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The genomic signal of local environmental adaptation in Aedes aegypti mosquitoesBennett, Kelly L.McMillan, W. OwenLoaiza, Jose R.DOI: info:10.1111/eva.13199
Bennett, Kelly L., McMillan, W. Owen, and Loaiza, Jose R. 2021. "The genomic signal of local environmental adaptation in Aedes aegypti mosquitoes." Evolutionary Applications
ID: 158773
Type: article
Authors: Bennett, Kelly L.; McMillan, W. Owen; Loaiza, Jose R.
Abstract: Local adaptation is important when predicting arthropod-borne disease risk because of its impacts on vector population fitness and persistence. However, the extent that vector populations are adapted to the environment generally remains unknown. Despite low population structure and high gene flow in Aedes aegypti mosquitoes across Panama, excepting the province of Bocas del Toro, we identified 128 candidate SNPs, clustered within 17 genes, which show a strong genomic signal of local environmental adaptation. This putatively adaptive variation occurred across fine geographical scales with the composition and frequency of candidate adaptive loci differing between populations in wet tropical environments along the Caribbean coast and dry tropical conditions typical of the Pacific coast. Temperature and vegetation were important predictors of adaptive genomic variation in Ae. aegypti with several potential areas of local adaptation identified. Our study lays the foundations of future work to understand whether environmental adaptation in Ae. aegypti impacts the arboviral disease landscape and whether this could either aid or hinder efforts of population control.
The role of heterogenous environmental conditions in shaping the spatiotemporal distribution of competing Aedes mosquitoes in Panama: implications for the landscape of arboviral disease transmissionBennett, Kelly L.Owen McMillan, W.Enriquez, VanessaBarraza, EliaDiaz, MarcelaBaca, BrendaWhiteman, AriCerro Medina, JaimeDucasa, MadeleineGomez Martinez, CarmeloAlmanza, AlejandroRovira, Jose R.Loaiza, Jose R.DOI: info:10.1007/s10530-021-02482-y
Bennett, Kelly L., Owen McMillan, W., Enriquez, Vanessa, Barraza, Elia, Diaz, Marcela, Baca, Brenda, Whiteman, Ari, Cerro Medina, Jaime, Ducasa, Madeleine, Gomez Martinez, Carmelo, Almanza, Alejandro, Rovira, Jose R., and Loaiza, Jose R. 2021. "The role of heterogenous environmental conditions in shaping the spatiotemporal distribution of competing Aedes mosquitoes in Panama: implications for the landscape of arboviral disease transmission." Biological Invasions
ID: 158769
Type: article
Authors: Bennett, Kelly L.; Owen McMillan, W.; Enriquez, Vanessa; Barraza, Elia; Diaz, Marcela; Baca, Brenda; Whiteman, Ari; Cerro Medina, Jaime; Ducasa, Madeleine; Gomez Martinez, Carmelo; Almanza, Alejandro; Rovira, Jose R.; Loaiza, Jose R.
Abstract: Monitoring the invasion process of the Asian tiger mosquito Aedes albopictus and its interaction with the contender Aedes aegypti, is critical to prevent and control the arthropod-borne viruses (i.e., Arboviruses) they transmit to humans. Generally, the superior ecological competitor Ae. albopictus displaces Ae. aegypti from most geographic areas, with the combining factors of biology and environment influencing the competitive outcome. Nonetheless, detailed studies asserting displacement come largely from sub-tropical areas, with relatively less effort being made in tropical environments, including no comprehensive research about Aedes biological interactions in Mesoamerica. Here, we examine contemporary and historical mosquito surveillance data to assess the role of shifting abiotic conditions in shaping the spatiotemporal distribution of competing Aedes species in the Republic of Panama. In accordance with prior studies, we show that Ae. albopictus has displaced Ae. aegypti under suboptimal wet tropical climate conditions and more vegetated environments within the southwestern Azuero Peninsula. Conversely, in the eastern Azuero Peninsula, Ae. aegypti persists with Ae. albopictus under optimal niche conditions in a dry and more seasonal tropical climate. While species displacement was stable over the course of two years, the presence of both species generally appears to fluctuate in tandem in areas of coexistence. Aedes albopictus was always more frequently found and abundant regardless of location and climatic season. The heterogenous environmental conditions of Panama shape the competitive outcome and micro-geographic distribution of Aedes mosquitoes, with potential consequences for the transmission dynamics of urban and sylvatic zoonotic diseases.
