BACKGROUNDAn increasing human population, the emergence of resistances against pesticides and their potential impact on the environment call for the development of new eco-friendly pest control strategies. RNA interference (RNAi)-based pesticides have emerged as a new option with the first products entering the market. Essentially, double-stranded RNAs targeting essential genes of pests are either expressed in the plants or sprayed on their surface. Upon feeding, pests mount an RNAi response and die. However, it has remained unclear whether RNAi-based insecticides should target the same pathways as classic pesticides or whether the different mode-of-action would favor other processes. Moreover, there is no consensus on the best genes to be targeted.RESULTSWe performed a genome-wide screen in the red flour beetle to identify 905 RNAi target genes. Based on a validation screen and clustering, we identified the 192 most effective target genes in that species. The transfer to oral application in other beetle pests revealed a list of 34 superior target genes, which are an excellent starting point for application in other pests. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses of our genome-wide dataset revealed that genes with high efficacy belonged mainly to basic cellular processes such as gene expression and protein homeostasis - processes not targeted by classic insecticides.CONCLUSIONOur work revealed the best target genes and target processes for RNAi-based pest control and we propose a procedure to transfer our short list of superior target genes to other pests. (c) 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Insecticide mode of action studies provide insights into how new insecticidal actives function and contribute to assessing safety to humans and nontarget organisms. Insect cell lines that express potential target sites can serve as valuable tools in this effort. In this paper, we report on the influence of two signaling molecules on protein expression in a nervous system cell line established from Spodoptera frugiperda (Bayer/BCIRL-SfNS2-0714-TR). We selected this line because we established it in our laboratory and we are experienced in using it. Cells were exposed to the insect developmental hormone (1 µg/mL 20-hydroxyecdysone, 20E) and/or a cyclooxygenase (COX) inhibitor (25 μM indomethacin, INDO; inhibits prostaglandin [PG] biosynthesis) for 24 h (Day 2), 72 h (Day 4), or 120 h (Day 6). We selected a PG biosynthesis inhibitor because PGs act in many aspects of insect biology, such as embryonic development, immunity, and protein phosphorylation. We selected the developmental hormone, 20E, because it also acts in fundamental aspects of insect biology. We identified specific proteins via in silico analysis. Changes in protein expression levels were determined using liquid chromatography-mass spectrometry (MS) + MS-MS. The largest number of changes in protein expression occurred on Day 2. The combination of 20E plus INDO led to 222 differentially expressed proteins, which documents the deep significance of PGs and 20E in insect biology. 20E and, separately, INDO led to changes in 30 proteins each (p value < 0.01; >2X or <0.5X-fold changes). We recorded changes in the expression of 9 or 12 proteins (20E), 10 or 6 proteins (INDO), and 21 or 20 proteins (20E + INDO) on D4 and D6, respectively. While the cell line was established from neuronal tissue, the differentially expressed proteins act in a variety of fundamental cell processes. In this paper, we moved beyond a list of proteins by providing detailed, Gene Ontology term analyses and enrichment, which offers an in-depth understanding of the influence of these treatments on the SfNS2 cells. Because proteins are active components of cell physiology in their roles as enzymes, receptors, elements of signaling transduction pathways, and cellular structures, changes in their expression levels under the influence of signaling molecules provide insights into their function in insect cell physiology.

Many plants produce chemical defense compounds as protection against antagonistic herbivores. However, how beneficial insects such as pollinators deal with the presence of these potentially toxic chemicals in nectar and pollen is poorly understood. Here, we characterize a conserved mechanism of plant secondary metabolite detoxification in the Hymenoptera, an order that contains numerous highly beneficial insects. Using phylogenetic and functional approaches, we show that the CYP336 family of cytochrome P450 enzymes detoxifies alkaloids, a group of potent natural insecticides, in honeybees and other hymenopteran species that diverged over 281 million years. We linked this function to an aspartic acid residue within the main access channel of CYP336 enzymes that is highly conserved within this P450 family. Together, these results provide detailed insights into the evolution of P450s as a key component of detoxification systems in hymenopteran species and reveal the molecular basis of adaptations arising from interactions between plants and beneficial insects.

