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While insecticide resistance presents a challenge for those intent on controlling insect populations, these challenges have also generated a set of tools that can be used to ask fundamental biological questions about that resistance. Numerous species of insects have evolved resistance to multiple classes of insecticides. Each one of these species and their respective resistant populations represent a potential tool for understanding the molecular basis of the evolution of resistance. However, in-laboratory maintenance of resistant insect populations (and their comparative susceptible populations) suitable for asking the needed set of questions around the molecular consequences of long-term pesticide exposure requires a significant, in places prohibitive, level of resources. Drosophila melanogaster (hereafter referred to as Drosophila) is a model insect system with populations easily selected with pesticides and readily maintainable over decades. Even within Drosophila, however, few populations exist where long-term pesticide selection has occurred along with contrasting non-selected population. As such, the Drosophila 91-C and 91-R populations, which exhibit insecticide resistance to DDT (91-R), compared to a non-selection population (91-C), represent a unique resource for the study of high level DDT resistance. Moreover, with the availability of “omics” technologies over the past several decades, this paired population has emerged as a useful tool for understanding both the molecular basis of pesticide resistance and the molecular consequences of long-term pesticide exposure. In this review, we summarize the studies with these aforementioned populations over the past several decades, addressing what has been learned from these efforts.

The role of miRNAs in mediating insecticide resistance remains largely unknown, even for the model species Drosophila melanogaster. Building on prior research, this study used microinjection of synthetic miR-310s mimics into DDT-resistant 91-R flies and observed both a significant transcriptional repression of computationally-predicted endogenous target P450 detoxification genes, Cyp6g1 and Cyp6g2, and also a concomitant increase in DDT susceptibility. Additionally, co-transfection of D. melanogaster S2 cells with dual luciferase reporter constructs validated predictions that miR-310s bind to target binding sites in the 3ʹ untranslated regions (3ʹ-UTR) of both Cyp6g1 and Cyp6g2 in vitro. Findings in the current study provide empirical evidence for a link between reduced miRNA expression and an insecticidal resistance phenotype through reduced targeted post-transcriptional suppression of transcripts encoding proteins involved in xenobiotic detoxification. These insights are important for understanding the breadth of adaptive molecular changes that have contributed to the evolution of DDT resistance in D. melanogaster.

Herbivorous insects encounter a variety of toxic environmental substances ranging from ingested plant defensive compounds to human-introduced insecticidal agents. Dietary antioxidants are known to reduce the negative physiological impacts of toxins in mammalian systems through amelioration of reactive oxygen-related cellular damage. The analogous impacts to insects caused by multigenerational exposure to pesticides and the effects on adaptive responses within insect populations, however, are currently unknown. To address these research gaps, we used Drosophila as a model system to explore adaptive phenotypic responses to acute dichlorodiphenyltrichloroethane (DDT) exposure in the presence of the dietary antioxidant vitamin C and to examine the structural genomic consequences of this exposure. DDT resistance increased significantly among four replicates exposed to a low concentration of DDT for 10 generations. In contrast, dietary intake of vitamin C significantly reduced DDT resistance after mutigenerational exposure to the same concentration of DDT. As to the genomic consequences, no significant differences were predicted in overall nucleotide substitution rates across the genome between any of the treatments. Despite this, replicates exposed to a low concentration of DDT without vitamin C showed the highest number of synonymous and non-synonymous variants (3196 in total), followed by the DDT plus vitamin C (1174 in total), and vitamin C alone (728 in total) treatments. This study demonstrates the potential role of diet (specifically, antioxidant intake) on adaptive genome responses, and thus on the evolution of pesticide resistance within insect populations.

The common bed bug, Cimex lectularius, possesses a cholinesterase expressed exclusively in the salivary gland (ClSChE). In this study, we investigated the molecular forms, tissue distribution patterns and biochemical properties of ClSChE and showed that ClSChE exists as a soluble monomeric form or a soluble dimeric form connected by a disulfide bridge. Immunohistochemical analysis confirmed that ClSChE was expressed in the epithelial cells of both the salivary gland and the duct. In addition, the secretion of monomeric ClSChE through the proboscis during feeding was confirmed by western blotting using a ClSChE-specific antibody. To predict the role of ClSChE injected into the tissue of an animal host, we analyzed the extent of hydrolysis of acetylcholine (ACh) by ClSChE by ultra-performance liquid chromatography-tandem mass spectrometry. ClSChE binding to ACh was not clearly resolved in the binding assay format used in this study, probably due to the weak but detectable ACh-hydrolytic activity of ClSChE. Nevertheless, kinetic analysis revealed that ClSChE possesses extremely low Km (high affinity to ACh) and Vmax values. These findings suggest that ClSChE functions virtually as an ACh-sequestering protein by having a very strong affinity to ACh but an extremely long turnover time. Given that ACh regulates a wide variety of host physiologies, we discuss the tentative roles of ClSChE in blood vessel constriction and itch/pain regulation in the host.

Background Cytochrome P450 monooxygenases (P450s) are involved in the biosynthesis of endogenous intracellular compounds and the metabolism of xenobiotics, including chemical insecticides. We investigated the structural and expression level variance across all P450 genes with respect to the evolution of insecticide resistance under multigenerational dichlorodiphenyltrichloroethane (DDT) selection. Results RNA-sequencing (RNA-seq) and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) indicated that the transcript levels of seven P450 genes were significantly up-regulated and three P450 genes were down-regulated in the DDT-resistant strain 91-R, as compared to the control strain 91-C. The overexpression of Cyp6g1 was associated with the presence of an Accord and an HMS-Beagle element insertion in the 5' upstream region in conjunction with copy number variation in the 91-R strain, but not in the 91-C strain. A total of 122 (50.2%) fixed nonsynonymous (amino acid-changing) mutations were found between 91-C and 91-R, and 20 (8.2%) resulted in amino acid changes within functional domains. Three P450 proteins were truncated as a result of premature stop codons and fixed between strains. Conclusion Our results demonstrate that a combination of changes in P450 protein-coding regions and transcript levels are possibly associated with DDT resistance, and thereby suggest that selection for variant function may occur within this gene family in response to chronic DDT exposure. © 2018 Society of Chemical Industry.

The brown pod-sucking bug, Clavigralla tomentosicollis Stal (Hemiptera: Coreidae), causes significant damage to cultivated cowpea, Vigna unguiculata Walp, a staple crop in sub-Saharan Africa. C. tomentosicollis pierce and suck sap from cowpea pods, resulting in reduced grain yield and quality. The complete, 16,089 bp mitogenome of C. tomentosicollis encodes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs) and an A + T rich control region, with gene order and orientation identical to that of the insect ancestral gene order. The initiation and termination codons for the PCGs used standard ATN codons and TAA or TAG codons respectively. All predicted tRNAs fold into a clover-leaf secondary structures with the exception of tRNA-Ser (AGN) with a semi-loop dihydrouridine arm. The 1509 bps A + T rich region contains a single 89 bp tandem repeat unit duplicated 3.7 times. When compared with other published Coreoidea mitogenomes, C. tomentosicollis was also highly A − T skewed, and similar in both size and A − T%; however, its longer tandem repeat within the A + T rich region was unique. The C. tomentosicollis mitogenome can serve as a foundation to combine molecular marker data with pest monitoring strategies to better understand the population dynamics of this species.

The fruit fly, Drosophila melanogaster, is predominantly found in overripe, rotten, fermenting, or decaying fruits and is constantly exposed to chemical stressors such as acetic acid, ethanol, and 2-phenylethanol. D. melanogaster has been employed as a model system for studying the molecular bases of various types of chemical-induced tolerance. Expression profiling using Illumina sequencing has been performed for identifying changes in gene expression that may be associated with evolutionary adaptation to exposure of acetic acid, ethanol, and 2-phenylethanol. We identified a total of 457 differentially expressed genes that may affect sensitivity or tolerance to three chemicals in the chemical treatment group as opposed to the control group. Gene-set enrichment analysis revealed that the genes involved in metabolism, multicellular organism reproduction, olfaction, regulation of signal transduction, and stress tolerance were over-represented in response to chemical exposure. Furthermore, we also detected a coordinated upregulation of genes in the Toll- and Imd-signaling pathways after the chemical exposure. Quantitative reverse transcription PCR analysis revealed that the expression levels of nine genes within the set of genes identified by RNA sequencing were up- or downregulated owing to chemical exposure. Taken together, our data suggest that such differentially expressed genes are coordinately affected by chemical exposure. Transcriptional analyses after exposure of D. melanogaster with three chemicals provide unique insights into subsequent functional studies on the mechanisms underlying the evolutionary adaptation of insect species to environmental chemical stressors.

