Cytotoxic and Immunological Effects of Terpinolene and 1-butyl-3,4-methylenedioxybenzene on Aedes aegypti Larvae.

Interactions between biomolecules and their target sites are fundamental to pest control, and such relationships can either intensify or inhibit their effects. A broader understanding of these complex interactions aids in interpreting toxicological responses, while physiological changes can elucidate insect metabolic pathways. Research involving terpinolene and 1-butyl-3,4-methylenedioxybenzene (BMDB) has demonstrated their insecticidal activity against Aedes aegypti. However, the effects of these substances on physiological and immunological parameters require further clarification; furthermore, the binary combination of these compounds may result in more significant and permanent damage. Thus, this study aimed to test the hypothesis that combining different modes of action would intensify histological and immunological alterations critical to insect survival, thereby enabling more effective control. To this end, LC50 values for the individual compounds were obtained from the literature (Silva et al. 2016), and a dose-response curve was established for the 1:1 combination, revealing an LC50 of 75.69 ± 2.37 ppm. The cytotoxic parameters evaluated included midgut histology, apoptosis, and cell proliferation in L4-stage larvae, while immunological parameters evaluated included phenoloxidase activity, nitric oxide levels, and oxidative stress markers (TBARS and GST). Histological changes in the midgut were observed in all treatment groups compared to the control; however, more severe histopathological alterations-such as vacuolization, cellular swelling, and loss of the peritrophic matrix-were observed in the combined treatment. Regarding apoptosis, a significant increase was observed only in treatments containing BMDB, whether alone (2.66 ± 0.66) or in combination (3.33 ± 0.33), compared to the control (0.5 ± 0.54). As for cell proliferation (PCNA), larvae in the combined group exhibited a lower rate (0.50 ± 0.11) compared to terpinolene (1.33 ± 0.12) and BMDB (1.40 ± 0.15). Reductions in phenoloxidase activity (1.026 ± 0.061 OD/min/mg) and nitric oxide levels (0.878 ± 0.034 µM/mL) were observed in the combined group. Regarding oxidative stress, there was no statistical difference in GST measurements (p > 0.05), whereas TBARS levels decreased in the combined group (2.497 ± 0.184 nmol MDA/mg/protein) compared to the control (3.401 ± 0.327 nmol MDA/mg/protein). Thus, despite exhibiting lower larvicidal activity compared to the isolated compounds, the binary combination caused more severe histopathological and immunological damage that compromises larval survival and recovery capacity, representing a promising strategy for integrated vector management.