Leishmaniases transmitted by bites of females of phlebotomine sand flies (Diptera: Psychodidae) belong among important and yet still neglected vector-borne diseases. Knowledge of feeding preferences towards potential reservoir animals is essential for understanding interactions of sand fly vectors with their hosts and setting correct and efficient control measures in endemic areas. Recently, MALDI-TOF mass spectrometry of host-specific hemoglobin peptides generated by trypsin digestion of the engorged blood was introduced as a rapid and cost-effective method for reliable blood meal identification in medically important arthropods including sand flies.

For routine use in field surveys, a protocol for identification of blood meals crushed on filter paper (dried blood spots) was developed and its performance assessed using laboratory-reared sand fly females of three genera (Phlebotomus perniciosus, Sergentomyia schwetzi, Lutzomyia longipalpis) fed on two hosts (mouse, rabbit), sampled at different time points after feeding and stored for varying times. The approach provided correct host assignment of experimentally acquired blood meals in the interval 0 – 48 hours post blood feeding in all tested sand fly species and from blood spots preserved for up to one year at room temperature. Practical usefullness of the protocol was further evaluated using naturally engorged sand flies (Phlebotomus tobbi, Ph. perfiliewi, Ph. simici) from an endemic locality in Greece, successfully analyzing 33/34 females and identifying human and domestic animal hosts (sheep, goats, pigs, horses, dogs and chickens) including a mixed blood meal. MALDI-TOF MS peptide mass mapping of blood meals spotted on filter paper proved to be a powerful and robust method for identification of blood meal origin of sand flies and potentially a valuable tool in entomological surveys targeting vectors of sand fly-borne pathogens. Moreover, dried blood spots represent simple and low-cost alternative sample storage enabling easy shipment of the blood meal samples at ambient temperatures from regions of collection to MS facilities for the analysis.