Molecular Identification of Aedes bahamensis (Diptera: Culicidae)

Aedes (Howardina) bahamensis Berlin (or Howardina bahamensis of Reinert et al. 2004) is an exotic species first detected in the United States in 1986 from eggs and adult females collected in Miami-Dade and Broward counties, Florida (Berlin 1969; Pafume et al. 1988; O’Meara et al. 1989). In the U.S., immature Ae. bahamensis are chiefly found in artificial containers (e.g., abandoned tires, cemetery vases, etc.) and exotic tank bromeliads (O’Meara et al. 1995a). Although Ae. bahamensis readily feeds on humans, it is not a major pest or disease vector. However, the immature stages are frequently found in habitats that are also known to harbor 2 medically important mosquito species: Ae. albopictus (Skuse) and Ae. aegypti (L.) (O’Meara et al. 1995b; Lounibos et al. 2010). Morphological differences between these 3 species provide useful characters that readily distinguish the egg, larval, and adult life stages (Linley 1989; Darsie & Ward 2005). Nevertheless, a molecular method to identify Ae. bahamensis would be useful to confirm the identity of damaged adult specimens collected in fan-based traps and would allow rapid identification of the species at any life stage. Here we report the rDNA second internal transcribed spacer (ITS2) sequence and a species-specific PCR primer for the identification of Ae. bahamensis. Specimens of Aedes bahamensis were obtained from colony material maintained by GFO at the Florida Medical Entomology Laboratory (Vero Beach, Florida) and field samples collected by BDB. Field collected specimens were identified using key characters described by Darsie (1992). Total DNA was obtained from adult specimens (8 colony and 14 field) using the Qiagen DNeasy kit (Qiagen, Valencia, California) or the DNAzol reagent (Molecular Research Center, Inc., Cincinnati, Ohio) per the manufacturers’ instructions. The resulting extractions were PCR amplified in 50 μL reactions using the Invitrogen PCR Supermix (Invitrogen, Carsbad, California). Each reaction mixture contained 3 μLs of DNA template (435 ng/μL), 1 μL of each forward and reverse primer (200 nM final concentration), and 45 μL of the PCR supermix. Amplification cycling conditions were 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, 54 °C for 30 s and 72 °C for 1 min. The CP-P1A/P1B primer pair was used to amplify the complete ITS2 (Fig. 1) (Wesson et al. 1992). A negative control (H2O en lieu of DNA template) was included in each run. PCR amplicons were visualized on a 1.5% agarose gel. There were no obvious intraspecific amplicon size polymorphisms. The PCR products were gel purified with the Qiaquick Gel Extraction kit (Qiagen) and subsequently cloned into the pCR 2.1 TOPO vector (Invitrogen). Purified plasmids were obtained using the Promega Wizard Plus SV miniprep kit (Promega, Madison, WI) and then sequenced (n = 10) using the Applied Biosystems (Carlsbad, CA) Big Dye Terminator V3.0 chemistry by the Davis Sequencing Facility, University of California (Davis, California). The sequences were verified as ITS2 after evaluating the results of an NCBI BLAST query, secondary structure analysis, and the identification of specific sequence motifs known to exist on the ITS2 of mosquitoes (Coleman 2007). Novel ITS2 sequences, partial 5.8S, and partial 28S sequences for Aedes bahamensis were annotated and representative samples were submitted to the NCBI GenBank (Accession numbers: JN020552, JN020553) (Keller et al. 2009). The CP-P1A/P1B primer pair produces a 380 base pair (bp) amplicon for Ae. bahamensis that differs from the amplicons of Ae. albopictus (600 bp), Ae. aegypti (360 bp), and Ae. triseriatus (Say)