Anopheles gambiae on remote islands in the Indian Ocean: origins and prospects for malaria elimination by genetic modification of extant populations.

The mosquito Anopheles gambiae s.s. is a primary malaria vector throughout sub-Saharan Africa including the islands of the Comoros archipelago (Anjouan, Grande Comore, Mayotte and Mohéli). These islands are located at the northern end of the Mozambique Channel in eastern Africa. Previous studies have shown a relatively high degree of genetic isolation between the Comoros islands and mainland populations of A. gambiae, but the origin of the island populations remains unclear. Here, we analyzed phylogenetic relationships among island and mainland populations using complete mitochondrial genome sequences of individual A. gambiae specimens. This work augments earlier studies based on analysis of the nuclear genome. We investigated the source population of A. gambiae for each island, estimated the number of introductions, when they occurred and explored evidence for contemporary gene flow between island and mainland populations. These studies are relevant to understanding historical patterns in the dispersal of this important malaria vector and provide information critical to assessing their potential for the exploration of genetic-based vector control methods to eliminate this disease. Phylogenetic analysis and haplotype networks were constructed from mitogenome sequences of 258 A. gambiae from the four islands. In addition, 112 individuals from seven countries across sub-Saharan Africa and Madagascar were included to identify potential source populations. Our results suggest that introduction events of A. gambiae into the Comoros archipelago were rare and recent events and support earlier claims that gene flow between the mainland and these islands is limited. This study is concordant with earlier work suggesting the suitability of these oceanic islands as appropriate sites for conducting field trial releases of genetically engineered mosquitoes (GEMs).

Genomic signatures of local adaptation in recent invasive Aedes aegypti populations in California.

BACKGROUND: Rapid adaptation to new environments can facilitate species invasions and range expansions. Understanding the mechanisms of adaptation used by invasive disease vectors in new regions has key implications for mitigating the prevalence and spread of vector-borne disease, although they remain relatively unexplored. RESULTS: Here, we integrate whole-genome sequencing data from 96 Aedes aegypti mosquitoes collected from various sites in southern and central California with 25 annual topo-climate variables to investigate genome-wide signals of local adaptation among populations. Patterns of population structure, as inferred using principal components and admixture analysis, were consistent with three genetic clusters. Using various landscape genomics approaches, which all remove the confounding effects of shared ancestry on correlations between genetic and environmental variation, we identified 112 genes showing strong signals of local environmental adaptation associated with one or more topo-climate factors. Some of them have known effects in climate adaptation, such as heat-shock proteins, which shows selective sweep and recent positive selection acting on these genomic regions. CONCLUSIONS: Our results provide a genome wide perspective on the distribution of adaptive loci and lay the foundation for future work to understand how environmental adaptation in Ae. aegypti impacts the arboviral disease landscape and how such adaptation could help or hinder efforts at population control.

Field Evaluation of In2Care Mosquito Traps to Control Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Hawai'i Island.

Aedes aegypti Linnaeus and Aedes albopictus Skuse are vectors of dengue virus and responsible for multiple autochthonous dengue outbreaks in Big Island, Hawai'i. Control of Ae. aegypti and Ae. albopictus has been achieved in In2Care trap trials, which motivated us to investigate this potential control approach in the Big Island. Our In2Care trial was performed in the coastal settlement of Miloli'i in the southwest of Big Island where both Ae. aegypti and Ae. albopictus are found. This trial starting in the second week of July and ending in the last week of October 2019 fell within the traditional wet season in Miloli'i. No significant reduction in egg or adult counts in our treatment areas following 12 wk of two In2Care trap placements per participating household were observed. In fact, an increase in numbers of adults during the trial reached levels that required the local mosquito abatement program to stop the In2Care trap trial and institute a thorough source reduction and treatment campaign. The source reduction campaign revealed a large variety and quantity of water sources competed with the oviposition cups we had placed, which likely lowered the chances of our oviposition cups being visited by pyriproxyfen-contaminated Aedes adults exiting the In2Care traps.

A highly expressed odorant receptor from the yellow fever mosquito, AaegOR11, responds to (+)- and (-)-fenchone and a phenolic repellent.

