Independent Exposure to the Fungicide Difenoconazole or the Herbicide Tebuthiuron Disrupts Survival, Behavior, and Alters Midgut-Associated Protein Detection in Aedes aegypti Larvae.

Agrochemicals used in agriculture have been detected in aquatic ecosystems, affecting the biota and potentially causing bioaccumulation. This work investigated the lethal and sublethal effects of the fungicide difenoconazole and the herbicide tebuthiuron on larvae of the yellow fever mosquito, Aedes aegypti. Mosquitoes are widespread and abundant throughout aquatic ecosystems, making them suitable models for toxicity studies, with A. aegypti being particularly easy to rear. We assessed the impact of exposure on larval survival, behavior, and physiology, including antioxidant enzyme activity and immunolabeling of key midgut proteins. Exposed larvae showed reduced survival, with a 50% mortality rate reached within 1 and 3 days after exposure to difenoconazole (75 mg/L) and tebuthiuron (2.85 × 104 mg/L), respectively. Both chemicals altered behavior, with difenoconazole reducing larval activity and tebuthiuron increasing it. Enzymatic assays indicated a reduction in key antioxidant enzymes (superoxide dismutase, catalase, and glutathione S-transferase) in the larval body, suggesting disruptions in the mechanisms that regulate redox homeostasis. Midgut immunolabeling showed a decrease in crucial proteins for tissue organization (cadherin, armadillo, FMRFamide), stem cell maintenance and differentiation (JNKs, WNT, Notch, Delta, and Prospero), and metamorphosis (PH3). Furthermore, exposure to difenoconazole caused an increase in markers of apoptosis and autophagy, caspase-3 and LC3. These results highlight the physiological impacts of agrochemical exposure on an essential organ for survival and development in a representative aquatic insect species, underscoring the broader ecological impacts of xenobiotic contamination in freshwater ecosystems.