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.

Infrared Photodissociation Spectroscopy of Water-Tagged Ions with a Widely Tunable Quantum Cascade Laser for Planetary Science Applications.

This work presents a benchtop method for collecting the room temperature gas phase infrared (IR) action spectra of protonated amino acids and their isomers. The adopted setup uses a minimally modified commercial electrospray ionization linear ion trap mass spectrometer (ESI-LIT-MS) coupled to a broadband continuous wave (cw) quantum cascade laser (QCL) source. This approach leverages messenger assisted action spectroscopic techniques using water-tagged molecular ions with complex formation, irradiation, and subsequent analysis, all taking place within a single linear ion trap stage. This configuration thus circumvents the use of multiple mass selection and analysis stages, cryogenic buffer cells, and complex high-power laser systems typically called upon to execute these techniques. The benchtop action spectrometer is used to collect the 935-1600 cm-1 (6.2-10.7 µm) IR action spectrum of a collection of amino acids and a dipeptide with results cross referenced against literature examples obtained with a free electron laser source. Recorded IR spectra are used for the analysis of binary mixture samples composed of constitutional isomers α-alanine and ß-alanine with ratios determined to ∼4% measurement uncertainty without the aid of a front-end separation stage. This turn-key QCL-based approach is a major step in showing the viability of tag-based action spectroscopic techniques for use in future in situ planetary science sensors and general analytical applications.

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.

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F2 populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.

The mustard leaf beetle, Phaedon cochleariae, as a screening model for exogenous RNAi-based control of coleopteran pests.

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.

Identification and functional characterization of a novel acetyl-CoA carboxylase mutation associated with ketoenol resistance in Bemisia tabaci.

Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations.

RNA-Seq transcriptome analysis to identify genes involved in metabolism-based diclofop resistance in Lolium rigidum.

Weed control failures due to herbicide resistance are an increasing and worldwide problem that significantly affect crop yields. Metabolism-based herbicide resistance (referred to as metabolic resistance) in weeds is not well characterized at the genetic level. An RNA-Seq transcriptome analysis was used to find candidate genes that conferred metabolic resistance to the herbicide diclofop in a diclofop-resistant population (R) of the major global weed Lolium rigidum. A reference cDNA transcriptome (19 623 contigs) was assembled and assigned putative annotations. Global gene expression was measured using Illumina reads from untreated control, adjuvant-only control, and diclofop treatment of R and susceptible (S). Contigs that showed constitutive expression differences between untreated R and untreated S were selected for further validation analysis, including 11 contigs putatively annotated as cytochrome P450 (CytP450), glutathione transferase (GST), or glucosyltransferase (GT), and 17 additional contigs with annotations related to metabolism or signal transduction. In a forward genetics validation experiment, nine contigs had constitutive up-regulation in R individuals from a segregating F2 population, including three CytP450, one nitronate monooxygenase (NMO), three GST, and one GT. Principal component analysis using these nine contigs differentiated F2 -R from F2 -S individuals. In a physiological validation experiment in which 2,4-D pre-treatment induced diclofop protection in S individuals due to increased metabolism, seven of the nine genetically validated contigs were induced significantly. Four contigs (two CytP450, NMO, and GT) were consistently highly expressed in nine field-evolved metabolic resistant L. rigidum populations. These four contigs were strongly associated with the resistance phenotype and are major candidates for contributing to metabolic diclofop resistance.

Mapping and Characterization of Target-Site Resistance to Cyclic Ketoenol Insecticides in Cabbage Whiteflies, Aleyrodes proletella (Hemiptera: Aleyrodidae).

