Rapid blood meal scoring in anthropophilic Aedes albopictus and application of PCR blocking to avoid pseudogenes Andrea Egizi a,b,⇑ , Sean P. Healy c , Dina M. Fonseca a,b a Center for Vector Biology, Department of Entomology, Rutgers University, 180 Jones Ave, New Brunswick, NJ 08901, United States b Graduate Program in Ecology and Evolution, Rutgers University, 14 College Farm Rd, New Brunswick, NJ 08901, United States c Monmouth County Mosquito Extermination Commission, P.O. Box 162, Eatontown, NJ 07724, United States article info Article history: Received 28 August 2012 Received in revised form 9 January 2013 Accepted 10 January 2013 Available online 24 January 2013 Keywords: Blood-feeding Mosquitoes Aedes Cytochrome b PCR multiplex Vector-borne disease abstract Blood meal analysis (BMA) is a useful tool for epidemiologists and vector ecologists to assess which vec- tor 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 con- sists of a nested PCR targeting the cytochrome b (cytb) mtDNA locus with a blocking primer in the inter- nal PCR. The blocking primer has a 3 0 inverted dT modification that when used with the Stoffel Taq fragment prevents amplification of nuclear cytochrome b pseudogenes in humans and allows for the con- tinued 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 appli- cation in other anthropophilic mosquitoes. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Aedes albopictus (Skuse), the Asian tiger mosquito, is native to Eastern Asia but has successfully expanded its range throughout much of the world especially into urban and suburban areas in both tropical and temperate regions (Benedict et al., 2007). Although on occasion an important vector of dengue to humans (Effler et al., 2005) and dog heartworm to dogs (Gratz, 2004), until recently this species was mainly considered to be a nuisance since it is a very aggressive human biter. After a single basepair mutation in the chikungunya virus (CHIKV) increased the vector competence of Ae. albopictus for the virus (Ng and Hapuarachchi, 2010; Tsetsarkin and Weaver, 2011) this mosquito has become the principal vector of the chikungunya epidemic occurring in Africa and the Indian Ocean Basin since 2004 (Enserink, 2007). Although chikungunya fever has not spread broadly in the temperate zone, an epidemic in northern Italy in 2007 sickened over 200 people (Moro et al., 2010) and small numbers of locally transmitted chikungunya fever cases have been identified in southern France since 2010 (Grand- adam et al., 2011). The European expansion of CHIKV would not have been possible without the invasion of that continent by Ae. albopictus (Lo Presti et al., 2012). Since the bloodfeeding habits of mosquitoes play a role in deter- mining the vectorial capacity of pathogens to wildlife and humans, it is especially important to assay what proportion of their meals are human-derived, or how anthropophilic they are, because this translates directly to their potential role in disease epidemics. Fur- ther, for invasive mosquitoes such as Ae. albopictus bloodfeeding patterns are critically important in understanding how they may alter disease transmission pathways and trigger epidemics in new areas. There are multiple techniques used for identifying blood meals in mosquitoes and other hematophagous arthropod species (see Kent, 2009 for a review) but the most recent methods use blood meal DNA. In this technique blood meal DNA is extracted, 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.01.008 ⇑ Corresponding author at: Center for Vector Biology, Department of Entomology, Rutgers University, 180 Jones Ave, New Brunswick, NJ 08901, United States. Tel.: +1 732 932 3388; fax: +1 888 504 2379. E-mail address: egizi@eden.rutgers.edu (A. Egizi). Infection, Genetics and Evolution 16 (2013) 122–128 Contents lists available at SciVerse ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid