Glyphosate (GY) is the most widely used herbicide in agriculture worldwide. Lead is a common heavy metal in the natural environment. Honeybees, as pollinators, are exposed to these pollutants. So far, few reports have evaluated the toxic effects of GY mixed with heavy metals on honeybees (Apis cerana cerana). This study found that the acute toxicity of lead (LC50 = 1083 mg/L) is much greater than that of GY (LC50 = 4764 mg/L) at 96 h. The acute toxicities of the mixed substances were as follows: LC50 = 621 mg/L of lead and LC50 = 946 mg/L of GY. The combination of lead and GY was more toxic than either of the individual substances alone. Compared to the individual toxicity, combined treatment significantly affected the bees’ learning and cognitive abilities and changed the relative expression of genes related to immune defense and detoxification metabolism in A. c. cerana. The combination of lead and GY seriously affected the behavior and physiology of the studied honeybees. This study provides basic data for further research on the combined effects of GY and heavy metals on bee health. It also serves as a reference for effective colony protection.

Pesticides such as neonicotinoids frequently harm beneficial insect pollinators and affect their survival, social behavior, digestive system, and metabolism. Investigating the mechanisms behind these impairments is crucial for enhancing pesticide risk assessments. Apis cerana, a native honeybee species in Asia, has received limited research attention regarding the toxicological mechanisms of thiamethoxam (TMX) exposure. We exposed newly emerged worker bees of A. cerana to a field-relevant dose of TMX (400 ng/g) under laboratory conditions to examine whether TMX exposure triggers similar or distinct effects in different biological processes and tissues. Our results demonstrate that TMX damages the gut cell structure and significantly increases mortality. Gut transcriptomic analysis revealed that the activation of signaling pathways such as glycosphingolipid biosynthesis, Notch signaling, and Wnt signaling likely contributed to structural damage in gut cells. Head transcriptomic results indicated that the activation of pathways including pyruvate metabolism, glycolysis/gluconeogenesis, thiamine metabolism, and riboflavin metabolism might negatively affect the stability of the neural system in A. cerana. The metabolic dysfunction of glycine, serine, threonine, as well as glycerophospholipids potentially impairs the neural system, leading to behavioral abnormalities and mortality. In summary, field-level TMX damages the gut cell structure, destabilizes the neural system, and increases the mortality rate of A. cerana. These findings demonstrate that TMX exposure induces complex, tissue-specific effects. This study provides a comprehensive understanding of the molecular and physiological impacts of TMX on A. cerana, offering valuable insights for the conservation and protection of this important pollinator species.