Trans-driven variation in expression is common among detoxification genes in the extreme generalist herbivore Tetranychus urticae.

The extreme adaptation potential of the generalist herbivore Tetranychus urticae (the two-spotted spider mite) to pesticides as well as diverse host plants has been associated with clade-specific gene expansions in known detoxifying enzyme families, and with extensive and rapid transcriptional responses. However, how this broad transcriptional potential is regulated remains largely unknown. Using a parental/F1 design in which four inbred strains were crossed to a common inbred strain, we assessed the genetic basis and inheritance of gene expression variation in T. urticae. Mirroring known phenotypic variation in the progenitor strains of the inbreds, we confirmed that the inbred strains we created were genetically distinct, varied markedly in pesticide resistance, and also captured variation in host plant fitness as is commonly observed in this species. By examining differences in gene expression between parents and allele-specific expression in F1s, we found that variation in RNA abundance was more often explained in trans as compared to cis, with the former associated with dominance in inheritance. Strikingly, in a gene ontology analysis, detoxification genes of the cytochrome P450 monooxygenase (CYP) family, as well as dioxygenases (DOGs) acquired from horizontal gene transfer from fungi, were specifically enriched at the extremes of trans-driven up- and downregulation. In particular, multiple CYPs and DOGs with broad substrate-specificities for pesticides or plant specialized compounds were exceptionally highly upregulated as a result of trans-regulatory variation, or in some cases synergism of cis and trans, in the most multi-pesticide resistant strains. Collectively, our findings highlight the potential importance of trans-driven expression variation in genes associated with xenobiotic metabolism and host plant use for rapid adaptation in T. urticae, and also suggests modular control of these genes, a regulatory architecture that might ameliorate negative pleiotropic effects.

Reduced proinsecticide activation by cytochrome P450 confers coumaphos resistance in the major bee parasite Varroa destructor.

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 [14C] 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.

Genome mapping coupled with CRISPR gene editing reveals a P450 gene confers avermectin resistance in the beet armyworm.

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.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness.

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.

The Role of Cytochrome P450s in Insect Toxicology and Resistance.

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.

Origin and evolution of the CYP4G subfamily in insects, cytochrome P450 enzymes involved in cuticular hydrocarbon synthesis.

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.

Knockdown of LmCYP303A1 alters cuticular hydrocarbon profiles and increases the susceptibility to desiccation and insecticides in Locusta migratoria.

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.

Analysis and preliminary characterisation of the cytochrome P450 monooxygenases from Frankia sp. EuI1c (Frankia inefficax sp.).

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.

Evolution of the Biosynthetic Pathway for Cyanogenic Glucosides in Lepidoptera.

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.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

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.

A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae.

Plants produce a wide range of allelochemicals to defend against herbivore attack, and generalist herbivores have evolved mechanisms to avoid, sequester, or detoxify a broad spectrum of natural defense compounds. Successful arthropod pests have also developed resistance to diverse classes of pesticides and this adaptation is of critical importance to agriculture. To test whether mechanisms to overcome plant defenses predispose the development of pesticide resistance, we examined adaptation of the generalist two-spotted spider mite, Tetranychus urticae, to host plant transfer and pesticides. T. urticae is an extreme polyphagous pest with more than 1,100 documented hosts and has an extraordinary ability to develop pesticide resistance. When mites from a pesticide-susceptible strain propagated on bean were adapted to a challenging host (tomato), transcriptional responses increased over time with ~7.5% of genes differentially expressed after five generations. Whereas many genes with altered expression belonged to known detoxification families (like P450 monooxygenases), new gene families not previously associated with detoxification in other herbivores showed a striking response, including ring-splitting dioxygenase genes acquired by horizontal gene transfer. Strikingly, transcriptional profiles of tomato-adapted mites resembled those of multipesticide-resistant strains, and adaptation to tomato decreased the susceptibility to unrelated pesticide classes. Our findings suggest key roles for both an expanded environmental response gene repertoire and transcriptional regulation in the life history of generalist herbivores. They also support a model whereby selection for the ability to mount a broad response to the diverse defense chemistry of plants predisposes the evolution of pesticide resistance in generalists.

An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis.

Insects use hydrocarbons as cuticular waterproofing agents and as contact pheromones. Although their biosynthesis from fatty acyl precursors is well established, the last step of hydrocarbon biosynthesis from long-chain fatty aldehydes has remained mysterious. We show here that insects use a P450 enzyme of the CYP4G family to oxidatively produce hydrocarbons from aldehydes. Oenocyte-directed RNAi knock-down of Drosophila CYP4G1 or NADPH-cytochrome P450 reductase results in flies deficient in cuticular hydrocarbons, highly susceptible to desiccation, and with reduced viability upon adult emergence. The heterologously expressed enzyme converts C(18)-trideuterated octadecanal to C(17)-trideuterated heptadecane, showing that the insect enzyme is an oxidative decarbonylase that catalyzes the cleavage of long-chain aldehydes to hydrocarbons with the release of carbon dioxide. This process is unlike cyanobacteria that use a nonheme diiron decarbonylase to make alkanes from aldehydes with the release of formate. The unique and highly conserved insect CYP4G enzymes are a key evolutionary innovation that allowed their colonization of land.