Acyrthosiphon pisum Harris (Hemiptera: Aphididae) is a globally distributed and economically important aphid pest of leguminous crops including peas, broad beans, and alfalfa. Infestations weaken plants directly by feeding, and indirectly, by the transmission of yield-affecting plant viruses. Although pea aphid control strategies largely rely on the application of insecticides, cases of insecticide resistance under applied conditions have not yet been described. Here we investigated the molecular basis of high levels of pyrethroid resistance (up to >180-fold) in two strains of A. pisum, PYR-R1 and VR, collected in the field in France and Italy, respectively. Foliar bioassays revealed significant synergistic effects between deltamethrin and piperonyl butoxide (a cytochrome P450 (P450) inhibitor), and, to a lesser extent, S,S,S-tributyl phosphorotrithioate (an esterase inhibitor). P450 and esterase activities in both resistant strains were significantly higher compared to an insecticide susceptible reference strain SUS-1. Target-site resistance mutations in the voltage-gated sodium channel known to confer pyrethroid resistance in other aphid pest species were not detected in either resistant strain. However, transcriptome profiling identified the P450 gene CYP6CY12 as highly over-expressed in the resistant PYR-R1 strain compared to the SUS-1 strain, and real-time quantitative PCR showed that this gene is also significantly overexpressed (>30-fold) in the VR strain. Functional expression of recombinant CYP6CY12 revealed its catalytic capacity to hydroxylate deltamethrin, thus confirming its causal role in pyrethroid resistance. Our study uncovered a P450-mediated mechanism of pyrethroid resistance in A. pisum for the first time and will inform future resistance management tactics to control this pest.

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 Caterpillars from the insect order Lepidoptera are some of the most widespread and destructive agricultural pests. Most of their impact is at the larval stage, where the midgut epithelium mediates the digestion and absorption of an astonishing amount of food. Although this tissue has been the subject of frequent investigation in Lepidoptera, a comprehensive expression atlas has yet to be generated. Results Here, we perform RNA-sequencing and proteomics on the gut of the polyphagous pest Helicoverpa armigera across, life stages, diet types, and compartments of the anterior-posterior axis. A striking relationship between the structural homology and expression pattern of a group of sugar transporters was observed in the early larval stages. Further comparisons were made among the spatial compartments of the midgut, which suggested a putative role for vATPases and SLC9 transporters in the generation of alkaline conditions in the H. armigera midgut. Conclusions This comprehensive resource will aid the scientific community in understanding lepidopteran gut physiology in unprecedented resolution. It is hoped that this study advances the understanding of the lepidopteran midgut and also facilitates functional work in this field.

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.

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.

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.

Background Spodoptera frugiperda, fall armyworm (FAW) is the major pest of maize in Brazil and has readily acquired field resistance to a broad range of synthetic insecticides and toBacillus thuringiensis(Bt) insecticidal proteins expressed in important crops. This study aims to understand patterns of cross-resistance in FAW by investigating the toxicological profile of a Bt-resistant Brazilian strain (Sf_Des) in comparison to a Bt-susceptible strain (Sf_Bra). Results Laboratory bioassays with 15 active substances of nine mode of action classes revealed that Sf_Des has a medium level of resistance to deltamethrin and chlorpyrifos. Very high cross-resistance was observed among Cry1 toxins, but high susceptibility against Vip3A. Strain Sf_Des exhibited - depending on the substrate - up to 19-fold increased cytochrome P450 activity in comparison to Sf_Bra. RNA-Seq data support a major role of P450 enzymes in the detoxification of insecticides because we detected 85 P450 transcripts upregulated in Sf_Des. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis confirmed thatCYP9A-like andCYP6B39are significantly upregulated (>200-fold) in Sf_Des in comparison to Sf_Bra strain. No target-site mutation linked to pyrethroid resistance was detected, but mutations in the AChE linked to organophosphate resistance were observed in Sf_Des. A Gene Ontology (GO) analysis of differentially expressed genes (DEG) categorized most of them into the biological process category, involved in oxidation-reduction and metabolic processes. Conclusion Our results indicate that multiple/cross-resistance mechanisms may have developed in the Sf_Des strain to conventional insecticides and Bt insecticidal proteins. The systematic toxicological analysis presented will help to guide recommendations for an efficient resistance management.

