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The genome of the cat flea, an ectoparasite of major veterinary importance and the first representative of the Siphonaptera, is highly unusual among arthropod genomes in showing a variable size and a very large number of gene duplications (Driscoll et al., 2020). The cat flea is the target of several classes of insecticides, justifying the description of its CYPome, the complement of P450s that are an important family of detoxification enzymes. 103 P450 genes were annotated on the nine chromosomes, with an additional 12 genes on small, extrachromosomal scaffolds. Only 34 genes were found as single sequences, with 47 duplicated two to four-fold. This included duplication of genes that are mostly single copy P450 genes in other arthropods. Large clusters of mitochondrial clan P450s were observed, resulting in a CYP12 bloom within this clan to 34 genes, a number of mitochondrial P450s not seen in other animals so far. The variable geometry of the cat flea CYPome poses a challenge to the study of P450 function in this species, and raises the question of the underlying causes of single copy control versus multicopy licence of P450 genes.

Insect cytochrome P450 monooxygenases (P450s) perform a variety of important physiological functions, but it is their role in the detoxification of xenobiotics, such as natural and synthetic insecticides, that is the topic of this review. Recent advances in insect genomics and postgenomic functional approaches have provided an unprecedented opportunity to understand the evolution of insect P450s and their role in insect toxicology. These approaches have also been harnessed to provide new insights into the genomic alterations that lead to insecticide resistance, the mechanisms by which P450s are regulated, and the functional determinants of P450-mediated insecticide resistance. In parallel, an emerging body of work on the role of P450s in defining the sensitivity of beneficial insects to insecticides has been developed. The knowledge gained from these studies has applications for the management of P450-mediated resistance in insect pests and can be leveraged to safeguard the health of important beneficial insects. Expected final online publication date for the Annual Review of Entomology, Volume 67 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

The genome of the cat flea, an ectoparasite of major veterinary importance and the first representative of the Siphonaptera, is highly unusual among arthropod genomes in showing a variable size and a very large number of gene duplications (Driscoll et al., 2020). The cat flea is the target of several classes of insecticides, justifying the description of its CYPome, the complement of P450s that are an important family of detoxification enzymes. 103 P450 genes were annotated on the nine chromosomes, with an additional 12 genes on small, extrachromosomal scaffolds. Only 34 genes were found as single sequences, with 47 duplicated two to four-fold. This included duplication of genes that are mostly single copy P450 genes in other arthropods. Large clusters of mitochondrial clan P450s were observed, resulting in a CYP12 bloom within this clan to 34 genes, a number of mitochondrial P450s not seen in other animals so far. The variable geometry of the cat flea CYPome poses a challenge to the study of P450 function in this species, and raises the question of the underlying causes of single copy control versus multicopy licence of P450 genes.

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt(−/−) larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox(−/−) mutants, which synthesized MF but no JH, completed the entire life cycle. While epox(−/−) adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

Frankia bacteria are nitrogen fixing species from the Actinobacterium phylum which live on the root nodules of plants. They have been hypothesised to have significant potential for natural product biosynthesis. The cytochrome P450 monooxygenase complement of Frankia sp. EuI1c (Frankia inefficax sp.), which comprises 68 members, was analysed. Several members belonged to previously uncharacterised bacterial P450 families. There was an unusually high number of CYP189 family members (21) suggesting that this family has undergone gene duplication events which are classified as "blooms". The likely electron transfer partners for the P450 enzymes were also identified and analysed. These consisted of predominantly [3Fe-4S] cluster containing ferredoxins (eight), a single [2Fe-2S] ferredoxin and a couple of ferredoxin reductases. Three of these CYP family members were produced and purified, using Escherichia coli as a host, and their substrate range was characterised. CYP1027H1 and CYP150A20 bound a broad range of norisoprenoids and terpenoids. CYP1074A2 was highly specific for certain steroids including testosterone, progesterone, stanolone and 4-androstene-3,17-dione. It is likely that steroids are the physiological substrates of CYP1074A2. These results also give an indication that terpenoids are the likely substrates of CYP1027H1 and CYP150A2. The large number of P450s belonging to distinct families as well as the associated electron transfer partners found in different Frankia strains highlights the importance of this family of enzymes has in the secondary metabolism of these bacteria.

Arthropod herbivores cause dramatic crop losses, and frequent pesticide use has led to widespread resistance in numerous species. One such species, the two-spotted spider mite, Tetranychus urticae, is an extreme generalist herbivore and a major worldwide crop pest with a history of rapidly developing resistance to acaricides. Mitochondrial Electron Transport Inhibitors of complex I (METI-Is) have been used extensively in the last 25 years to control T. urticae around the globe, and widespread resistance to each has been documented. METI-I resistance mechanisms in T. urticae are likely complex, as increased metabolism by cytochrome P450 monooxygenases as well as a target-site mutation have been linked with resistance. To identify loci underlying resistance to the METI-I acaricides fenpyroximate, pyridaben and tebufenpyrad without prior hypotheses, we crossed a highly METI-I-resistant strain of T. urticae to a susceptible one, propagated many replicated populations over multiple generations with and without selection by each compound, and performed bulked segregant analysis genetic mapping. Our results showed that while the known H92R target-site mutation was associated with resistance to each compound, a genomic region that included cytochrome P450-reductase (CPR) was associated with resistance to pyridaben and tebufenpyrad. Within CPR, a single nonsynonymous variant distinguished the resistant strain from the sensitive one. Furthermore, a genomic region linked with tebufenpyrad resistance harbored a non-canonical member of the nuclear hormone receptor 96 (NHR96) gene family. This NHR96 gene does not encode a DNA-binding domain (DBD), an uncommon feature in arthropods, and belongs to an expanded family of 47 NHR96 proteins lacking DBDs in T. urticae. Our findings suggest that although cross-resistance to METI-Is involves known detoxification pathways, structural differences in METI-I acaricides have also resulted in resistance mechanisms that are compound-specific.

