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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.

The study was conducted to evaluate whether Bemisia tabaci (Gennadius) (MEAM1, formerly B biotype) populations in Turkey have developed resistance to neonicotinoid insecticides. We collected B. tabaci from vegetable and cotton growing areas in the Mediterranean coastal region of Turkey. B. tabaci populations were collected from the following crops (and areas): Solanum lycopersicum L. (Aydıncık and Erdemli-Mersin), Gossypium hirsutum L. (Karataş-Adana), Capsicum annuum L. (Kumluca-Antalya), and Cucumis sativus L. (Samandağ-Hatay). We performed insecticide bioassays and biochemical assays to determine levels of susceptibility to acetamiprid, imidacloprid, thiacloprid, and thiamethoxam. The bioassays showed that most of the B. tabaci populations were resistant to all the neonicotinoids tested when compared with the laboratory insecticide-susceptible strain, SUD-S. The highest resistance factor was 2060 for imidacloprid at Kumluca and the lowest was 5.36 for thiamethoxam at Samandağ. Furthermore, the highest and lowest monooxygenase enzyme activity level of B. tabaci was in the Kumluca and Samandağ populations, respectively. The CYP6CM1 protein lateral flow assay results supported those of the biochemical assays. Our results support those reported elsewhere that enhanced monooxygenase activity, at least in part, is responsible for neonicotinoid resistance in B. tabaci populations. © 2018, Department of Agricultural and Food Sciences. All rights reserved. D3BAP2012 GS and MRU conceived and designed research. GS conducted experiments. RN contributed new reagents. GS, KD and RN analyzed data. GS wrote the manuscript. GS thanks the Cukurova University BAP Department for their support of the doctorate project (D3BAP2012). The authors thanks Martin Williamson (Rothamsted Research, UK) for supplying SUD-S susceptible whiteflies strain. We also thank Recep Ay (Suleyman Demirel University, Plant Protection Department, Isparta, Turkey), Graham Moores, Selcan Alptekin (Rothamsted Research, UK), Sedef Nehir El and Şebnem Şimşek (Ege University, Food Engineering Department, İzmir, Turkey) and Osman Gülnaz (Cuku-rova University, Department of Biology, Faculty of Arts and Sciences, Turkey). The authors also thank Gregory T. Sullivan (School of Earth and Environmental Sciences, Brisbane, Australia) for editing the English in an earlier version of this manuscript.

The pollen beetle Meligethes aeneus is the most important coleopteran pest in European oilseed rape cultivation, annually infesting millions of hectares and responsible for substantial yield losses if not kept under economic damage thresholds. This species is primarily controlled with insecticides but has recently developed high levels of resistance to the pyrethroid class. The aim of the present study was to provide a transcriptomic resource to investigate mechanisms of resistance. cDNA was sequenced on both Roche (Indianapolis, IN, USA) and Illumina (LGC Genomics, Berlin, Germany) platforms, resulting in a total of similar to 53 m reads which assembled into 43 396 expressed sequence tags (ESTs). Manual annotation revealed good coverage of genes encoding insecticide target sites and detoxification enzymes. A total of 77 nonredundant cytochrome P450 genes were identified. Mapping of Illumina RNAseq sequences (from susceptible and pyrethroid-resistant strains) against the reference transcriptome identified a cytochrome P450 (CYP6BQ23) as highly overexpressed in pyrethroid resistance strains. Single-nucleotide polymorphism analysis confirmed the presence of a target-site resistance mutation (L1014F) in the voltage-gated sodium channel of one resistant strain. Our results provide new insights into the important genes associated with pyrethroid resistance in M. aeneus. Furthermore, a comprehensive EST resource is provided for future studies on insecticide modes of action and resistance mechanisms in pollen beetle.

Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme A carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.

