Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster.

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala(301) to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala(301) to Ser resistance mutation and Met(360) to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser(301) homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser(301) change into an Ala(301) background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

Dissecting the insect metabolic machinery using twin ion mass spectrometry: a single P450 enzyme metabolizing the insecticide imidacloprid in vivo.

Insecticide resistance is one of the most prevalent examples of anthropogenic genetic change, yet our understanding of metabolic-based resistance remains limited by the analytical challenges associated with rapidly tracking the in vivo metabolites of insecticides at nonlethal doses. Here, using twin ion mass spectrometry analysis of the extracts of whole Drosophila larvae and excreta, we show that (i) eight metabolites of the neonicotinoid insecticide, imidacloprid, can be detected when formed by susceptible larval genotypes and (ii) the specific overtranscription of a single gene product, Cyp6g1, associated with the metabolic resistance to neonicotinoids, results in a significant increase in the formation of three imidacloprid metabolites that are formed in C-H bond activation reactions; that is, Cyp6g1 is directly linked to the enhanced metabolism of imidacloprid in vivo. These results establish a rapid and sensitive method for dissecting the metabolic machinery of insects by directly linking single gene products to insecticide metabolism.

Copy number variation and transposable elements feature in recent, ongoing adaptation at the Cyp6g1 locus.

The increased transcription of the Cyp6g1 gene of Drosophila melanogaster, and consequent resistance to insecticides such as DDT, is a widely cited example of adaptation mediated by cis-regulatory change. A fragment of an Accord transposable element inserted upstream of the Cyp6g1 gene is causally associated with resistance and has spread to high frequencies in populations around the world since the 1940s. Here we report the existence of a natural allelic series at this locus of D. melanogaster, involving copy number variation of Cyp6g1, and two additional transposable element insertions (a P and an HMS-Beagle). We provide evidence that this genetic variation underpins phenotypic variation, as the more derived the allele, the greater the level of DDT resistance. Tracking the spatial and temporal patterns of allele frequency changes indicates that the multiple steps of the allelic series are adaptive. Further, a DDT association study shows that the most resistant allele, Cyp6g1-[BP], is greatly enriched in the top 5% of the phenotypic distribution and accounts for approximately 16% of the underlying phenotypic variation in resistance to DDT. In contrast, copy number variation for another candidate resistance gene, Cyp12d1, is not associated with resistance. Thus the Cyp6g1 locus is a major contributor to DDT resistance in field populations, and evolution at this locus features multiple adaptive steps occurring in rapid succession.

CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis.

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.

Characterization of Drosophila melanogaster cytochrome P450 genes.

Cytochrome P450s form a large and diverse family of heme-containing proteins capable of carrying out many different enzymatic reactions. In both mammals and plants, some P450s are known to carry out reactions essential for processes such as hormone synthesis, while other P450s are involved in the detoxification of environmental compounds. In general, functions of insect P450s are less well understood. We characterized Drosophila melanogaster P450 expression patterns in embryos and 2 stages of third instar larvae. We identified numerous P450s expressed in the fat body, Malpighian (renal) tubules, and in distinct regions of the midgut, consistent with hypothesized roles in detoxification processes, and other P450s expressed in organs such as the gonads, corpora allata, oenocytes, hindgut, and brain. Combining expression pattern data with an RNA interference lethality screen of individual P450s, we identify candidate P450s essential for developmental processes and distinguish them from P450s with potential functions in detoxification.

Fighting fly genes.

Fighting by organisms such as mice and Drosophila provides model systems for investigating the genetic basis of aggression. Recent experiments to dissect male aggressive behaviour in Drosophila melanogaster, using gene expression analysis of selected lines followed by mutant analysis, have identified new candidate genes associated with male aggression, including one strong candidate that encodes a cytochrome P450 enzyme. Here, we describe the study of aggressive behaviour in flies and explore the possibility that cytochrome P450 is involved in aggression.

