MALDI-TOF MS identification of Glossina from Senegal and Guinea, and detection of associated Trypanosomes

• MALDI-TOF MS provided reliable and reproducible identification of Glossina species from Senegal and Guinea using legs and wings. • A new reference spectral database was generated, correctly classifying all specimens with high log-score values (≥1.8). • Molecular screening revealed circulation of Trypanosoma congolense riverine/forest type and T. theileri in Glossina p. gambiensis and G. m. submorsitans . • Partial proteomic discrimination was observed between infected and uninfected flies, especially in legs of G. m. submorsitans . • This study confirms MALDI-TOF MS as a rapid, low-cost, and effective tool for Glossina identification and pathogen monitoring. Tsetse flies ( Glossina spp., Diptera: Glossinidae ) are key vectors of African trypanosomes, causing human and animal African trypanosomiasis. Accurate species identification is crucial for surveillance and control, but morphological and molecular methods can be challenging. This study evaluated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species-level identification of tsetse flies and assessed their infection status using molecular approaches. A total of 144 Glossina specimens were collected from Senegal and Guinea, morphologically assigned to G. palpalis (n = 100), G. morsitans (n = 36), and G. fuscipes (n = 8), with molecular analysis of a subset confirming G. palpalis gambiensis and G. morsitans submorsitans . Legs and wings were analyzed by MALDI-TOF MS, yielding high-quality spectra for 93.7% of legs and 92.3% of wings. Spectral profiles exhibited strong intra-species reproducibility and clear inter-species specificity, enabling reliable discrimination. Using an internal reference database, all spectra were correctly identified (log-score values 1.8–2.8 for wings, 1.9–2.7 for legs). Screening for microorganisms detected Trypanosoma congolense and T. theileri , with partial discrimination observed between infected and uninfected flies, particularly in legs of G. morsitans submorsitans . These results demonstrate that MALDI-TOF MS is a robust tool for tsetse fly species identification and has potential for monitoring vector-borne pathogens.