Fig 1.
Map of Senegal showing the snail collection sites and the list of freshwater snails collected from two sites in Senegal.
(I) The map of Senegal showing the sites from which the sample collections were conducted. “R” refers to species collected in Richard Toll; Biomphalaria pfeifferi (239), Bulinus truncatus (127), Lymnaea natalensis (38), Melanoides tuberculata (37), Bellamya unicolor (20), Cleopatra bulimoides (1). “N” refers to Bulinus senegalensis (68) collected in Niakhar and morphologically identified. (II) shows the shells of freshwater snails confirmed molecularly: Bu. forskalii (A), Bu. truncatus (B), M. tuberculata (C), Be. unicolor (D), L. natalensis (E), C. bulimoides (F) and Bi. pfeifferi (G). The map used in this study are not subject to copyright. Map of Senegal: QGIS.org, 2021. QGIS Geographic Information System. QGIS Association. http://www.qgis.org.
Table 1.
The list of protocols and optimized parameters for all sample storage conditions.
Fig 2.
Flowchart showing the study design and all samples collected from the first and second field collection [2018–2019] and those used for each step in the study.
The study design is divided into three sections: (A) protocol optimisation, (B) database creation and (C) the validation with a blind test. The number and the species of field and laboratory-reared freshwater snails are mentioned.
Fig 3.
Comparison of MALDI-TOF MS spectra of Bi. pfeifferi using different protocols (H: head, FH: foot-head, F: foot).
(A) Representative MS spectra from Bi. pfeifferi head (H1, H2, H3), foot-head (FH) and foot (F). (B) MALDI-TOF MS spectra distinction from Bi. pfeifferi between protocols compared by principal component analysis using ClinProTools v.2.2; protocol H1(40μl Mix+ tungsten beads), protocol H2 (40μl Mix+ glass beads), protocol H3 (30μl Mix+ glass beads), protocol FH (30μl Mix+ glass beads) and protocol F (30μl Mix+ glass beads). (C) Evaluation of MS spectra reproducibility generated by the five protocols using composite correlation index (CCI). The MS parameters: intensity ≥ 3.000 [a.u.], absence of background noise and the reproducibility of MS spectra. a.u.: arbitrary units; m/z: mass-to-charge ratio. The protocol F was selected to build the MS reference database and perform the analysis.
Fig 4.
MALDI-TOF MS spectra obtained from different snail species to create the database.
(A) Spectral alignment of seven snail species showing discriminative peaks using Flex Analysis software; smoothed spectra with baseline subtracted. (B) dendrogram of MALDI-TOF MS spectra of snail species collected in Senegal. Cluster analysis was performed using Biotyper software v.3.0. (C) Graphical representation showing the log-score values classification according to the following species: Bu. forskalii, Bu. truncatus, M. tuberculata, Be. unicolor, L. natalensis and Bi. pfeifferi.
Table 2.
Laboratory-reared and field species selected to create a MALDI-TOF MS reference database, identified molecularly using COI.
Fig 5.
Comparison of MALDI-TOF MS spectra from the foot of ethanol-stored specimens.
(A) Comparison of MALDI-TOF MS spectra of three different protocols on principal component analysis using ClinProTools v.2.2; protocol F1 (30μl Mix+ glass beads), protocol F2 (30μl Mix+ tungsten beads), and protocol F3 (15μl Mix+ glass beads). (B) Representative MS spectra of Bi. pfeifferi obtained for each protocol, analysed by Flex analysis. (C) Hierarchical clustering dendrogram of Bi. pfeifferi, Bu. truncatus, Bu. globosus, Bu. forskalii, and Bu. senegalensis performed by Biotyper software v.3.0. (D) Representative MALDI-TOF MS profiles of Bi. pfeifferi, Bu. truncatus, Bu. globosus, Bu. forskalii and Bu. senegalensis using protocol F3, performed using Flex Analysis Software. a.u.: arbitrary units; m/z: mass-to-charge ratio. The x-axis represents PC1 and the y-axis represents PC2.
Table 3.
Identification results of ethanol-stored snail specimens including schistosomes detection results.
Fig 6.
Phylogenetic tree showing the position of nine snail species (Bi. pfeifferi, Bu. truncatus, Bu. forskalii, Bu. senegalensis, Bu. globosus, Be. unicolor, M. tuberculata, L. natalensis, and C. bulimoides) used in this study.
The tree was constructed using the maximum likelihood method based on the Kimura 2-parameter distance (MEGA7). The values on the branches are bootstrap support values based on 1000 replications. Branches are colour-coded to the bootstrap values. The identity of each taxon is colour-coded conforming to the species.