Proteomic fingerprinting of Neotropical hard tick species (Acari: Ixodidae) using a self-curated mass spectra reference libraryGittens, Rolando A.Almanza, AlejandroBennett, Kelly L.Mejia, Luis C.Sanchez-Galan, Javier E.Merchan, FernandoKern, JonathanMiller, Matthew J.Esser, Helen J.Hwang, RobertDong, MayDe Leon, Luis F.alvarez, EricLoaiza, Jose R.DOI: info:10.1371/journal.pntd.0008849v. 14No. 10e0008849–e0008849
Gittens, Rolando A., Almanza, Alejandro, Bennett, Kelly L., Mejia, Luis C., Sanchez-Galan, Javier E., Merchan, Fernando, Kern, Jonathan, Miller, Matthew J., Esser, Helen J., Hwang, Robert, Dong, May, De Leon, Luis F., alvarez, Eric, and Loaiza, Jose R. 2020. "Proteomic fingerprinting of Neotropical hard tick species (Acari: Ixodidae) using a self-curated mass spectra reference library." Plos Neglected Tropical Diseases 14 (10):e0008849– e0008849.
ID: 157640
Type: article
Authors: Gittens, Rolando A.; Almanza, Alejandro; Bennett, Kelly L.; Mejia, Luis C.; Sanchez-Galan, Javier E.; Merchan, Fernando; Kern, Jonathan; Miller, Matthew J.; Esser, Helen J.; Hwang, Robert; Dong, May; De Leon, Luis F.; alvarez, Eric; Loaiza, Jose R.
Abstract: Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry is an analytical method that detects macromolecules that can be used for proteomic fingerprinting and taxonomic identification in arthropods. The conventional MALDI approach uses fresh laboratory-reared arthropod specimens to build a reference mass spectra library with high-quality standards required to achieve reliable identification. However, this may not be possible to accomplish in some arthropod groups that are difficult to rear under laboratory conditions, or for which only alcohol preserved samples are available. Here, we generated MALDI mass spectra of highly abundant proteins from the legs of 18 Neotropical species of adult field-collected hard ticks, several of which had not been analyzed by mass spectrometry before. We then used their mass spectra as fingerprints to identify each tick species by applying machine learning and pattern recognition algorithms that combined unsupervised and supervised clustering approaches. Both Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) classification algorithms were able to identify spectra from different tick species, with LDA achieving the best performance when applied to field-collected specimens that did have an existing entry in a reference library of arthropod protein spectra. These findings contribute to the growing literature that ascertains mass spectrometry as a rapid and effective method to complement other well-established techniques for taxonomic identification of disease vectors, which is the first step to predict and manage arthropod-borne pathogens. Author summary Hard ticks (Ixodidae) are external parasites that feed on the blood of almost every species of terrestrial vertebrate on earth, including humans. Due to a complete dependency on blood, both sexes and even immature stages, are capable of transmitting disease agents to their hosts, causing distress and sometimes death. Despite the public health significance of ixodid ticks, accurate species identification remains problematic. Vector species identification is core to developing effective vector control schemes. Herein, we provide the first report of MALDI identification of several species of field-collected Neotropical tick specimens preserved in ethanol for up to five years. Our methodology shows that identification does not depend on a commercial reference library of lab-reared samples, but with the help of machine learning it can rely on a self-curated reference library. In addition, our approach offers a complimentary tool with good accuracy and lower cost per sample than conventional and modern identification approaches such as morphology and molecular barcoding, provided that financial resources are available to invest in the acquisition of the MALDI equipment.
Habitat disturbance and the organization of bacterial communities in Neotropical hematophagous arthropodsBennett, Kelly L.Almanza, AlejandroMcMillan, W. O.Saltonstall, KristinVdovenko, Evangelina LópezVinda, Jorge S.Mejía, LuisDriesse, KaitlinDe León, Luis F.Loaiza, José R.DOI: info:10.1371/journal.pone.0222145v. 141–19
Bennett, Kelly L., Almanza, Alejandro, McMillan, W. O., Saltonstall, Kristin, Vdovenko, Evangelina López, Vinda, Jorge S., Mejía, Luis, Driesse, Kaitlin, De León, Luis F., and Loaiza, José R. 2019. "Habitat disturbance and the organization of bacterial communities in Neotropical hematophagous arthropods." PLOS ONE 14:1– 19.