Ten constitutively differentially expressed miRNAs were previously described between DDT-resistant 91-R and -susceptible control Drosophila melanogaster strains, and among their predicted target genes were those associated with metabolic DDT resistance mechanisms. The present study evaluated the inducibility of miRNA expression and putative downstream regulation of cytochrome P450s in response to DDT exposure in a time-dependent manner in 91-R and the susceptible Canton-S strain. Specifically, RT-qPCR analysis showed that DDT exposures led to the significant down-regulation (repression) of miR-310-3p, miR-311-3p, miR-312-3p, miR-313-3p, and miR-92a-3p levels in Canton-S. This is contrasted with the lack of significant changes in 91-R at most time-points following DDT exposure. The levels of expression among miRNAs exhibited opposite expression patterns compared to their corresponding putative target cytochrome P450s at the same time points after DDT exposure. Collectively, results from this study suggest that miR-310-3p, miR-311-3p, miR-312-3p, miR-313-3p, and miR-92a-3p might have a potential role in the control of DDT detoxification through the post-transcriptional regulation of target cytochrome P450s in Canton-S. Conversely, the lack of significant changes of these same miRNAs in 91-R following DDT-exposure suggests a possible adaptive mutation that removes repressive control mechanisms. These data are important for the understanding impact of adaptive changes in miRNA expression on post-transcriptional regulatory mechanism involved in the evolution of DDT resistance in 91-R.

Insecticide resistance is an ongoing challenge in agriculture and disease vector control. Here, we demonstrate a novel strategy to attenuate resistance. We used genomics tools to target fundamental energy-associated pathways and identified a potential "Achilles' heel" for resistance, a resistance-associated protein that, upon inhibition, results in a substantial loss in the resistance phenotype. Specifically, we compared the gene expression profiles and structural variations of the insulin/insulin-like growth factor signaling (IIS) pathway genes in DDT-susceptible (91-C) and -resistant (91-R) Drosophila melanogaster (Drosophila) strains. A total of eight and seven IIS transcripts were up- and down-regulated, respectively, in 91-R compared to 91-C. A total of 114 nonsynonymous mutations were observed between 91-C and 91-R, of which 51.8% were fixed. Among the differentially expressed transcripts, phosphoenolpyruvate carboxykinase (PEPCK), down-regulated in 91-R, encoded the greatest number of amino acid changes, prompting us to perform PEPCK inhibitor-pesticide exposure bioassays. The inhibitor of PEPCK, hydrazine sulfate, resulted in a 161- to 218-fold decrease in the DDT resistance phenotype (91-R) and more than a 4- to 5-fold increase in susceptibility in 91-C. A second target protein, Glycogen synthase kinase 3β (GSK3β-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3β-PO inhibitor, lithium chloride, likewise reduced the resistance but to a lesser extent than did hydrazine sulfate for PEPCK. We demonstrate the potential role of IIS genes in DDT resistance and the potential discovery of an "Achilles' heel" against pesticide resistance in this pathway.

Ten constitutively differentially expressed miRNAs were previously described between DDT-resistant 91-R and -susceptible control Drosophila melanogaster strains, and among their predicted target genes were those associated with metabolic DDT resistance mechanisms. The present study evaluated the inducibility of miRNA expression and putative downstream regulation of cytochrome P450s in response to DDT exposure in a time-dependent manner in 91-R and the susceptible Canton-S strain. Specifically, RT-qPCR analysis showed that DDT exposures led to the significant down-regulation (repression) of miR-310-3p, miR-311-3p, miR-312-3p, miR-313-3p, and miR-92a-3p levels in Canton-S. This is contrasted with the lack of significant changes in 91-R at most time-points following DDT exposure. The levels of expression among miRNAs exhibited opposite expression patterns compared to their corresponding putative target cytochrome P450s at the same time points after DDT exposure. Collectively, results from this study suggest that miR-310-3p, miR-311-3p, miR-312-3p, miR-313-3p, and miR-92a-3p might have a potential role in the control of DDT detoxification through the post-transcriptional regulation of target cytochrome P450s in Canton-S. Conversely, the lack of significant changes of these same miRNAs in 91-R following DDT-exposure suggests a possible adaptive mutation that removes repressive control mechanisms. These data are important for the understanding impact of adaptive changes in miRNA expression on post-transcriptional regulatory mechanism involved in the evolution of DDT resistance in 91-R.

