The study was conducted using six experimental huts located at Asendabo, Jimma Zone, Oromia Regional State, Ethiopia, located between latitudes 7° 42′ 50″ N and 07° 53′ 50″ N and longitudes 37° 11′ 22″ E and 37° 20′ 36″ E, at altitudes ranging from 1672 to 1864 m above sea level. The huts were used previously for evaluation of vector control tools , but the roofs, walls, floors, ceilings and verandahs of each hut were completely renovated for use in this study. The huts are located approximately 0.3 km from the reservoir shore of Gilgel Gibe hydroelectric power dam in Nada District, Oromia Regional State, Ethiopia. Earlier work showed that An. arabiensis population from the study area was resistant to DDT, pyrethroids and malathion, while they were susceptible to primiphos-methyl and some of the carbamates used in malaria vector control .
The six treatments in this study included:
Control hut (no IRS, untreated net only).
Hut with PermaNet® 2.0 (deltamethrin) only (no IRS).
Hut with PermaNet® 3.0 (deltamethrin with PBO) only (no IRS).
Hut sprayed with Actellic 300CS (1 g/m2) and untreated net.
Hut sprayed with Actellic 300CS (1 g/m2) and PermaNet® 2.0.
Hut sprayed with Actellic 300CS (1 g/m2) and PermaNet® 3.0.
The huts used for this trial were the West African type . They were built approximately 3 m apart from one another on a concrete foundation which has a moat filled with water to prevent ants and other scavengers that may potentially enter the hut and feed on dead mosquitoes. The walls were made of bricks and the roofs of corrugated iron. Each hut had four louvred windows made of iron sheets with 1 cm between slits which allows free flying mosquitoes to enter the hut but restricts them from exiting the hut. A ceiling made of white sheet cloth allowed for easy capture of mosquitoes resting inside the huts. Huts had a central room and a verandah. The verandah was left open from the inside to allow for entry of mosquitoes from the central room and the walls were screened partly with white plastic sheets and partly with metal mesh to capture exiting mosquitoes.
Use of nets for the study
During the study, two types of nets (PermaNet® 2.0 and PermaNet® 3.0) with and without IRS were assessed. Moreover, an untreated net was used with IRS and untreated net without IRS was used as a negative control. Both PermaNet® 2.0 and PermaNet® 3.0 nets are treated with the pyrethroid deltamethrin, but PermaNet 3.0 has also PBO synergist. During the study, each ITN in each hut was replaced by a new replicate net every 3 weeks since the trial had no replicate huts. Eighteen holes (4 cm × 4 cm) were cut in each net purposefully to simulate a torn net .
Indoor residual spraying (IRS)
Actellic® 300CS, a pirimiphos-methyl-based IRS insecticide, was applied to the three IRS treatment huts 1 week before the start of the trial. Pirimiphos-methyl is an organophosphate insecticide being used for IRS by the malaria elimination program of Ethiopia. During the spraying, filter papers were affixed to the wall surfaces at three heights (high, middle and low) to ensure the application was properly and uniformly applied and determine the concentration of the applied insecticide. The spray operators were trained on the spraying techniques and the wall surfaces of huts were lined with chalk to properly maintain the swath and avoid overlap following the WHO protocol.
Mosquitoes were collected 6 days per week over 9 weeks (from 16th November 2020 to 16th January 2021), for a total of 54 collection nights from each of the six experimental huts following the WHO Pesticide Evaluation Scheme (WHOPES) recommendations .
Six volunteer mosquito collectors from the local community were recruited and trained to sleep in each hut every night for a total of 54 nights provided with a bed and mattress every night. Sleepers were rotated among the six huts every night, completing one rotation every week. This was done to address differential mosquito attraction to individual sleepers. The sleepers entered the huts each evening at 6:00 p.m. and started mosquito collection in each hut at dawn at 6:00 a.m.
