# Glossina from the Republic of the Congo: species identification by MALDI-TOF MS and research of associated micro-organisms

## TL;DR

This study shows that MALDI-TOF MS can quickly and reliably identify tsetse fly species, but cannot detect infections.

## Contribution

The study introduces MALDI-TOF MS as a novel, rapid method for identifying Glossina species with high accuracy.

## Key findings

- MALDI-TOF MS achieved high accuracy in identifying Glossina species using wings and legs.
- The method could not distinguish infected from uninfected tsetse flies.
- Wings provided the highest quality spectra for species identification.

## Abstract

Human African trypanosomiasis (HAT) and Animal African trypanosomiasis (AAT), transmitted by Glossina species, remain major health and economic burdens in Africa. Accurate vector identification is essential for effective control strategies. However, current identification methods of Glossina species based on morphological and/or molecular techniques have several limitations that often hinder reliable species-level classification. This study assessed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as an alternative or complementary approach to morphological and molecular methods for Glossina species identification and explored its ability to detect infection status. A total of 265 tsetse flies were collected and morphologically classified into the Glossina palpalis group (n = 200) and the Glossina fuscipes group (n = 65), later confirmed by molecular analysis as Glossina palpalis palpalis and Glossina fuscipes quanzensis, respectively. Spectra were generated from wings, legs, and thoraxes to identify the most suitable body parts. For G. p. palpalis, high-quality spectra were obtained from wings (98.0%), legs (96.5%), and thoraxes (93.5%); for G. f. quanzensis, corresponding values were 89.2%, 87.7%, and 72.3%. Blind testing showed that 89.5% of spectra for G. p. palpalis and 95.2% for G. f. quanzensis matched morphological identification, with 87.0% and 94.6%, respectively, reaching relevant score thresholds. Molecular screening detected Trypanosoma congolense DNA in nine specimens, but MALDI-TOF MS spectra could not distinguish infected from uninfected flies. These findings demonstrate that MALDI-TOF MS is a rapid, reliable tool for Glossina species identification, particularly using wings and legs, but is unsuitable for infection status determination.