GPCR-MAPK signaling pathways underpin fitness trade-offs in whitefly.

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

Dual mutations in the whitefly nicotinic acetylcholine receptor ß1 subunit confer target-site resistance to multiple neonicotinoid insecticides.

Neonicotinoid insecticides, which target insect nicotinic acetylcholine receptors (nAChRs), have been widely and intensively used to control the whitefly, Bemisia tabaci, a highly damaging, globally distributed, crop pest. This has inevitably led to the emergence of populations with resistance to neonicotinoids. However, to date, there have been no reports of target-site resistance involving mutation of B. tabaci nAChR genes. Here we characterize the nAChR subunit gene family of B. tabaci and identify dual mutations (A58T&R79E) in one of these genes (BTß1) that confer resistance to multiple neonicotinoids. Transgenic D. melanogaster, where the native nAChR Dß1 was replaced with BTß1A58T&R79E, were significantly more resistant to neonicotinoids than flies where Dß1 were replaced with the wildtype BTß1 sequence, demonstrating the causal role of the mutations in resistance. The two mutations identified in this study replace two amino acids that are highly conserved in >200 insect species. Three-dimensional modelling suggests a molecular mechanism for this resistance, whereby A58T forms a hydrogen bond with the R79E side chain, which positions its negatively-charged carboxylate group to electrostatically repulse a neonicotinoid at the orthosteric site. Together these findings describe the first case of target-site resistance to neonicotinoids in B. tabaci and provide insight into the molecular determinants of neonicotinoid binding and selectivity.

Phylogenomic and functional characterization of an evolutionary conserved cytochrome P450-based insecticide detoxification mechanism in bees.

The regulatory process for assessing the risks of pesticides to bees relies heavily on the use of the honeybee, Apis mellifera, as a model for other bee species. However, the validity of using A. mellifera as a surrogate for other Apis and non-Apis bees in pesticide risk assessment has been questioned. Related to this line of research, recent work on A. mellifera has shown that specific P450 enzymes belonging to the CYP9Q subfamily act as critically important determinants of insecticide sensitivity in this species by efficiently detoxifying certain insecticide chemotypes. However, the extent to which the presence of functional orthologs of these enzymes is conserved across the diversity of bees is unclear. Here we used a phylogenomic approach to identify > 100 putative CYP9Q functional orthologs across 75 bee species encompassing all major bee families. Functional analysis of 26 P450s from 20 representative bee species revealed that P450-mediated detoxification of certain systemic insecticides, including the neonicotinoid thiacloprid and the butenolide flupyradifurone, is conserved across all major bee pollinator families. However, our analyses also reveal that CYP9Q-related genes are not universal to all bee species, with some Megachilidae species lacking such genes. Thus, our results reveal an evolutionary conserved capacity to metabolize certain insecticides across all major bee families while identifying a small number of bee species where this function may have been lost. Furthermore, they illustrate the potential of a toxicogenomic approach to inform pesticide risk assessment for nonmanaged bee species by predicting the capability of bee pollinator species to break down synthetic insecticides.

P450 gene duplication and divergence led to the evolution of dual novel functions and insecticide cross-resistance in the brown planthopper Nilaparvata lugens.

The sustainable control of many highly damaging insect crop pests and disease vectors is threatened by the evolution of insecticide resistance. As a consequence, strategies have been developed that aim to prevent or delay resistance development by rotating or mixing insecticides with different modes of action (MoA). However, these approaches can be compromised by the emergence of mechanisms that confer cross-resistance to insecticides with different MoA. Despite the applied importance of cross-resistance, its evolutionary underpinnings remain poorly understood. Here we reveal how a single gene evolved the capacity to detoxify two structurally unrelated insecticides with different MoA. Using transgenic approaches we demonstrate that a specific variant of the cytochrome P450 CYP6ER1, previously shown to confer resistance to the neonicotinoid imidacloprid in the brown planthopper, N. lugens, also confers cross-resistance to the phenylpyrazole ethiprole. CYP6ER1 is duplicated in resistant strains, and we show that while the acquisition of mutations in two encoded substrate recognition sites (SRS) of one of the parologs led to resistance to imidacloprid, a different set of mutations, outside of known SRS, are primarily responsible for resistance to ethiprole. Epistatic interactions between these mutations and their genetic background suggest that the evolution of dual resistance from the same gene copy involved functional trade-offs in respect to CYP6ER1 catalytic activity for ethiprole versus imidacloprid. Surprisingly, the mutations leading to ethiprole and imidacloprid resistance do not confer the ability to detoxify the insecticide fipronil, another phenylpyrazole with close structural similarity to ethiprole. Taken together, these findings reveal how gene duplication and divergence can lead to the evolution of multiple novel functions from a single gene. From an applied perspective they also demonstrate how cross-resistance to structurally unrelated insecticides can evolve, and illustrate the difficulty in predicting cross-resistance profiles mediated by metabolic mechanisms.