ID: 152577
Type: article
Authors: Bennett, Kelly L.; Almanza, Alejandro; McMillan, W. O.; Saltonstall, Kristin; Vdovenko, Evangelina López; Vinda, Jorge S.; Mejía, Luis; Driesse, Kaitlin; De León, Luis F.; Loaiza, José R.
Abstract: The microbiome plays a key role in the biology, ecology and evolution of arthropod vectors of human pathogens. Vector-bacterial interactions could alter disease transmission dynamics through modulating pathogen replication and/or vector fitness. Nonetheless, our understanding of the factors shaping the bacterial community in arthropod vectors is incomplete. Using large-scale 16S amplicon sequencing, we examine how habitat disturbance structures the bacterial assemblages of field-collected whole-body hematophagous arthropods that vector human pathogens including mosquitoes (Culicidae), sand flies (Psychodidae), biting midges (Ceratopogonidae) and hard ticks (Ixodidae). We found that all comparisons of the bacterial community among species yielded statistically significant differences, but a difference was not observed between adults and nymphs of the hard tick, Haemaphysalis juxtakochi. While Culicoides species had the most distinct bacterial community among dipterans, tick species were composed of entirely different bacterial OTU's. We observed differences in the proportions of some bacterial types between pristine and disturbed habitats for Coquillettidia mosquitoes, Culex mosquitoes, and Lutzomyia sand flies, but their associations differed within and among arthropod assemblages. In contrast, habitat quality was a poor predictor of differences in bacterial classes for Culicoides biting midges and hard tick species. In general, similarities in the bacterial communities among hematophagous arthropods could be explained by their phylogenetic relatedness, although intraspecific variation seems influenced by habitat disturbance.
High infestation of invasive Aedes mosquitoes in used tires along the local transport network of PanamaBennett, Kelly L.Gómez Martínez, CarmeloAlmanza, AlejandroRovira, Jose R.McMillan, W. O.Enriquez, VanessaBarraza, EliaDiaz, MarcelaSanchez-Galan, JavierWhiteman, AriGittens, Rolando A.Loaiza, Jose R.DOI: info:10.1186/s13071-019-3522-8v. 12No. 1264
Bennett, Kelly L., Gómez Martínez, Carmelo, Almanza, Alejandro, Rovira, Jose R., McMillan, W. O., Enriquez, Vanessa, Barraza, Elia, Diaz, Marcela, Sanchez-Galan, Javier, Whiteman, Ari, Gittens, Rolando A., and Loaiza, Jose R. 2019. "High infestation of invasive Aedes mosquitoes in used tires along the local transport network of Panama." Parasites & Vectors 12 (1):264.
ID: 151372
Type: article
Authors: Bennett, Kelly L.; Gómez Martínez, Carmelo; Almanza, Alejandro; Rovira, Jose R.; McMillan, W. O.; Enriquez, Vanessa; Barraza, Elia; Diaz, Marcela; Sanchez-Galan, Javier; Whiteman, Ari; Gittens, Rolando A.; Loaiza, Jose R.
Abstract: BACKGROUND: The long-distance dispersal of the invasive disease vectors Aedes aegypti and Aedes albopictus has introduced arthropod-borne viruses into new geographical regions, causing a significant medical and economic burden. The used-tire industry is an effective means of Aedes dispersal, yet studies to determine Aedes occurrence and the factors influencing their distribution along local transport networks are lacking. To assess infestation along the primary transport network of Panama we documented all existing garages that trade used tires on the highway and surveyed a subset for Ae. aegypti and Ae. albopictus. We also assess the ability of a mass spectrometry approach to classify mosquito eggs by comparing our findings to those based on traditional larval surveillance. RESULTS: Both Aedes species had a high infestation rate in garages trading used tires along the highways, providing a conduit for rapid dispersal across Panama. However, generalized linear models revealed that the presence of Ae. aegypti is associated with an increase in road density by a log-odds of 0.44 (0.73 ± 0.16; P = 0.002), while the presence of Ae. albopictus is associated with a decrease in road density by a log-odds of 0.36 (0.09 ± 0.63; P = 0.008). Identification of mosquito eggs by mass spectrometry depicted similar occurrence patterns for both Aedes species as that obtained with traditional rearing methods. CONCLUSIONS: Garages trading used tires along highways should be targeted for the surveillance and control of Aedes-mosquitoes and the diseases they transmit. The identification of mosquito eggs using mass spectrometry allows for the rapid evaluation of Aedes presence, affording time and cost advantages over traditional vector surveillance; this is of importance for disease risk assessment.
Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictusBennett, Kelly L.Gómez-Martínez, CarmeloChin, YamilethSaltonstall, KristinMcMillan, W. OwenRovira, José R.Loaiza, José R.DOI: info:10.1038/s41598-019-48414-8v. 9Article 12160
Bennett, Kelly L., Gómez-Martínez, Carmelo, Chin, Yamileth, Saltonstall, Kristin, McMillan, W. Owen, Rovira, José R., and Loaiza, José R. 2019. "Dynamics and diversity of bacteria associated with the disease vectors Aedes aegypti and Aedes albopictus." Scientific Reports 9:Article 12160.
ID: 152248
Type: article
Authors: Bennett, Kelly L.; Gómez-Martínez, Carmelo; Chin, Yamileth; Saltonstall, Kristin; McMillan, W. Owen; Rovira, José R.; Loaiza, José R.
Abstract: Aedes aegypti and Aedes albopictus develop in the same aquatic sites where they encounter microorganisms that influence their life history and capacity to transmit human arboviruses. Some bacteria such as Wolbachia are currently being considered for the control of Dengue, Chikungunya and Zika. Yet little is known about the dynamics and diversity of Aedes-associated bacteria, including larval habitat features that shape their tempo-spatial distribution. We applied large-scale 16S rRNA amplicon sequencing to 960 adults and larvae of both Ae. aegypti and Ae. albopictus mosquitoes from 59 sampling sites widely distributed across nine provinces of Panama. We find both species share a limited, yet highly variable core microbiota, reflecting high stochasticity within their oviposition habitats. Despite sharing a large proportion of microbiota, Ae. aegypti harbours higher bacterial diversity than Ae. albopictus, primarily due to rarer bacterial groups at the larval stage. We find significant differences between the bacterial communities of larvae and adult mosquitoes, and among samples from metal and ceramic containers. However, we find little support for geography, water temperature and pH as predictors of bacterial associates. We report a low incidence of natural Wolbachia infection for both Aedes and its geographical distribution. This baseline information provides a foundation for studies on the functions and interactions of Aedes-associated bacteria with consequences for bio-control within Panama.
Comparative phylogeography of Aedes mosquitoes and the role of past climatic change for evolution within AfricaBennett, Kelly LouiseKaddumukasa, MarthaShija, FortunateDjouaka, RousseauMisinzo, GeraldLutwama, JuliusLinton, Yvonne-MarieWalton, CatherineDOI: info:10.1002/ece3.3668v. 8No. 53019–3036
Bennett, Kelly Louise, Kaddumukasa, Martha, Shija, Fortunate, Djouaka, Rousseau, Misinzo, Gerald, Lutwama, Julius, Linton, Yvonne-Marie, and Walton, Catherine. 2018. "Comparative phylogeography of Aedes mosquitoes and the role of past climatic change for evolution within Africa." Ecology and Evolution 8 (5):3019– 3036.
ID: 145933
Type: article
Authors: Bennett, Kelly Louise; Kaddumukasa, Martha; Shija, Fortunate; Djouaka, Rousseau; Misinzo, Gerald; Lutwama, Julius; Linton, Yvonne-Marie; Walton, Catherine
Abstract: The study of demographic processes involved in species diversification and evolution ultimately provides explanations for the complex distribution of biodiversity on earth, indicates regions important for the maintenance and generation of biodiversity, and identifies biological units important for conservation or medical consequence. African and forest biota have both received relatively little attention with regard to understanding their diversification, although one possible mechanism is that this has been driven by historical climate change. To investigate this, we implemented a standard population genetics approach along with Approximate Bayesian Computation, using sequence data from two exon-primed intron-crossing (EPIC) nuclear loci and mitochondrial cytochrome oxidase subunit I, to investigate the evolutionary history of five medically important and inherently forest dependent mosquito species of the genus Aedes. By testing different demographic hypotheses, we show that Aedes bromeliae and Aedes lilii fit the same model of lineage diversification, admixture, expansion, and recent population structure previously inferred for Aedes aegypti. In addition, analyses of population structure show that Aedes africanus has undergone lineage diversification and expansion while Aedes hansfordi has been impacted by population expansion within Uganda. This congruence in evolutionary history is likely to relate to historical climate-driven habitat change within Africa during the late Pleistocene and Holocene epoch. We find differences in the population structure of mosquitoes from Tanzania and Uganda compared to Benin and Uganda which could relate to differences in the historical connectivity of forests across the continent. Our findings emphasize the importance of recent climate change in the evolution of African forest biota.
