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

BACKGROUND: Malaria 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. METHODS: A 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. RESULTS: Regardless 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. CONCLUSION: The 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.

Morphological and proteomic characterization of midgut of the malaria vector Anopheles albimanus at early time after a blood feeding.

The midgut of anopheline mosquito is the entry of Plasmodium, the causative agent of malaria.When the mosquito feeds on parasite infected host, Plasmodium parasites reach the midgut and must confront digestive enzymes, the innate immune response and go across the peritrophic matrix (PM), a thick extracellular sheath secreted by the mosquito midgut epithelial cells. Then, to continue its development, the parasite must reach the salivary glands to achieve transmission to a vertebrate host. We report here the morphological and biochemical descriptions of the midgut changes after a blood meal in Anopheles albimanus. Before blood feeding, midgut epithelial cells contained numerous electrondense vesicles distributed in the central to apical side. These vesicles were secreted to the luminal side of the midgut after a blood meal. At early times after blood ingest, the PM is formed near microvilli as a granulous amorphous material and after it consolidates forming a highly organized fibrillar structure, constituted by layers of electrondense and electronlucent regions. Proteomic comparative analysis of sugar and blood fed midguts showed several molecules that modify their abundance after blood intake; these include innate immunity, cytoskeletal, stress response, signaling, and digestive, detoxifying and metabolism enzymes. Biological significance In the midgut of mosquitoes during bloodfeeding, many simultaneous processes occur, including digestion, innate immune activities, cytoskeleton modifications, construction of a peritrophic matrix and hormone production, between others. Mechanical forces are very intense during bloodfeeding and epithelial and muscular cells must resist the stress, modifying the actin cytoskeleton and coordinating intracellular responses by signaling. Microorganisms present in midgut contents reproduce and interact with epithelial cells triggering innate immune response. When infectious agents are present in the blood meal they must traverse the peritrophic matrix, an envelope formed from secretion products of epithelial cells, and evade the immune system in order to reach the epithelium and continue their journey towards salivary glands, in preparation for the transmission to the new hosts. During all these processes, proteins of mosquitoes are modified in order to deal with mechanical and biological challenges, and the aim of this work is to study these changes.

Genetic Evidence of Importation of Drug-Resistant Plasmodium falciparum to Guatemala from the Democratic Republic of the Congo.

Imported malaria threatens control and elimination efforts in countries that have low rates of transmission. In 2010, an outbreak of Plasmodium falciparum malaria was reported among United Nations peacekeeping soldiers from Guatemala who had recently returned from the Democratic Republic of the Congo (DRC). Epidemiologic evidence suggested that the soldiers were infected in the DRC, but local transmission could not be ruled out in all cases. We used population genetic analyses of neutral microsatellites to determine the outbreak source. Genetic relatedness was compared among parasites found in samples from the soldiers and parasite populations collected in the DRC and Guatemala; parasites identified in the soldiers were more closely related to those from the DRC. A phylogenetic clustering analysis confirms this identification with >99.9% confidence. Thus, results support the hypothesis that the soldiers likely imported malaria from the DRC. This study demonstrates the utility of molecular genotyping in outbreak investigations.