Identification of blood meals imbibed by phlebotomine sand flies using cytochrome b PCR and reverse line blotting

Blood meal identification is important for determining the host preferences and the vectorial capacity of hematophagous arthropods. In the past, mostly serological techniques using host-specific antibodies were used, but in recent years more sensitive and accurate polymerase chain reaction (PCR)-based molecular approaches for identifying blood meals have been developed. Here, a vertebrate-specific PCR is combined with reverse line blot analysis for identifying blood meals ingested by female phlebotomine sand fly vectors of leishmaniasis. Species-specific oligonucleotides were covalently linked to nylon membranes, and biotinylated PCR products of the mitochondrial cytochrome b gene were used as probes in a hybridization reaction revealed using colorimetric or enhanced chemiluminescent detection systems. This combination identified blood meals up to 96 hours after ingestion containing minimal amounts of DNA (>0.1 pg). The specific probes discriminated between putative host species in several study areas. The source of blood was identified in 68 of 89 wild-caught sand flies tested (76%). Mixed blood meals were identified in 15 (17%) of those. The advantages and limitations of this method are discussed.