Efficacy of (R)-limonene, α-terpinene, terpinolene and β-pinene against Cochliomyia hominivorax (New World screwworm) in vitro and in vivo evaluations.

Cochliomyia hominivorax (screwworm fly) has a veterinary and public health importance, as it is the primary agent of myiasis in both livestock and humans across the Americas. This fly oviposits in fresh, open wounds, where the larvae feed on living tissue, compromising animal health and livestock productivity. Furthermore, the efficacy of conventional insecticides has decreased, suggesting the development of parasite resistance. Terpenes are major constituents of essential oils known for their insecticidal properties. The objective of this study was to evaluate the terpenes (R)-limonene, α-terpinene, terpinolene, and β-pinene against the first (L1), second (L2), and third (L3) larval instars of C. hominivorax. Among the evaluated terpenes, α-terpinene exhibited the highest lethality, with the lowest concentration required to kill 95% of the larvae (LC95) and was incorporated into an ointment at a concentration of 200 mg/g for in vivo testing. Twelve Holstein cattle were used in the study. Six control animals received bilateral 3 cm incisions in the gluteal region: one side was inoculated with 50 L1 larvae (untreated control), while the contralateral side received 50 L1 larvae plus the ointment vehicle (negative control). Another six cattle, each with a single gluteal lesion inoculated with 50 L1 larvae, were treated with the α-terpinene ointment, which was reapplied after 24 and 48 h. After 72 h, the surviving L3 larvae were recovered, counted, and measured. The α-terpinene ointment treatment showed no significant efficacy, as no differences were found in larval recovery rates between groups. However, larvae exposed to α-terpinene were significantly smaller compared to those in the untreated control and the vehicle control groups suggesting a relevant sub-lethal effect on parasite development. The discrepancy between the in vitro larvicidal activity and the limited in vivo efficacy may stem from the rapid volatilization of α-terpinene or its uneven distribution within the wound environment highlighting further optimization of the vehicle to maintain therapeutic concentrations and achieve full efficacy.