Research Article: Molecular Differentiation of the African Yellow Fever Vector Aedes bromeliae (Diptera: Culicidae) from Its Sympatric Non-vector Sister Species, Aedes lilii

Date Published: December 7, 2015

Publisher: Public Library of Science

Author(s): Kelly Louise Bennett, Yvonne-Marie Linton, Fortunate Shija, Martha Kaddumukasa, Rousseau Djouaka, Gerald Misinzo, Julius Lutwama, Yiau-Min Huang, Luke B. Mitchell, Miriam Richards, Eric Tossou, Catherine Walton, Michael J Turell.

Abstract: IntroductionYellow 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 FindingsDNA 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/SignificanceOur 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.

Partial Text: Correctly identifying the vector species involved in mosquito-borne disease transmission is fundamental to predicting disease outbreaks, ascertaining general risk to the human population and targeting control efforts. Despite this necessity, the reliable identification of mosquitoes is problematic in many cases, including the medically important Aedes simpsoni complex. This complex comprises three known species including Ae. simpsoni, Ae. lilii and Ae. bromeliae. Among these three species, Ae. bromeliae is an important vector of yellow fever virus (YFV) and potentially other arboviruses [1]. Yellow fever has increased in incidence as a result of urbanisation and changes to public health policy [2, 3]. The burden of yellow fever in Africa is estimated to be 130,000 cases a year, 85,000 of which result in deaths, despite the availability of a vaccine [4]. After mass immunisation campaigns at the beginning of the 20th Century, YFV was successfully reduced in targeted countries. However, YFV outbreaks are causing renewed attention. In West Africa, 13 out of 14 countries known to host YFV now report cases regularly and have experienced epidemics since 2000 [5, 6]. Further concern has arisen over identification of a novel YFV genotype implicated in recent outbreaks including the first reported outbreak in Kenya, East Africa (1992–1993) [7]. Related genotypes have also been reported in Sudan in 2003 and 2005 and more recently in northern Uganda in 2010 [8–10]. Although much attention has been given to understanding yellow fever disease epidemiology [11], relatively little is known about the mosquito vectors.

Here we used multiple markers to confirm there are two closely related species of the Simpsoni Complex that occur both allopatrically and sympatrically across the sampled range in sub-Saharan Africa. These species correspond to Ae. bromeliae and Ae. lilii of the Simpsoni Complex as characterised by Mukwaya et al. [22, 27]. We infer that a third forest taxon originally reported as Ae. lilii in Walter et al. [28] relates to another species, possibly a another member of the Simpsoni Group. The development of a molecular species identification method for the YFV vector Ae. bromeliae and non-vector Ae. lilii using variation at the ITS region allowed us to make for the first time reliable inferences about mosquito ecology and distribution. Such information is vital for a complete understanding of disease transmission by these species.



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