A novel MALDI-TOF MS-based method for blood meal identification in insect vectors: A proof of concept study on phlebotomine sand flies
Abstract
Author summary Leishmaniases belong among the most important and yet neglected vector-borne diseases, transmitted mostly by bite of female phlebotomine sand flies. To understand role of different reservoir hosts in the transmission cycles, it is important to determine blood meal sources of bloodfeeding females.
Most of currently used methods face challenges due to tiny volumes of engorged blood, in case of mammals also enucleated, as well as quick progress of blood digestion which leads to rapid DNA and protein degradation. New approach towards blood source determination presented in this study is based on MALDI-TOF mass spectrometry that identifies unique peptide sequences of host hemoglobins, showing high precision and sensitivity together with a longer time period for successful host determination when compared to nowadays standardly used DNA sequencing.
It was tested and verified on engorged phlebotomine sand flies from both laboratory colonies and natural endemic areas and also on Culex mosquitoes and shall be universal to hematophagous insects. Beside blood meal identification, it allows also the use of both morphological and molecular methods (DNA- or protein-based) for the species identification of the analysed specimen.
Background 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, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. Methodology/Principal findings 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.