The organochloride insecticide dichlorodiphenyltrichloroethane (DDT) and its metabolites can increase cellular levels of reactive oxygen species (ROS), cause mitochondrial dysfunction, and induce apoptosis. The highly DDT-resistant Drosophila melanogaster Meigen 1830 (Drosophila) strain, 91-R, and its susceptible control, 91-C, were used to investigate functional and structural changes among mitochondrial-derived pathways. Resequencing of mitochondrial genomes (mitogenomes) detected no structural differences between 91-R and 91-C, whereas RNA-seq suggested the differential expression of 221 mitochondrial-associated genes. Reverse transcriptase-quantitative PCR validation of 33 candidates confirmed that transcripts for six genes (Cyp12d1-p, Cyp12a4, cyt-c-d, COX5BL, COX7AL, CG17140) were significantly upregulated and two genes (Dif, Rel) were significantly downregulated in 91-R. Among the upregulated genes, four genes are duplicated within the reference genome (cyt-c-d, CG17140, COX5BL, and COX7AL). The predicted functions of the differentially expressed genes, or known functions of closely related genes, suggest that 91-R utilizes existing ROS regulation pathways of the mitochondria to combat increased ROS levels from exposure to DDT. This study represents, to our knowledge, the initial investigation of mitochondrial genome sequence variants and functional adaptations in responses to intense DDT selection and provides insights into potential adaptations of ROS management associated with DDT selection in Drosophila.

The adaptation of insect populations to insecticidal control is a continual threat to human health and sustainable agricultural practices, but many complex genomic mechanisms involved in this adaption remain poorly understood. This study applied a systems approach to investigate the interconnections between structural and functional variance in response to dichlorodiphenyltrichloroethane (DDT) within the Drosophila melanogaster strain 91-R. Directional selection in 6 selective sweeps coincided with constitutive gene expression differences in DDT resistant flies, including the most highly upregulated transcript, Unc-115 b, which plays a central role in axon guidance, and the most highly downregulated transcript, the angiopoietin-like CG31832, which is involved in directing vascular branching and dendrite outgrowth but likely may be under trans-regulatory control. Direct functions and protein–protein interactions mediated by differentially expressed transcripts control changes in cell migration, signal transduction, and gene regulatory cascades that impact the nervous system. Although changes to cellular stress response pathways involve 8 different cytochrome P450s, stress response, and apoptosis is controlled by a multifacetted regulatory mechanism. These data demonstrate that DDT selection in 91-R may have resulted in genome-wide adaptations that impacts genetic and signal transduction pathways that converge to modify stress response, cell survival, and neurological functions. This study implicates the involvement of a multigenic mechanism in the adaptation to a chemical insecticide, which impact interconnected regulatory cascades. We propose that DDT selection within 91-R might act systemically, wherein pathway interactions function to reinforce the epistatic effects of individual adaptive changes on an additive or nonadditive basis.

We examined the molecular and enzymatic properties of two acetylcholinesterases (AChEs; ClAChE1 and ClAChE2) from the common bed bug, Cimex lectularius. Native polyacrylamide gel electrophoresis followed by activity staining and Western blotting revealed that ClAChE1 is the main catalytic enzyme and is abundantly expressed in various tissues. Both ClAChEs existed in dimeric form connected by a disulfide bridge and were attached to the membrane via a glycophosphatidylinositol anchor. To determine their kinetic and inhibitory properties, both ClAChE1 and ClAChE2 were in vitro expressed in Sf9 cells using a baculovirus expression system. ClAChE1 showed higher catalytic efficiency toward acetylcholine, supporting the hypothesis that ClAChE1 plays a major role in postsynaptic transmission. An inhibition assay revealed that ClAChE1 is generally more sensitive to organophosphates and carbamates examined although ClAChE2 was >4000-fold more sensitive to malaoxon than ClAChE1. The relatively higher correlation between the in vitro ClAChE1 inhibition and the in vivo toxicity suggested that ClAChE1 is the more relevant toxicological target for organophosphates and carbamates. Although the physiological function of ClAChE2 remains to be elucidated, ClAChE2 also appears to have neuronal functions, as judged by its tissue distribution and molecular and kinetic properties. Our findings help expand our knowledge on insect AChEs and their toxicological properties.