Cuticular hydrocarbon (CHC)biosynthesis is a major pathway of insect physiology. In Drosophila melanogaster the cytochrome P450 CYP4G1 catalyses the insect-specific oxidative decarbonylation step, while in the malaria vector Anopheles gambiae, two CYP4G paralogues, CYP4G16 and CYP4G17 are present. Analysis of the subcellular localization of CYP4G17 and CYP4G16 in larval and pupal stages revealed that CYP4G16 preserves its PM localization across developmental stages analyzed; however CYPG17 is differentially localized in two distinct types of pupal oenocytes, presumably oenocytes of larval and adult developmental specificity. Western blot analysis showed the presence of two CYP4G17 forms, potentially associated with each oenocyte type. Both An. gambiae CYP4Gs were expressed in D. melanogaster flies in a Cyp4g1 silenced background in order to functionally characterize them in vivo. CYP4G16, CYP4G17 or their combination rescued the lethal phenotype of Cyp4g1-knock down flies, demonstrating that CYP4G17 is also a functional decarbonylase, albeit of somewhat lower efficiency than CYP4G16 in Drosophila. Flies expressing mosquito CYP4G16 and/or CYP4G17 produced similar CHC profiles to ‘wild-type’ flies expressing the endogenous CYP4G1, but they also produce very long-chain dimethyl-branched CHCs not detectable in wild type flies, suggesting that the specificity of the CYP4G enzymes contributes to determine the complexity of the CHC blend. In conclusion, both An. gambiae CYP4G enzymes contribute to the unique Anopheles CHC profile, which has been associated to defense, adult desiccation tolerance, insecticide penetration rate and chemical communication.

Varroa destructor is one of the main problems in modern beekeeping. Highly selective acaricides with low toxicity to bees are used internationally to control this mite. One of the key acaricides is the organophosphorus (OP) proinsecticide coumaphos, that becomes toxic after enzymatic activation inside Varroa. We show here that mites from the island Andros (AN-CR) exhibit high levels of coumaphos resistance. Resistance is not mediated by decreased coumaphos uptake, target-site resistance, or increased detoxification. Reduced proinsecticide activation by a cytochrome P450 enzyme was the main resistance mechanism, a powerful and rarely encountered evolutionary solution to insecticide selection pressure. After treatment with sublethal doses of [(14)C] coumaphos, susceptible mite extracts had substantial amounts of coroxon, the activated metabolite of coumaphos, while resistant mites had only trace amounts. This indicates a suppression of the P450 (CYP)-mediated activation step in the AN-CR mites. Bioassays with coroxon to bypass the activation step showed that resistance was dramatically reduced. There are 26 CYPs present in the V. destructor genome. Transcriptome analysis revealed overexpression in resistant mites of CYP4DP24 and underexpression of CYP3012A6 and CYP4EP4. RNA interference of CYP4EP4 in the susceptible population, to mimic underexpression seen in the resistant mites, prevented coumaphos activation and decreased coumaphos toxicity.

The evolution of insecticide resistance represents a global constraint to agricultural production. Because of the extreme genetic diversity found in insects and the large numbers of genes involved in insecticide detoxification, better tools are needed to quickly identify and validate the involvement of putative resistance genes for improved monitoring, management, and countering of field-evolved insecticide resistance. The avermectins, emamectin benzoate (EB) and abamectin are relatively new pesticides with reduced environmental risk that target a wide number of insect pests, including the beet armyworm, Spodoptera exigua, an important global pest of many crops. Unfortunately, field resistance to avermectins recently evolved in the beet armyworm, threatening the sustainable use of this class of insecticides. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15–16 Mbp of chromosome 17. Knockout of the CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored EB susceptibility, implicating this gene in avermectin resistance. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of EB and abamectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for effective monitoring and adaptive management of insecticide resistance., Author summary Insecticide resistance is a global constraint to agricultural production, and rapid identification of resistance genes is critical for better monitoring and management of resistant insect pests. Identification of metabolic resistance genes has always been a challenging task due to the high diversity of insect detoxification enzyme genes. Here, we report a high-quality chromosome-level assembly of the beet armyworm genome and use bulked segregant analysis (BSA) to identify the locus of avermectin resistance, which mapped on 15–16 Mbp of chromosome 17. Knockout of the CYP9A186 gene that maps within this region by CRISPR/Cas9 gene editing fully restored avermectin susceptibility. Heterologous expression and in vitro functional assays further confirm that a natural substitution (F116V) found in the substrate recognition site 1 (SRS1) of the CYP9A186 protein results in enhanced metabolism of avermectin. Hence, the combined approach of coupling gene editing with BSA allows for the rapid identification of metabolic resistance genes responsible for insecticide resistance, which is critical for field monitoring of such mutations, for making improved decisions on appropriate use of effective chemistries, as well for improvements in the design of future compounds that target S. exigua.

