An. stephensi, an efficient malaria vector in parts of Asia and Africa, was observed in 75.3% of water sources surveyed, and contributed to 80.9% of wild-caught Anopheles mosquitoes in Awash Sebat Kilo, Ethiopia. High susceptibility of these mosquitoes to Plasmodium falciparum and vivax infection presents a challenge for malaria control in the Horn of Africa. This study was conducted in Awash Sebat Kilo, Ethiopia, an area of perennial malaria transmission, from April to September 2019. We examined aquatic habitats for immature-stage Anopheles mosquitoes by standard dipping (10x/site) for 5 consecutive days. We assessed mosquito resting, feeding, and host-seeking behavior.

We assessed mosquito resting, feeding, and host-seeking behavior by 5 methods: CDC miniature light traps model 512 (John W. Hock Company, https://www.johnwhock.com), human landing catches, pyrethrum spray sheet collection, aspiration from animal shelters, and cattle-baited traps (5). We identified adult mosquitoes by using standard keys and confirmed identification by targeted sequencing of nuclear internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome oxidase subunit 1 gene (COI) (6). An. stephensi mosquito DNA sequences were compared with publicly available libraries to generate clade topologies (7). We determined mosquito blood meal sources by using multiplex PCR targeting cytochrome b (8) and infection status by using 18S rRNA-based nested PCR (9).

Adult An. stephensi mosquitoes reared from immatures from local water sources and a colony of An. arabiensis mosquitoes (~120 each) were fed in the dark for 30 min on membrane feeders containing fresh blood from patients with microscopy-confirmed P. vivax and P. falciparum mono- and mixed-species infections that were attending Adama malaria clinic (10). Unfed and partially fed mosquitoes were removed; fully engorged mosquitoes were maintained on sugar solution. At 7 or 12 days after feeding, mosquitoes were dissected and their midguts were examined for oocysts and salivary glands for sporozoites. To compare infection status between An. arabiensis and An. stephensi mosquitoes, we performed logistic regression. We used individual mosquito data and a fixed effect for each patient to account for correlations between mosquito observations from the same donor. Bland-Altmann plots were generated for differences in infectivity between mosquito sources by using the Pitman test of difference in variance. For analyses, we used STATA version 13 (StataCorp., https://www.stata.com/company) and GraphPad Prism 5.3 (GraphPad Software Inc., https://www.graphpad.com).

The supplemental section of the work describes the data included here and how it was collected.