Multiple toxins and a protease contribute to the aphid-killing ability of Pseudomonas fluorescens PpR24.

Aphids are globally important pests causing damage to a broad range of crops. Due to insecticide resistance, there is an urgent need to develop alternative control strategies. In our previous work, we found Pseudomonas fluorescens PpR24 can orally infect and kill the insecticide-resistant green-peach aphid (Myzus persicae). However, the genetic basis of the insecticidal capability of PpR24 remains unclear. Genome sequencing of PpR24 confirmed the presence of various insecticidal toxins such as Tc (toxin complexes), Rhs (rearrangement hotspot) elements, and other insect-killing proteases. Upon aphids infection with PpR24, RNA-Seq analysis revealed 193 aphid genes were differentially expressed with down-regulation of 16 detoxification genes. In addition, 1325 PpR24 genes (542 were upregulated and 783 downregulated) were subject to differential expression, including genes responsible for secondary metabolite biosynthesis, the iron-restriction response, oxidative stress resistance, and virulence factors. Single and double deletion of candidate virulence genes encoding a secreted protease (AprX) and four toxin components (two TcA-like; one TcB-like; one TcC-like insecticidal toxins) showed that all five genes contribute significantly to aphid killing, particularly AprX. This comprehensive host-pathogen transcriptomic analysis provides novel insight into the molecular basis of bacteria-mediated aphid mortality and the potential of PpR24 as an effective biocontrol agent.

Characterisation of lepidopteran geranylgeranyl diphosphate synthase as a putative pesticide target.

Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.

MAPK-directed activation of the whitefly transcription factor CREB leads to P450-mediated imidacloprid resistance.

The evolution of insect resistance to pesticides poses a continuing threat to agriculture and human health. While much is known about the proximate molecular and biochemical mechanisms that confer resistance, far less is known about the regulation of the specific genes/gene families involved, particularly by trans-acting factors such as signal-regulated transcription factors. Here we resolve in fine detail the trans-regulation of CYP6CM1, a cytochrome P450 that confers resistance to neonicotinoid insecticides in the whitefly Bemisia tabaci, by the mitogen-activated protein kinase (MAPK)-directed activation of the transcription factor cAMP-response element binding protein (CREB). Reporter gene assays were used to identify the putative promoter of CYP6CM1, but no consistent polymorphisms were observed in the promoter of a resistant strain of B. tabaci (imidacloprid-resistant, IMR), which overexpresses this gene, compared to a susceptible strain (imidacloprid-susceptible, IMS). Investigation of potential trans-acting factors using in vitro and in vivo assays demonstrated that the bZIP transcription factor CREB directly regulates CYP6CM1 expression by binding to a cAMP-response element (CRE)-like site in the promoter of this gene. CREB is overexpressed in the IMR strain, and inhibitor, luciferase, and RNA interference assays revealed that a signaling pathway of MAPKs mediates the activation of CREB, and thus the increased expression of CYP6CM1, by phosphorylation-mediated signal transduction. Collectively, these results provide mechanistic insights into the regulation of xenobiotic responses in insects and implicate both the MAPK-signaling pathway and a transcription factor in the development of pesticide resistance.

Functional orthologs of honeybee CYP6AQ1 in stingless bees degrade the butenolide insecticide flupyradifurone.