The cornerstone of the reverse chemical ecology approach is the identification of odorant receptors (OR) sensitive to compounds in a large panel of odorants. In this approach, we de-orphanize ORs and, subsequently, measure behaviors elicited by these semiochemicals. After that, we evaluate behaviorally active compounds for applications in insect vector management. Intriguingly, multiple ORs encoded by genes highly expressed in mosquito antennae do not respond to any test odorant. One such case is CquiOR125 from the southern house mosquito, Culex quinquefasciatus Say. To better understand CquiOR125's role in Culex mosquito olfaction, we have cloned a CquiOR125 orthologue in the genome of the yellow fever mosquito, Aedes aegypti (L.), AaegOR11. Unlike the unresponsive nature of the orthologue in Cx. quinquefasciatus, oocytes co-expressing AaegOR11 and AaegOrco elicited robust responses when challenged with fenchone, 2,3-dimethylphenol, 3,4-dimethylphenol, 4-methycyclohexanol, and acetophenone. Interestingly, AaegOR11 responded strongly and equally to (+)- and (-)-fenchone, with no chiral discrimination. Contrary to reports in the literature, fenchone did not show any repellency activity against Ae. aegypti or Cx. quinquefasciatus. Laboratory and field tests did not show significant increases in egg captures in cups filled with fenchone solutions compared to control cups. The second most potent ligand, 2,3-dimethylphenol, showed repellency activity stronger than that elicited by DEET at the same dose. We, therefore, concluded that AaegOR11 is a mosquito repellent sensor. It is feasible that CquiOR125 responds to repellents that remain elusive.

Efficacy of Trapping Methods in the Collection of Eretmapodites (Diptera: Culicidae) Mosquitoes in an Afrotropical Rainforest Region, South western Cameroon.

Very little data exist on the biology of an afrotropical rainforest mosquito Eretmapodites (Er.) in a world undergoing dramatic changes due to deforestation. The aim was to assess the efficacy of different trapping methods in the collection of Er. mosquito in forested area. This was a longitudinal study involving collection of mosquitoes for over two years. Multiple collection methods (grouped into two categories), were used; i) net baited and un-baited traps to collect adults, ii) techniques that target immature stages subsequently reared to adults. All males were identified by genitalia dissection. Five thousand seven hundred and four mosquitoes representing 11 genera among which 2,334 Er. were identified. Mosquito abundance was highest in the net traps (n = 1276 (56.4%)) and sweep nets (n = 393(17.4%)) respectively. The abundance was highest in green colored net traps (435(34.09%)) with significant value of χ2= 40.000, P < 0.001 and in pigeons baited traps (473 (37.06%)) with significant value of χ2= 42.000, P = 0.003. The diversity ranges from H' = 2.65; DS = 0.84; SR = 24; ACE = 24.77 in sweep net to H' = 0; DS = 0; SR = 1; ACE = 1 in rock pool among males mosquitoes. While for females, H = 1.14; DS = 0.71; SR = 5; ACE = 5.16, in sweep net to H = 0; DS = 0; SR = 1; ACE = 1 in rock pool, tarpaulin, resting cage. Net traps, bamboo pot, and sweep netting are efficient in collecting high abundance of forest mosquitoes in the Talanagaye rainforest.

Population Genetics of Anopheles pretoriensis in Grande Comore Island.

Anopheles pretoriensis is widely distributed across Africa, including on oceanic islands such as Grande Comore in the Comoros. This species is known to be mostly zoophylic and therefore considered to have low impact on the transmission of human malaria. However, A. pretoriensis has been found infected with Plasmodium, suggesting that it may be epidemiologically important. In the present study, we sequenced and assembled the complete mitogenome of A. pretoriensis and inferred its phylogenetic relationship among other species in the subgenus Cellia. We also investigated the genetic structure of A. pretoriensis populations on Grande Comore Island, and between this island population and sites in continental Africa, using partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Seven haplotypes were found on the island, one of which was ubiquitous. There was no clear divergence between island haplotypes and those found on the continent. The present work contributes knowledge on this understudied, yet abundant, Anopheles species.

Selection of sites for field trials of genetically engineered mosquitoes with gene drive.

