Evolution of MALDI-TOF MS Profiles from Lice and Fleas Preserved in Alcohol over Time

SIMPLE SUMMARY: Arthropods form an extremely diverse group, including millions of species. Some arthropods, transmitting pathogenic agents, are qualified vectors. To prevent infectious disease outbreaks, accurate identification of arthropods at the species level and distinguishing vectors from non-vectors remain essential. Over the last two decades, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a relevant tool for arthropod identification. Scarce data are available regarding lice and fleas ectoparasites, which present a huge veterinary and human public health problem. These two arthropod species are often collected from livestock in the field and stored in alcohol. However, previous works reported that the duration and conditions of specimen ethanol storage could impede their MS classification. The present study’s aim was to evaluate the performance of the MALDI-TOF MS tool for correct identification of Pediculus humanus corporis lice and Ctenocephalides felis fleas preserved in alcohol from one to four years. This study highlighted that a correct rate of identification by MS could be obtained for lice and fleas preserved in alcohol for up to four years on the condition that the drying period was sufficiently long enough for accurate identification. The strategy described in the present work could be used as a guideline for MS identification of other arthropod species stored for a long time in ethanol. ABSTRACT: MALDI-TOF is now considered a relevant tool for the identification of arthropods, including lice and fleas. However, the duration and conditions of storage, such as in ethanol, which is frequently used to preserve these ectoparasites, could impede their classification. The purpose of the present study was to assess the stability of MS profiles from Pediculus humanus corporis lice and Ctenocephalides felis fleas preserved in alcohol from one to four years and kinetically submitted to MALDI-TOF MS. A total of 469 cephalothoraxes from lice (n = 170) and fleas (n = 299) were tested. The reproducibility of the MS profiles was estimated based on the log score values (LSVs) obtained for query profiles compared to the reference profiles included in the MS database. Only MS spectra from P. humanus corporis and C. felis stored in alcohol for less than one year were included in the reference MS database. Approximately 75% of MS spectra from lice (75.2%, 94/125) and fleas (74.4%, 122/164) specimens stored in alcohol for 12 to 48 months, queried against the reference MS database, obtained relevant identification. An accurate analysis revealed a significant decrease in the proportion of identification for both species stored for more than 22 months in alcohol. It was hypothesized that incomplete drying was responsible for MS spectra variations. Then, 45 lice and 60 fleas were subjected to longer drying periods from 12 to 24 h. The increase in the drying period improved the proportion of relevant identification for lice (95%) and fleas (80%). This study highlighted that a correct rate of identification by MS could be obtained for lice and fleas preserved in alcohol for up to four years on the condition that the drying period was sufficiently long for accurate identification.