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

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

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

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

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

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

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.

The role of ATP-binding cassette transporters in arthropod pesticide toxicity and resistance.

Pesticide resistance in arthropods threatens agricultural productivity and the control of vector-borne diseases. The ATP-binding cassette (ABC) transporters have emerged as important factors in the toxicity of synthetic pesticides, as well as for Bacillus thuringiensis insecticidal Cry protein binding. Depending on the localization of expression, both higher and lower expression of ABCs have been linked with pesticide resistance. The recent development of genetic-based approaches such as RNAi and CRISPR/Cas9 gene editing in nonmodel species, has greatly contributed to unveil their functional importance in pesticide toxicity and resistance. Using these tools, we are now poised to further unravel the molecular genetic mechanisms of gene regulation uncovering more elusive regulatory resistance genes.

The molecular determinants of pesticide sensitivity in bee pollinators.

Bees carry out vital ecosystem services by pollinating both wild and economically important crop plants. However, while performing this function, bee pollinators may encounter potentially harmful xenobiotics in the environment such as pesticides (fungicides, herbicides and insecticides). Understanding the key factors that influence the toxicological outcomes of bee exposure to these chemicals, in isolation or combination, is essential to safeguard their health and the ecosystem services they provide. In this regard, recent work using toxicogenomic and phylogenetic approaches has begun to identify, at the molecular level, key determinants of pesticide sensitivity in bee pollinators. These include detoxification systems that convert pesticides to less toxic forms and key residues in insecticide target-sites that underlie species-specific insecticide selectivity. Here we review this emerging body of research and summarise the state of knowledge of the molecular determinants of pesticide sensitivity in bee pollinators. We identify gaps in our knowledge for future research and examine how an understanding of the genetic basis of bee sensitivity to pesticides can be leveraged to, a) predict and avoid negative bee-pesticide interactions and facilitate the future development of pest-selective bee-safe insecticides, and b) inform traditional effect assessment approaches in bee pesticide risk assessment and address issues of ecotoxicological concern.

Presence of Multiple Genetic Mutations Related to Insecticide Resistance in Chinese Field Samples of Two Phthorimaea Pest Species.

Potatoes hold the distinction of being the largest non-cereal food crop globally. The application of insecticides has been the most common technology for pest control. The repeated use of synthetic insecticides of the same chemical class and frequent applications have resulted in the emergence of insecticide resistance. Two closely related pests that feed on potato crops are the potato tuber moth, Phthorimaea operculella, and the tomato leafminer, Phthorimaea absoluta (syn. Tuta absoluta). Previous studies indicated the existence of insecticide resistance to various classes of insecticides including organophosphates, carbamates, and pyrethroids in field populations of P. operculella and P. absoluta. However, the exact mechanisms of insecticide resistance in P. operculella and to a lesser extent P. absoluta remain still poorly understood. Detecting resistance genotypes is crucial for the prediction and management of insecticide resistance. In this study, we identified multiple genetic mutations related to insecticide resistance in two species of Phthorimaea. An unexpected genetic divergence on target-site mutations was observed between P. operculella and P. absoluta. Three mutations (A201S, L231V, and F290V) in Ace1 (acetylcholinesterase), four mutations (M918T, L925M, T928I, and L1014F) in VGSC (voltage-gated sodium channel), and one mutation (A301S) in RDL (GABA-gated chloride channel) have been detected with varying frequencies in Chinese P. absoluta field populations. In contrast, P. operculella field populations showed three mutations (F158Y, A201S, and L231V) in Ace1, one mutation (L1014F) in VGSC at a lower frequency, and no mutation in RDL. These findings suggest that pyrethroids, organophosphates, and carbamates are likely to be ineffective in controlling P. absoluta, but not P. operculella. These findings contributed to a deeper understanding of the presence of target-site mutations conferring resistance to commonly used (and cheap) classes of insecticides in two closely related potato pests. It is recommended to consider the resistance status of both pests for the implementation of resistance management strategies in potatoes.

Evidence of active oviposition avoidance to systemically applied imidacloprid in the Colorado potato beetle.

Agricultural pests can develop behavioral resistance to insecticides by choosing to feed or oviposit on insecticide-free hosts. As young larvae have relatively low mobility, oviposition preferences from female adults may play a critical role in shaping the evolutionary trajectory of pest populations. While oviposition avoidance of insecticide-treated hosts was found in different agriculture pests, it remains unclear whether female adults actively choose to occupy insecticide-free hosts. To address this question, we investigated feeding and oviposition preferences between imidacloprid-treated and imidacloprid-free plants in the Colorado potato beetle, Leptinotarsa decemlineata Say, a major potato pest. We performed behavioral choice assays on two strains that differed in both fecundity and insecticide resistance. We found that one strain preferred to feed on the insecticide-free plants and that this preference is not innate. Meanwhile, the other strain chose plants for feeding and oviposition randomly. Further analyses of the moving patterns of the beetles suggested that the oviposition preference in the first strain is likely due to active learning.

A cytochrome P450 insecticide detoxification mechanism is not conserved across the Megachilidae family of bees.

Recent work has demonstrated that many bee species have specific cytochrome P450 enzymes (P450s) that can efficiently detoxify certain insecticides. The presence of these P450s, belonging or closely related to the CYP9Q subfamily (CYP9Q-related), is generally well conserved across the diversity of bees. However, the alfalfa leafcutter bee, Megachile rotundata, lacks CYP9Q-related P450s and is 170-2500 times more sensitive to certain insecticides than bee pollinators with these P450s. The extent to which these findings apply to other Megachilidae bee species remains uncertain. To address this knowledge gap, we sequenced the transcriptomes of four Megachile species and leveraged the data obtained, in combination with publicly available genomic data, to investigate the evolution and function of P450s in the Megachilidae. Our analyses reveal that several Megachilidae species, belonging to the Lithurgini, Megachilini and Anthidini tribes, including all species of the Megachile genus investigated, lack CYP9Q-related genes. In place of these genes Megachile species have evolved phylogenetically distinct CYP9 genes, the CYP9DM lineage. Functional expression of these P450s from M. rotundata reveal they lack the capacity to metabolize the neonicotinoid insecticides thiacloprid and imidacloprid. In contrast, species from the Osmiini and Dioxyini tribes of Megachilidae have CYP9Q-related P450s belonging to the CYP9BU subfamily that are able to detoxify thiacloprid. These findings provide new insight into the evolution of P450s that act as key determinants of insecticide sensitivity in bees and have important applied implications for pesticide risk assessment.