Toxicity and Synergistic Interactions of Citrus limon Essential Oil and Limonene with Chemical Acaricides Against Haemaphysalis doenitzi (Acari: Ixodidae)

Haemaphysalis (H.) doenitzi is a vector for the transmission of pathogens to endangered and domesticated animals, and the need for its control is becoming increasingly urgent.This study evaluated plant essential oils (EOs) and their major components as eco-friendly alternatives to chemical acaricides, including synergistic effects with acaricides.GC-MS analysis identified limonene (86.76%) and α-hexylcinnamaldehyde (21.71%) as the main components of Citrus (C.) limon and Jasminum (J.) sambac EO, respectively.Immersion tests showed both EOs are highly effective against H. doenitzi nymphs and adults.Citrus limon EO showed higher acaricidal toxicity than J. sambac EO, whereas the major component of J. sambac EO was more potent than limonene.Citrus limon EO and limonene inhibited CarE activity and upregulated ABC transporter expression.Molecular docking revealed limonene has a stronger binding affinity to ABC transporters, GSTs, and CYP450 enzymes.Tick control trials showed J. sambac EO increased engorgement weight, whereas C. limon EO reduced both engorgement and egg mass weight.RNAi knockdown of chitin binding protein (HDCBPa) and fumarylacetoacetase (HDFAHa) significantly increased tick sensitivity to C. limon EO and limonene, leading to higher mortality.Synergistically, a 1:3 mixture of C. limon EO and pyridaben produced a co-toxicity coefficient (CTC) of 129.71.In conclusion, C. limon EO and limonene may exert acaricidal effects by inhibiting CarE, upregulating ABC transporter gene, and binding enzymes, while reducing tick weight and egg mass and enhancing pyridaben efficacy, highlighting their superior potential for tick control.