Application of matrix-assisted laser desorption/ionization mass spectrometry to identify species of Neotropical Anopheles vectors of malaria

Abstract

BackgroundMalaria control in Panama is problematic due to the high diversity of morphologically similar Anopheles mosquito species, which makes identification of vectors of human Plasmodium challenging. Strategies by Panamanian health authorities to bring malaria under control targeting Anopheles vectors could be ineffective if they tackle a misidentified species.MethodsA rapid mass spectrometry identification procedure was developed to accurately and timely sort out field-collected Neotropical Anopheles mosquitoes into vector and non-vector species. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of highly-abundant proteins were generated from laboratory-reared mosquitoes using different extraction protocols, body parts, and sexes to minimize the amount of material from specimen vouchers needed and optimize the protocol for taxonomic identification. Subsequently, the mass spectra of field-collected Neotropical Anopheles mosquito species were classified using a combination of custom-made unsupervised (i.e., Principal component analysis—PCA) and supervised (i.e., Linear discriminant analysis—LDA) classification algorithms.ResultsRegardless of the protocol used or the mosquito species and sex, the legs contained the least intra-specific variability with enough well-preserved proteins to differentiate among distinct biological species, consistent with previous literature. After minimizing the amount of material needed from the voucher, one leg was enough to produce reliable spectra between specimens. Further, both PCA and LDA were able to classify up to 12 mosquito species, from different subgenera and seven geographically spread localities across Panama using mass spectra from one leg pair. LDA demonstrated high discriminatory power and consistency, with validation and cross-validation positive identification rates above 93% at the species level.ConclusionThe selected sample processing procedure can be used to identify field-collected Anopheles species, including vectors of Plasmodium, in a short period of time, with a minimal amount of tissue and without the need of an expert mosquito taxonomist. This strategy to analyse protein spectra overcomes the drawbacks of working without a reference library to classify unknown samples. Finally, this MALDI approach can aid ongoing malaria eradication efforts in Panama and other countries with large number of mosquito’s species by improving vector surveillance in epidemic-prone sites such as indigenous Comarcas.Electronic supplementary materialThe online version of this article (10.1186/s12936-019-2723-0) contains supplementary material, which is available to authorized users.

Proteomic fingerprinting of Neotropical hard tick species (Acari: Ixodidae) using a self-curated mass spectra reference library

“…Thus, our findings confirm that our novel analytical approach using MALDI and PCA/ LDA clustering algorithms is robust for species classification regardless of the arthropod assemblage, sample storing conditions, and the lack of a high-quality reference library. In fact, the percentage of automatic spectra acquisition from the processed tick species was much higher (Table 1) than that obtained in our previous publication using mosquitoes, which ranged from 41.8% in Anopheles albimanus to 70.3% in Anopheles triannulatus [27].…”

“…Our results herein also show that both classification algorithms, PCA and LDA, were capable of clustering and recognizing spectra from up to 18 different tick species, including roughly 50% of ixodid taxa (e.g., both ecologically dominant and rare species) reported for Panama [27,42]. LDA outcomes were more discriminant and robust than PCA overall, but PCA also classified species from different genera with over 91% accuracy and consistency.…”

“…Although more challenging than identifying bacteria and fungi due to the size and heterogeneity of the specimen, MALDI has also been used to discriminate among species of invertebrates, including mosquitoes (Culicidae-Anopheles), fleas (Pulicidae-Ctenocephalide), biting midges (Ceratopogonidae-Culicoides), sandflies (Psychodidae-Phlebotomus, Lutzomyia) and ticks (Ixodidae-Rhipicephalus) [19][20][21][22][23][24][25][26][27]. A key finding from these studies is that protein spectra obtained from body sections or whole specimens were similar among individuals of the same morphological species but differed noticeably across different species.…”

Identification of Aedes mosquitoes by MALDI-TOF MS biotyping using protein signatures from larval and pupal exuviae

“…gambiae on SDS-PAGE gel revealed distinct profiles with a major band of low molecular weight for larval samples. Likewise, the specificity of MS spectra according to the body part used from the same species was recently reported [20,29]. The advantages of being able to query independently protein profiles from exuviae of fourth-instar larvae and pupae from the same specimens against the reference MS spectra DB, allow a double control for the identification of the mosquito.…”

“…This MS biotyping tool was successfully applied for the identification of mosquitoes at the adult [20,29] but also at the immature stages [21,22]. Nevertheless, the rates of correct identification at the pupal stages were lower than at late-instar stages using MALDI-TOF MS biotyping [22,30].…”