Flupyradifurone (FPF), a novel butenolide insecticide binding to nicotinic acetylcholine receptors (nAChRs), has been shown to be less acutely toxic to western honey bees (Apis mellifera) than other insecticides such as neonicotinoids sharing the same target-site. A previous study revealed that this is due to enhanced oxidative metabolism of FPF, mediated by three cytochrome P450 monooxygenases (P450s), including CYP6AQ1. Therefore, we followed a toxicogenomics approach and investigated the potential role of functional CYP6AQ1 orthologs in FPF metabolism from eight different bee species, including stingless bees (Tribe: Meliponini). We conducted a phylogenetic analysis on four stingless bee species, including Frieseomelitta varia, Heterotrigona itama, Melipona quadrifasciata and Tetragonula carbonaria to identify CYP6AQ1-like functional orthologs. Three non-Meliponini, but tropical bee species, i.e., Ammobates syriacus, Euglossa dilemma and Megalopta genalis were analyzed as well. We identified candidate P450s in all (neo)tropical species with greater than 61% and 67% predicted protein sequence identities when compared to A. mellifera CYP6AQ1 and Bombus terrestris CYP6AQ26, respectively. Heterologous expression in High Five insect cells of these functional orthologs revealed a common coumarin substrate profile and a preference for the O-debenzylation of bulkier substrates. Competition assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) with these enzymes indicated inhibition of BOMFC metabolism by increasing concentrations of FPF. Furthermore, UPLC-MS/MS analysis revealed the capacity of all CYP6AQ1-like orthologs to metabolize FPF by hydroxylation in vitro at various levels, indicating a conserved FPF detoxification potential in different (neo)tropical bee species including Meliponini. This research, employing a toxicogenomics approach, provides important insights into the potential of stingless and other tropical bee species to detoxify FPF, and highlights the significance of investigating the detoxification mechanisms of insecticides in non-Apis bee species by molecular tools to inform risk assessment and conservation efforts.

Resilience of transfluthrin to oxidative attack by duplicated CYP6P9 variants known to confer pyrethroid resistance in the major malaria mosquito Anopheles funestus.

Resistance to common pyrethroids, such as deltamethrin and permethrin is widespread in the malaria mosquito Anopheles funestus and mainly conferred by upregulated cytochrome P450 monooxygenases (P450s). In the pyrethroid resistant laboratory strain An. funestus FUMOZ-R the duplicated genes CYP6P9a and CYP6P9b are highly upregulated and have been shown to metabolize various pyrethroids, including deltamethrin and permethrin. Here, we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus using a baculovirus expression system and evaluated the interaction of the multifluorinated benzyl pyrethroid transfluthrin with these enzymes by different approaches. First, by Michaelis-Menten kinetics in a fluorescent probe assay with the model substrate 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC), we showed the inhibition of BOMFC metabolism by increasing concentrations of transfluthrin. Second, we tested the metabolic capacity of recombinantly expressed CYP6P9 variants to degrade transfluthrin utilizing UPLC-MS/MS analysis and detected low depletion rates, explaining the virtual lack of resistance of strain FUMOZ-R to transfluthrin observed in previous studies. However, as both approaches suggested an interaction of CYP6P9 variants with transfluthrin, we analyzed the oxidative metabolic fate and failed to detect hydroxylated transfluthrin, but low amounts of an M-2 transfluthrin metabolite. Based on the detected metabolite we hypothesize oxidative attack of the gem-dimethyl substituted cyclopropyl moiety, resulting in the formation of an allyl cation upon ring opening. In conclusion, these findings support the resilience of transfluthrin to P450-mediated pyrethroid resistance, and thus, reinforces its employment as an important resistance-breaking pyrethroid in resistance management strategies to control the major malaria vector An. funestus.

Expression profile of the entire detoxification gene inventory of the western honeybee, Apis mellifera across life stages.

The western honeybee, Apis mellifera, is a managed pollinator of many crops and potentially exposed to a wide range of foreign compounds, including pesticides throughout its life cycle. Honeybees as well as other insects recruit molecular defense mechanisms to facilitate the detoxification of xenobiotic compounds. The inventory of detoxification genes (DETOXome) is comprised of five protein superfamilies: cytochrome P450 monooxygenases (P450), carboxylesterases, glutathione S-transferases (GST), UDP-glycosyl transferases (UGT) and ATP-binding cassette (ABC) transporters. Here we characterized the gene expression profile of the entire honeybee DETOXome by analyzing 47 transcriptomes across the honeybee life cycle, including different larval instars, pupae, and adults. All life stages were well separated by principal component analysis, and K-means clustering revealed distinct temporal patterns of gene expression. Indeed, >50% of the honeybee detoxification gene inventory is found in one cluster and follows strikingly similar expression profiles, i.e., increased expression during larval development, followed by a sharp decline after pupation and a steep increase again in adults. This cluster includes 29 P450 genes dominated by CYP3 and CYP4 clan members, 15 ABC transporter genes mostly belonging to the ABCC subfamily and 13 carboxylesterase genes including almost all members involved in dietary/detox and hormone/semiochemical processing. RT-qPCR analysis of selected detoxification genes from all families revealed high expression levels in various tissues, especially Malpighian tubules, fatbody and midgut, supporting the view that these tissues are essential for metabolic clearance of environmental toxins and pollutants in honeybees. Our study is meant to spark further research on the molecular basis of detoxification in this critical pollinator to better understand and evaluate negative impacts from potentially toxic substances. Additionally, the entire gene set of 47 transcriptomes collected and analyzed provides a valuable resource for future honeybee research across different disciplines.