Genotype to phenotype, the molecular and physiological dimensions of resistance in arthropods.

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.

Establishment and analysis of a reference transcriptome for Spodoptera frugiperda.

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.

Insights into unique features of Drosophila CYP4G enzymes.

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most intriguing insect P450s in terms of structure and function. In Drosophila, CYP4G1 is highly expressed in the oenocytes and is the last enzyme in the biosynthesis of cuticular hydrocarbons, while CYP4G15 is expressed in the brain and is of unknown function. Both proteins have a CYP4G-specific and characteristic amino acid sequence insertion corresponding to a loop between the G and H helices whose function is unclear. Here we address these enigmatic structural and functional features of Drosophila CYP4Gs. First, we used reverse genetics to generate D. melanogaster strains in which all or part of the CYP4G-specific loop was removed from CYP4G1. We showed that the full loop was not needed for proper folding of the P450, but it is essential for function, and that just a short stretch of six amino acids is required for the enzyme's ability to make hydrocarbons. Second, we confirmed by immunocytochemistry that CYP4G15 is expressed in the brain and showed that it is specifically associated with the cortex glia cell subtype. We then expressed CYP4G15 ectopically in oenocytes, revealing that it can produce of a blend of hydrocarbons, albeit to quantitatively lower levels resulting in only a partial rescue of CYP4G1 knockdown flies. The CYP4G1 structural variants studied here should facilitate the biochemical characterization of CYP4G enzymes. Our results also raise the question of the putative role of hydrocarbons and their synthesis by cortex glial cells.

Aliens in the CYPome of the black fungus gnat, Bradysia coprophila.

The diverse cytochrome P450 enzymes of insects play essential physiological roles and also play important roles in the metabolism of environmental chemicals such as insecticides. We manually curated the complement of P450 (CYP) genes, or CYPome, of the black fungus gnat, Bradysia (Sciara) coprophila (Diptera, Sciaroidea), a species with a variable number of chromosomes. This CYPome carries two types of "alien" P450 genes. The first type of alien P450s was found among the 163 CYP genes of the core genome (autosomes and X). They consist of 28 sequences resulting from horizontal gene transfer, with closest sequences not found in insects, but in other arthropods, often Collembola. These genes are not contaminants, because they are expressed genes with introns, found in synteny with regular dipteran genes, also found in B. odoriphaga and B. hygida. Two such "alien" genes are representatives of CYP clans not otherwise found in insects, a CYP53 sequence related to fungal CYP53 genes, and a CYP19-like sequence similar to some collembolan sequences but of unclear origin. The second type of alien P450s are represented by 99 sequences from germline-restricted chromosomes (GRC). While most are P450 pseudogenes, 33 are apparently intact, with half being more closely related to P450s from Cecidomyiidae than from Sciaridae, thus supporting the hypothesis of a cross-family hybridization origin of the GRC.

A nuclear receptor HR96-related gene underlies large trans-driven differences in detoxification gene expression in a generalist herbivore.

The role, magnitude, and molecular nature of trans-driven expression variation underlying the upregulation of detoxification genes in pesticide resistant arthropod populations has remained enigmatic. In this study, we performed expression quantitative trait locus (eQTL) mapping (n = 458) between a pesticide resistant and a susceptible strain of the generalist herbivore and crop pest Tetranychus urticae. We found that a single trans eQTL hotspot controlled large differences in the expression of a subset of genes in different detoxification gene families, as well as other genes associated with host plant use. As established by additional genetic approaches including RNAi gene knockdown, a duplicated gene with a nuclear hormone receptor HR96-related ligand-binding domain was identified as causal for the expression differences between strains. The presence of a large family of HR96-related genes in T. urticae may enable modular control of detoxification and host plant use genes, facilitating this species' known and rapid evolution to diverse pesticides and host plants.

Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster.

BACKGROUND: The benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(M)BOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8) form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5) belonging to the same CYP71C subfamily. The origin of this cluster is unknown. RESULTS: We show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase) and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region. CONCLUSIONS: These results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2) at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.

The P450 genes of the cat flea, Ctenocephalides felis: a CYPome in flux.

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.

Pyrethroid metabolism by eleven Helicoverpa armigera P450s from the CYP6B and CYP9A subfamilies.

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.