The tomato russet mite, Aculops lycopersici, is among the smallest animals on earth. It is a worldwide pest on tomato and can potently suppress the host’s natural resistance. We sequenced its genome, the first of an eriophyoid, and explored whether there are genomic features associated with the mite’s minute size and lifestyle. At only 32.5 Mb, the genome is the smallest yet reported for any arthropod and, reminiscent of microbial eukaryotes, exceptionally streamlined. It has few transposable elements, tiny intergenic regions, and is remarkably intron-poor, as more than 80% of coding genes are intronless. Furthermore, in accordance with ecological specialization theory, this defense-suppressing herbivore has extremely reduced environmental response gene families such as those involved in chemoreception and detoxification. Other losses associate with this species’ highly derived body plan. Our findings accelerate the understanding of evolutionary forces underpinning metazoan life at the limits of small physical and genome size., eLife digest Arthropods are a group of invertebrates that include insects – such as flies or beetles – arachnids – like spiders or scorpions – and crustaceans – including shrimp and woodlice. One of the tiniest species of arthropods, measuring less than 0.2 millimeters, is the tomato russet mite Aculops lycopersici. This arachnid is among the smallest animals on Earth, even smaller than some single-celled organisms, and only has four legs, unlike other arachnids. It is a major pest on tomato plants, which are toxic to many other animals, and it feeds on the top cell layer of the stems and leaves. Tomato growers need a way to identify and treat tomato russet mite infestations, but this tiny species remains something of a mystery. One way to tackle this pest may be to take a closer look at its genome, as this could reveal what genes the mite uses to detoxify its diet. Examining the mite’s genome could also reveal information about how evolution handles creatures becoming smaller. An area of particular interest is the overall size of its genome. Not all of the DNA in a genome is part of genes that code for proteins; there are also sections of so-called ‘non-coding’ DNA. These sequences play important roles in controlling how and when cells use their genes. In the human genome, for example, just 1% of the DNA codes for protein. In fact, most human protein-coding genes are interrupted by sequences of non-coding DNA, called introns. Here, Greenhalgh, Dermauw et al. sequence the entire tomato russet mite genome and reveal that not only is the mite's body size miniature: these tiny animals have the smallest arthropod genome reported to date, almost a hundred times smaller than the human genome. Part of this genetic miniaturization seems to be down to massive loss of non-coding DNA. Around 40% of the mite genome codes for protein, and 80% of its protein coding genes contain no introns. The rest of the miniaturization involves loss of genes themselves. The mites have lost some of the genes that determine body structure, which could explain why they have fewer legs than other arachnids. Additionally, they only carry a small set of genes involved in sensing chemicals and clearing toxins, which could explain why they are mostly found on tomato plants. Greenhalgh, Dermauw et al.’s findings shed light on what may happen to the genome at the extremes of size evolution. Sequencing the genomes of other mites could reveal when in evolutionary history this genetic miniaturization occurred. Furthermore, a better understanding of the tomato russet mite genome could lead to the development of methods to detect the infestation of plants earlier and be highly beneficial for tomato agriculture.

Background Cuticular hydrocarbons (CHCs) have a critical role in preventing desiccation and penetration of xenobiotics in insects. Previous studies have shown that cytochrome P450 subfamily 4G (CYP4G) enzymes are oxidative decarbonylases, essential for CHC biosynthesis. However, it is unclear whether there are functional differences between the two CYP4G genes in most insects. In Locusta migratoria, we identified two CYP4G genes (LmCYP4G62 and LmCYP4G102). LmCYP4G102 plays a critical role in the synthesis of CHCs, but the function of LmCYP4G62 is unknown. Results We identified, characterized, and compared two LmCYP4G genes, based on L. migratoria transcriptomic and genomic databases. RT-qPCR showed that both were highly expressed in tissues with which oenocytes are associated, the integument and fat body. Immunostaining indicated that LmCYP4G62 and LmCYP4G102 were highly abundant in oenocytes in these tissues. However, the two enzymes had a different subcellular distribution, with LmCYP4G62 localized on the plasma membrane and LmCYP4G102 dispersed throughout the oenocyte cytoplasm, presumably on the endoplasmic reticulum. RNA interference-mediated gene silencing against each of the two genes resulted in reduced CHC contents, in all classes for LmCYP4G102, but mostly shorter chain CHCs for LmCYP4G62. Silencing of both genes resulted in increased insecticide penetration through the cuticle, and increased locust susceptibility to desiccation and insecticides. Conclusion Our studies suggest that both LmCYP4G62 and LmCYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration, but they are not fully redundant. Further, the two LmCYP4G genes might be used as new targets for insect pest management. © 2020 Society of Chemical Industry.

The P450 family (CYP genes) of arthropods encodes diverse enzymes involved in the metabolism of foreign compounds and in essential endocrine or ecophysiological functions. The P450 sequences (CYPome) from 40 arthropod species were manually curated, including 31 complete CYPomes, and a maximum likelihood phylogeny of nearly 3000 sequences is presented. Arthropod CYPomes are assembled from members of six CYP clans of variable size, the CYP2, CYP3, CYP4 and mitochondrial clans, as well as the CYP20 and CYP16 clans that are not found in Neoptera. CYPome sizes vary from two dozen genes in some parasitic species to over 200 in species as diverse as collembolans or ticks. CYPomes are comprised of few CYP families with many genes and many CYP families with few genes, and this distribution is the result of dynamic birth and death processes. Lineage-specific expansions or blooms are found throughout the phylogeny and often result in genomic clusters that appear to form a reservoir of catalytic diversity maintained as heritable units. Among the many P450s with physiological functions, six CYP families are involved in ecdysteroid metabolism. However, five so-called Halloween genes are not universally represented and do not constitute the unique pathway of ecdysteroid biosynthesis. The diversity of arthropod CYPomes has only partially been uncovered to date and many P450s with physiological functions regulating the synthesis and degradation of endogenous signal molecules (including ecdysteroids) and semiochemicals (including pheromones and defense chemicals) remain to be discovered. Sequence diversity of arthropod P450s is extreme, and P450 sequences lacking the universally conserved Cys ligand to the heme have evolved several times. A better understanding of P450 evolution is needed to discern the relative contributions of stochastic processes and adaptive processes in shaping the size and diversity of CYPomes.

