strains. Heat-shocked flies were also resistant to a discriminating dose of 10 μg of DDT per vial, whereas controls lacking the GAL4 drivers, and thus lacking overtranscription of the inserted copy of the gene, were susceptible (Fig. 4B). These transgenic experiments demonstrate that overtranscription ofCyp6g1 alone is both necessary and sufficient for P450-mediated DDT resistance.

Figure 4

Heat shock–mediated overtranscription ofCyp6g1 is both necessary and sufficient for DDT resistance. (A) An inserted transgenic copy of Cyp6g1, under the control of GAL4/UAS with a heat-shock driver, can be overtranscribed after heat shock. Ten 20-min heat shock (37°C) treatments (with a 20-min 25°C recovery period between heat shocks) of two different transgenic fly lines (GAL4/UAS-Cyp6g1X andGAL4/UAS-Cyp6g12 , on the X and 2nd chromosome, respectively) up-regulated the Cyp6g1 transcript about 100 times in both strains. (B) Heat shock–induced overtranscription of Cyp6g1 allows the transgenic strains to survive a discriminating dose of 10 μg of DDT per vial. Adult flies were exposed to DDT applied to the inside of 20-ml glass scintillation vials as described previously (18).

Although several studies have implicated the overexpression or alteration of individual P450s in insecticide resistance (3,5, 7), our results raise several important new conclusions for the molecular basis and origins of metabolic resistance. First, given the number of P450 genes present inD. melanogaster (19) and the potential complexity of their interactions (3), it was not expected that a single allele of a single gene could be associated with such widespread insecticide resistance. Second, it was also not expected that resistance alleles mapping to the same region as DDT-Rshould show cross-resistance to such a wide range of compounds, including organochlorine, organophosphorus, carbamate, neonicotinoid, and insect growth regulator insecticides. Such broad cross-resistance would make a pest insect difficult to control with available insecticides and, although we have not formally demonstrated that this cross-resistance is strictly correlated with Cyp6g1overtranscription, this is now our working hypothesis. In this respect, we note that the insect CYP6 P450 family is phylogenetically related to the mammalian CYP3 family of which CYP3A4, expressed in the human liver, is also noted for its broad substrate specificity (26). Third, the possibility that insecticide resistance might involve transposable elements has been raised previously (27). However, a previous putative association of a transposable element with a P450 gene thought to be associated with resistance (28) was subsequently disproved (29). In the current study, although linkage between resistance and the Accord element is complete, the causal relation between the element and overtranscription remains to be proven. Fourth, and finally, earlier work on amplified esterase genes in mosquitoes suggested that a single global spread of one specific amplicon accounted for insecticide resistance in global populations ofCulex pipiens mosquitoes (30). Further analysis of mosquito populations, however, showed that numerous different mutational events and their resulting amplicons make up the extant global population of resistance alleles in mosquitoes (24,31). Our description of an identical resistant allele in 20 DDT-resistant strains of D. melanogaster of diverse geographic origin represents the global spread of a single insecticide-resistance allele.

Supporting Online Material

Materials and Methods

Fig. S1

Table S1

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