Unexpected diversity of Anopheles species in Eastern Zambia: implications for evaluating vector behavior and interventions using molecular tools

Abstract

The understanding of malaria vector species in association with their bionomic traits is vital for targeting malaria interventions and measuring effectiveness. Many entomological studies rely on morphological identification of mosquitoes, limiting recognition to visually distinct species/species groups. Anopheles species assignments based on ribosomal DNA ITS2 and mitochondrial DNA COI were compared to morphological identifications from Luangwa and Nyimba districts in Zambia. The comparison of morphological and molecular identifications determined that interpretations of species compositions, insecticide resistance assays, host preference studies, trap efficacy, and Plasmodium infections were incorrect when using morphological identification alone. Morphological identifications recognized eight Anopheles species while 18 distinct sequence groups or species were identified from molecular analyses. Of these 18, seven could not be identified through comparison to published sequences. Twelve of 18 molecularly identified species (including unidentifiable species and species not thought to be vectors) were found by PCR to carry Plasmodium sporozoites - compared to four of eight morphological species. Up to 15% of morphologically identified Anopheles funestus mosquitoes in insecticide resistance tests were found to be other species molecularly. The comprehension of primary and secondary malaria vectors and bionomic characteristics that impact malaria transmission and intervention effectiveness are fundamental in achieving malaria elimination.

DNA barcoding mosquitoes: advice for potential prospectors

“…But this journey towards to what we term 'species', and their genetic discontinuity, is rarely clean-cut: there can be fascinating genetic exchanges between closely related so-called 'species ' (Mallet et al 2016). Additionally mosquitoes often exist rather as closely related species complexes that, when studied intimately -as with the Anopheles gambiae complex in Africa -can be observed to be diverging at some parts of the genome under low recombination while other parts of the genome undergo gene flow (Besansky et al 2003;Reidenbach et al 2012).…”

Novel Wolbachia strains in Anopheles malaria vectors from Sub-Saharan Africa

“…The molecular phylogeny of the ITS2 gene revealed Wolbachia -infected individuals from Lwiro and Katana are the same An. species A ( Figure 3) previously collected in Eastern Zambia 43 and Western Kenya 63 . All ITS2 sequences were deposited in GenBank (accession numbers MH598414–MH598445; listed in Supplementary Table 1).…”

“…require species-diagnostic PCR assays. Historically, malaria parasite entomology studies in Africa have focused predominantly on species from these complexes, likely due to the fact that mosquitoes from these complexes dominate the collections 43 . In our study, we used ITS2 sequencing to confirm secondary vector species that were P. falciparum -infected given the difficulties of morphological identification and recent studies demonstrating the inaccuracy of diagnostic species PCR-based molecular identification 69 .…”

“…Globally, there is a large variety of Anopheles vector species (~70) that have the capacity to transmit malaria 41 and could potentially contain resident Wolbachia strains. Additionally, this number of malaria vector species may be an underestimate given that recent studies using molecular barcoding have also revealed a larger diversity of Anopheles species than would have be identified using morphological identification alone 42, 43 . …”

Implicating Cryptic and Novel Anophelines as Malaria Vectors in Africa

“…vaneedeni [55] and An. leesoni , are largely zoophilic and although they have been shown to carry sporozoites [56,57], they are usually not considered to play a major role in malaria transmission due to low human contact rates. An additional complication is that the morphological similarities of these species are not limited just to the Funestus group.…”