The cotton bollworm, Helicoverpa armigera, is a generalist herbivore widely distributed over the world and is a major lepidopteran pest on cotton. Studies, especially from Asia, show that it relies on cytochrome P450 monooxygenases with broad substrate specificities to protect itself from pesticides. The number of P450s may have expanded in the processes of coping with the wide diversity of phytochemicals that the insect encounters among its numerous host plants. In order to examine the metabolic capabilities of these P450s, we focused here on all ten P450s of the Helicoverpa armigera CYP6AE subfamily, which can be easily induced by plant toxins and pyrethroids. These P450s, along with cytochrome P450 reductase, were heterologously expressed in insect cells and compared functionally. In vitro metabolism showed that all CYP6AE subfamily members can convert esfenvalerate to 4′-hydroxyesfenvalerate efficiently except CYP6AE20. In contrast, none of the recombinant CYP6AE enzymes could metabolise gossypol under our experimental conditions. Epoxidation capabilities were observed in the CYP6AE subfamily, aldrin can be converted to dieldrin at rates up to 0.45 ± 0.04 pmol/min/pmol P450. Seven P450s in this subfamily can metabolise imidacloprid, but with lower efficiency than Bemisia tabaci CYP6CM1vQ. CYP6AE20 had virtually no metabolic competence to these four compounds but could metabolise several model fluorogenic substrates. These results showed the broad substrate spectrum of H. armigera CYP6AE P450s and suggest a limited role of gossypol upon the evolution of H. armigera CYP6AE genes.

Lepidopteran P450s of the CYP6B and CYP9A subfamilies are thought to play important roles in host plant adaptation and insecticide resistance. An increasing number of paralogs and orthologs with high levels of sequence identity have been found in these subfamilies by mining recent genome projects. However, the biochemical function of most of them remains unknown. A better understanding of the evolution of P450 genes and of the catalytic competence of the enzymes they encode is needed to facilitate studies of host plant use and insecticide resistance. Here, we focused on the full complement of CYP6B (4 genes) and CYP9A (7 genes) in the generalist herbivore, Helicoverpa armigera. These P450s were heterologously expressed in Sf9 cells and compared functionally. In vitro assays showed that all CYP6B and CYP9A P450s can metabolize esfenvalerate efficiently, except for the evolutionarily divergent CYP6B43. A new 2'-hydroxy-metabolite of esfenvalerate was identified and found to be the main metabolite produced by CYP9A12. All tested P450s showed only low induction responses to esfenvalerate. To put these results from H. armigera P450s in perspective, 158 complete CYP6B and 100 complete CYP9A genes from 34 ditrysian species were manually curated. The CYP9A subfamily was more widespread than the CYP6B subfamily and the latter showed dramatic gains and losses, with ten species lacking CYP6B genes. Two adjacent CYP6B loci were found on chromosome 21, with different fates during the evolution of Lepidoptera. The diversity and functional redundancy of CYP6B and CYP9A genes challenge resistance management and pest control strategies as many P450s are available to insects to cope with chemical stresses they encounter.

The neonicotinoid insecticides have raised concerns regarding the health of bee pollinators. New research has identified a P450 enzyme that protects honey bees and bumble bees from the toxicity of two neonicotinoids, thiacloprid and acetamiprid. This P450 enzyme provides a margin of safety to bees.

The large and diverse P450 (CYP) superfamily encodes enzymes with a wide spectrum of monooxygenase and related activities. Insect P450 enzymes of the CYP4G subfamily are known to catalyze the synthesis of cuticular hydrocarbons that serve multiple functions from desiccation resistance to chemical communication. These functions are essential for survival. In order to understand the evolution of insect CYP4G genes, 368 sequences from 24 insect orders and 167 species were mined and analyzed. The genomes of most species of Neoptera carry at least two CYP4G genes that are paralogs of the two Drosophila CYP4G genes. The duplication of the original CYP4G is basal to Neoptera and no CYP4G is found in Paleoptera, or beyond the class Insecta. The sequences of CYP4G and particularly their active site have been highly conserved over 400 MY, but all CYP4G sequences are characterized by a +44 residue insertion between the G and H helices, which protrudes from the globular structure of the enzyme distally from the membrane anchor. Although it is generally considered that genes with highly conserved sequence and function are evolutionarily "stable", the evidence from the CYP4G subfamily shows that since their initial duplication over 400 MYA, these genes have experienced many gene births and deaths. The CYP4G1 homolog has been lost several times, and is missing in five orders of insects. These losses are both ancient, as in all Hemiptera and Thysanoptera, and more recent as in honey bees. Serial duplications leading to CYP4G gene clusters have also been observed, as in house flies and in fireflies. The detailed evolutionary history of CYP4G genes does not support the "stability" of these essential genes, but rather a "revolving door" pattern where their essential function is maintained despite an apparently random birth and death process. The dual function of cuticular hydrocarbons, in desiccation resistance achieved mainly by the quantity of hydrocarbons produced and in chemical communication, achieved by the blend of hydrocarbons produced, may explain the apparently paradoxical evolution of CYP4G genes.

