Diversity and Insecticide Susceptibility of Anopheles Mosquitoes in Bushbuckridge, a Residual Malaria Transmission Setting in South Africa

¹ Centre for Emerging and Zoonotic and Parasitic Diseases, National Institute for Communicable Diseases and Wits Research Institute for Malaria, School of Pathology, University of the Witwatersrand, Johannesburg, South Africa
² Wits Research Institute for Malaria, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
³ Mpumalanga Department of Health, Malaria Control Programme, Mbombela, South Africa

Academic Editor: John Frean

Published: 26 June 2026 by MDPI in 2026 International Online Conference on Tropical Medicine and Infectious Disease session Vector-Borne Diseases

Abstract:

Introduction:
Bushbuckridge subdistrict, located in the northeastern lowveld of South Africa, lies within the country’s malaria-endemic belt and borders Kruger National Park, as well as being near Mozambique and Eswatini. Despite significant progress toward malaria elimination, the area remains vulnerable to residual and cross-border transmission. Understanding local Anopheles vector dynamics and insecticide susceptibility is therefore essential for sustaining malaria control. This study assessed Anopheles species diversity and insecticide susceptibility to inform entomological surveillance priorities aligned with the WHO Entomological Surveillance Planning Tool (ESPT).

Methods:
Larval Anopheles mosquitoes were collected from natural habitats between June 2022 and April 2024 and reared to adulthood. Adult males and females underwent WHO tube bioassays with deltamethrin (0.05%), DDT (4%), and pirimiphos-methyl (0.25%). Species were identified using morphological keys and PCR assays.

Results:
A total of 4454 adults emerged (45.1% males, 54.9% females), representing 18 taxa, including primary, secondary, and non-vector species, highlighting ecological complexity. Anopheles pretoriensis was most abundant (33.4%), followed by An. quadriannulatus (20.7%) and An. rufipes (20.0%). The primary malaria vector An. arabiensis represented 7.1%, demonstrating its persistence in residual transmission areas and the need for targeted control. Two specimens indicated potential hybridisation between An. arabiensis and An. quadriannulatus. PCR identification failed in 3% of An. gambiae complex and 16% of An. funestus group specimens. Bioassays showed high susceptibility overall, but An. funestus group males exposed to DDT had 94.6% mortality, suggesting possible resistance.

Conclusions:
This study highlights a diverse Anopheles community in a residual transmission setting. While susceptibility remains high, emerging resistance and hybridisation underscore the need for continued species-specific, ESPT-informed surveillance to guide adaptive malaria vector control strategies.

Keywords: Anopheles mosquitoes; malaria vectors; insecticide susceptibility; vector surveillance; malaria elimination.

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Comments on this paper

Mario Olivera

1 July 2026

Comment:
This is a well-structured abstract addressing an important aspect of malaria elimination. The combination of species diversity and insecticide susceptibility data provides valuable information for vector surveillance in a residual transmission setting, and the linkage to the WHO ESPT strengthens its operational relevance.

Question:
Given that Anopheles arabiensis represented only 7.1% of the collections while An. pretoriensis and An. quadriannulatus predominated, could the authors briefly discuss the potential epidemiological role of these abundant secondary or non-vector species and whether their high abundance may influence future surveillance or vector control priorities in the study area?

Thabo Mashatola

1 July 2026

Thank you for this valuable comment. Although Anopheles arabiensis accounted for only 7.1% of the collections, the predominance of An. pretoriensis and An. quadriannulatus provides important ecological context for malaria surveillance in the study area. Anopheles quadriannulatus, although generally regarded as a non-vector because of its predominantly zoophilic behaviour, is morphologically indistinguishable from An. arabiensis as both belong to the An. gambiae complex. Consequently, molecular identification remains essential to accurately distinguish these species and ensure reliable estimates of vector abundance. Likewise, An. pretoriensis has historically been considered a secondary or non-vector species with limited epidemiological importance. However, the high abundance of these species may reflect environmental conditions that also support competent malaria vectors, and changes in ecological or behavioural patterns could alter their epidemiological significance over time. In addition, abundant non-vector and secondary vector species provide valuable baseline information for insecticide susceptibility monitoring and may inform the development of future genetic vector control strategies or insecticide formulations. Therefore, while current vector control efforts should remain focused on proven malaria vectors such as An. arabiensis, continued surveillance of the broader Anopheles community is important for detecting changes in species composition, accurately identifying members of the An. gambiae complex, and anticipating shifts that could influence malaria transmission.