Maternal invasion history of Aedes aegypti and Aedes albopictus into the Isthmus of Panama: Implications for the control of emergent viral disease agentsEskildsen, Gilberto A.Rovira, Jose R.Smith, OctavioMiller, Matthew J.Bennett, Kelly L.McMillan, W. O.Loaiza, JoseDOI: info:10.1371/journal.pone.0194874v. 13No. 3
Eskildsen, Gilberto A., Rovira, Jose R., Smith, Octavio, Miller, Matthew J., Bennett, Kelly L., McMillan, W. O., and Loaiza, Jose. 2018. "Maternal invasion history of Aedes aegypti and Aedes albopictus into the Isthmus of Panama: Implications for the control of emergent viral disease agents." PloS One 13 (3):
ID: 146206
Type: article
Authors: Eskildsen, Gilberto A.; Rovira, Jose R.; Smith, Octavio; Miller, Matthew J.; Bennett, Kelly L.; McMillan, W. O.; Loaiza, Jose
Abstract: Despite an increase in dengue outbreaks and the arrival of chikungunya and Zika disease in Panama, studies on the demographic history of the invasive Aedes mosquitoes that are the principle vectors of these diseases are still lacking in this region. Here, we assess the genetic diversity of these mosquitoes in order to decipher their invasion histories into the Isthmus of Panama. DNA sequences from the mitochondrial cytochrome C oxidase I gene obtained from 30 localities in 10 provinces confirmed the presence of more than one mitochondrial haplogroup (i.e., maternal lineage) in each species. The invasion of Aedes albopictus was likely from temperate European countries, as the most frequent and widespread haplogroup in Panama harbored variants that are uncommon elsewhere in the Americas. Two infrequent and geographically restricted Ae. albopictus haplotypes appear to have subsequently invaded Panama from neighboring Costa Rica and the USA, respectively. In addition, we recovered two deeply divergent mitochondrial clades in Panamanian Aedes aegypti. The geographic origins of these clades is unknown, given that divergence in the mitochondrial genome is probably due to ancient population processes within the native range of Ae. aegypti, rather than due to its global expansion out of Africa. However, Panamanian Ae. aegypti mitochondrial sequences within the first clade were closely related to others from Colombia, Bolivia, Brazil, Mexico and the USA, suggesting two separate invasions from Western Hemisphere source populations. The pattern of increased genetic diversity in Aedes mosquitoes in Panama is likely facilitated by the numerous land and water inter-connections across the country, which allows them to enter via sea- and land-transportation from Europe, North, Central and South America. Our results here should be considered in disease mitigation programs if emergent arboviruses are to be effectively diminished in Panama through vector suppression.
Historical environmental change in Africa drives divergence and admixture of Aedes aegypti mosquitoes: a precursor to successful worldwide colonization?Bennett, Kelly LouiseShija, FortunateLinton, Yvonne-MarieMisinzo, GeraldKaddumukasa, MarthaDjouaka, RousseauAnyaele, OkorieHarris, AngelaIrish, SethHlaing, ThaungPrakash, AnilLutwama, JuliusWalton, CatherineDOI: info:10.1111/mec.13762v. 25No. 174337–4354
Bennett, Kelly Louise, Shija, Fortunate, Linton, Yvonne-Marie, Misinzo, Gerald, Kaddumukasa, Martha, Djouaka, Rousseau, Anyaele, Okorie, Harris, Angela, Irish, Seth, Hlaing, Thaung, Prakash, Anil, Lutwama, Julius, and Walton, Catherine. 2016. "Historical environmental change in Africa drives divergence and admixture of Aedes aegypti mosquitoes: a precursor to successful worldwide colonization?." Molecular ecology 25 (17):4337– 4354.
