Identification of ticks from North of Italy by MALDI-TOF MS

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged as a promising method for arthropods identification. A total of 125 ticks collected from 2013 until 2023 in Italy either stored frozen at -20 °C (n = 75) or preserved in 70% ethanol (n = 50) were identified using morphological keys before being analyzed by MALDI-TOF MS and, when necessary, by molecular biology in Marseille, France. Of the 125 samples analyzed by MALDI-TOF MS, 106 (84.8%) yielded good quality MS spectra (i.e., intensity>3000 arbitrary units with low background noise). The spectra of 21 specimens from eight tick species, including Dermacentor marginatus, Haemaphysalis parva, Haemaphysalis punctata, Ixodes hexagonus, Ixodes ricinus, Rhipicephalus pusillus, Rhipicephalus sanguineus s.s., and Rhipicephalus turanicus whose morphological identities were confirmed by molecular methods, were used to update an in-house MALDI-TOF MS arthropod spectra database. Blind testing of the remaining 85 good quality spectra against the updated database produced the log score values (LSVs) ranging from 1.56 to 2.57. Overall, identification by MALDI-TOF MS matched morphological identification for 59/85 (69.4%) spectra, whereas 26/85 (30.6%) spectra were discordant with morphological identification. Molecular identification using 16S rRNA gene sequencing was available for 17/26 discordant samples, and 16/17 (94.1%) supported the MALDI-TOF MS result. Considering only samples confirmed by both MALDI-TOF MS and molecular methods, 71/72 (98.6%) were correctly identified by MALDI-TOF MS, with 68/72 (94.4%) showing LSVs > 1.8, a value previously reported as relevant for arthropod identification. This study confirms the effectiveness of MALDI-TOF MS for tick identification and highlights its potential as a new tool for tick identification in Italy.