Chemometric electrochemical fingerprinting of thermal stress in honeybee larvae: a tool for discrimination and welfare assessment.

Thermal stress is a major environmental factor affecting honeybee (Apis mellifera) physiology, colony stability, and overall welfare. Here, we report a novel electrochemical fingerprinting strategy based on differential pulse voltammetry (DPV) combined with supervised chemometric modeling for the discrimination of thermally stressed honeybee larvae. Larvae were exposed to optimal brood temperature (34.5 °C) or mild hyperthermic conditions (36.0 °C for 1 h), and their electrochemical responses were recorded using glassy carbon electrodes modified with a carboxylated multiwalled carbon nanotube/Kolliphor dispersion. The resulting voltammetric profiles exhibited two reproducible anodic signals, including a peak associated with octopamine (OA), a key neurochemical mediator of stress. Partial least squares discriminant analysis (PLS-DA) models were constructed using the complete voltammetric profile and selected signal regions. The model based on the full DPV fingerprint achieved the best predictive performance, reaching an accuracy of 0.96 in the independent test set, whereas models based on individual peak regions showed lower discrimination capability. These results demonstrate that the complete electrochemical fingerprint provides a more comprehensive representation of the physiological response to thermal stress than single-analyte analysis alone. The proposed strategy introduces a shift from targeted electrochemical quantification toward multivariate physiological fingerprinting, offering a rapid and robust tool for environmental stress monitoring and honeybee welfare assessment. To the best of our knowledge, this is the first study to combine DPV-based electrochemical fingerprinting with supervised chemometric classification for the assessment of thermal stress in honeybee larvae.