Cis-regulatory elements in the Accord retrotransposon result in tissue-specific expression of the Drosophila melanogaster insecticide resistance gene Cyp6g1.

Transposable elements are a major mutation source and powerful agents of adaptive change. Some transposable element insertions in genomes increase to a high frequency because of the selective advantage the mutant phenotype provides. Cyp6g1-mediated insecticide resistance in Drosophila melanogaster is due to the upregulation of the cytochrome P450 gene Cyp6g1, leading to the resistance to a variety of insecticide classes. The upregulation of Cyp6g1 is correlated with the presence of the long terminal repeat (LTR) of an Accord retrotransposon inserted 291bp upstream of the Cyp6g1 transcription start site. This resistant allele (DDT-R) is currently at a high frequency in D. melanogaster populations around the world. Here, we characterize the spatial expression of Cyp6g1 in insecticide-resistant and -susceptible strains. We show that the Accord LTR insertion is indeed the resistance-associated mutation and demonstrate that the Accord LTR carries regulatory sequences that increase the expression of Cyp6g1 in tissues important for detoxification, the midgut, Malpighian tubules, and the fat body. This study provides a significant example of how changes in tissue-specific gene expression caused by transposable-element insertions can contribute to adaptation.

Evidence for activation of nitenpyram by a mitochondrial cytochrome P450 in Drosophila melanogaster.

BACKGROUND: Nitenpyram is a member of the economically important neonicotinoid class of insecticides. The in vivo metabolism of nitenpyram is not well characterised, but cytochrome P450 activity is the major mechanism of resistance to neonicotinoids identified in insect pests, and P450s metabolise other neonicotinoids including imidacloprid. RESULTS: Here, we used the GAL4-UAS targeted expression system to direct RNA interference (RNAi) against the cytochrome P450 redox partners to interrupt P450 functions in specific tissues in Drosophila melanogaster. RNAi of the mitochondrial redox partner defective in the avoidance of repellents (dare) in the digestive tissues reduced nitenpyram mortality, suggesting an activation step in the metabolism of nitenpyram carried out by a mitochondrial P450. RNAi of the mitochondrial cytochrome P450 Cyp12a5, which is expressed in the digestive tissues, resulted in the same phenotype, and transgenic overexpression of Cyp12a5 increased nitenpyram sensitivity. CONCLUSION: These results suggest that in vivo metabolism of nitenpyram by the mitochondrial P450 CYP12A5 results in the formation of a product with higher toxicity than the parent compound. © 2018 Society of Chemical Industry.

Towards genetic markers in animal populations as biomonitors for human-induced environmental change.

Genetic markers provide potentially sensitive indicators of changes in environmental conditions because the genetic constitution of populations is normally altered well before populations become extinct. Genetic indicators in populations include overall genetic diversity, genetic changes in traits measured at the phenotypic level, and evolution at specific loci under selection. While overall genetic diversity has rarely been successfully related to environmental conditions, genetically based changes in traits have now been linked to the presence of toxins and both local and global temperature shifts. Candidate loci for monitoring stressors are emerging from information on how specific genes influence traits, and from screens of random loci across environmental gradients. Drosophila research suggests that chromosomal regions under recent intense selection can be identified from patterns of molecular variation and a high frequency of transposable element insertions. Allele frequency changes at candidate loci have been linked to pesticides, pollutants and climate change. Nevertheless, there are challenges in interpreting allele frequencies in populations, particularly when a large number of loci control a trait and when interactions between alleles influence trait expression. To meet these challenges, population samples should be collected for longitudinal studies, and experimental programmes should be undertaken to link variation at candidate genes to ecological processes.

The genetics and genomics of insecticide resistance.