This article discusses the insect CYP genes and P450 enzymes. Cytochrome P450, or CYP genes, constitutes one of the largest family of genes, with representatives in virtually all living organisms, from bacteria to protists, plants, fungi, and animals. The multiple functions of insect P450 enzymes, their complex biochemistry, and their toxicological and physiological importance are presented. Physiological functions are not restricted to one branch of the P450 evolutionary tree, and the ramifications that seem typical of “environmental response genes” are found in both microsomal and mitochondrial P450 encoding genes. The CYP3 and CYP4 clans seem the most diverse, and the smaller CYP2 and mitochondrial CYP clans seem to have proportionally more genes found as orthologs across species. There are many physiological functions for P450s that just are not known or suspected. CYP4C7 was shown to be selectively expressed in the corpora allata and to metabolize JH and its precursors to new metabolites.

The cotton bollworm Helicoverpa armigera, is one of the world’s major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance., Cotton bollworm is an important agricultural pest with widespread resistance to insecticides. Here Wang et al. identifies CYP6AEs from cotton bollworm involved in detoxifying plant toxins and chemical insecticides through the CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism.

Cytochrome P450 monooxygenases (CYPs) serve many functions in insects, from the regulation of development to xenobiotic detoxification. Several conserved CYPs have been shown to play a role in insect growth and development. CYP303A1 is a highly conserved CYP with a single ortholog in most insects, but its underlying molecular characteristics and specific physiological functions remain poorly understood. In Drosophila melanogaster and Locusta migratoria, CYP303A1 is indispensable for eclosion to adult. Here, we report additional functions of the locust gene LmCYP303A1 in nymphal molts, cuticular lipid deposition and insecticide penetration. RT-qPCR revealed that LmCYP303A1 had a high expression level before ecdysis and was highly expressed in integument, wing pads, foregut and hindgut. Suppression of LmCYP303A1 expression by RNA interference (RNAi) caused a lethal phenotype with molting defect from nymph to nymph. In addition, LmCYP303A1 RNAi resulted in locusts being more susceptible to desiccation and to insecticide toxicity. Furthermore, knockdown of LmCYP303A1 efficiently suppressed the transcript level of key genes (ELO7, FAR15 and CYP4G102) responsible for cuticular hydrocarbon (CHC) synthesis, which led to a decrease in some CHC levels. Taken together, our results suggest that one of the functions of LmCYP303A1 is to regulate the biosynthesis of CHC, which plays critical roles in protecting locusts from water loss and insecticide penetration.

Cyanogenic glucosides are widespread defence compounds in plants, and they are also found in some arthropods, especially within Lepidoptera. The aliphatic linamarin and lotaustralin are the most common cyanogenic glucosides in Lepidoptera, and they are biosynthesised de novo, and/or sequestered from food plants. Their biosynthetic pathway was elucidated in the burnet moth, Zygaena filipendulae, and consists of three enzymes: two cytochrome P450 enzymes, CYP405A2 and CYP332A3, and a glucosyl transferase, UGT33A1. Heliconius butterflies also produce linamarin and lotaustralin and have close homologs to CYP405A2 and CYP332A3. To unravel the evolution of the pathway in Lepidoptera, we performed phylogenetic analyses on all available CYP405 and CYP332 sequences. CYP332 sequences were present in almost all Lepidoptera, while the distribution of CYP405s among butterflies and moths was much more limited. Negative purifying selection was found in both CYP enzyme families, indicating that the biosynthesis of CNglcs is an old trait, and not a newly evolved pathway. We compared CYP405A2 to its close paralog, CYP405A3, which is not involved in the biosynthetic pathway. The only significant difference between these two enzymes is a smaller substrate binding pocket in CYP405A2, which would make the enzyme more substrate specific. We consider it likely that the biosynthetic pathway of CNglcs in butterflies and moths have evolved from a common pathway, perhaps based on a predisposition for detoxifying aldoximes by way of a CYP332. Later the aldoxime metabolising CYP405s evolved, and a UGT was recruited into the pathway to establish de novo biosynthesis of CNglcs.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

International audience; The recent accumulation of molecular studies on mutations in insects, ticks and mites conferring resistance to insecticides, acaricides and biopesticides is reviewed. Resistance is traditionally classified by physiological and biochemical criteria, such as target-site insensitivity and metabolic resistance. However, mutations are discrete molecular changes that differ in their intrinsic frequency, effects on gene dosage and fitness consequences. These attributes in turn impact the population genetics of resistance and resistance management strategies, thus calling for a molecular genetic classification. Mutations in structural genes remain the most abundantly described, mostly in genes coding for target proteins. These provide the most compelling examples of parallel mutations in response to selection. Mutations causing upregulation and downregulation of genes, both in cis (in the gene itself) and in trans (in regulatory processes) remain difficult to characterize precisely. Gene duplications and gene disruption are increasingly reported. Gene disruption appears prevalent in the case of multiple, hetero-oligomeric or redundant targets.

BACKGROUND: The withdrawal of malathion in the European Union in 2009 resulted in a large increase in lambda-cyhalothrin applications for the control of the Mediterranean fruit fly, Ceratitis capitata, in Spanish citrus crops. RESULTS: Spanish field populations of C. capitata have developed resistance to lambda-cyhalothrin (6–14-fold), achieving LC50 values (129–287 ppm) higher than the recommended concentration for field treatments (125 ppm). These results contrast with the high susceptibility to lambda-cyhalothrin found in three Tunisian field populations. We have studied the mechanism of resistance in the laboratory-selected resistant strain W-1K�� (205-fold resistance). Bioassays with synergists showed that resistance was almost completely suppressed by the P450 inhibitor PBO. The study of the expression of 53 P450 genes belonging to the CYP4, CYP6, CYP9 and CYP12 families in C. capitata revealed that CYP6A51 was overexpressed (13–18-fold) in the resistant strain. The W-1K�� strain also showed high levels of cross-resistance to etofenprox (240-fold) and deltamethrin (150-fold). CONCLUSION: Field-evolved resistance to lambda-cyhalothrin has been found in C. capitata. Metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant strain W-1K�� . The levels of cross-resistance found may compromise the effectiveness of other pyrethroids for the control of this species. © 2014 Society of Chemical Industry Supporting information may be found in the online version of this article.