Cabbage whitefly, Aleyrodes proletella L., is an invasive hemipteran pest of cruciferous plants, particularly field brassica crops. Its importance has been increased over the last decade, particularly in European countries. The control of cabbage whiteflies largely relies on the application of synthetic insecticides, including tetronic and tetramic acid derivatives such as spiromesifen and spirotetramat (cyclic ketoenol insecticides), acting as insect growth regulators targeting acetyl-CoA carboxylase (ACC). In 2019, reduced efficacy against cabbage whiteflies of ketoenol insecticides at recommended label rates has been reported. Subsequently we collected field samples of A. proletella in different European countries and confirmed the presence of ketoenol resistance in laboratory bioassays. Reciprocal crossing experiments revealed an autosomal dominant trait, i.e., heterozygotes express a fully resistant phenotype. Transcriptome sequencing and assembly of ACC variants from resistant strains revealed the presence of an ACC target-site mutation, A2083V, as previously described and functionally validated in Bemisia tabaci (A2084V in A. proletella). Next, we employed a molecular genotyping assay to investigate the geographic spread of resistance and analyzed 49 populations collected in eight European countries. Resistance allele frequency was highest in the Netherlands, followed by Germany. Finally, we provide a proposal for the implementation of appropriate resistance management strategies.

A conserved hymenopteran-specific family of cytochrome P450s protects bee pollinators from toxic nectar alkaloids.

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.

Toxicological and molecular profiling of insecticide resistance in a Brazilian strain of fall armyworm resistant to Bt Cry1 proteins.

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 to Bacillus 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 that CYP9A-like and CYP6B39 are 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. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sampling Accelerated Micron Scale Ice Particles with a Quadrupole Ion Trap Mass Spectrometer.

The Enceladus plume is a target of astrobiological interest in planetary science since it may carry signs of extraterrestrial life entrapped in ice grains formed from the subsurface ocean of this moon of Saturn. Fly-by mission concepts have been proposed to perform close investigations of the plume, including detailed in situ measurements of chemical composition with a new generation of mass spectrometer instrumentation. Such a scenario involves high-velocity collisions (typically around 5 km/s or higher) of the instrument with the encountered ice grains. Postimpact processes may include molecular fragmentation, impact ionization, and various subsequent chemical reactions that could alter the original material prior to analysis. In order to simulate Enceladus plume fly through conditions, we are developing an ice grain accelerator and have coupled it to the quadrupole ion trap mass spectrometer (QITMS) developed for flight applications. Our experimental setup enables the creation and acceleration of ice particles with well-defined size, charge, and velocity, which are subsequently directed into the QITMS, where they impact the surface of the mass analyzer and the analysis of postimpact, volatilized molecules takes place. In this work, we performed mass spectral analysis of ice grains of ca. 1.3 µm in diameter, accelerated and impacted at velocities up to 1000 m/s, with an upgrade of the accelerator in progress that will enable velocities up to 5000 m/s. We report the first observations of ice grain impacts measured by the QITMS, which were recorded as brief increases in the abundance of water molecules detected within the instrument.

Unique features of a global human ectoparasite identified through sequencing of the bed bug genome.

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.

Demonstration of a room temperature 2.48-2.75 THz coherent spectroscopy source.

We report the first demonstration of a continuous wave coherent source covering 2.48-2.75 THz, with greater than 10% instantaneous tuning bandwidth and having 1-14 µW of output power at room temperature. This source is based on a 91.8-101.8 GHz synthesizer followed by a power amplifier and three cascaded frequency triplers. It demonstrates for the first time that purely electronic solid-state sources can generate a useful amount of power in a region of the electromagnetic spectrum where lasers (solid state or gas) were previously the only available coherent sources. The bandwidth, agility, and operability of this THz source have enabled wideband, high resolution spectroscopic measurements of water, methanol, and carbon monoxide with a resolution and signal-to-noise ratio unmatched by any other existing system, providing new insight in the physics of these molecules. Furthermore, the power and optical beam quality are high enough to observe the Lamb-dip effect in water. The source frequency has an absolute accuracy better than 1 part in 10(12) and the spectrometer achieves sub-Doppler frequency resolution better than 1 part in 10(8). The harmonic purity is better than 25 dB. This source can serve as a coherent signal for absorption spectroscopy, a local oscillator for a variety of heterodyne systems and can be used as a method for precision control of more powerful but much less frequency agile quantum mechanical terahertz sources.