Investigating the role of the ROS/CncC signaling pathway in the response to xenobiotics in Spodoptera frugiperda using Sf9 cells.

Spodoptera frugiperda (fall armyworm, FAW) is an invasive polyphagous lepidopteran pest that has developed sophisticated resistance mechanisms involving detoxification enzymes to eliminate toxic compounds it encounters in its diet including insecticides. Although its inventory of detoxification enzymes is known, the mechanisms that enable an adapted response depending on the toxic compound remain largely unexplored. Sf9 cells were used to investigate the role of the transcription factors, Cap n' collar isoform C (CncC) and musculoaponeurotic fibrosarcoma (Maf) in the regulation of the detoxification response. We overexpressed CncC, Maf or both genes, and knocked out (KO) CncC or its repressor Kelch-like ECH associated protein 1 (Keap1). Joint overexpression of CncC and Maf is required to confer increased tolerance to indole 3-carbinol (I3C), a plant secondary metabolite, and to methoprene, an insecticide. Both molecules induce reactive oxygen species (ROS) pulses in the different cell lines. The use of an antioxidant reversed ROS pulses and restored the tolerance to I3C and methoprene. The activity of detoxification enzymes varied according to the cell line. Suppression of Keap1 significantly increased the activity of cytochrome P450s, carboxylesterases and glutathione S-transferases. RNAseq experiments showed that CncC mainly regulates the expression of detoxification genes but is also at the crossroads of several signaling pathways (reproduction and immunity) maintaining homeostasis. We present new data in Sf9 cell lines suggesting that the CncC:Maf pathway plays a central role in FAW response to natural and synthetic xenobiotics. This knowledge helps to better understand detoxification gene expression and may help to design next-generation pest insect control measures.

The complete genome assemblies of 19 insect pests of worldwide importance to agriculture.

There are many insect pests worldwide that damage agricultural crop and reduce yield either by direct feeding or by the transmission of plant diseases. To date, control of pest insects has been achieved largely by applying synthetic insecticides. However, insecticide use can be seriously impacted by legislation that limits their use or by the evolution of resistance in the target pest. Thus, there is a move towards less use of insecticides and increased adoption of integrated pest management strategies using a wide range of non-chemical and chemical control methods. For good pest control there is a need to understand the mode of action and selectivity of insecticides, the life cycles of the pests and their biology and behaviours, all of which can benefit from good quality genome data. Here we present the complete assembled (chromosome level) genomes (incl. mtDNA) of 19 insect pests, Agriotes lineatus (click beetle/wireworm), Aphis gossypii (melon/cotton aphid), Bemisia tabaci (cotton whitefly), Brassicogethes aeneus (pollen beetle), Ceutorhynchus obstrictus (seedpod weevil), Chilo suppressalis (striped rice stem borer), Chrysodeixis includens (soybean looper), Diabrotica balteata (cucumber beetle), Diatraea saccharalis (sugar cane borer), Nezara viridula (green stink bug), Nilaparvata lugens (brown plant hopper), Phaedon cochleariae (mustard beetle), Phyllotreta striolata (striped flea beetle), Psylliodes chrysocephala (cabbage stem flea beetle), Spodoptera exigua (beet army worm), Spodoptera littoralis (cotton leaf worm), Diabrotica virgifera (western corn root worm), Euschistus heros (brown stink bug) and Phyllotreta cruciferae (crucifer flea beetle). For the first 15 of these we also present the annotation of genes encoding potential xenobiotic detoxification enzymes. This public resource will aid in the elucidation and monitoring of resistance mechanisms, the development of highly selective chemistry and potential techniques to disrupt behaviour in a way that limits the effect of the pests.

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.

A near-chromosome level genome assembly of the European hoverfly, Sphaerophoria rueppellii (Diptera: Syrphidae), provides comparative insights into insecticide resistance-related gene family evolution.

BACKGROUND: Sphaerophoria rueppellii, a European species of hoverfly, is a highly effective beneficial predator of hemipteran crop pests including aphids, thrips and coleopteran/lepidopteran larvae in integrated pest management (IPM) programmes. It is also a key pollinator of a wide variety of important agricultural crops. No genomic information is currently available for S. rueppellii. Without genomic information for such beneficial predator species, we are unable to perform comparative analyses of insecticide target-sites and genes encoding metabolic enzymes potentially responsible for insecticide resistance, between crop pests and their predators. These metabolic mechanisms include several gene families - cytochrome P450 monooxygenases (P450s), ATP binding cassette transporters (ABCs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs) and carboxyl/choline esterases (CCEs). METHODS AND FINDINGS: In this study, a high-quality near-chromosome level de novo genome assembly (as well as a mitochondrial genome assembly) for S. rueppellii has been generated using a hybrid approach with PacBio long-read and Illumina short-read data, followed by super scaffolding using Hi-C data. The final assembly achieved a scaffold N50 of 87Mb, a total genome size of 537.6Mb and a level of completeness of 96% using a set of 1,658 core insect genes present as full-length genes. The assembly was annotated with 14,249 protein-coding genes. Comparative analysis revealed gene expansions of CYP6Zx P450s, epsilon-class GSTs, dietary CCEs and multiple UGT families (UGT37/302/308/430/431). Conversely, ABCs, delta-class GSTs and non-CYP6Zx P450s showed limited expansion. Differences were seen in the distributions of resistance-associated gene families across subfamilies between S. rueppellii and some hemipteran crop pests. Additionally, S. rueppellii had larger numbers of detoxification genes than other pollinator species. CONCLUSION AND SIGNIFICANCE: This assembly is the first published genome for a predatory member of the Syrphidae family and will serve as a useful resource for further research into selectivity and potential tolerance of insecticides by beneficial predators. Furthermore, the expansion of some gene families often linked to insecticide resistance and selectivity may be an indicator of the capacity of this predator to detoxify IPM selective insecticides. These findings could be exploited by targeted insecticide screens and functional studies to increase effectiveness of IPM strategies, which aim to increase crop yields by sustainably and effectively controlling pests without impacting beneficial predator populations.

A scaffold-level genome assembly of a minute pirate bug, Orius laevigatus (Hemiptera: Anthocoridae), and a comparative analysis of insecticide resistance-related gene families with hemipteran crop pests.

BACKGROUND: Orius laevigatus, a minute pirate bug, is a highly effective beneficial predator of crop pests including aphids, spider mites and thrips in integrated pest management (IPM) programmes. No genomic information is currently available for O. laevigatus, as is the case for the majority of beneficial predators which feed on crop pests. In contrast, genomic information for crop pests is far more readily available. The lack of publicly available genomes for beneficial predators to date has limited our ability to perform comparative analyses of genes encoding potential insecticide resistance mechanisms between crop pests and their predators. These mechanisms include several gene/protein families including cytochrome P450s (P450s), ATP binding cassette transporters (ABCs), glutathione S-transferases (GSTs), UDP-glucosyltransferases (UGTs) and carboxyl/cholinesterases (CCEs). METHODS AND FINDINGS: In this study, a high-quality scaffold level de novo genome assembly for O. laevigatus has been generated using a hybrid approach with PacBio long-read and Illumina short-read data. The final assembly achieved a scaffold N50 of 125,649 bp and a total genome size of 150.98 Mb. The genome assembly achieved a level of completeness of 93.6% using a set of 1658 core insect genes present as full-length genes. Genome annotation identified 15,102 protein-coding genes - 87% of which were assigned a putative function. Comparative analyses revealed gene expansions of sigma class GSTs and CYP3 P450s. Conversely the UGT gene family showed limited expansion. Differences were seen in the distributions of resistance-associated gene families at the subfamily level between O. laevigatus and some of its targeted crop pests. A target site mutation in ryanodine receptors (I4790M, PxRyR) which has strong links to diamide resistance in crop pests and had previously only been identified in lepidopteran species was found to also be present in hemipteran species, including O. laevigatus. CONCLUSION AND SIGNIFICANCE: This assembly is the first published genome for the Anthocoridae family and will serve as a useful resource for further research into target-site selectivity issues and potential resistance mechanisms in beneficial predators. Furthermore, the expansion of gene families often linked to insecticide resistance may be an indicator of the capacity of this predator to detoxify selective insecticides. These findings could be exploited by targeted pesticide screens and functional studies to increase effectiveness of IPM strategies, which aim to increase crop yields by sustainably, environmentally-friendly and effectively control pests without impacting beneficial predator populations.

