Field-evolved resistance to nitenpyram is associated with fitness costs in whitefly.

BACKGROUND: Elucidating fitness cost associated with field-evolved insect resistance to insecticide is of particular importance to current sustainable pest control. The global pest whitefly Bemisia tabaci has developed resistance to many members of neonicotinoids, but little is known about whitefly resistance to neonicotinoid nitenpyram and its associated fitness cost. Using insecticide bioassay and life-table approach, this study aims to investigate nitenpyram resistance status in field-collected whitefly populations, and to explore whether such resistance is accompanied by a fitness cost. RESULTS: The bioassay results revealed that 14 of 29 whitefly populations displayed moderate to extremely high resistance to nitenpyram, demonstrating a widespread field-evolved resistance to nitenpyram. This field-evolved resistance in the whitefly has increased gradually over the past 3 years from 2021 to 2023. Further life-table study showed that two resistant whitefly populations exhibited longer developmental time, shorter lifespans of adult, and lower fecundity compared with the most susceptible population. The relative fitness cost of the two resistant populations was calculated as 0.69 and 0.56 by using net productive rate R0, which suggests that nitenpyram resistance comes with fitness cost in the whitefly, especially on reproduction. CONCLUSION: Overall, these results represent field-evolved high resistance to nitenpyram in the whitefly. The existing fitness costs associated with nitenpyram resistance are helpful to propose a suitable strategy for sustainable control of whiteflies by rotation or mixture of insecticide with different modes of action. © 2024 Society of Chemical Industry.

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.

CYP4CS5-mediated thiamethoxam and clothianidin resistance is accompanied by fitness cost in the whitefly Bemisia tabaci.

BACKGROUND: Understanding the trade-offs between insecticide resistance and the associated fitness is of particular importance to sustainable pest control. One of the most devastating pest worldwide, the whitefly Bemisia tabaci, has developed resistance to various insecticides, especially the neonicotinoid group. Although neonicotinoid resistance often is conferred by P450s-mediated metabolic resistance, the relationship between such resistance and the associated fitness phenotype remains largely elusive. By gene cloning, quantitative reverse transcription (qRT)-PCR, RNA interference (RNAi), transgenic Drosophila melanogaster, metabolism capacity in vitro and 'two sex-age stage' life table study, this study aims to explore the molecular role of a P450 gene CYP4CS5 in neonicotinoid resistance and to investigate whether such resistance mechanism carries fitness costs in the whitefly. RESULTS: Our bioassay tests showed that a total of 13 field-collected populations of B. tabaci MED biotype displayed low-to-moderate resistance to thiamethoxam and clothianidin. Compared to the laboratory susceptible strain, we then found that an important P450 CYP4CS5 was remarkably upregulated in the field resistant populations. Such overexpression of CYP4CS5 had a good match with the resistance level among the whitefly samples. Further exposure to the two neonicotinoids resulted in an increase in CYP4CS5 expression. These results implicate that overexpression of CYP4CS5 is closely correlated with thiamethoxam and clothianidin resistance. RNAi knockdown of CYP4CS5 increased mortality of the resistant and susceptible populations after treatment with thiamethoxam and clothianidin in bioassay, but obtained an opposite result when using a transgenic line of D. melanogaster expressing CYP4CS5. Metabolic assays in vitro revealed that CYP4CS5 exhibited certain capacity of metabolizing thiamethoxam and clothianidin. These in vivo and in vitro assays indicate an essential role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance in whitefly. Additionally, our life-table analysis demonstrate that the field resistant whitefly exhibited a prolonged development time, shortened longevity and reduced fecundity compared to the susceptible, suggesting an existing fitness cost as a result of the resistance. CONCLUSION: Collectively, in addition to the important role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance, this resistance mechanism is associated with fitness costs in the whitefly. These findings not only contribute to the development of neonicotinoids resistance management strategies, but also provide a new target for sustainable whitefly control. © 2023 Society of Chemical Industry.

Adipokinetic hormone signaling regulates cytochrome P450-mediated chlorantraniliprole sensitivity in Spodoptera frugiperda (Lepidoptera: Noctuidae).

BACKGROUND: Fall armyworm (FAW, Spodoptera frugiperda) is one of the most destructive and invasive pests worldwide and causes significant economic losses. Intensive and frequent use of insecticides has led to the development of resistance in FAW. Adipokinetic hormone (AKH) have been proven to be involved in insecticide resistance in insects. However, the molecular mechanism underlying chlorantraniliprole resistance mediated by AKH signaling in FAW remains unclear. RESULTS: The expression of SpfAKHR was highest in male adults and lowest in 1st instar larvae. SpfAKH was expressed the highest in eggs and the lowest in 6th instar larvae. AKH signaling was involved in the sensitivity of FAW to chlorantraniliprole through a toxicological bioassay, and the combination of chlorantraniliprole and bithionol (an inhibitor of key enzymes in the AKH pathway) significantly increased the mortality of FAW. Chlorantraniliprole significantly induced the expression of ten P450s, SpfAKH and SpfAKHR in FAW. RNA interference against SpfAKHR significantly decreased the P450 content, downregulated the expression of three P450 genes (SpfCYP6B50, SpfCYP321A9 and SpfCYP9A58) and inhibited the resistance of FAW to chlorantraniliprole. The topical application of AKH peptide significantly increased the P450 content, upregulated the expression of five P450 genes (SpfCYP321A9, SpfCY321A8, SpfCYP321A10, SpfCYP321A7 and SpfCYP6AB12), and enhanced the survival of FAW against chlorantraniliprole. CONCLUSIONS: AKH plays an important role in enhancing chlorantraniliprole resistance in FAW by exerting a positive influence on P450 gene expression and P450 content. These results provide valuable insights into insecticide resistance regulation and FAW control strategies. © 2022 Society of Chemical Industry.