Bifenazate is a recently developed acaricide that is mainly used to control spider mites on a variety of crops. Although first thought to be a neurotoxin, genetic evidence obtained from bifenazate resistant Tetranychus urticae strains suggested an alternative mode of action as a Qo pocket inhibitor of the mitochondrial complex III. In this study, we reveal how bifenazate resistance in strains of Panonychus citri is maternally inherited and can confer cross-resistance to the known Qo inhibitor acequinocyl. The mitochondrial genome of P. citri was sequenced and Qo pocket mutations were shown to be linked with the resistant trait. Parallel evolution of cytochrome b mediated bifenazate resistance corroborates the alternative mode of action and yet again illustrates that care should be taken when employing Qo inhibitors as crop protection compounds.

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid.

BACKGROUND: Rice brown planthopper, Nilaparvata lugens Stal, is a primary insect pest of cultivated rice, and effective control is essential for economical crop production. Resistance to neonicotinoid insecticides, in particular imidacloprid, has been reported as an increasing constraint in recent years. In order to investigate the extent of resistance, 24 samples of N. lugens were collected from China, India, Indonesia, Malaysia, Thailand and Vietnam during 2005 and 2006. Their responses to two diagnostic doses of imidacloprid (corresponding approximately to the LC95 and 5 x LC95 of a susceptible strain) were examined. RESULTS: Ten of the 12 samples collected during 2005 were found to be susceptible to imidacloprid, but two late-season samples from India showed reduced mortality at both diagnostic doses. All 13 strains collected in 2006 showed reduced mortality at both doses when compared with the susceptible strain. Dose-response lines showed resistance in one of the most resistant field strains to be approximately 100-fold compared with the susceptible standard. CONCLUSION: The data demonstrate the development and spread of neonicotinoid resistance in N. lugens in Asia and support reports of reduced field efficacy of imidacloprid. (c) 2008 Society of Chemical Industry

The pollen beetle, Brassicogethes aeneus, is an economically important pest of oilseed rape (Brassica napus) throughout Europe. The control of B. aeneus has relied heavily on the use of chemical insecticides leading to the evolution of resistance. However, investigation of the molecular basis of resistance has been hampered by an absence of genomic resources for this species, including the lack of a reference genome assembly. Here we address this need by generating a chromosome-scale genome assembly for B. aeneus. A combination of long-read single-molecule sequencing and in vivo chromatin conformation capture (Hi-C) sequencing was used to generate an assembly of 585 Mb, comprising 11 chromosome sized scaffolds (scaffold N50 of 61.6 Mb) and containing 13,381 protein-coding genes. We leveraged the new assem- bly, in combination with post-genomic functional approaches to investigate the molecular basis of metabolic resistance to pyrethroid insecticides in B. aeneus. Our data confirmed that two P450s, CYP6BQ23 and CYP6BQ25, have the capacity to metabolise the pyrethroid deltamethrin in B. aeneus and thus have the potential to confer resistance. However, the relative expression of these P450s in pyrethroid susceptible and resistant strains suggests that CYP6BQ23 plays a much more significant role in resistance than CYP6BQ25. In summary, the high-quality genome assembly for B. aeneus reported here provides a valuable resource for future research on this species. Our findings on P450-mediated resistance to insecticides are of applied relevance for the development of strategies for the sustainable control of this important pest.

Neonicotinoid insecticides retain a crucial role within many chemical and integrated control strategies for the tobacco whitefly, Bemisia tabaci Gernnadius, in spite of the establishment of potent and widespread resistance in many areas. Metabolic resistance mechanisms mediated by overexpression of P450-dependent monooxygenases have been implicated in neonicotinoid resistance in the two most prevalent B. tabaci biotypes. Further characterisation of resistance to the neonicotinoid imidacloprid in populations of both these 13- and Q-types is reported. Expression of resistance to imidacloprid was age specific in 13- and Q-type strains of B. tabaci. The highest observed resistance ratio at LC50 expressed in prepupal nymphs was 13, compared with at least 580 in their adult counterparts. For all strains, resistance expressed in immatures was not sufficiently potent to compromise recommended imidacloprid application rates. Targeting neonicotinoids towards immature life stages of B. tabaci may circumvent the protection conferred by current mechanisms of resistance, simultaneously reducing the selection pressures imposed. However, such tactics may enhance the expression of existing resistance mechanisms in immatures, or promote the establishment of novel ones expressed in all life stages. (c) 2008 Society of Chemical Industry

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.

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.

Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait.