The invasive Asian Tiger Mosquito, Aedes albopictus, a container-inhabiting species, was first discovered in the United States in 1985 (Sprenger and Wuithiranyagool 1986). Currently, Ae. albopictus has invaded 36 states in the continental United States (Enserink 2008). Literature suggests that Ae. albopictus will expand its range throughout much of the eastern United States(Nawrocki and Hawley 1987). Currently, the northern most distribution of Ae. albopictus has reached New York state and has been found in all five boroughs of New York City, Nassau, Suffolk, Westchester and Rockland counties.
Mitochondrial DNA (mtDNA) is thought to be useful in phylogenetic analyses, and has previously been used to investigate inter- and intraspecific phylogenetics of mosquito species (Avise et al. 1987, Besansky et al. 1997, Birungi and Munstermann 2002, Mousson et al. 2005, Zitko et al. 2011). This study will characterize and assess the utility of 3 mitochondrial genes in phylogenetic investigation of Ae. albopictus on the east coast of the United States and locally within New York State. Mitochondial genes used for this study are cytochrome b (cyt b), cytochrome oxidase I (COI), and NADH dehydrogenase subunit 5 (ND5).
Materials and Methods
Adult Aedes albopictus (Ae. albopictus-NY were obtained as frozen specimens from Yonkers, NY in Westchester county, and as dried specimens from Tennessee (Ae. albopictus-TN). Fresh larval samples of Ochlerotatus japonicus were collected from Larchmont, NY in Westchester county. One specimen from each location was used for analysis. DNA was extracted from each of the samples and a 10ng stock solution was made.
Primers used to amplify the 3 mitochondrial genes were obtained from the literature. A second primer was found for cytochrome oxidase I, COI-UEA. Target regions were amplified and run on a 1% agarose gel to visualize the PCR products. The most robust samples were chosen and PCR purified. These were sent out for sequencing, aligned, and pairwise similarities were determined.
PCR amplification using the Cyt b and ND5 primers resulted in products of about 350 and 450 bp, respectively (Figure 1). Amplification using COI and COI-UEA primers yielded no bands for any of the samples.
Samples chosen for PCR purification were the 10ng sample for Ae. albopictus-NY and Oc. japonicus for Cyt b and ND5, and the 5ng sample for Ae. albopictus-TN for Cyt b and ND5. Specimen sequences were paired and aligned using Clustal Omega to determine the number of base pair differences. A 211 bp sequence was aligned for Cyt b, and all pairs showed no base differences (Figure 2). A 402 bp sequence was aligned for ND5 (Figure 3). Ae. albopictus-NY and Ae. albopictus-TN had no base differences. When paired with Oc. japonicus, there were 48 base differences. Pairwise similarity showed all three specimens were 100% homologous for Cyt b (Figure 4). Both Ae. albopictus specimens had 100% similarity for ND5, but had 88% pairwise similarity when paired with Oc. japonicus (Figure 4).
The difference in base pairs was used to classify MOTUs for each set of comparisons (Figure 4). Ae. albopictus and Ae. albopictus-TN are considered 1 MOTU for Cyt b. ND5 classifies the Ae. albopictus specimens as 1 MOTU and Oc. japonicus as another unit.
Primer pairs for regions of cytochrome b and NADH dehydrogenase subunit 5 were successful at amplifying mosquito DNA for all specimens. Pairwise similarities showed a 100% homology between Ae. albopictus and Oc. japonicus for Cyt b, and 88% homology for ND5, suggesting that ND5 may be a more useful region for future phylogenetic studies. Cyt b may be a more useful marker at higher taxonomic levels.
The initial primer pair COI yielded no bands when PCR products were run on a 1% agarose gel. The forward and reverse primers contained 13% and 17% GC content, respectively, and could cause improper or no annealing to occur, thus resulting in no amplification. The GC content of COI-UEA were also low, 29% and 25%, and PCR temperatures were adjusted accordingly. However, again no bands were produced. Measures should be taken to either optimize the COI-UEA primer pair, or develop a primer pair with a GC content closer to 50%.
The utility of these primers as markers for intraspecific phylogenetic analysis could not be determined from this study. As only one sample from each location was used, no reliable inferences can be made. Future studies should include larger sample sizes from each location to see if variability occurs among the geographic distribution of Ae. albopictus within the United States, and more locally in New York State.
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