| Summary: | Ethyl formate (EF), a naturally occurring fumigant, has attracted widespread attention owing to its low toxicity in mammals. Here, Direct Immersion Solid-Phase Microextraction (DI-SPME) was employed for sample preparation in mass spectrometry-based untargeted metabolomics to evaluate the effects on <i>Tribolium castaneum</i> (Herbst) strains with different levels of PH<sub>3</sub> resistance (sensitive, TC-S; moderately resistant, TC-M; strongly resistant, TC-SR) when exposed to a sub-lethal concentration (LC<sub>30</sub>) of EF. The bioassay indicated that <i>T. castaneum</i> strains with varying PH<sub>3</sub> resistance levels did not confer cross-resistance to EF. A metabolomic analysis revealed that exposure to sublethal doses of EF significantly altered 23 metabolites in <i>T. castaneum</i>, including 2 that are unique to the species which remained unaffected by external conditions, while 11 compounds showed a strong response. A pathway topology analysis indicated that EF caused changes to several metabolic pathways, mainly involving fatty acids and their related metabolic pathways. This study showed that EF can induce highly similar metabolic responses in insects across varying levels of PH<sub>3</sub> resistance, suggesting that the mechanisms driving the toxicity of EF and PH<sub>3</sub> are distinct. These insights significantly extend our knowledge of the toxic mechanisms of EF and provide direct evidence for the efficacy of EF treatment for managing PH<sub>3</sub> resistance in insects.
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