Aedes aegypti is the primary vector of dengue, the most common arboviral disease, across most tropical and subtropical regions of the world. It is also a major vector of chikungunya, a rapidly emerging virus that is causing significant disease throughout the Caribbean islands and much of the Central and South America.1 In the continental United States, autochthonous transmission of dengue has been observed in Florida since 2009.2 No licensed vaccines or therapeutic drugs are available for preventing or treating either disease. Aedes aegypti is a highly anthropophilic invasive species that is well adapted for wide-spread colonization of urban environments, as it readily breeds in water in natural and man-made containers around and within human dwellings. Although Ae. aegypti has likely maintained an intermittent presence in the United States for ∼375 years, its distribution has been assumed to be limited by cold temperatures to boundaries roughly southward of the average 10°C winter isotherm.3 However, two potentially synergistic phenomena may be at play, which promote sustained northward range expansion: 1) the influence of climate changes that will allow for expansion into areas not previously colonized4 and 2) this species readily adapts to breeding in subterranean structures that provide a suitable and protective environment from adverse climatic conditions.5 Although Ae. aegypti collections have been reported from Virginia and Maryland, they typically comprised relatively small numbers and have been considered to be seasonal introductions, and restricted from overwintering by cold-season extremes,3 as no life stage is tolerant of even short-term (e.g., overnight) temperatures below 0°C.6 Here, we report on summer-season collections and genotypic analyses of Ae. aegypti larvae and adults across four contiguous years in the Capitol Hill neighborhood in Washington, DC (WDC), and conclude that this is likely an overwintering population maintained in as yet undefined subterranean habitats.
After the initial serendipitous discovery of a single adult female Ae. aegypti on October 22, 2011, in the Capitol Hill neighborhood, multiple subsequent surveys were performed to collect Ae. aegypti larvae from container habitats within ∼200 m of the original collection site at the square fountain (Figure 1A). Larvae were collected from a birdbath, square fountain, trash can, potted plant saucer, and a small, grated stormwater basin in an alleyway (Figure 1B and C). Collection dates (sample sizes) were October 22 and 25, 2011 (6 and 20, respectively); September 26, 2012 (17); July 13, August 25, October 19, and November 22, 2013 (6, 1, 8, and 1, respectively); and August 18 and September 14, 2014 (49 and 7, respectively). A total of 115 Ae. aegypti larvae were identified using standard taxonomic keys.7 Adults were occasionally reared from larvae, killed by freezing, and dried by placing them in open glass vials. Larvae were killed with hot water then stored in 2.5-mL Eppendorf tubes containing 70% ethyl alcohol. Samples were shipped to University of Notre Dame for genetic analysis.
Ethanol-preserved larval and dried adult samples were subjected to DNA extraction as previously described,8 and DNA was suspended in final volumes of 200 μL for larvae and 1,000 μL for adults containing 0.02 M NaOH and 0.036 M Tris-HCl, pH 8.0. A 710-bp amplicon from the mitochondrial cytochrome oxidase I (mtCOI) gene was obtained using LCO1490 and HCO2199 primers in 25 μL PCR reactions containing 1 μL of genomic DNA and was subjected to thermocycling conditions as previously reported.9 Amplicons were purified (Qiagen QIAquick PCR Purification Kit) and direct sequenced with the same primers using BigDye on a 3730xl DNA Analyzer (Applied Biosystems). Multiplexes of primers for 10 microsatellite markers with fluorochrome-labeled forward primers (Integrated DNA Technologies) were prepared in 25 μL PCR reactions, amplified, and subjected to fragment analysis as previously described.8
Fifty-nine mtCOI sequences were obtained and aligned in Clustal Omega10 with 20 Ae. aegypti mtCOI sequences from various geographic regions of the world downloaded from VectorBase.11 All sequences were trimmed to a 441-bp consensus and used to construct a neighbor-joining phylogenetic tree in MEGA6,12 using a mtCOI sequence from Aedes albopictus (GenBank accession no. KC690960) as an outgroup. For microsatellite analysis, samples were divided by year of collection (YOC) and tested for Hardy–Weinberg equilibrium (HWE) using Arlequin v3.5.13 The Bayesian clustering method in the STRUCTURE program v2.3.414 was used to assign individuals to genetic clusters. We implemented the Evanno method15 to determine the most likely number of clusters (K) after five independent runs of STRUCTURE for K = 1 to K = 5 on individuals from four YOC, using 100,000 burn-in steps followed by 500,000 iterations.
