Anopheles stephensi Mosquitoes as Vectors of Plasmodium vivax and falciparum, Horn of Africa, 2019

Anopheles stephensi mosquitoes, efficient vectors in parts of Asia and Africa, were found 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.

outdoors on 15 randomly selected households for two nights that makes a total of 60 traps in 30 nights. Indoor traps were hung at the foot edge of the person who slept under an untreated bed net (1). Other occupants in the houses were left to use LLINs provided by the control program as part of the routine malaria control. The traps were switched on at 6:00PM and off at 6:00AM the next morning in each sampling night. PSC were conducted from 6:00 AM to 2:00 AM on five randomly selected households' per-day and 20 households were included in each round of sampling, thus a total of 60 households were sampled in three rounds. The HLC were conducted in nine selected households both in and outdoors that was repeated the next day. Locally trained entomology technicians were employed to collect female Anopheles by standard mouth aspirator from 6:00 PM to 6:00 AM from both indoors and outdoors. Two collectors were assigned at a time for each house (one outdoors and one indoors) in shifts of 6 hours (the first shift being 6:00PM -12:00PM and the second from 12:00 PM to 6:00 AM). Collectors in the same shift changed with each other between outdoors and indoors every hour after recording their findings on the checklist to avoid bias due to individual variation in attraction and competence. In addition to this, animal sheds were inspected using HC and cattle bait trap (2) were conducted for collecting mosquitos biting and resting in animal shelters. Mosquitoes resting in animal shelters and cattle bait traps were collected using standard mouth aspirator for 30 minutes in each, from 5:30 AM to 6:00 AM. All collected Anopheles mosquitoes were counted and sorted out morphologically to species level (3,4) and by their abdominal stage into unfed, freshly fed, half-gravid or gravid (5), except those collected by HLC. In animal shelter with high number of mosquito collection was repeated the next morning.
Of the five methods used for mosquito collection in the 2 monthly studies of 6 days each and therefore 12 days in total, Anopheles were caught only by the three methods: CDC light traps, aspiration from animal shelter (hand collection), and human landing catches.

Plasmodium infection status of individual wild-caught morphologically-confirmed adult
An. stephensi mosquitoes was assessed using nested polymerase chain reaction (nPCR) targeting the small 18S subunit (6) using genomic DNA extracted from homogenate of mosquito's headthorax and abdomen separately (7), indicating sporozoite and oocyst-stage infections, respectively. Multiplex PCR that targets the mitochondrial cytochrome b gene and produces species-specific fragments of varying sizes was used to assess blood meal sources of individual mosquitoes (8). For confirmation of morphologically identified An. stephensi, DNA was extracted from whole mosquito bodies using the DNeasy Blood and Tissue kit (Qiagen, UK).
PCR was performed for each individual mosquito, targeting the nuclear internal transcribed spacer 2 region (ITS2) and the mitochondrial cytochrome oxidase subunit 1 gene (COI) (9

Sporozoite quantification
Sporozoites were quantified on day 12 post feeding in salivary glands of mosquitoes that remained from the batch where high oocysts were detected during midgut dissection on day 7 post feeding and categorized into four (with a grade from 1-4) following protocol reported before (14).