Molecular characterisation of understudied Anopheles species using morphological and DNA-based approaches

Abstract

Accurate identification of Anopheles species is critical for effective malaria vector surveillance and control. A priori morphological identification forms the basis for species identification. However, it is time-consuming, and identification is incomplete if specimens have lost important external taxonomic features. Therefore, it is vital to use molecular techniques in conjunction with morphology for identification. This study aimed to use both morphological and molecular methods for the identification of understudied Anopheles species. Focus was placed on An. marshallii group and An. maculipalpis specimens from KwaZulu-Natal (KZN), South Africa, as well as An. hargreavesi and An. gibbinsi from Nigeria and Zambia, given their potential roles as overlooked or secondary malaria vectors. Morphological identification of An. marshallii group and An. maculipalpis individual female specimens and their first filial generation (F1) progeny was conducted using established taxonomic keys. Molecular analysis was performed by sequencing the Internal Transcribed Spacer 2 (ITS2) and Cytochrome Oxidase I (COI) regions of these species. Phylogenetic trees were constructed using the Neighbor-Joining (NJ) method to assess the evolutionary relationships among the different species. Additionally, the presence of Plasmodium falciparum in female specimens of the An. marshallii group and An. maculipalpis from KZN was assessed using a hydrolysis probe assay to determine their potential role as vectors in the region. The morphological identification of An. hargreavesi and An. gibbinsi specimens was based on their distinctive traits. There was low genetic variation observed in An. hargreavesi ITS2 and COI sequences. Anopheles hargreavesi ITS2 and COI sequences from this study could not be aligned with previously published reference sequences due to the absence of available sequence data for this species in existing databases. The generated An. hargreavesi sequences from this study represent the first published molecular data for this species, highlighting the importance of expanding genetic reference databases. The ITS2 region of An. gibbinsi revealed high homology with a 100% match to a previously published "Anopheles sp. 6" from Kenya, suggesting that they are the same species. Morphological analysis of An. marshallii group adults from KZN isofemale families confirmed their classification within the An. marshallii group, with no notable differences observed between families. However, the use of egg and larval morphology to identify An. marshallii group specimens from KZN presented several challenges, limiting their usefulness as a reliable diagnostic tool in this study. Molecular sequencing of these specimens revealed unique ITS2 and COI sequences, vi indicating that the families belonged to the same species. The An. marshallii group specimens from KZN exhibited a low level of DNA variation and formed distinct clades in phylogenetic trees, indicating their novelty and divergence from other known species. Misidentification posed a significant practical challenge as specimens initially identified as An. pretoriensis were later confirmed through molecular analysis to be An. maculipalpis, highlighting the limitations of morphological techniques in distinguishing closely related or cryptic species. Moreover, high intraspecific genetic variation in sequences complicated species delineation in certain groups, underlining the need for multi-locus approaches in future studies. None of the female specimens tested positive for Plasmodium falciparum, indicating no evidence of infection among the tested populations at the time of collection. The findings have significant implications for malaria control, as secondary vectors or understudied species may be overlooked in surveillance efforts due to misidentification or underrepresentation in reference databases. This study highlights the importance of using morphological and molecular identification techniques to intensify vector surveillance and improve our understanding of the An. marshallii group and An. maculipalpis species, given their potential role in malaria transmission and the difficulties associated with accurately identifying these species using morphological methods alone. While generating large-scale DNA barcode data for anophelines is comparatively easy, this information becomes valuable when linked to accurate morphological species identification. By establishing this linkage between morphology and molecular data, this research provides the foundation for developing rapid identification assays, such as multiplex PCR, to streamline and enhance vector surveillance efforts for species like the An. marshallii group and An. maculipalpis