Characterization of a novel pesticide transporter and P-glycoprotein orthologues in Drosophila melanogaster.

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge about mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect Drosophila melanogaster. An RNAi screen was performed, which knocked down individual ABCs in specific epithelial tissues and examined the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This implicated a novel ABC drug transporter, CG4562, in spinosad transport, but also highlighted the P-glycoprotein orthologue Mdr65 as the most impactful ABC in terms of chemoprotection. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, transgenic Drosophila lines expressing P-glycoprotein from the major malaria vector Anopheles gambiae were used to establish a system for in vivo characterization of this transporter in non-model insects. This study provides the basis for establishing Drosophila as a model for toxicology research on drug transporters.

Genomic insights into neonicotinoid sensitivity in the solitary bee Osmia bicornis.

The impact of pesticides on the health of bee pollinators is determined in part by the capacity of bee detoxification systems to convert these compounds to less toxic forms. For example, recent work has shown that cytochrome P450s of the CYP9Q subfamily are critically important in defining the sensitivity of honey bees and bumblebees to pesticides, including neonicotinoid insecticides. However, it is currently unclear if solitary bees have functional equivalents of these enzymes with potentially serious implications in relation to their capacity to metabolise certain insecticides. To address this question, we sequenced the genome of the red mason bee, Osmia bicornis, the most abundant and economically important solitary bee species in Central Europe. We show that O. bicornis lacks the CYP9Q subfamily of P450s but, despite this, exhibits low acute toxicity to the N-cyanoamidine neonicotinoid thiacloprid. Functional studies revealed that variation in the sensitivity of O. bicornis to N-cyanoamidine and N-nitroguanidine neonicotinoids does not reside in differences in their affinity for the nicotinic acetylcholine receptor or speed of cuticular penetration. Rather, a P450 within the CYP9BU subfamily, with recent shared ancestry to the Apidae CYP9Q subfamily, metabolises thiacloprid in vitro and confers tolerance in vivo. Our data reveal conserved detoxification pathways in model solitary and eusocial bees despite key differences in the evolution of specific pesticide-metabolising enzymes in the two species groups. The discovery that P450 enzymes of solitary bees can act as metabolic defence systems against certain pesticides can be leveraged to avoid negative pesticide impacts on these important pollinators.

Biochemical profiling of functionally expressed CYP6P9 variants of the malaria vector Anopheles funestus with special reference to cytochrome b5 and its role in pyrethroid and coumarin substrate metabolism.

Cytochrome P450 monooxygenases (P450s) are well studied enzymes catalyzing the oxidative metabolism of xenobiotics in insects including mosquitoes. Their duplication and upregulation in agricultural and public health pests such as anopheline mosquitoes often leads to an enhanced metabolism of insecticides which confers resistance. In the laboratory strain Anopheles funestus FUMOZ-R the duplicated P450s CYP6P9a and CYP6P9b are highly upregulated and proven to confer pyrethroid resistance. Microsomal P450 activity is regulated by NADPH cytochrome P450 oxidoreductase (CPR) required for electron transfer, whereas the modulatory role of cytochrome b5 (CYB5) on insect P450 activity is less clear. In previous studies CYP6P9a and CYP6P9b were recombinantly expressed in tandem with An. gambiae CPR using E. coli-expression systems and CYB5 added to the reaction mix to enhance activity. However, the precise role of CYB5 on substrate turn-over when combined with CYP6P9a and CYP6P9b remains poorly investigated, thus one objective of our study was to address this knowledge gap. In contrast to the CYP6P9 variants, the expression levels of both CYB5 and CPR were not upregulated in the pyrethroid resistant FUMOZ-R strain when compared to the susceptible FANG strain, suggesting no immediate regulatory role of these genes in pyrethroid resistance in FUMOZ-R. Here, for the first time we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus in a baculovirus expression system using High-5 insect cells. Co-expression of each enzyme with CPR from either An. gambiae or An. funestus did not reveal noteworthy differences in catalytic capacity. Whereas the co-expression of An. funestus CYB5 - tested at different multiplicity of infection (MOI) ratios - resulted in a significantly higher metabolization of coumarin substrates as measured by fluorescence assays. This was confirmed by Michaelis-Menten kinetics using the most active substrate, 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC). We observed a similar increase in coumarin substrate turnover by adding human CYB5 to the reaction mix. Finally, we compared by UPLC-MS/MS analysis the depletion rate of deltamethrin and the formation of 4'OH-deltamethrin by recombinantly expressed CYP6P9a and CYP6P9b with and without CYB5 and detected no difference in the extent of deltamethrin metabolism. Our results suggest that co-expression (or addition) of CYB5 with CYP6P9 variants, recombinantly expressed in insect cells, can significantly enhance their metabolic capacity to oxidize coumarins, but not deltamethrin.