International audience; Background: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. Results: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. Conclusions: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis., Biomphalaria glabrata is a fresh water snail that acts as a host for trematode Schistosoma mansoni that causes intestinal infection in human. This work describes the genome and transcriptome analyses from 12 different tissues of B glabrata, and identify genes for snail behavior and evolution.

Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment.

Insect cytochrome P450 monooxygenases (CYPs) play essential roles in both xenobiotic metabolism and developmental processes. However, the exact physiological function of many CYP genes remains largely unknown. Screening the expression of the CYP genes from the CYP2 and mitochondrial CYP clans of Drosophila melanogaster revealed that Cyp303a1 is highly expressed in the pupal stage. Knockdown of CYP303A1 transcripts by RNAi using the Gal4/UAS system with a ubiquitous driver (tubulin-Gal4) in Drosophila or by dsRNA injection in the last nymph stage of Locusta migratoria resulted in severe defects in eclosion and lethality during and after adult emergence. In Drosophila, tissue-specific RNAi of Cyp303a1 with a wing-specific driver (MS1096-Gal4) revealed that Cyp303a1 was essential for wing extension. Stage-specific RNAi of Cyp303a1 using Gal80ts for thermal-dependent-suppression found that the expression of Cyp303a1 at the middle pupal stage was absolutely required. Meanwhile, Cyp303a1 mutants exhibited more than 80% lethality at the late embryonic development stages. Embryonic lethality of the Cyp303a1 mutants was fully rescued by the ubiquitous overexpression of exogenous Cyp303a1. Taken together, we conclude that Cyp303a1 is indispensable for embryonic development and adult eclosion in D. melanogaster, the latter role being conserved over 400 million years of insect evolution.

The complete genome of the two-spotted spider mite, Tetranychus urticae, has been reported. This is the first sequenced genome of a highly polyphagous and resistant agricultural pest. The question as to what the genome offers the community working on spider mite control is addressed.

International audience; BACKGROUND: To reduce rates of synthetic insecticide applications, natural product alternatives and synergists are needed. A study has been made of the toxicity of ethanolic senescent leaf extracts (SLEs) of Jatropha gossypifolia and Melia azedarach on larvae of the noctuid pest Spodoptera frugiperda. Their effects as syngergists and inhibitors of several enzyme activities are also reported. RESULTS: When added to the diet, M. azedarach SLE showed lower toxicity than J. gossypifolia SLE. However, after 2 weeks on the diet, the M. azedarach SLE proved to be lethal to 100% of the larval population. Artificial diets with both SLEs have an antifeedant effect on armyworm larvae. Acute toxicity after topical application in a dipping assay was relatively low for both J. gossypifolia and M. azedarach SLEs (LC50 of 2.6 and 1.4 g L-1, respectively, after 24 h). However, mixtures of the SLEs of M. azedarach and J. gossypifolia had a strong synergistic effect with cypermethrin. Synergism was higher with the J. gossypifolia SLE, perhaps because it contains several natural products with a methylenedioxyphenyl moiety. Both extracts inhibited P450, general esterase and acetylcholinesterase activities in vitro and in vivo. CONCLUSION: Both J. gossypifolia and M. azedarach SLEs are antifeedants to armyworm larvae when present in the food, and also have a synergistic effect with cypermethrin in topical assays. Although the synergistic effect is less than with piperonyl butoxide, both SLEs have some inhibitor activity against detoxification enzymes and acetylcholinesterase. Thus J. gossypifolia and M. azedarach SLEs may be considered as ecofriendly approaches for the control of S. frugiperda in order to reduce cypermethrin usage. Copyright (c) 2012 Society of Chemical Industry

7 páginas, 2 figuras, 4 tablas -- PAGS nros. 756-762, Target site insensitivity and metabolic resistance mediated by esterases have been previously suggested to be involved in resistance to malathion in a field-derived strain (W) of Ceratitis capitata. In the present study, we have obtained the coding sequence for acetylcholinesterase (AChE) gene (Ccace) of C. capitata. An allele of Ccace carrying only a point mutation Gly328Ala (Torpedo numbering) adjacent to the glutamate of the catalytic triad was found in individuals of the W strain. Adult flies homozygotes for this mutant allele showed reduced AChE activity and less sensitivity to inhibition by malaoxon, showing that target site insensitivity is one of the factors of malathion resistance. In addition, all individuals from the resistant W strain showed reduced aliesterase activity, which has been associated with specific malathion resistance in higher Diptera. However, the αE7 gene (CcαE7), sequenced in susceptible and resistant individuals, did not carry any of the mutations associated with organophosphorus insecticide resistance in other Diptera. Another esterase mechanism, perhaps a carboxylesterase selective for malathion, in addition to mutant AChE, thus contributes to malathion resistance in C. capitata, This study received financial support from CICYT (AGL2004-05169).

