Malaria vector composition and insecticide resistance in Vhembe district, a high disease burden region of South Africa

Abstract

South Africa’s malaria endemic provinces (Limpopo, Mpumalanga & KwaZulu-Natal) continue to face residual malaria transmission that is partly attributed to outdoor biting and resting Anopheles species. This reduces effectiveness of control interventions as they mostly target indoor vector species. Limpopo Province remains the most affected region in the country, carrying the highest disease burden, particularly in the north-eastern Vhembe district. However, the factors sustaining residual transmission in the area remain unknown. Vectors responsible for transmitting malaria and other potential secondary species have been reported in the area, but in low numbers, as has the presence of species implicated in malaria transmission in other African countries. The role of vectors and other factors, including insecticide resistance, in the persistence of residual malaria remain to be determined. Therefore, this study investigated species composition, feeding preference and insecticide resistant status in mosquitoes from Vhembe, towards understanding the cause of the persistent and residual malaria. Anopheles mosquitoes were collected in Bende Mutale, Tshivaloni, and Gwakwani, some of the areas in the Vhembe district, Limpopo Province, with the highest malaria burden. Mosquitoes were collected using carbon dioxide- and animal-baited traps, dippers and sweep nets over four collection events during the 2022-2023 and 2023-2024 malaria season. Species were morphologically identified to species level, with those belonging to the An. gambiae complex and Anopheles funestus group being identified by species-specific polymerase chain reaction (PCR). For those specimens that could not be identified morphologically, amplification and sequencing of the cytochrome oxidase subunit 1 (COI) barcoding region was performed. Insecticide susceptibility testing was carried out on Anopheles mosquitoes using standard WHO tube bioassays, testing against DDT, deltamethrin, bendiocarb and malathion. PCR and sequencing were used to investigate knockdown resistance (kdr) mutations using a typing approach. Finally, host preference was investigated through bloodmeal analysis on wild-caught blood fed females using PCR and sequencing of the cytochrome b (cyt b) gene. A total of 6,134 Anopheles specimens were collected of which 4,772 were used for insecticide susceptibility testing. Species composition assessment (Chapter 2) revealed that Anopheles gambiae s.l. (n=2,160) comprised of three sibling species in Vhembe: An. arabiensis, An. merus and An. quadriannulatus. The 357 An. funestus s.l. collected consisted of An. rivulorum-like, An. rivulorum, An. leesoni and An. vaneedeni. Other species collected included An. coustani, An. rufipes, An. pretoriensis, An. listeri, An. nili and An. gibbinsi. Nine Anopheles spp could not be assigned to species based on COI sequencing. Insecticide susceptibility testing (Chapter 3) detected potential resistance to bendiocarb and deltamethrin in An. quadriannulatus and An. rivulorum, respectively. Resistance to DDT, deltamethrin and bendiocarb was detected in An. coustani, An. rufipes, and An. pretoriensis. Full insecticide susceptibility to all insecticides was reported in An. arabiensis, An. merus, An. vaneedeni, An. rivulorum-like and An. leesoni. The sodium channel mutation associated with kdr resistance was not detected in any of the An. gambiae s.l. specimens tested. Host-specificity assessed through bloodmeal analyses (Chapter 4) revealed that An. quadriannulatus and An. pretoriensis favoured cattle and goats. Comparative analysis of two cyt b assays confirmed that both amplify mosquito vector DNA in the absence of bloodmeal DNA of adequate integrity. The results of this study support continued entomological monitoring to further investigate the roles of these species in malaria transmission and to monitor insecticide resistance in these populations. Successful implementation of targeted interventions relies on research evidence to effectively reduce vector and malaria burden.