MALDI-TOF MS Identification of Mosquitoes

Each week, the legs from six specimens morphologically identified per species and per collection site were submitted to MALDI-TOF MS analysis for identification, as previously reported [32 (link)]. Protein mass profiles were obtained using a Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics, Germany). The Resulting MS profiles were visualized with Flex analysis v.3.3 software and exported to ClinProTools version v.2.2 and MALDI-Biotyper v.3.0 (Bruker Daltonics, Germany) for data processing (smoothing, baseline subtraction, peak picking). The MS spectra from selected adult mosquitoes were tested against the homemade MS reference spectra database, including, among other arthropods reference spectra, 201 MS reference spectra from 31 distinct adult mosquito species [33 (link)]. The reliability of species identification was estimated using the log score values (LSVs) obtained from the MALDI Biotyper software v.3.3, which ranged from 0 to 3. These LSVs corresponded to the degree of similarity between spectra being tested and the MS reference spectra database. A specimen was considered to be correctly and relevantly identified at the species level when the queried spectrum reached the threshold log score value (LSV) of 1.8 [28 (link)].