Generalization of transcriptomics results can be achieved by comparison across experiments. This generalization is based on integration of interrelated transcriptomics studies into a compendium. Such a focus on the bigger picture enables both characterizations of the fate of an organism and distinction between generic and specific responses. Numerous methods for analyzing transcriptomics datasets exist. Yet, most of these methods focus on gene-wise dimension reduction to obtain marker genes and gene sets for, for example, pathway analysis. Relying only on isolated biological modules might result in missing important confounders and relevant contexts. We developed a method called Plant PhysioSpace, which enables researchers to compute experimental conditions across species and platforms without a priori reducing the reference information to specific gene sets. Plant PhysioSpace extracts physiologically relevant signatures from a reference dataset (i.e. a collection of public datasets) by integrating and transforming heterogeneous reference gene expression data into a set of physiology-specific patterns. New experimental data can be mapped to these patterns, resulting in similarity scores between the acquired data and the extracted compendium. Because of its robustness against platform bias and noise, Plant PhysioSpace can function as an inter-species or cross-platform similarity measure. We have demonstrated its success in translating stress responses between different species and platforms, including single-cell technologies. We have also implemented two R packages, one software and one data package, and a Shiny web application to facilitate access to our method and precomputed models.

Background: Sphaerophoria rueppellii, a European species of hoverfly, is a highly effective beneficial predator of 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 at the subfamily levels between S. rueppellii and some crop pests. 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.

RNA interference (RNAi) is a promising, selective pest control technology based on the silencing of targeted genes mediated by the degradation of mRNA after the ingestion of double-stranded (ds) RNA. However, the identification of the best target genes remains a challenge, because large scale screening is only feasible in lab model systems and it remains unclear, to what degree such data can be transferred to pest species. Here, we report on our efforts to transfer target genes found in a lab model to the mustard leaf beetle, Phaedon cochleariae. The mustard leaf beetle can be reared easily and resource-efficient in large quantities all year round and is an established chrysomelid pest for higher throughput screening approaches in the crop protection industry. Mustard leaf beetle transcriptome sequencing and assembly revealed genes orthologous to those previously described as highly efficient RNAi targets in the model beetle Tribolium castaneum. First, we observed mortality after injection of dsRNA targeting the respective orthologous genes in 2nd instar mustard beetle larvae. Next, we adopted a robust, automated multi-well plate foliar RNAi screening procedure with 2nd instar larvae of the mustard leaf beetle to assess those genes. Indeed, foliar application and oral uptake of dsRNA targeting the same genes resulted in larval mortality as well. The most effective target genes with a strong (lethal) phenotype - at dsRNA doses as low as 300 ng/leaf disc (equal to 9.6 g/ha) - were srp54k, rop, αSNAP, rpn7 and rpt3. Rather limited effects were observed after application of dsRNA targeting cactus, shibire and PP-α, though they had previously been shown to be highly lethal in red flour beetle. Importantly, our experiments demonstrated that the overall efficacy pattern obtained after oral dsRNA application was well correlated with the results obtained after dsRNA injection. RT-qPCR confirmed significant target gene knock-down after normalization by employing three reference genes shown to be stably expressed across life stages. In summary, several RNAi targeted genes elicited a strong lethal phenotype and significant target gene knock-down after feeding, suggesting P. cochleariae as a potential coleopteran screening model for foliarly applied exogenous RNAi.

BackgroundThe 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.ResultsHere, 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.ConclusionsWe 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.

