A novel MALDI-TOF MS-based method for blood meal identification in insect vectors: a proof of concept study on phlebotomine sand flies

Authors: HLAVACKOVA, KRISTYNA - CHARLES UNIVERSITY, CZECH REPUBLIC; DVORAK, VIT - CHARLES UNIVERSITY, CZECH REPUBLIC; CHASKOPOULOU, ALEXANDRA - EUROPEAN BIOLOGICAL CONTROL LABORATORY (EBCL); VOLF, PETR - CHARLES UNIVERSITY, CZECH REPUBLIC; HALADA, PETR - CZECH ACADEMY OF SCIENCES

Submitted to: PLOS Neglected Tropical Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/29/2019
Publication Date: 9/9/2019
Citation: Hlavackova, K., Dvorak, V., Chaskopoulou, A., Volf, P., Halada, P. 2019. A novel MALDI-TOF MS-based method for blood meal identification in insect vectors: a proof of concept study on phlebotomine sand flies. PLOS Neglected Tropical Diseases. 13(9): e0007669. https://doi.org/10.1371/journal.pntd.0007669.
DOI: https://doi.org/10.1371/journal.pntd.0007669

Interpretive Summary: Leishmaniases are greatly important and yet neglected vector-borne diseases caused by parasites, usually transmitted by the bite of certain female sand flies. To better understand the role of different parasite hosts during parasite transmission cycles, it is important to determine blood meal sources of bloodfeeding females. Most currently-used methods for host blood identification encounter challenges that stem from tiny volumes of injested blood, as well as quick blood digestion which leads to inevitable and rapid DNA and protein degradation. To overcome this problem, a novel approach for blood meal source identification of parasitic arthropods was developed based on MALDI-TOF peptide mass mapping of host-specific hemoglobin peptides. The method was developed using lab-reared sand flies and further validated using field-collected samples. Although tested on sand flies, the method could be universally applied to other blood-feeding insects as demonstrated by analysis of certain experimentally bloodfed mosquitoes. The method represents an efficient tool for accurate identification of host blood as it requires a minimal material input and employs simple and fast sample preparation, especially useful for high numbers of specimens collected during field surveys. This method will be of interest to professionals involved in the surveillance, monitoring or control of vector-borne diseases. It may also be of interest to decision-makers and stakeholders in public health, and those involved in sand fly or other blood-feeding insect disease vector surveillance, monitoring and control.

Technical Abstract: Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA- and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification based on peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool for large-scale screening of host preferences studies. Unlike other methods including MALDI-TOF protein profiling, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies.