Enhanced procedures for mosquito identification by MALDI-TOF MS

Abstract Background. The increasing interest in arthropod identification by MALDI-TOF MS conducted to the emergence of heterogenic procedures, including sample preparation and selection of body parts to use. The absence of a consensual strategy hampers direct inter-studies comparisons and brings confusion to new users. Establishing optimized procedures and standardized protocols for mosquito identification by MALDI-TOF MS becomes compulsory, allowing notably the sharing of reference MS databases. Objective. Then, here, we propose to assess the optimal conditions for mosquito identification using MALDI-TOF MS profiling. Methods. Three homogenization methods, two manuals and one automatic, applied on three distinct body parts, legs, thorax and head, were evaluated on two mosquito laboratory strains, Anopheles coluzzii and Aedes aegypti. MS profiles reproducibility, identification rate with relevant scores and the suitable procedures for high-throughput analyses were the main criteria for establishing optimized guidelines. Additionally, the consequences of blood feeding and geographical origin were evaluated using both laboratory strain and field collected mosquitoes. Results. The three body parts exhibited relevant score values for mosquito identification using MALDI-TOF MS profiling; however, thorax and legs appeared the most suitable, independently of homogenization methods or species. Although manual grindings displayed high identification performance for the three body parts, this homogenization mode is not adapted to process a large number of samples. Then, the automatic procedure was selected as the reference homogenization method. Blood feeding status did not hamper the identification of mosquito species, despite the presence of MS peaks from blood origin in MS profiles from three body parts tested on both species. Finally, a significant improvement in identification scores was obtained for specimens from the field when MS spectra of species from the same geographical area were added to the database. Conclusion. The present work established guidelines concerning the selection of mosquito anatomic part and modality of sample preparation (e.g., homogenization) for future specimen identification by MALDI-TOF MS profiling. These standardized operational protocols could be used as references for creating an international MS database.