Additional file 8:Â Table S4. Full proteome. A full table of all unigenes identified in this study with an indication of whether the unigene was detected in the proteome of a given tissue. Corresponding sequences for transcripts can be found in the bioproject PRJNA557118.

Formic acid (FA) has been used for decades to control Varroa destructor , one of the most important parasites of the western honey bee, Apis mellifera . The rather unselective molecular mode of action of FA and its possible effects on honeybees have long been a concern of beekeepers, as it has undesirable side effects that affect the health of bee colonies. This study focuses on short-term transcriptomic changes as analysed by RNAseq in both larval and adult honey bees and in mites after FA treatment under applied conditions. Our study aims to identify those genes in honey bees and varroa mites differentially expressed upon a typical FA hive exposure scenario. Five detoxification-related genes were identified with significantly enhanced and one gene with significantly decreased expression under FA exposure. Regulated genes in our test setting included members of various cytochrome P450 subfamilies, a flavin-dependent monooxygenase and a cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH), known to be involved in formate metabolism in mammals. We were able to detect differences in the regulation of detoxification-associated genes between mites and honey bees as well as between the two different developmental stages of the honey bee. Additionally, we detected repressed regulation of Varroa genes involved in cellular respiration, suggesting mitochondrial dysfunction and supporting the current view on the mode of action of FA—inhibition of oxidative phosphorylation. This study shows distinct cellular effects induced by FA on the global transcriptome of both host and parasite in comparison. Our expression data might help to identify possible differences in the affected metabolic pathways and thus make a first contribution to elucidate the mode of detoxification of FA.

Background Stink bugs are an emerging threat to crop security in many parts of the globe, but there are few genetic resources available to study their physiology at a molecular level. This is especially true for tissues such as the midgut, which forms the barrier between ingested material and the inside of the body. Results Here, we focus on the midgut of the southern green stink bug Nezara viridula and use both transcriptomic and proteomic approaches to create an atlas of expression along the four compartments of the anterior-posterior axis. Estimates of the transcriptome completeness were high, which led us to compare our predicted gene set to other related stink bugs and Hemiptera, finding a high number of species-specific genes in N. viridula. To understand midgut function, gene ontology and gene family enrichment analyses were performed for the most highly expressed and specific genes in each midgut compartment. These data suggested a role for the anterior midgut (regions M1-M3) in digestion and xenobiotic metabolism, while the most posterior compartment (M4) was enriched in transmembrane proteins. A more detailed characterization of these findings was undertaken by identifying individual members of the cytochrome P450 superfamily and nutrient transporters thought to absorb amino acids or sugars. Conclusions These findings represent an initial step to understand the compartmentalization and physiology of the N. viridula midgut at a genetic level. Future studies will be able to build on this work and explore the molecular physiology of the stink bug midgut.

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.

Interaction of plant roots with arbuscular mycorrhizal fungi (AMF) is a complex trait resulting in cooperative interactions among the two symbionts including bidirectional exchange of resources. To study arbuscular mycorrhizal symbiosis (AMS) trait variation in the model plant Lotus japonicus, we performed an integrated multi-omics analysis with a focus on plant and fungal phospholipid (PL) metabolism and biological significance of lysophosphatidylcholine (LPC). Our results support the role of LPC as a bioactive compound eliciting cellular and molecular response mechanisms in Lotus. Evidence is provided for large interspecific chemical diversity of LPC species among mycorrhizae with related AMF species. Lipid, gene expression and elemental profiling emphasize the Lotus-Glomus intraradices interaction as distinct from other arbuscular mycorrhizal (AM) interactions. In G. intraradices, genes involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs were enhanced, while in Lotus, FA synthesis genes were up-regulated during AMS. Furthermore, FAS protein localization to mitochondria suggests FA biosynthesis and elongation may also occur in AMF. Our results suggest the existence of interspecific partitioning of PL resources for generation of LPC and novel candidate bioactive PLs in the Lotus-G. intraradices symbiosis. Moreover, the data advocate research with phylogenetically diverse Glomeromycota species for a broader understanding of the molecular underpinnings of AMS.