Interactions between a soluble form of microsomal cytochrome b(5) (b(5)) from Musca domestica (housefly) and Bacillus megaterium flavocytochrome P450 BM3 and its component reductase (CPR), heme (P450) and FAD/NADPH-binding (FAD) domains were analyzed by a combination of steady-state and stopped-flow kinetics methods, and optical spectroscopy techniques. The high affinity binding of b(5) to P450 BM3 induced a low-spin to high-spin transition in the P450 heme iron (K(d) for b(5) binding = 0.44 microM and 0.72 microM for the heme domain and intact flavocytochrome, respectively). The b(5) had modest inhibitory effects on steady-state turnover of P450 BM3 with fatty acids, and the ferrous-carbon monoxy P450 complex was substantially stabilized on binding b(5). Single turnover reduction of b(5) by BM3 using stopped-flow absorption spectroscopy (k(lim) = 116 s(-1)) was substantially faster than steady-state reduction of b(5) by P450 BM3 (or its CPR and FAD domains), indicating rate-limiting step(s) other than BM3 flavin-to-b(5) heme electron transfer in the steady-state reaction. Steady-state b(5) reduction by P450 BM3 was considerably accelerated at high ionic strength. Pre-reduction of P450 BM3 by NADPH decreased the k(lim) for b(5) reduction approximately 10-fold, and also resulted in a lag phase in steady-state b(5) reduction that was likely due to BM3 conformational perturbations sensitive to the reduction state of the flavocytochrome. Ferrous b(5) could not reduce the ferric P450 BM3 heme domain under anaerobic conditions, consistent with heme iron reduction potentials of the two proteins. However, rapid oxidation of both hemoproteins occurred on aeration of the ferrous protein mixture (and despite the much slower autoxidation rate of b(5) in isolation), consistent with electron transfer occurring from b(5) to the oxyferrous P450 BM3 in the complex. The results demonstrate that strong interactions occur between a eukaryotic b(5) and a model prokaryotic P450. Binding of b(5) perturbs BM3 heme iron spin-state equilibrium, as is seen in many physiologically relevant b(5) interactions with eukaryotic P450s. These results are consistent with the conservation of structure of P450s (particularly at the heme proximal face) between prokaryotes and eukaryotes, and may point to as yet undiscovered roles for b(5)-like proteins in the control of activities of certain prokaryotic P450s.

Complete analysis of the phosphorylation of serine and threonine residues directly from biological extracts is still at an early stage and will remain a challenging goal for many years. Analysis of phosphorylated proteins and identification of the phosphorylated sites in a crude biological extract is a major topic in proteomics, since phosphorylation plays a dominant role in post-translational protein modification. Beta elimination of the serine/threonine-bound phosphate by alkali action generates (methyl)dehydroalanine. The reactivity of this group susceptible of nucleophilic attacks might be used as a tool for phosphoproteome analysis. Most of the known serine/threonine kinases recognize motifs in protein targets that are rich in lysine(s) and/or arginine(s). The (methyl)dehydroalanine resulting from beta elimination of the serine/threonine-bound phosphate by alkali action is likely to react with the amino groups of these neighboring amino acids. Furthermore, the addition reaction of dehydroalanine-peptides with a nucleophilic group more likely generates diastereoisomers derivatives. The internal cyclic bonds and/or the stereoisomer peptide derivatives thus generated confer resistance to trypsin cleavage and/or constitute stop signal s for exopeptidases such as carboxypeptidase. This might form the basis of a method to facilitate the systematic identification of phosphorylated peptides.

The first fully sequenced insect genomes were those of the fruitfly and the mosquito, both from the order Diptera. Now, with an increasing number and diversity of insect genomes becoming available, the diversity of insect P450 genes can be better appreciated and tentative ideas about the evolution of the CYP (cytochrome P450) superfamily in insects can be proposed. There are four large clades of insect P450 genes that existed before the divergence of the class Insecta and that are also represented by CYP families in vertebrates: the CYP2 clade, the CYP3 clade, the CYP4 clade and the mitochondrial P450 clade. P450s with known or suspected physiological functions are present in each of these clades and only a dozen genes appear to have orthologues or very close paralogues in each insect genome. P450 enzymes from each of these clades have been linked to insecticide resistance or to the metabolism of natural products and xenobiotics. In particular, insects appear to maintain a repertoire of mitochondrial P450 paralogues devoted to the response to environmental challenges.

Recent studies on jasmonic acid (JA) biosynthetic mutants have shown that jasmonates play essential roles in pollen maturation and dehiscence and wound-induced defence against biotic attacks. To better understand the biosynthetic mechanisms of this essential plant hormone, we isolated an Arabidopsis knock-out mutant defective in the JA biosynthetic gene CYP74A (allene oxide synthase, AOS) using reverse genetics screening methods. This enzyme catalyses dehydration of the hydroperoxide to an unstable allene oxide in the JA biosynthetic pathway. Endogenous JA levels, which increase 100-fold 1 h after wounding in wild-type plants, do not increase after wounding in the aos mutant. In addition, the mutant showed severe male sterility due to defects in anther and pollen development. The male-sterile phenotype was completely rescued by exogenous application of methyl jasomonate and by complementation with constitutive expression of the AOS gene. RT-PCR analysis showed that the induction of transcripts for vegetative storage protein and lipoxygenase genes, previously shown to be inducible by wound and jasmonate application in the wild-type, was absent in the aos mutant. In transgenic plants constitutively expressing AOS, wound-induced JA levels were 50-100% higher compared to wild-type plants. Taken together with JA deficiency in the aos mutant, our results show that AOS is critical for the biosynthesis of all biologically active jasmonates. Our results also suggest that AOS expression is limiting JA levels in wounded plants, but that the AOS hydroperoxide substrate levels, controlled by upstream enzymes (lipoxygenase and phospholipase), determine JA levels in unwounded plants.