Novel malaria control strategies using genetically engineered mosquitoes (GEMs) are on the horizon. Population modification is one approach wherein mosquitoes are engineered with genes rendering them refractory to the malaria parasite, Plasmodium falciparum, coupled with a low-threshold, Cas9-based gene drive. When released into a wild vector population, GEMs preferentially transmit these parasite-blocking genes to their offspring, ultimately modifying a vector population into a nonvector one. Deploying this technology awaits ecologically contained field trial evaluations. Here, we consider a process for site selection, the first critical step in designing a trial. Our goal is to identify a site that maximizes prospects for success, minimizes risk, and serves as a fair, valid, and convincing test of efficacy and impacts of a GEM product intended for large-scale deployment in Africa. We base site selection on geographic, geological, and biological, rather than social or legal, criteria. We recognize the latter as critically important but not as a first step in selecting a site. We propose physical islands as being the best candidates for a GEM field trial and present an evaluation of 22 African islands. We consider geographic and genetic isolation, biological complexity, island size, and topography and identify two island groups that satisfy key criteria for ideal GEM field trial sites.

The origin of island populations of the African malaria mosquito, Anopheles coluzzii.

Anopheles coluzzii is a major malaria vector throughout its distribution in west-central Africa. Here we present a whole-genome study of 142 specimens from nine countries in continental Africa and three islands in the Gulf of Guinea. This sample set covers a large part of this species' geographic range. Our population genomic analyses included a description of the structure of mainland populations, island populations, and connectivity between them. Three genetic clusters are identified among mainland populations and genetic distances (FST) fits an isolation-by-distance model. Genomic analyses are applied to estimate the demographic history and ancestry for each island. Taken together with the unique biogeography and history of human occupation for each island, they present a coherent explanation underlying levels of genetic isolation between mainland and island populations. We discuss the relationship of our findings to the suitability of São Tomé and Príncipe islands as candidate sites for potential field trials of genetic-based malaria control strategies.

Frequency of sodium channel genotypes and association with pyrethrum knockdown time in populations of Californian Aedes aegypti.

BACKGROUND: Since their detection in 2013, Aedes aegypti has become a widespread urban pest in California. The availability of cryptic larval breeding sites in residential areas and resistance to insecticides pose significant challenges to control efforts. Resistance to pyrethroids is largely attributed to mutations in the voltage gated sodium channels (VGSC), the pyrethroid site of action. However, past studies have indicated that VGSC mutations may not be entirely predictive of the observed resistance phenotype. METHODS: To investigate the frequencies of VGSC mutations and the relationship with pyrethroid insecticide resistance in California, we sampled Ae. aegypti from four locations in the Central Valley, and the Greater Los Angeles area. Mosquitoes from each location were subjected to an individual pyrethrum bottle bioassay to determine knockdown times. A subset of assayed mosquitoes from each location was then analyzed to determine the composition of 5 single nucleotide polymorphism (SNP) loci within the VGSC gene. RESULTS: The distribution of knockdown times for each of the five Californian populations sampled was non-parametric with potentially bimodal distributions. One group succumbs to insecticidal effects around 35-45 min and the second group lasts up to and beyond the termination of the assay (120+ min). We detected 5 polymorphic VGSC SNPs within the sampled California populations. One is potentially new and alternatively spliced (I915K), and four are documented and associated with resistance: F1534C, V1016I, V410L and S723T. The Central Valley populations (Clovis, Dinuba, Sanger and Kingsburg) are fairly homogenous with only 5% of the mosquitoes showing heterozygosity at any given position. In the Greater LA mosquitoes, 55% had at least one susceptible allele at any of the five SNP loci. The known resistance allele F1534C was detected in almost all sampled mosquitoes (99.4%). We also observe significant heterogeneity in the knockdown phenotypes of individuals with the identical VGSC haplotypes suggesting the presence of additional undefined resistance mechanisms. CONCLUSIONS: Resistance associated VGSC SNPs are prevalent, particularly in the Central Valley. Interestingly, among mosquitoes carrying all 4 resistance associated SNPs, we observe significant heterogeneity in bottle bioassay profiles suggesting that other mechanisms are important to the individual resistance of Ae. aegypti in California.

Evidence for Divergent Selection on Immune Genes between the African Malaria Vectors, Anopheles coluzzii and A. gambiae.

