MALDI-TOF mass spectrometry: a new tool for rapid identification of cercariae (Trematoda, Digenea)

Identification of cercariae was long based on morphological and morphometric features, but these approaches remain difficult to implement and require skills that have now become rare. Molecular tools have become the reference even though they remain relatively time-consuming and expensive. We propose a new approach for the identification of cercariae using MALDI-TOF mass spectrometry. Snails of different genera (Radix, Lymnaea, Stagnicola, Planorbis, and Anisus) were collected in the field to perform emitting tests in the laboratory. The cercariae they emitted (Trichobilharzia anseri, Diplostomum pseudospathaceum, Alaria alata, Echinostoma revolutum, Petasiger phalacrocoracis, Tylodelphys sp., Australapatemon sp., Cotylurus sp., Posthodiplostomum sp., Parastrigea sp., Echinoparyphium sp. and Plagiorchis sp.) were characterized by sequencing the D2, ITS2 and ITS1 domains of rDNA, and by amplification using specific Alaria alata primers. A sample of each specimen, either fresh or stored in ethanol, was subjected to a simple preparation protocol for MALDI-TOF analysis. The main spectral profiles were analyzed by Hierarchical Clustering Analysis. Likewise, the haplotypes were analyzed using the maximum likelihood method. Analytical performance and the log-score value (LSV) cut-off for species identification were then assessed by blind testing. The clusters obtained by both techniques were congruent, allowing identification at a species level. MALDI-TOF enables identification at an LSV cut-off of 1.7 without false-positives; however, it requires more data on closely related species. The development of a "high throughput" identification system for all types of cercariae would be of considerable interest in epidemiological surveys of trematode infections.