Honey bees (Apis mellifera) are major pollinators, playing a critical role in global food production, biodiversity, and ecosystem stability. However, their populations are increasingly threatened by multiple interacting stressors, including pesticide exposure. Among these, agricultural insecticides and anti-Varroa acaricides such as dinotefuran and amitraz can persist in hive matrices, resulting in chronic and combined exposure. This study investigates the low lethal (LC₁₀ and LC₃₀) effects of these compounds, individually and in combination, on honey bee survival, immune function, oxidative stress responses, detoxification pathways, and hypopharyngeal gland morphology. Both pesticides negatively affected honey bee health at low lethal concentrations, with dinotefuran showing higher toxicity. Exposure led to the reduced survival, suppression of vitellogenin expression, and dysregulation of genes related to antioxidant defense, immunity, and detoxification. Additionally, high concentrations of dinotefuran and its combination with amitraz impaired hypopharyngeal gland morphology. Notably, co-exposure resulted in synergistic toxic effects, exacerbating physiological damage beyond individual treatments. These findings emphasize the potential risks of combined exposure to agricultural and beekeeping pesticides. A more comprehensive risk assessment and stricter regulations are urgently needed.

Although Cacopsylla pyri Forster and Cacopsylla pyricola Linnaeus have long been considered as more significant pests of pear trees around the world, Cacopsylla permixta Burckhardt and Hodkinson is the most significant pest in some parts of Iran, especially in pear gardens of Karaj. Current control strategies against this pest in Iran generally involve five or six insecticide treatments each year, despite unsatisfactory results are reported at many localities. So, it is crucial to know the most susceptible generation of the pest to apply a good strategy for its control. The aim of this study was to explore the seasonal variation in susceptibility of C. permixta to four commonly used insecticides. The results showed that winter forms (February) were 2.71‐fold, 4.58‐fold, 3.26 fold and 3.38‐fold more tolerant to diazinon, imidacloprid, acetamiprid and abamectin, respectively, compared with summer forms. Also, Esterase, GST and P450 monooxygenase activity was highest during February. Moreover, the content of lipid, carbohydrate, glycogen and protein was significantly higher in February compared with other months. Based on these results, the best period for insecticide treatment for efficient control is treatment against the first generation, at the time when eggs are laid by females as well as during the egg hatching and the larvae appearance. At this time of year, psylla are more susceptible, which would likely lead to better results and the reduction in damage during the next summer. However, further studies are needed to test this in farm settings, and to whether this holds true for other psylla species.

Enzymatic detoxification of organophosphate (OP) insecticides can confer resistance in some insects, yet the precise molecular basis of this trait, and how it has evolved, remains poorly understood. In certain dipteran species, a G→D mutation in the oxyanion hole of α-carboxylesterases (CBEs) enhances OP hydrolysis, yet this adaptation is not widespread despite the presence of orthologous CBEs in other insect species that are also exposed to OPs. The extent, and molecular basis, of evolutionary contingency and epistasis in this catalytic OP resistance has not been explored, and how further mutations might optimize OP detoxification in the future is not clear. Here, we systematically compare OP hydrolysis and analyze structures of CBE orthologs across several dipteran species, revealing that the success of the G137D mutation is sequence context-dependent. We employed laboratory-directed evolution to enhance OP turnover over 1,000-fold vs. the wildtype enzyme and tested these variants in transgenic Drosophila melanogaster , demonstrating that improved catalytic rates do not directly translate to increased resistance. By highlighting the trade-off between organophosphate affinity and turnover, this work further clarifies the complex evolutionary trajectories determining why a particular resistance mechanism may evolve in some species but not others.