We identified two mtCOI haplotypes from a total of 59 sequences obtained from Ae. aegypti individuals collected during 2011–2014. The two haplotypes were shared across all four YOC, and were often identified in larvae collected from the same breeding container such as the birdbath (Figure 1B). Our sequence data were submitted to GenBank under accession numbers KM362421 and KM362422. The phylogenetic tree based on a combined analysis of the WDC sequences with 20 mtCOI haplotype sequences from various geographic regions placed the WDC haplotypes within two distinct phylogenetic groups (Figure 2A), reflective of the two African origin ancestral clades associated with extant global Ae. aegypti populations.16 The most common WDC haplotype A showed close relationships with Ae. aegypti populations from South America and Asia, whereas WDC haplotype B showed close relationships with Ae. aegypti populations from the Caribbean, South America, and sub-Saharan Africa. This finding is completely consistent with results obtained from multiple studies on Ae. aegypti populations throughout the Caribbean and Central and South America and with data from multiple mitochondrial genes.17–19 This and the fact that the two haplotype sequences are identical to the haplotype sequences reported for Ae. aegypti populations in Brazil and Guyana support the conclusion that the WDC Ae. aegypti population is derived from lineages resident in the Americas, and were likely transported into WDC via human-mediated travel. These data also support the expected outcome of a persistent low genetic diversity founder population; for example, in contrast, analyses of Ae. aegypti mtND4 and mtND5 haplotypes across southern Florida in 2006 revealed 68 different haplotypes among 362 individuals examined.19
We determined genotypes at 10 microsatellite loci for 70 individuals from the WDC population collected in 2011 (N = 22), 2012 (N = 12), 2013 (N = 8), and 2014 (N = 28). These loci fit HWE expectations with only two exceptions across all years, with the total number of alleles observed per locus across all years ranging from two to 10 (Figure 3). Examination of individual allele presence and frequency across years clearly reflect no evidence of new allele introgression across all 10 loci, further supporting our conclusion that this represents an overwintering population in WDC. Bayesian cluster analysis using STRUCTURE identified the likely number of clusters as K = 2, but failed to detect any evidence for clustering across YOC as the WDC annual populations represented similar mixtures of the identified genetic clusters (Figure 2B).
Taken together, our mtCOI haplotype and microsatellite marker data confirm consistent genetic identities among Ae. aegypti WDC populations across all YOC, with no evidence of introgression by novel genotypes. The most parsimonious conclusion is that this represents a small continuous resident WDC population that reflects founder effects since the original introduction during or prior to our initial sampling in 2011. This raises interesting questions about how the population has persisted despite the obvious extreme winter seasonal conditions, with an average January isotherm for WDC of only 2.2°C.20 A historical review of A. aegypti presence across the continental United States does include documented collections in the northern Virginia/Maryland/WDC area, but concludes that this represents the extreme northern edge and that overwintering populations were unlikely.3 January isotherms between collection years had considerable variation, with two consecutive mild years followed by extraordinary cold (2012: 4.9°C, 2013: 4.6°C, and 2014: 0.17°C). The most likely scenario is that an Ae. aegypti population has colonized a subterranean habitat with suitable climate, periodic standing water, and potentially access to blood-meal sources for year-round breeding and survival. However, as Ae. aegypti eggs and larvae can survive much lower temperatures than adults,6 a subterranean habitat may be compatible for winter survival as diapausing eggs. We speculate that presently unknown point(s) of access from the subterranean site(s) to the surface must exist within the immediate area around the collection sites (Figure 1B and C) and that small numbers of adults are finding the route to the surface and engaging in limited breeding each summer.
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