Characterization of molecular and kinetic properties of two acetylcholinesterases from the Colorado potato beetle, Leptinotarsa decemlineata.

The molecular and biochemical properties of two acetylcholinesterases (LdAChE1 and LdAChE2) from the Colorado potato beetle, Leptinotarsa decemlineata, were investigated in this study. Polyacrylamide gel electrophoresis in conjunction with western blotting with LdAChE1- or LdAChE2-specific antibodies suggested that LdAChE1 exists in a soluble form, whereas LdAChE2 exists in both soluble and amphiphilic forms with a glycophosphatidylinositol anchor. Both LdAChEs exist as homodimers with each monomer connected with a disulfide bond. LdAChE1 was the most highly expressed in the thorax followed by the head, leg, and abdomen, whereas LdAChE2 was the most highly expressed in the head, followed by the thorax, leg, and abdomen. The overall expression levels of LdAChE1, however, were higher than those of LdAChE2 in all examined tissues. Kinetic analysis using recombinant LdAChE1 and LdAChE2 showed that LdAChE2 has a 4.8-fold higher catalytic efficiency toward acetylthiocholine iodide compared to LdAChE1. LdAChE2 was more sensitive to organophosphorus and carbamate insecticides than LdAChE1. The addition of irreversibly phosphorylated LdAChE1 via paraoxon titration significantly reduced LdAChE2 inhibition by insecticides and glycoalkaloids, suggesting a sequestration role of soluble LdAChE1 in the chemical defense against xenobiotics. Taken together, LdAChE2 may be the main enzyme for synaptic transmission, thus serving as a toxicologically more relevant target, whereas the soluble LdAChE1 may function as a bioscavenger.

Comparative and functional genomics of the ABC transporter superfamily across arthropods.

BACKGROUND: The ATP-binding cassette (ABC) transporter superfamily is comprised predominantly of proteins which directly utilize energy from ATP to move molecules across the plasma membrane. Although they have been the subject of frequent investigation across many taxa, arthropod ABCs have been less well studied. While the manual annotation of ABC transporters has been performed in many arthropods, there has so far been no systematic comparison of the superfamily within this order using the increasing number of sequenced genomes. Furthermore, functional work on these genes is limited. RESULTS: Here, we developed a standardized pipeline to annotate ABCs from predicted proteomes and used it to perform comparative genomics on ABC families across arthropod lineages. Using Kruskal-Wallis tests and the Computational Analysis of gene Family Evolution (CAFE), we were able to observe significant expansions of the ABC-B full transporters (P-glycoproteins) in Lepidoptera and the ABC-H transporters in Hemiptera. RNA-sequencing of epithelia tissues in the Lepidoptera Helicoverpa armigera showed that the 7 P-glycoprotein paralogues differ substantially in their tissue distribution, suggesting a spatial division of labor. It also seems that functional redundancy is a feature of these transporters as RNAi knockdown showed that most transporters are dispensable with the exception of the highly conserved gene Snu, which is probably due to its role in cuticular formation. CONCLUSIONS: We have performed an annotation of the ABC superfamily across > 150 arthropod species for which good quality protein annotations exist. Our findings highlight specific expansions of ABC transporter families which suggest evolutionary adaptation. Future work will be able to use this analysis as a resource to provide a better understanding of the ABC superfamily in arthropods.