During their life cycles, microbes infecting mosquitoes encounter components of the mosquito anti-microbial innate immune defenses. Many of these immune responses also mediate susceptibility to malaria parasite infection. In West Africa, the primary malaria vectors are Anopheles coluzzii and A. gambiae sensu stricto, which is subdivided into the Bamako and Savanna sub-taxa. Here, we performed whole genome comparisons of the three taxa as well as genotyping of 333 putatively functional SNPs located in 58 immune signaling genes. Genome data support significantly higher differentiation in immune genes compared with a randomly selected set of non-immune genes among the three taxa (permutation test p < 0.001). Among the 58 genes studied, the majority had one or more segregating mutations (72.9%) that were significantly diverged among the three taxa. Genes detected to be under selection include MAP2K4 and Raf. Despite the genome-wide distribution of immune genes, a high level of linkage disequilibrium (r2 > 0.8) was detected in over 27% of SNP pairs. We discuss the potential role of immune gene divergence as adaptations to the different larval habitats associated with A. gambiae taxa and as a potential force driving ecological speciation in this group of mosquitoes.

New species of Culex (Culiciomyia) (Diptera: Culicidae) from Talangaye Forest in Cameroon and descriptions and identification keys for males of the Afrotropical species of the subgenus.

Species of Culex (Diptera: Culicidae) belonging to the subgenus Culiciomyia were collected in partially logged areas and in surrounding pristine forest (Talangaye Forest) in the Nguti Subdivision in the South-West Region of Cameroon. Mosquitoes were collected mainly by sweep netting through forest floor vegetation. Morphological species identification of African Culiciomyia relies almost exclusively on the structure of the male genitalia and the shapes of comb scales on the maxillary palpi of males. Other features of males and the habitus of females are largely indistinguishable between the species of this subgenus. In total, seven currently described species and three new species were collected in the forest. The males of the three new species are described and named as Culex apicopilosus Cornel Mayi, sp. n., Culex lanzaroi Cornel Mayi, sp. n. and Culex pseudosubaequalis Cornel Mayi, sp. n. More detailed descriptions of males of the other currently known species that were collected in the Talangaye Forest and pictorial keys to the males of all Afrotropical species of Culiciomyia, including the new species, are provided.

Malaria Vectors and Vector Surveillance in Limpopo Province (South Africa): 1927 to 2018.

Despite the annual implementation of a robust and extensive indoor residual spraying programme against malaria vectors in Limpopo Province (South Africa), significant transmission continues and is a serious impediment to South Africa's malaria elimination objectives. In order to gain a better understanding regarding possible causes of this residual malaria, we conducted a literature review of the historical species composition and abundance of malaria vector mosquitoes in the Limpopo River Valley region of the Vhembe District, northern Limpopo Province, the region with the highest remaining annual malaria cases in South Africa. In addition, mosquito surveys were carried out in the same region between October 2017 and October 2018. A total of 2225 adult mosquitoes were collected using CO2-baited tent and light traps, human landing catches and cow-baited traps. Of the 1443 Anopheles collected, 516 were members of the An. gambiae complex and 511 An. funestus group. In the malaria endemic rural areas outside the Kruger National Park, one specimen each of An. gambiae s.s. and An. funestus and only three of An. arabiensis were collected. The latter species was abundant at a remote hot spring in the neighboring Kruger National Park. Eighteen other species of Anopheles were collected. Our survey results support the historical findings that An. arabiensis, the species widely held to be the prime malaria vector in South Africa, is a rare species in the malaria endemic Limpopo River Valley. The implications of the mosquito surveys for malaria transmission, elimination and vector control in northern Limpopo Province and neighboring regions are discussed.

Habitat and Seasonality Affect Mosquito Community Composition in the West Region of Cameroon.

To identify potential sylvatic, urban and bridge-vectors that can be involved in current or future virus spillover from wild to more urbanised areas, entomological field surveys were conducted in rural, peri-urban and urban areas spanning the rainy and dry seasons in western Cameroon. A total of 2650 mosquitoes belonging to 37 species and eight genera were collected. Mosquito species richness was significantly influenced by the specific combination of the habitat type and the season. The highest species richness was found in the peri-urban area (S = 30, Chao1 = 121 ± 50.63, ACE = 51.97 ± 3.88) during the dry season (S = 28, Chao1 = 64 ± 25.7, ACE = 38.33 ± 3.1). Aedes (Ae.) africanus and Culex (Cx.) moucheti were only found in the rural and peri-urban areas, while Cx. pipiens s.l. and Ae. aegypti were only found in the urban area. Cx. (Culiciomyia) spp., Cx. duttoni and Ae. albopictus were caught in the three habitat types. Importantly, approximately 52% of the mosquito species collected in this study have been implicated in the transmission of diverse arboviruses. This entomological survey provides a catalogue of the different mosquito species that may be involved in the transmission of arboviruses. Further investigations are needed to study the vectorial capacity of each mosquito species in arbovirus transmission.