The ATP-binding cassette (ABC) transporters represent a superfamily of proteins that have important physiological roles in both prokaryotes and eukaryotes. In insects, ABC transporters have previously been implicated in insecticide resistance. The 91-R strain of Drosophila melanogaster has been intensely selected with DDT over six decades. A recent selective sweeps analysis of 91-R implicated the potential role of MDR49, an ABC transporter, in DDT resistance, however, to date the details of how MDR49 may play a role in resistance have not been elucidated. In this study, we investigated the impact of structural changes and an alternative splicing event in MDR49 on DDT-resistance in 91-R, as compared to the DDT susceptible strain 91-C. We observed three amino acid differences in MDR49 when 91-R was compared with 91-C, and only one isoform (MDR49B) was implicated in DDT resistance. A transgenic Drosophila strain containing the 91-R-MDR49B isoform had a significantly higher LD50 value as compared to the 91-C-MDR49B isoform at the early time points (6 h to 12 h) during DDT exposure. Our data support the hypothesis that the MDR49B isoform, with three amino acid mutations, plays a role in the early aspects of DDT resistance in 91-R.

We identified and characterized the full-length cDNA sequences encoding two acetylcholinesterases (ClAChE1 and ClAChE2) and a salivary gland-specific cholinesterase-like protein (ClSChE) from the common bed bug, Cimex lectularius. All three cholinesterase genes (Clac1, Clace2 and Clsce) have conserved motifs, including a catalytic triad, a choline-binding site and an acyl pocket. Phylogenetic analysis showed that ClAChE1 belongs to the insect AChE1 clade, whereas ClAChE2 belongs to the insect AChE2 clade. ClSChE was grouped into the clade containing all AChE1s, suggesting a paralogous relationship to ClAChE1. Transcription levels of Clace1 were higher than those of Clace2 in all tissues examined, including the central nervous system (CNS). In contrast, the Clsce transcript was not detected in the CNS but specifically found in the salivary gland at much higher levels (>3000-fold) than those of Clace1 and Clace2. Western blot analysis using anti-ClAChE antibodies, in conjunction with activity staining, revealed that ClAChE1 is more active than ClAChE2, whereas ClSChE has little enzyme activity. Three-dimensional structure modelling suggested that ClAChEs and ClSChE shared structural similarities, but had some differences in the residues forming the acyl pocket and oxyanion hole. The current findings should provide valuable insights into the evolution and functional diversification of insect cholinesterase.

Dichloro-diphenyl-trichloroethane (DDT) resistance among arthropod species is a model for understanding the molecular adaptations in response to insecticide exposures. Previous studies reported that DDT resistance may involve one or multiple detoxification genes, such as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), esterases, and ATP binding cassette (ABC) transporters, or changes in the voltage-sensitive sodium channel. However, the possible involvement of microRNAs (miRNAs) in the post-transcriptional regulation of genes associated with DDT resistance in the Drosophila melanogaster strain 91-R remains poorly understood. In this study, the majority of the resulting miRNAs discovered in small RNA libraries from 91-R and the susceptible control strain, 91-C, ranged from 16-25 nt, and contained 163 precursors and 256 mature forms of previously-known miRNAs along with 17 putative novel miRNAs. Quantitative analyses predicted the differential expression of ten miRNAs between 91-R and 91-C, and, based on Gene Ontology and pathway analysis, these ten miRNAs putatively target transcripts encoding proteins involved in detoxification mechanisms. RT-qPCR validated an inverse correlation between levels of differentially-expressed miRNAs and their putatively targeted transcripts, which implies a role of these miRNAs in the differential regulation of detoxification pathways in 91-R compared to 91-C. This study provides evidence associating the differential expression of miRNAs in response to multigenerational DDT selection in Drosophila melanogaster and provides important clues for understanding the possible roles of miRNAs in mediating insecticide resistance traits.