The past ten years have seen the elucidation of the molecular basis of insect resistance to many chemical insecticides. Target genes, mostly in the nervous system, have been identified and cloned from Drosophila melanogaster and resistance-associated mutations have been examined in a range of pest insects. More recently, with the advent of annotated insect genomes, resistance mediated by complex multi-gene enzyme systems such as esterases, cytochrome p450s and glutathione-S-transferases has also been elucidated. In this article, we review the impact of Drosophila genetics on the field of insect resistance and focus on the current and future impact of genomics. These studies enable us to address three fundamental questions in the evolution of resistance. How many genes are involved? How many mutations are there within these genes? How often do these mutations arise in natural populations?

Two independent duplications forming the Cyp307a genes in Drosophila.

The conserved relationship between orthologs of many cytochrome P450 genes involved in ecdysone synthesis is not reflected in the evolution of the Drosophila Cyp307a genes. In Drosophila melanogaster Cyp307a1 (spook) and Cyp307a2 (spookier) both play essential roles in ecdysone synthesis and may possess biochemically redundant functions. Using phylogenetic analyses we show that the Drosophila Cyp307a genes were formed from two independent duplication events depicting a complicated evolutionary scenario. An initial duplication, from a Cyp307a2 ancestral gene produced the Cyp307a1 gene that has been maintained only in the Sophophoran subgenus. A second duplication in the Drosophila subgenus formed an additional paralog, Cyp307a3. Microsynteny is conserved for Cyp307a2 throughout the Drosophila species, but is not conserved between Cyp307a1 and Cyp307a3. These are located in different genomic positions in the Sophophora and Drosophila subgenera, respectively. Cyp307a3 appears to encode a functional gene product and is expressed in a different spatial and temporal manner to Cyp307a1. This suggests some level of functional divergence between the Cyp307a paralogs in different Drosophila species.

Evaluating the insecticide resistance potential of eight Drosophila melanogaster cytochrome P450 genes by transgenic over-expression.

In Drosophila melanogaster, the increased expression of Cyp6g1 results in resistance to chemically unrelated insecticides including DDT, neonicotinoids and insect growth regulator insecticides. To determine the insecticide resistance capacity of other D. melanogaster cytochrome P450s, we used the GAL4/UAS system to express individual P450s in the midgut, Malpighian tubules and fat body of transgenic flies. Drosophila over-expressing Cyp6g1, Cyp6g2, Cyp6t3, Cyp6a2, Cyp6a8, Cyp6a19, Cyp6a23 and Cyp12d1 were screened for resistance to four insecticides--DDT, nitenpyram, dicyclanil and diazinon. Increased survival on insecticides is detected for Cyp6g1 (DDT, nitenpyram and dicyclanil), Cyp6g2 (nitenpyram and diazinon) and Cyp12d1 (DDT and dicyclanil) over-expression lines. No increased survival on any insecticide was detected for flies over-expressing either Cyp6a2, Cyp6a8, Cyp6t3, Cyp6a19 or Cyp6a23.

Piperonyl butoxide induces the expression of cytochrome P450 and glutathione S-transferase genes in Drosophila melanogaster.

Piperonyl butoxide (PBO) is an insecticide synergist known to inhibit the activity of cytochrome P450 enzymes. PBO is currently used in some insecticide formulations, and has also been suggested as a pretreatment for some pesticide applications. Little is known about how insects respond to PBO exposure at the gene transcription level. The authors have characterised the transcriptional response of the Drosophila melanogaster genome after PBO treatment, using both a custom-designed 'detox' microarray, containing cytochrome P450 (P450), glutathione S-transferase (GST) and esterase genes, and a full genome microarray. A subset of P450 and GST genes is identified, along with additional metabolic genes, that are induced by PBO. The gene set is an extremely similar gene set to that induced by phenobarbital, a compound for which pretreatment is known to confer tolerance to a range of insecticide compounds. The implications of the induction of gene families known to metabolise insecticides and the use of PBO in pest management programs are discussed.

A comparison of Drosophila melanogaster detoxification gene induction responses for six insecticides, caffeine and phenobarbital.

Modifications of metabolic pathways are important in insecticide resistance evolution. Mutations leading to changes in expression levels or substrate specificities of cytochrome P450 (P450), glutathione-S-transferase (GST) and esterase genes have been linked to many cases of resistance with the responsible enzyme shown to utilize the insecticide as a substrate. Many studies show that the substrates of enzymes are capable of inducing the expression of those enzymes. We investigated if this was the case for insecticides and the enzymes responsible for their metabolism. The induction responses for P450s, GSTs and esterases to six different insecticides were investigated using a custom designed microarray in Drosophila melanogaster. Even though these gene families can all contribute to insecticide resistance, their induction responses when exposed to insecticides are minimal. The insecticides spinosad, diazinon, nitenpyram, lufenuron and dicyclanil did not induce any P450, GST or esterase gene expression after a short exposure to high lethal concentrations of insecticide. DDT elicited the low-level induction of one GST and one P450. These results are in contrast to induction responses we observed for the natural plant compound caffeine and the barbituate drug phenobarbital, both of which highly induced a number of P450 and GST genes under the same short exposure regime. Our results indicate that, under the insecticide exposure conditions we used, constitutive over-expression of metabolic genes play more of a role in insect survival than induction of members of these gene families.

Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster.

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

Genomic islands in Photorhabdus.

Genomic islands are responsible for unique aspects of bacterial behavior such as symbiosis and pathogenicity. Photorhabdus luminescens is a pathogen of insects that spends part of its lifecycle in symbiosis with a nematode. Here, we describe novel genomic islands from Photorhabdus that are involved in symbiosis and pathogenicity, and discuss the inter-relationship between virulence factors used against invertebrates and vertebrates.

The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen.

The Photorhabdus luminescens W14 toxin encoding gene makes caterpillars floppy (mcf) was discovered due to its ability to kill caterpillars when expressed in Escherichia coli. Here we describe a homologue of mcf (renamed as mcf1), termed mcf2, discovered in the same genome. The mcf2 gene predicts another large toxin whose central domain, like Mcf1, also shows limited homology to Clostridium cytotoxin B. However, the N-terminus of Mcf2 shows significant similarity to the type-III secreted effector HrmA from the plant pathogen Pseudomonas syringae and no similarity to the N-terminus of Mcf1. HrmA is a plant avirulence gene whose transient expression in tobacco cells results in cell death. Here we show that E. coli expressing Mcf2 can, like E. coli expressing Mcf1, kill insects. Further, expression of the c-Myc tagged N-terminus of Mcf2, the region showing similarity to HrmA, results in nuclear localisation of the fusion protein and subsequent destruction of transfected mammalian cells. The Mcf1 and Mcf2 toxins therefore belong to a family of high molecular mass toxins, differing at their N-termini, which encode different effector domains.

Whole-genome expression analysis in the third instar larval midgut of Drosophila melanogaster.

Survival of insects on a substrate containing toxic substances such as plant secondary metabolites or insecticides is dependent on the metabolism or excretion of those xenobiotics. The primary sites of xenobiotic metabolism are the midgut, Malpighian tubules, and fat body. In general, gene expression in these organs is reported for the entire tissue by online databases, but several studies have shown that gene expression within the midgut is compartmentalized. Here, RNA sequencing is used to investigate whole-genome expression in subsections of third instar larval midguts of Drosophila melanogaster. The data support functional diversification in subsections of the midgut. Analysis of the expression of gene families that are implicated in the metabolism of xenobiotics suggests that metabolism may not be uniform along the midgut. These data provide a starting point for investigating gene expression and xenobiotic metabolism and other functions of the larval midgut.

The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster.

Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. We examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala(301) site influenced resistance levels, with glycine showing greater survival than serine replacement. We confirm that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. We also emphasize the beneficial aspects of testing predicted mutations in a whole organism to validate a candidate gene approach.

Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila.

Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.