Complete mitogenome sequence of Anopheles coustani from São Tomé island.

We report the first complete mitogenome (Mt) sequence of Anopheles coustani, an understudied malaria vector in Africa. The sequence was extracted from one individual mosquito from São Tomé island. The length of the A. coustani Mt genome was 15,408 bp with 79.3% AT content. Phylogenetic analysis revealed that A. coustani is most closely related to A. sinensis (93.5% of identity); and 90.1% identical to A. gambiae complex members.

Effect of deforestation on prevalence of avian haemosporidian parasites and mosquito abundance in a tropical rainforest of Cameroon.

Habitat change caused by deforestation can modify the interactions of many biotic and abiotic factors, and in turn influence patterns of diseases in wild birds. Whether deforestation directly or indirectly affects the prevalence of avian haemosporidian parasites through their hosts and/or vectors is still not well understood. We sampled understory bird communities (insectivorous, frugivorous, granivorous and nectarivorous birds) and mosquitoes in three habitats showing a gradient of deforestation (pristine forest, fragmented forest, and young palm oil plantation), to assess the effects of habitat changes on avian haemosporidian (Plasmodium and Haemoproteus) prevalence and its relationship to bird feeding guilds and mosquito abundance. Blood samples of 845 individual birds belonging to 85 species and 27 families were collected in the three habitat types and screened using microscopy and PCR. Plasmodium infections were detected in 136 individuals (16.09%) and varied significantly among habitat types while Haemoproteus infections were detected in 98 individuals (11.60%) and did not vary significantly among habitat types. However, the prevalence of Plasmodium and Haemoproteus in bird feeding groups varied significantly among habitats. Nectarivorous and granivorous birds had the highest Plasmodium and Haemoproteus prevalence, respectively. The abundance of mosquitoes varied significantly among habitat types and the prevalence of Plasmodium significantly and positively correlated with mosquito abundance in fragmented forest. This study highlights the importance of host and mosquito determinants in the transmission dynamics of avian Plasmodium and Haemoproteus infections following habitat changes. Selective logging favored an increase in the prevalence of Plasmodium in insectivores, the prevalence of Haemoproteus in nectarivores and the abundance of female mosquitoes while, the establishment of the palm oil plantation favored an increase in the prevalence of Plasmodium in granivores and Haemoproteus in nectarivores. Species feeding behavior is also an important determinant to consider for a better understanding of patterns of parasite infections in a changing environment.

Transcontinental dispersal of Anopheles gambiae occurred from West African origin via serial founder events.

The mosquito Anopheles gambiae s.s. is distributed across most of sub-Saharan Africa and is of major scientific and public health interest for being an African malaria vector. Here we present population genomic analyses of 111 specimens sampled from west to east Africa, including the first whole genome sequences from oceanic islands, the Comoros. Genetic distances between populations of A. gambiae are discordant with geographic distances but are consistent with a stepwise migration scenario in which the species increases its range from west to east Africa through consecutive founder events over the last ~200,000 years. Geological barriers like the Congo River basin and the East African rift seem to play an important role in shaping this process. Moreover, we find a high degree of genetic isolation of populations on the Comoros, confirming the potential of these islands as candidate sites for potential field trials of genetically engineered mosquitoes for malaria control.

Impact of deforestation on the abundance, diversity, and richness of Culex mosquitoes in a southwest Cameroon tropical rainforest.

Deforestation is a major threat to biodiversity but little data exist on how deforestation in real-time affects the overall mosquito species community despite its known role in the transmission of diseases. We compared the abundance and diversity of Culex mosquitoes before and after deforestation along a gradient of three different anthropogenic disturbance levels in a tropical rainforest in southwestern Cameroon. The collections were conducted in unlogged forest (January, 2016), selectively logged forest (January, 2017), and within a young palm plantation (October, 2017) using net traps, sweep nets, resting traps, and dipping for immature stages in water bodies. Mosquitoes were morphologically identified to subspecies, groups, and species. A total of 2,556 mosquitoes was collected of which 1,663 (65.06%) belong to the genus Culex, (n=427 (25.68%) in the unlogged forest; n=900 (54.12%) in the selectively logged forest; and n=336 (20.2%) in the young palm plantation) with a significant difference among the habitats. Diversity and richness of mosquitoes varied significantly among habitats with the highest values found in the selectively logged forest (H=2.4; DS=0.87; S=33) and the lowest value in the unlogged forest (H=1.37; DS=0.68; S=13). The results of this study showed that deforestation affects the abundance and diversity of Culex mosquitoes and favors the invasion of anthropophilic mosquitoes. Higher mosquito abundance and diversity in the selectively logged forest than in the pristine forest is notable and some explanations for these differences are discussed.

