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.
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.
In Africa, epidemic outbreaks of yellow fever continue despite the availability of an effective vaccine. Effective understanding of disease epidemiology and control requires the ability to reliably identify vectors of yellow fever. The mosquito Ae. bromeliae, a competent vector of yellow fever virus, cannot be reliably morphologically differentiated from its sister species Ae. lilii, which does not bite humans and so does not transmit yellow fever. DNA sequencing of four molecular markers allowed comparisons of how they perform at distinguishing these species. We found that the mitochondrial cytochrome c oxidase ( COI) barcoding region and nuclear apolipophorin 2 ( apoLp2) and cytochrome p450 ( CYPJ92) were unable to reliably distinguish these species. Conversely, genetic variation at the internal transcribed spacer region ( ITS) was able to confirm the vector Ae. bromeliae and non-vector Ae. lilii as distinct species. Based on ITS sequence differences, we developed a robust molecular method to identify the vector Ae. bromeliae from its sister species Ae. lilii. Consequently, we find that these species use the same larval habitats including banana, cocoyam and Dracena spp. in Uganda. Whereas only Ae. lilii appears to be present in Benin and only Ae. bromeliae in Tanzania, we confirm that both species occur in Uganda. Reliable species designation will promote more detailed studies of distribution, ecology and vector status essential for disease risk assessment and mosquito control.