Enzymatic biomarkers reveal sublethal effects of thiamethoxam and fipronil on the stingless bee Melipona scutellaris.

Bees are key pollinators in natural and agricultural ecosystems, yet they are increasingly affected by the intensive use of pesticides. Although not primary targets of these compounds, bees may be exposed during foraging activities. Thiamethoxam and fipronil are widely used insecticides and have been implicated in pollinator decline. Stingless bees are essential pollinators in Neotropics, making the assessment of pesticide effects on these species particularly important. This study evaluated the sublethal effects of thiamethoxam and fipronil on neural and detoxification-related enzymes in the stingless bee Melipona scutellaris. Forager bees were exposed to fipronil (LC₅₀/2 = 0.0055 and LC₅₀/5 = 0.0022 ng a.i./μL) and thiamethoxam (LC₅₀/2 = 0.027 and LC₅₀/5 = 0.010 ng a.i./μL). AChE and CaE-3 activities were assessed in the heads, while GST activity was measured in the abdomens after 1, 6, 12, 24, 48, and 96 h of exposure. Thiamethoxam promoted more pronounced alterations in AChE, CaE-3, and GST activities than fipronil, particularly at longer exposure periods. Significant Treatment vs Time interactions demonstrated that enzymatic modulation varied according to insecticide type and exposure duration, although response profiles differed among biomarkers. Alterations in AChE activity indicate disruption of cholinergic regulation, whereas changes in CaE-3 and GST activities suggest activation of detoxification and oxidative stress-related pathways. The results demonstrate that M. scutellaris exhibits marked enzymatic sensitivity to sublethal concentrations of thiamethoxam and fipronil, reinforcing the importance of incorporating sublethal endpoints into pesticide risk assessments and highlight the potential of enzymatic biomarkers as early indicators of pesticide-induced stress in stingless bees.