Application of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry to Identify Species of Neotropical Anopheles Vector
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Loaiza et al. Malar J (2019) 18:95 https://doi.org/10.1186/s12936-019-2723-0 Malaria Journal RESEARCH Open Access Application of matrix-assisted laser desorption/ionization mass spectrometry to identify species of Neotropical Anopheles vectors of malaria Jose R. Loaiza1,2,3 , Alejandro Almanza1, Juan C. Rojas1, Luis Mejía1,2, Norma D. Cervantes4, Javier E. Sanchez‑Galan1,5, Fernando Merchán6, Arnaud Grillet6,7, Matthew J. Miller8, Luis F. De León1,9 and Rolando A. Gittens1,10* Abstract Background: Malaria control in Panama is problematic due to the high diversity of morphologically similar Anopheles mosquito species, which makes identifcation of vectors of human Plasmodium challenging. Strategies by Panama‑ nian health authorities to bring malaria under control targeting Anopheles vectors could be inefective if they tackle a misidentifed species. Methods: A rapid mass spectrometry identifcation procedure was developed to accurately and timely sort out feld‑collected Neotropical Anopheles mosquitoes into vector and non‑vector species. Matrix‑assisted laser desorp‑ tion/ionization (MALDI) mass spectra of highly‑abundant proteins were generated from laboratory‑reared mosquitoes using diferent extraction protocols, body parts, and sexes to minimize the amount of material from specimen vouch‑ ers needed and optimize the protocol for taxonomic identifcation. Subsequently, the mass spectra of feld‑collected Neotropical Anopheles mosquito species were classifed using a combination of custom‑made unsupervised (i.e., Principal component analysis—PCA) and supervised (i.e., Linear discriminant analysis—LDA) classifcation algorithms. Results: Regardless of the protocol used or the mosquito species and sex, the legs contained the least intra‑specifc variability with enough well‑preserved proteins to diferentiate among distinct biological species, consistent with pre‑ vious 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 diferent 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 identif‑ cation rates above 93% at the species level. Conclusion: The selected sample processing procedure can be used to identify feld‑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 with‑ out a reference library to classify unknown samples. Finally, this MALDI approach can aid ongoing malaria eradication *Correspondence: [email protected] 1 Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de Investigaciones Científcas y Servicios de Alta Tecnología (INDICASAT AIP), City of Knowledge, Panama 0843‑01103, Republic of Panama Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Loaiza et al. Malar J (2019) 18:95 Page 2 of 12 eforts in Panama and other countries with large number of mosquito’s species by improving vector surveillance in epidemic‑prone sites such as indigenous Comarcas. Keywords: Anopheles mosquito, Taxonomic identifcation, MALDI, Mass spectrometry, Malaria vector, Panama Background However, generating DNA barcodes requires advanced Despite historical and ongoing eradication eforts, human sample preparation and proper laboratory facilities to malaria transmitted by Anopheles mosquitoes continues extract, amplify and sequence nucleic acids, most of to be a major public health concern around the world [1]. which are rarely found in developing countries where Malaria was one of the leading causes of death during arthropod-borne infections like malaria prevail [14]. the construction of the Panamanian Interoceanic Canal In recent years, matrix–assisted laser desorption/ioni- in the early 1900s. In Panama, malaria prevalence oscil- zation (MALDI) mass spectrometry has become an alter- lated dramatically during the last 50 years, with sporadic native for arthropod taxonomic identifcation [10, 15, 16]. and/or cyclical epidemics every fve to 10 years [2–4]. Tis method has been used efectively to study several Recently however, from 2001 to 2005, a malaria outbreak aspects of vector biology, including taxonomic status (i.e., was documented in indigenous territories known as species boundaries), pathogen infection rates and food “Comarcas” where a sixfold increase in the number of source identity [17–21]. MALDI mass spectrometry uses cases was observed [5, 6]. Tis epidemic was controlled a profle of the most abundant proteins to “fngerprint” during subsequent years, and the number of sympto- biological samples, and thus, is conceptually similar to matic cases in the country has dropped considerably DNA barcoding, but possibly cheaper on a per sample since this event. Nonetheless, malaria is still endemic in basis. Furthermore, MALDI can generate accurate iden- Panama, and there is potential for future outbreaks par- tifcations in just a few hours, rather than 5 to 10 days as ticularly in indigenous Comarcas with health, social and in the case of DNA barcoding even in “rush” cases [22]. demographic disparities [3, 6]. Previous eforts with MALDI to taxonomically classify Malaria control in Panama is done mainly through members of family Culicidae were successful using both eradication of mosquito vectors using toxic insecti- laboratory-reared and feld-collected specimens, and cides. Tis strategy requires that the Anopheles species specifc body parts (e.g., thorax, cephalothorax and/or responsible for transmission be promptly and accurately legs) plus samples from diferent regions of the world [9, identifed. Nonetheless, identifcation in Panama is prob- 10, 23–25]. Yssouf et al. [9, 10] used MALDI with all six lematic due to a high number of morphologically similar legs to classify mosquito species from Africa, Europe and Anopheles species [7]. Control strategies to bring malaria the US while Mewara et al. [24], using the same approach, down targeting Anopheles vectors could be inefective accurately identifed specimens of four diferent mos- if they tackle a misidentifed non-vector species. Tis quito genera in Northern India. More recently in France, is likely the case in Panamanian indigenous Comarcas Vega-Rúa et al. [25] designed a double entry query proto- where as many as 10 Anopheles species occur in a single col with MALDI protein spectra obtained from thoraxes locality, and where 40% of these are expected to transmit and legs to improve the identifcation of morphologically both Plasmodium vivax and P. falciparum to humans [5, compromised specimens. Hence, MALDI’s accurate and 8]. Te identifcation of Anopheles mosquitoes in Panama rapid identifcation capabilities might prove ideal to solve is done using traditional morphological approaches (e.g., the shortcomings of taxonomically classifying Anopheles dichotomic keys), but this approach requires meticu- mosquitoes in Panama, thus assisting ongoing malaria lous taxonomic training and a great deal of entomo- eradication eforts by improving the vector surveillance logical expertise [9, 10]. Also, it is time consuming and system in indigenous Comarcas. could be impractical when inspecting numerous samples Here, a methodology based on previously published [11]. Hence, Panamanian health authorities require new extraction protocols was adjusted and assessed the accu- approaches to accurately and timely sort out vector from racy of MALDI identifcation with a small portion of non-vector Anopheles species. tissue from the mosquito body to otherwise preserve a DNA barcoding is a valid alternative to identify arthro- specimen voucher. Diferent statistical procedures were pod species because it has better taxonomic resolution also explored to analyse and classify protein spectra from than morphological approaches, even if the promise of feld-collected mosquitoes, which are difcult to evalu- being less expensive has not yet materialized [12]. DNA ate with currently available strategies from commercial barcodes work well with a small amount of tissue, and do vendors, including working without a reference library of not require prior knowledge of insect morphology [13]. well curated protein spectra. Specifcally, the authors ask Loaiza et al. Malar J (2019) 18:95 Page 3 of 12 Table 1 Description of three diferent MALDI mass spectrometry protein-extraction protocols used in the present study Protocol #1 Protocol #2 Protocol #3 Selected mosquito body parts were placed in separate Selected mosquito body parts were rinsed with Selected mosquito body parts were microcentrifuge tubes, rinsed with 300 μL ultrapure distilled water and dried with paper homogenized with