Carvacrol as a dual-target acaricidal candidate against the cattle tick Rhipicephalus (Boophilus) microplus: molecular docking, mechanistic validation, and early field testing

The blood-sucking ectoparasite Rhipicephalus B. microplus represents one of the greatest economic threats to livestock worldwide. The discovery of novel acaricidal molecules based on environmentally sustainable technologies is therefore required, particularly those suitable for use in dairy and meat-producing livestock without generating contaminant residues. This study aimed to identify and validate plant-derived metabolites with dual activity on acetylcholinesterase and mitochondrial targets as sustainable acaricidal agents against R. microplus, integrating computational screening, biochemical assays and field evaluation. Following in silico screening of 1.300 plant metabolites targeting acetylcholinesterase and mitochondrial function, fourteen compounds were selected for in vitro and in vivo mortality assays using immersion and larval vessel methods. Carvacrol was identified as the most promising metabolite and showed an inhibitory mechanism of action on acetylcholinesterase and mitochondrial complexes I, III, and IV. Its acaricidal efficacy was subsequently confirmed under field conditions using ethanolic and oily formulations (spray and pour-on) applied to cattle (Bos taurus). The spray formulation significantly reduced tick infestation, decreasing tick counts by 20% every 10 days (p < 0.001), whereas the pour-on formulation showed no significant reduction (p = 0.093). These results demonstrate that an integrated discovery pipeline from computational screening to field validation provides a robust strategy for identifying plant-derived acaricidal agents targeting key physiological pathways in R. microplus. HighlightsO_LIStructured and sequential workflow (pipeline) from in silico to Bos taurus C_LIO_LIIdentification and validation of phytometabolites against R. microplus C_LIO_LICarvacrol multitarget activity on acetylcholinesterase and bioenergetic in ticks C_LIO_LIThe spray formulation reduces tick infestation by 20% every 10 days C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/727743v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@11c9931org.highwire.dtl.DTLVardef@8d6088org.highwire.dtl.DTLVardef@1bcc09aorg.highwire.dtl.DTLVardef@108d1d1_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Avendano, G. (2026) https://BioRender.com/7pm1ffj