Field-evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia

This is the final version of the article. Available from Wiley via the DOI in this record.BACKGROUND: We report on the status of imidacloprid and ethiprole resistance in Nilaparvata lugens Stål collected from across South and East Asia over the period 2005-2012. RESULTS: A resistance survey found that field populations had developed up to 220-fold resistance to imidacloprid and 223-fold resistance to ethiprole, and that many of the strains collected showed high levels of resistance to both insecticides. We also found that the cytochrome P450 CYP6ER1 was significantly overexpressed in 12 imidacloprid-resistant populations tested when compared with a laboratory susceptible strain, with fold changes ranging from ten- to 90-fold. In contrast, another cytochrome P450 CYP6AY1, also implicated in imidacloprid resistance, was underexpressed in ten of the populations and only significantly overexpressed (3.5-fold) in a single population from India compared with the same susceptible strain. Further selection of two of the imidacloprid-resistant field strains correlated with an approximate threefold increase in expression of CYP6ER1. CONCLUSIONS: We conclude that overexpression of CYP6ER1 is associated with field-evolved resistance to imidacloprid in brown planthopper populations in five countries in South and East Asia.This work was funded by Bayer CropScience. Rothamsted Research receives grant-aided support from the Biotechnology and Biosciences Research Council of the United Kingdom

Driving down malaria transmission with engineered gene drives

The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings

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

This is the final version. Available on open access from Public Library of Science via the DOI in this recordData Availability: All relevant data are within the manuscript and its Supporting Information files.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.European Union Horizon 2020Medical Research Council (MRC

Fly-Tox: A panel of transgenic flies expressing pest and pollinator cytochrome P450s

This is the final version. Available from the publisher via the DOI in this record.There is an on-going need to develop new insecticides that are not compromised by resistance and that have improved environmental profiles. However, the cost of developing novel compounds has increased significantly over the last two decades. This is in part due to increased regulatory requirements, including the need to screen both pest and pollinator insect species to ensure that pre-existing resistance will not hamper the efficacy of a new insecticide via cross-resistance, or adversely affect non-target insect species. To add to this problem the collection and maintenance of toxicologically relevant pest and pollinator species and strains is costly and often difficult. Here we present Fly-Tox, a panel of publicly available transgenic Drosophila melanogaster lines each containing one or more pest or pollinator P450 genes that have been previously shown to metabolise insecticides. We describe the range of ways these tools can be used, including in predictive screens to avoid pre-existing cross-resistance, to identify potential resistance-breaking inhibitors, in the initial assessment of potential insecticide toxicity to bee pollinators, and identifying harmful pesticide-pesticide interactions.European Research Council (ERC)European Union's Horizon 2020 research and innovation programmeBiotechnology and Biological Sciences Research Council (BBSRC

Field-evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia

BACKGROUND We report on the status of imidacloprid and ethiprole resistance in Nilaparvata lugens Stål collected from across South and East Asia over the period 2005–2012. RESULTS A resistance survey found that field populations had developed up to 220‐fold resistance to imidacloprid and 223‐fold resistance to ethiprole, and that many of the strains collected showed high levels of resistance to both insecticides. We also found that the cytochrome P450 CYP6ER1 was significantly overexpressed in 12 imidacloprid‐resistant populations tested when compared with a laboratory susceptible strain, with fold changes ranging from ten‐ to 90‐fold. In contrast, another cytochrome P450 CYP6AY1, also implicated in imidacloprid resistance, was underexpressed in ten of the populations and only significantly overexpressed (3.5‐fold) in a single population from India compared with the same susceptible strain. Further selection of two of the imidacloprid‐resistant field strains correlated with an approximate threefold increase in expression of CYP6ER1. CONCLUSIONS We conclude that overexpression of CYP6ER1 is associated with field‐evolved resistance to imidacloprid in brown planthopper populations in five countries in South and East Asia

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

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations

Quantitative MRI and loss of free ambulation in Duchenne muscular dystrophy

The purpose of this ethics approved trial was to correlate quantitative MRI with functional abilities in both ambulant and non-ambulant Duchenne muscular dystrophy (DMD). Twenty patients with genetically confirmed DMD were recruited. Physical assessment was performed using the motor function measurement (MFM) scale. Axial 3T MRI scans of the thighs were acquired using T1-weighted in- and opposed-phase images (TR = 20 ms, TE1 = 2.45 ms, TE2 = 3.68 ms, flip angle = 15°) to calculate the relative fat fraction according to the two-point Dixon method in the knee extensors, flexors, and adductor muscles. The average MFM was 65.3 % and correlated negatively to age (r (2) = 0.60). Overall mean fat fraction correlated positively to age (r (2) = 0.51-0.64). An average of 5 % increase in mean fat fraction per year was calculated. Mean fat fraction of the quadriceps showed a high negative correlation (r (2) = 0.93) to the D1 (standing position and transfers) component of the MFM. A cutoff for mean fat fraction of 50 % predicted loss of ambulation with a sensitivity of 100 % and a specificity of 91 %. Therefore, quantitative muscle MRI seems to be a promising endpoint for short clinical trials evaluating the effect of newer treatments on the time of loss of ambulation in DMD