Pereira_2026_Pathogens_15_

Rhipicephalus microplus is a cattle ectoparasite responsible for economic losses in livestock systems. The present study aimed to evaluate the essential oil (EO) of Piper hemmendorffii and its major constituent, limonene, for their activity against R. microplus larvae. The EO of P. hemmendorffii and its major compound, limonene, were evaluated against pyrethroid-resistant R. microplus larvae using larval immersion bioassays, biochemical analyses of detoxification enzymes and acetylcholinesterase, and scanning electron microscopy (SEM). The EO showed higher larvicidal potency (LC(50) = 10.46 mg/mL; 95% CI: 9.86-11.09) compared to limonene (LC(50) = 31.62 mg/mL; 95% CI: 30.86-32.40). Piper hemmendorffii EO affected the following classes of enzymes investigated: alpha-Esterase (alpha-EST) activity increased at LC(25) and LC(50), reaching its highest level at LC(50), whereas beta-esterase (beta-EST) activity increased only at LC(25). Cytochrome P450 (CYP450)-associated heme content activity showed a significant increase exclusively at LC(50), suggesting a response of the surviving larvae to EO exposure, although this increase does not by itself demonstrate the activation of detoxification pathways. In contrast, limonene did not significantly affect the activity of the three evaluated enzymes. The inhibition of AChE by the EO suggests a possible effect on the cholinergic system, which may be associated with neurotoxic activity, whereas limonene had no significant effect. The absence of detectable ultrastructural changes by SEM suggests that treatment with either EO or limonene did not induce evident external morphological damage to the larval cuticle under the evaluated conditions. EO toxicity seems to be associated with changes in detoxification enzyme and AChE activities, suggesting the involvement of these biochemical pathways in larval response.