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

Abstract

BackgroundIdentification 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 findingsTo 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.Conclusions/SignificancePMM-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.

New assessment of Anopheles vector species identification using MALDI-TOF MS

“…Thus, they postulated that the abdomen blood content somehow negatively influenced the frozen preservation of the engorged specimens. Similarly, for MALDI-TOF identification of sand fly species, the thorax of engorged specimens led to blood contaminations during the separation from the abdomen, after frozen storage [27][28][29]. Here, visually engorged Anopheles displayed specific patterns in thorax protein profiles and mass spectra reproducibility level of field-caught specimens was lower to that of laboratoryreared ones.…”

Ecology, Seasonality and Host Preferences of Austrian Phlebotomus (Transphlebotomus) Mascittii Grassi, 1908, Populations

“…Only ve of a total of 15 collected engorged females could be successfully analyzed by either MALDI-TOF mass spectrometry or DNA sequencing, probably due to late stages of blood meal digestion in the remaining specimens. Degraded DNA makes PCR challenging already 24 hours after the blood meal [52] and even though MALDI-TOF peptide mass mapping is possible for longer periods after the blood meal [26], observed blood bolus in some specimens suggest a too advanced stage of digestion, making molecular identi cation of the blood meal host impossible.…”

“…MALDI-TOF peptide mass mapping analysis of host-speci c hemoglobin peptides was performed according to a protocol by Hlavackova et al [26]. Blood from engorged abdomens was digested using trypsin (Promega) and the resulting peptides were mixed with an α-cyano-4-hydroxycinnamic acid matrix (Bruker Daltonics).…”

Phlebotomine Sand Fly Survey in the Republic of Moldova: Species Composition, Distribution and Host Preferences

“…These ndings were con rmed by a sequencing analysis of chosen engorged specimens. Since peptide mass mapping using MALDI-TOF mass spectrometry was recently introduced as a new and effective tool for bloodmeal identi cation of haematophagous arthropods (Hlavackova et al, 2019), it was also used to identify the blood source of some engorged P. papatasi females. Obtained results con rmed both humans and chickens, emphasizing the apparent tight trophic connection of P. papatasi with human dwellings.…”

“…Bloodmeals of chosen engorged females collected in 2016 and 2017, all identi ed by morphology as P. papatasi, were analyzed by peptide mass mapping of host-speci c haemoglobin peptides using MALDI-TOF mass spectrometry. In total, 25 females originating from Ceadir-Lunga (n=5), Corjova (n=2) Malaesti Noi (n=4) and Slobozia Mare (n=14) were prepared as described by Hlavackova et al (2019). From dissected specimens, thoraxes were stored for con rmation of species identi cation by MALDI-TOF MS protein pro ling and abdomens were homogenized in 50 μl of distilled water (Merck KGaA, Darmstadt, Germany) by BioVortexer homogenizer (BioSpec), 10 μl of the homogenate was then incubated with 10 μl of 50 mM N-ethylmorpholine acetate buffer (pH 8.1; Sigma-Aldrich) and 100 ng of trypsin (Promega) at 37˚C for 30 minutes.…”