Nature Communications 8: Article number: 15451 (2017); Published 16 May 2017; Updated 23 August 2017 The original version of this Article contained an error in the spelling of the author Leon Di Stefano, which was incorrectly given as Leon di Stephano. This has now been corrected in both the PDF andHTML versions of the Article.

P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing.

Background Spodoptera frugiperda (Noctuidae) is a major agricultural pest throughout the American continent. The highly polyphagous larvae are frequently devastating crops of importance such as corn, sorghum, cotton and grass. In addition, the Sf9 cell line, widely used in biochemistry for in vitro protein production, is derived from S. frugiperda tissues. Many research groups are using S. frugiperda as a model organism to investigate questions such as plant adaptation, pest behavior or resistance to pesticides. Results In this study, we constructed a reference transcriptome assembly (Sf_TR2012b) of RNA sequences obtained from more than 35 S. frugiperda developmental time-points and tissue samples. We assessed the quality of this reference transcriptome by annotating a ubiquitous gene family - ribosomal proteins - as well as gene families that have a more constrained spatio-temporal expression and are involved in development, immunity and olfaction. We also provide a time-course of expression that we used to characterize the transcriptional regulation of the gene families studied. Conclusion We conclude that the Sf_TR2012b transcriptome is a valid reference transcriptome. While its reliability decreases for the detection and annotation of genes under strong transcriptional constraint we still recover a fair percentage of tissue-specific transcripts. That allowed us to explore the spatial and temporal expression of genes and to observe that some olfactory receptors are expressed in antennae and palps but also in other non related tissues such as fat bodies. Similarly, we observed an interesting interplay of gene families involved in immunity between fat bodies and antennae. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-704) contains supplementary material, which is available to authorized users.

Cyanogenic glucosides are among the most widespread defense chemicals of plants. Upon plant tissue disruption, these glucosides are hydrolyzed to a reactive hydroxynitrile that releases toxic hydrogen cyanide (HCN). Yet many mite and lepidopteran species can thrive on plants defended by cyanogenic glucosides. The nature of the enzyme known to detoxify HCN to β-cyanoalanine in arthropods has remained enigmatic. Here we identify this enzyme by transcriptome analysis and functional expression. Phylogenetic analysis showed that the gene is a member of the cysteine synthase family horizontally transferred from bacteria to phytophagous mites and Lepidoptera. The recombinant mite enzyme had both β-cyanoalanine synthase and cysteine synthase activity but enzyme kinetics showed that cyanide detoxification activity was strongly favored. Our results therefore suggest that an ancient horizontal transfer of a gene originally involved in sulfur amino acid biosynthesis in bacteria was co-opted by herbivorous arthropods to detoxify plant produced cyanide. DOI: http://dx.doi.org/10.7554/eLife.02365.001, eLife digest Hydrogen cyanide is a poison that is deadly for most forms of life. Also known as prussic acid, it has killed countless humans throughout history in accidents and during the Holocaust. Hydrogen cyanide is also used by plants to defend themselves against insects and other herbivorous animals. Many plants produce chemicals called cyanogenic glycosides that can be converted into hydrogen cyanide when the plant is eaten. This is an ancient and efficient defense against all sorts of herbivores, including humans. For instance, cassava is a key source of food in sub-Saharan Africa and South America, but it contains cyanogenic glucosides and is highly toxic if eaten in unprocessed form. However, some insects and mites can thrive on cyanogenic plants, often to the extent of becoming pests on these plants. Certain moths, such as burnet moths, have gone further and now depend on cyanogenic glucosides for their own defenses against predators such as birds. How these mites and insects are capable of fending off cyanide toxicity has long remained a mystery. Now Wybouw et al. have identified a mite enzyme that detoxifies hydrogen cyanide to produce a compound called beta-cyanoalanine. Remarkably, the DNA that encodes this enzyme did not evolve in animals but originally belonged to a bacterium. Wybouw et al. show that the gene was transferred to the genome of the spider mite Tetranychus urticae perhaps a few hundred million years ago. An equivalent gene was also found in moths and butterflies, which explains why these insects can thrive on plants that produce hydrogen cyanide. This lateral gene transfer from bacteria to animals is a remarkable coalition of two kingdoms against another, and illustrates a new aspect of the chemical warfare between plants and animals. This study also increases our awareness of the importance of laterally transferred genes in the genomes of higher organisms. DOI: http://dx.doi.org/10.7554/eLife.02365.002

Up-regulation of detoxifying enzymes in insecticide-resistant strains of the house fly is a common mechanism for metabolic resistance. However, the molecular basis of this increased insecticide metabolism is not well understood. In the multiresistant Rutgers strain, several cytochromes P450 and glutathione S-transferases are constitutively overexpressed at the transcriptional level. Overexpression is the result of trans-regulation, and a regulatory gene has been located on chromosome 2. A Gly137 to Asp point mutation in alphaE7 esterase gene, leading to the loss of carboxylesterase activity, has been associated with organophosphate resistance in the house fly and the sheep blowfly. We show here that purified recombinant CYP6A1 is able to detoxify diazinon with a high efficiency. We also show that either the Gly137 to Asp point mutation in alphaE7 esterase gene or a deletion at this locus confer resistance and overproduction of the CYP6A1 protein. Based on these findings, we propose it is the absence of the wild-type Gly137 allele of the alphaE7 gene that releases the transcriptional repression of genes coding for detoxification enzymes such as CYP6A1, thereby leading to metabolic resistance to diazinon.