In 2010, the initial annotations of the genomes of the body louse (Pediculus humanus humanus Linnaeus) and its primary endosymbiont, “Candidatus Riesia pediculicola,” were completed. The body louse had the smallest genome of any insect sequenced to that point. Prior to the proposal for the sequencing project, there was a dearth of information about louse genes, with no more than around 500–600 inferred open reading frames in public databases. Since the publishing of this genome project, the field of louse genomics has experienced significant advances in our understanding of the taxonomic relationship and the differences in vector competence between head and body lice. To date, the louse system has emerged as a model system to understand xenobiotic induction responses. Finally, a louse RNAi-based reverse genetic system has been developed with the potential to study the functional role of louse genes in vector competence.

The Drosophila melanogaster 91-R and 91-C strains are of common origin, however, 91-R has been intensely selected for dichlorodiphenyltrichloroethane (DDT) resistance over six decades while 91-C has been maintained as the non-selected control strain. These fly strains represent a unique genetic resource to understand the accumulation and fixation of mutations under laboratory conditions over decades of pesticide selection. Considerable research has been done to investigate the differential expression of genes associated with the highly DDT resistant strain 91-R, however, with the advent of whole genome sequencing we can now begin to develop an in depth understanding of the genomic changes associated with this intense decades-long xenobiotic selection pressure. Here we present the first whole genome sequencing analysis of the 91-R and 91-C fly strains to identify genome-wide structural changes within the open reading frames. Between-strain changes in allele frequencies revealed a higher percent of new alleles going to fixation for the 91-R strain, as compared to 91-C (P

Adaptation of insect phenotypes for survival after exposure to xenobiotics can result from selection at multiple loci with additive genetic effects. To the authors’ knowledge, no selective sweep analysis has been performed to identify such loci in highly dichlorodiphenyltrichloroethane (DDT) resistant insects. Here we compared a highly DDT resistant phenotype in the Drosophila melanogaster (Drosophila) 91-R strain to the DDT susceptible 91-C strain, both of common origin. Whole genome re-sequencing data from pools of individuals was generated separately for 91-R and 91-C, and mapped to the reference Drosophila genome assembly (v. 5.72). Thirteen major and three minor effect chromosome intervals with reduced nucleotide diversity (π) were identified only in the 91-R population. Estimates of Tajima's D (D) showed corresponding evidence of directional selection in these same genome regions of 91-R, however, no similar reductions in π or D estimates were detected in 91-C. An overabundance of non-synonymous proteins coding to synonymous changes were identified in putative open reading frames associated with 91-R. Except for NinaC and Cyp4g1, none of the identified genes were the ‘usual suspects’ previously observed to be associated with DDT resistance. Additionally, up-regulated ATP-binding cassette transporters have been previously associated with DDT resistance; however, here we identified a structurally altered MDR49 candidate resistance gene. The remaining fourteen genes have not previously been shown to be associated with DDT resistance. These results suggest hitherto unknown mechanisms of DDT resistance, most of which have been overlooked in previous transcriptional studies, with some genes having orthologs in mammals.

Insecticide-resistant Drosophila melanogaster strains represent a resource for the discovery of the underlying molecular mechanisms of cytochrome P450 constitutive over-expression, even if some of these P450s are not directly involved in the resistance phenotype. For example, in select 4,4’-dichlorodiphenyltrichloroethane (DDT) resistant strains the glucocorticoid receptor-like (GR-like) potential transcription factor binding motifs (TFBMs) have previously been shown to be associated with constitutively differentially-expressed cytochrome P450s, Cyp12d1, Cyp6g2 and Cyp9c1. However, insects are not known to have glucocorticoids. The only ortholog to the mammalian glucocorticoid receptor (GR) in D. melanogaster is an estrogen-related receptor (ERR) gene, which has two predicted alternative splice isoforms (ERRa and ERRb). Sequencing of ERRa and ERRb in select DDT susceptible and resistant D. melanogaster strains has revealed a glycine (G) codon insertion which was only observed in the ligand binding domain of ERR from the resistant strains tested (ERR-G). Transgenic flies, expressing the ERRa-G allele, constitutively over-expressed Cyp12d1, Cyp6g2 and Cyp9c1. Only Cyp12d1 and Cyp6g2 were over-expressed in the ERRb-G transgenic flies. Phylogenetic studies show that the G-insertion appeared to be located in a less conserved domain in ERR and this insertion is found in multiple species across the Sophophora subgenera.