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.

Figure 1. Phylogenetic Tree of ITS2 sequences.

A majority rule consensus tree was generated from Bayesian analysis of dataset of ITS2 sequences. Bayesian posterior probabilities are shown above branches. AN18 is the only outlier and should cluster with Series Myzomia. Underlined species indicate those found positive with Plasmodium spp.

Figure 2. Comparison of morphological and molecular identifications.

The number of specimens identified as specific Anopheline species by morphological and molecular techniques (n = 2024), and percentage accuracy of morphology compared to molecular identity are presented. Molecular identification was determined by sequencing of ITS2 and COI regions and comparisons to the database.