Standardization of Bioassays for Evaluating Insecticide Efficacy on Cochliomyia hominivorax Larvae (Diptera, Calliphoridae)
Control; screwworm; in vitro test; larvicide.
Cochliomyia hominivorax is the main etiological agent of myiasis in Latin America, and although a wide range of drugs is currently available for the control of its parasitic stages, reduced efficacy of some compounds has been reported; therefore, in vitro bioassays are essential for detecting resistance and for evaluating the most effective exposure methods for each insecticide. This study aimed to evaluate the efficacy of different in vitro bioassay methodologies against third-instar larvae (L3) of C. hominivorax. Four active ingredients—chlorpyrifos, fipronil, fluralaner, and doramectin—were tested using two methodologies: filter paper impregnation and larval immersion for 1, 3, and 5 minutes. In the impregnation assays, nine concentrations of each active ingredient (1000–1 µg/mL for chlorpyrifos, doramectin and fluralaner, and 5000–1 µg/mL for fipronil) were applied to filter paper discs, along with a non-treated control and a placebo group containing only the diluent; after drying, the discs were placed in Petri dishes and ten L3 larvae of C. hominivorax were added to each disc. In the immersion assays, groups of ten L3 larvae were submerged in solutions containing the same nine concentrations used in the impregnation tests, in addition to control and placebo groups, and all assays were performed in sextuplicate. After treatment, the assays were incubated at 28 °C and 70% relative humidity, larvicidal effects were evaluated at 24, 48, and 72 h after exposure, and adult emergence inhibition was assessed 10 days after incubation; median lethal concentrations (LC50) were estimated using Probit analysis in the RStudio software. At 72 h, chlorpyrifos LC50 values were 100.5, 777.3, 259.3, and 302.3 µg/mL for the impregnation and the 1, 3, and 5 min immersion methodologies, respectively; for fipronil, the corresponding values were 76.8, 14.5, 1.2, and 2.4 µg/mL; for fluralaner, LC50 values were 267.5, 2068.9, 872.9, and 635.5 µg/mL; and for doramectin, 676.3, 334.3, 255.1, and 215.3 µg/mL, respectively. Regarding emergence inhibition, LC50 values for fluralaner were 15.4, 302.7, 92.0, and 67.7 µg/mL, and for doramectin were 4.2, 19.7, 15.1, and 7.0 µg/mL using the impregnation and the 1, 3, and 5 min immersion methodologies, respectively; for chlorpyrifos, LC50 values could be calculated only for the immersion methodology, resulting in 541.2, 114.7, and 120.1 µg/mL for 1, 3, and 5 min, respectively, whereas for fipronil, LC50 values were estimated only for the impregnation and 1 min immersion methodologies, yielding 13.5 and 4.6 µg/mL, respectively. The results demonstrate that the efficacy of insecticidal compounds varies significantly according to the in vitro methodology employed, highlighting the importance of bioassay standardization for the accurate interpretation of larvicidal activity.