Aedes aegypti Males as Vehicles for Insecticide Delivery.

Aedes aegypti continues to spread globally and remains a challenge to control, in part due to its 'cryptic behavior' in that it often deposits eggs (oviposits) in larval habitats that are difficult to find and treat using traditional methods. Auto-dissemination strategies target these cryptic breeding sites by employing mosquitoes to deliver lethal doses of insecticide. This report describes the initial field trials of an application known as Autodissemination Augmented by Males (ADAM), utilizing A. aegypti males dusted with pyriproxyfen (PPF). Findings presented here are drawn from both caged and field trial studies. Together, these trials examined for the ability of A. aegypti males to disseminate PPF and to impact field populations. PPF-dusted males were able to effectively deliver lethal doses of PPF to oviposition sites under the conditions tested. Results from field trials in Florida and California demonstrated reduced A. aegypti populations in treated areas, compared to areas where PPF-treated males were not released. These results indicate that the release of PPF-dusted A. aegypti males can impact A. aegypti populations as measured by both reduced larval survival and lower numbers of adult female A. aegypti. We propose the ADAM approach as an addition to existing mosquito control techniques targeting A. aegypti and other mosquitoes that utilize cryptic larval habitats.

Odorant Inhibition in Mosquito Olfaction.

How chemical signals are integrated at the peripheral sensory system of insects is still an enigma. Here we show that when coexpressed with Orco in Xenopus oocytes, an odorant receptor from the southern house mosquito, CquiOR32, generated inward (regular) currents when challenged with cyclohexanone and methyl salicylate, whereas eucalyptol and fenchone elicited inhibitory (upward) currents. Responses of CquiOR32-CquiOrco-expressing oocytes to odorants were reduced in a dose-dependent fashion by coapplication of inhibitors. This intrareceptor inhibition was also manifested in vivo in fruit flies expressing the mosquito receptor CquiOR32, as well in neurons on the antennae of the southern house mosquito. Likewise, an orthologue from the yellow fever mosquito, AaegOR71, showed intrareceptor inhibition in the Xenopus oocyte recording system and corresponding inhibition in antennal neurons. Inhibition was also manifested in mosquito behavior. Blood-seeking females were repelled by methyl salicylate, but repellence was significantly reduced when methyl salicylate was coapplied with eucalyptol.

Genome-wide divergence among invasive populations of Aedes aegypti in California.

BACKGROUND: In the summer of 2013, Aedes aegypti Linnaeus was first detected in three cities in central California (Clovis, Madera and Menlo Park). It has now been detected in multiple locations in central and southern CA as far south as San Diego and Imperial Counties. A number of published reports suggest that CA populations have been established from multiple independent introductions. RESULTS: Here we report the first population genomics analyses of Ae. aegypti based on individual, field collected whole genome sequences. We analyzed 46 Ae. aegypti genomes to establish genetic relationships among populations from sites in California, Florida and South Africa. Based on 4.65 million high quality biallelic SNPs, we identified 3 major genetic clusters within California; one that includes all sample sites in the southern part of the state (South of Tehachapi mountain range) plus the town of Exeter in central California and two additional clusters in central California. CONCLUSIONS: A lack of concordance between mitochondrial and nuclear genealogies suggests that the three founding populations were polymorphic for two main mitochondrial haplotypes prior to being introduced to California. One of these has been lost in the Clovis populations, possibly by a founder effect. Genome-wide comparisons indicate extensive differentiation between genetic clusters. Our observations support recent introductions of Ae. aegypti into California from multiple, genetically diverged source populations. Our data reveal signs of hybridization among diverged populations within CA. Genetic markers identified in this study will be of great value in pursuing classical population genetic studies which require larger sample sizes.