ID: 141698
Type: article
Authors: Bennett, Kelly Louise; Shija, Fortunate; Linton, Yvonne-Marie; Misinzo, Gerald; Kaddumukasa, Martha; Djouaka, Rousseau; Anyaele, Okorie; Harris, Angela; Irish, Seth; Hlaing, Thaung; Prakash, Anil; Lutwama, Julius; Walton, Catherine
Abstract: Increasing globalization has promoted the spread of exotic species, including disease vectors. Understanding the evolutionary processes involved in such colonizations is both of intrinsic biological interest and important to predict and mitigate future disease risks. The Aedes aegypti mosquito is a major vector of dengue, chikungunya and Zika, the worldwide spread of which has been facilitated by Ae. aegypti's adaption to human-modified environments. Understanding the evolutionary processes involved in this invasion requires characterization of the genetic make-up of the source population (s). The application of approximate Bayesian computation (ABC) to sequence data from four nuclear and one mitochondrial marker revealed that African populations of Ae. aegypti best fit a demographic model of lineage diversification, historical admixture and recent population structuring. As ancestral Ae. aegypti were dependent on forests, this population history is consistent with the effects of forest fragmentation and expansion driven by Pleistocene climatic change. Alternatively, or additionally, historical human movement across the continent may have facilitated their recent spread and mixing. ABC analysis and haplotype networks support earlier inferences of a single out-of-Africa colonization event, while a cline of decreasing genetic diversity indicates that Ae. aegypti moved first from Africa to the Americas and then to Asia. ABC analysis was unable to verify this colonization route, possibly because the genetic signal of admixture obscures the true colonization pathway. By increasing genetic diversity and forming novel allelic combinations, divergence and historical admixture within Africa could have provided the adaptive potential needed for the successful worldwide spread of Ae. aegypti.
Molecular Differentiation of the African Yellow Fever Vector Aedes bromeliae (Diptera: Culicidae) from Its Sympatric Non-vector Sister Species, Aedes liliiBennett, Kelly LouiseLinton, Yvonne-MarieShija, FortunateKaddumukasa, MarthaDjouaka, RousseauMisinzo, GeraldLutwama, JuliusHuang, Yiau-MinMitchell, Luke B.Richards, MiriamTossou, EricWalton, CatherineDOI: info:10.1371/journal.pntd.0004250v. 9No. 12
Bennett, Kelly Louise, Linton, Yvonne-Marie, Shija, Fortunate, Kaddumukasa, Martha, Djouaka, Rousseau, Misinzo, Gerald, Lutwama, Julius, Huang, Yiau-Min, Mitchell, Luke B., Richards, Miriam, Tossou, Eric, and Walton, Catherine. 2015. "Molecular Differentiation of the African Yellow Fever Vector Aedes bromeliae (Diptera: Culicidae) from Its Sympatric Non-vector Sister Species, Aedes lilii." PLoS neglected tropical diseases 9 (12):
ID: 138376
Type: article
Authors: Bennett, Kelly Louise; Linton, Yvonne-Marie; Shija, Fortunate; Kaddumukasa, Martha; Djouaka, Rousseau; Misinzo, Gerald; Lutwama, Julius; Huang, Yiau-Min; Mitchell, Luke B.; Richards, Miriam; Tossou, Eric; Walton, Catherine
Abstract: INTRODUCTION: Yellow fever continues to be a problem in sub-Saharan Africa with repeated epidemics occurring. The mosquito Aedes bromeliae is a major vector of yellow fever, but it cannot be readily differentiated from its non-vector zoophilic sister species Ae. lilii using morphological characters. Genetic differences have been reported between anthropophilic Ae. bromeliae and zoophilic Ae. lilii and between forest and domestic populations. However, due to the application of different molecular markers and non-overlapping populations employed in previous studies, interpretation of species delimitation is unclear. METHODOLOGY/PRINCIPLE FINDINGS: DNA sequences were generated from specimens of Ae. simpsoni s.l. from the Republic of Benin, Tanzania and Uganda for two nuclear genes apolipophorin 2 (apoLp2) and cytochrome p450 (CYPJ92), the ribosomal internal transcribed spacer region (ITS) and the mitochondrial cytochrome c oxidase (COI) barcoding region. Nuclear genes apoLp2 and CYPJ92 were unable to differentiate between species Ae. bromeliae and Ae. lilii due to ancestral lineage sorting, while ITS sequence data provided clear topological separation on a phylogeny. The standard COI barcoding region was shown to be subject to species introgression and unable to clearly distinguish the two taxa. Here we present a reliable direct PCR-based method for differentiation of the vector species Ae. bromeliae from its isomorphic, sympatric and non-biomedically important sister taxon, Ae. lilii, based on the ITS region. Using molecular species verification, we describe novel immature habitats for Ae. lilii and report both sympatric and allopatric populations. Whereas only Ae. lilii is found in the Republic of Benin and only Ae. bromeliae in Tanzania, both species are sympatric in Uganda. CONCLUSIONS/SIGNIFICANCE: Our accurate identification method will allow informed distribution and detailed ecological studies that will facilitate assessment of arboviral disease risk and development of future targeted vector control.