In Vitro and In Silico Investigations of the Acaricidal Activity of Carvacrol Against Rhipicephalus annulatus

Carvacrol, a naturally occurring monoterpenic phenol, has attracted considerable interest as a potential botanical acaricide against hard ticks. This study evaluated the acaricidal efficacy and molecular mode of action of carvacrol against Rhipicephalus annulatus, using phoxim as a positive control.

An in vitro Adult Immersion Test (AIT) was conducted to assess the effects of different concentrations of carvacrol, phoxim, and their combinations on adult ticks. In addition, acetylcholinesterase (AChE) activity and reduced glutathione (GSH) levels were measured to investigate potential biochemical mechanisms underlying acaricidal activity. Molecular docking analyses were performed to identify potential molecular targets and compare the binding affinities of carvacrol and phoxim.

Both carvacrol and phoxim exhibited concentration-dependent acaricidal activity against adult R. annulatus, with LC50 values of 43.2 and 0.22 mg/mL, respectively. Among the tested combinations, carvacrol (25 mg/mL) combined with phoxim (0.25 mg/mL) achieved 100% mortality and demonstrated a synergistic interaction. Biochemical analyses revealed that carvacrol, similar to phoxim, significantly inhibited AChE activity and depleted GSH levels, indicating disruption of both cholinergic neurotransmission and antioxidant defense mechanisms. Molecular docking provided mechanistic support for these findings, demonstrating favorable binding affinities of carvacrol toward several key tick targets, including acetylcholinesterase, glutathione-related proteins, pheromone and odorant-related receptors, and P6S tyrosinase. Notably, carvacrol exhibited binding affinities comparable to or greater than those of phoxim for several targets, supporting a multi-target mode of action.

Collectively, these findings indicate that carvacrol exerts acaricidal activity through coordinated disruption of neural, antioxidant, signaling, and chemosensory pathways. Furthermore, its synergistic interaction with phoxim highlights its potential as a botanical acaricide that could complement conventional synthetic compounds and contribute to sustainable control strategies against R. annulatus.