Rapid blood meal scoring in anthropophilic Aedes albopictus and application of PCR blocking to avoid pseudogenes

Blood meal analysis (BMA) is a useful tool for epidemiologists and vector ecologists to assess which vector species are critical to disease transmission. In most current BMA assays vertebrate primers amplify DNA from a blood meal, commonly an abundant mitochondrial (mtDNA) locus, which is then sequenced and compared to known sequences in GenBank to identify its source. This technique, however, is time consuming and costly as each individual sample must be sequenced for species identification and mixed blood meals cloned prior to sequencing. Further, we found that several standard BMA vertebrate primers match sequences of the mtDNA of the Asian tiger mosquito, Aedes albopictus, making their use for blood meal identification in this species impossible. Because of the importance of Ae. albopictus as a vector of dengue and chikungunya viruses to humans, we designed a rapid assay that allows easy identification of human blood meals as well as mixed meals between human and nonhuman mammals. The assay consists of a nested PCR targeting the cytochrome b (cytb) mtDNA locus with a blocking primer in the internal PCR. The blocking primer has a 3′ inverted dT modification that when used with the Stoffel Taq fragment prevents amplification of nuclear cytochrome b pseudogenes in humans and allows for the continued use of cytb in BMA studies, as it is one of the most species-rich loci in GenBank. We used our assay to examine 164 blooded specimens of Ae. albopictus from suburban coastal New Jersey and found 62% had obtained blood from humans with 7.6% mixes between human and another mammal species. We also confirmed the efficiency of our assay by comparing it with standard BMA primers on a subset of 62 blooded Ae. albopictus. While this assay was designed for use in Ae. albopictus, it will have broader application in other anthropophilic mosquitoes.

► We developed a multiplex PCR assay to identify blood meals in Aedes albopictus. ► The assay distinguishes human blood meals from nonhuman mammals and mixes. ► A PCR blocking primer prevents amplification of human nuclear pseudogenes. ► We discuss the ubiquity of pseudogenes and how they may affect blood meal analysis.