At 6:00 a.m., mosquito collection (both dead and alive) commenced using mouth aspiration, starting from inside the nets, then from inside the hut (floor, wall, and ceilings) and finally from the verandah. Mosquitoes were sorted, counted, and scored by location (inside net, inside the central room of the hut, and verandah), physiological stage (unfed or blood-fed) and whether they were dead or alive. Mosquitoes were identified using the standard African Anopheles morphological key . Dead mosquitoes were kept in Eppendorf tubes over silica gel until further processing in the laboratory. Live mosquitoes were kept in paper cups in an insecticide-free room and provided with 10% sugar solution for 24 h at which point delayed mortality was recorded.
Cone bioassays were conducted twice during the trial period to assess the bio-efficacy of Actellic 300CS. At each time point, three cones were fixed to the wall surfaces at three heights from the ground (high 160 cm, medium 100 cm and low 40 cm) of each hut and ten mosquitoes were used per cone with a total of 30 mosquitoes per hut, for each of the three sprayed huts and a control hut (120 mosquitoes in total). Mosquitoes were exposed for 30 min after which immediate knockdown was recorded and mortality was recorded after a 24 h holding period. Non-blood-fed, 3–5 day old adult female Anopheles gambiae sensu lato (s.l.) were used for bioassays.
The first cone bioassay was conducted 3 weeks after spray using a confirmed susceptible An. arabiensis strain from Sekoru Insectary without pre-exposure to PBO synergist. The second bioassay was conducted at the end of the trial (10 weeks after spray). In the second bioassay, both susceptible An. arabiensis laboratory strain from Sekoru Insectary and wild An. gambiae s.l. were used. The assay was carried out with and without PBO pre-exposure for both strains. Wild populations of An. gambiae s.l. used for the bioassays were raised from larvae and pupae collected from semi-permanent and permanent breeding habitats around Wolkite area, central Ethiopia. They were reared to adults at Asendabo Field Insectary. Mosquitoes were exposed to papers treated with PBO (2%) in a WHO cylinder for 1 h.
Blood meal host source analysis and mosquito identification
All the fed mosquitoes collected from the different huts were assayed to determine the blood meal host source using direct ELISA . Moreover, a sub-sample of An. gambiae s.l. was also molecularly identified to species using species-specific PCR following established protocol .
Four study outcomes measured were:
Deterrence—the reduction in hut entry by mosquitoes in treatment huts relative to the control hut (hut with untreated net and no IRS).
Exophily—the proportion of mosquitoes found resting on the walls of the verandah.
Blood-feeding inhibition—the reduction in blood-feeding in treatment huts in comparison with control hut (hut with untreated net and no IRS).
Immediate and delayed mortality—the proportion of mosquitoes entering the hut that were dead in the morning (immediate mortality) or after being caught alive and held for 24 h with access to a sugar solution (delayed mortality).
Data from the five treatment huts were compared with data from the control hut to determine the insecticidal effects on the variables listed above.
Personal protection, killing effect, and exophily were estimated using the following formulas [9, 11].
where Bt is the total number of freshly blood-fed mosquitoes in the huts with treated nets. Bu is the total number of freshly blood-fed mosquitoes in the huts with untreated nets.
where Kt is number of mosquitoes killed (immediate) in the huts with treated nets. Ku is number of mosquitoes killed (immediate) in the huts with untreated nets. Tu is the total number of mosquitoes collected from the huts with untreated nets.
where Ev is the number of mosquitoes found in verandah. Et is the total number of mosquitoes found inside the hut and verandah
For all three parameters, mosquito entry was estimated by adding the number of dead and alive mosquitoes observed inside the hut and the verandah. Estimates of personal protection and killing effect were determined using mosquitoes from all collection nights.
To test differences in mean mosquito density in each of the treatment huts, ANOVA was employed after log-transformation of non-normal distributed mosquito count data. T-tests were employed to determine the differences in mean proportions of blood-fed mosquitoes and mosquitoes exiting early from the different test huts and control hut.
In all analyses, the hut with the untreated net and without IRS was used as a negative control, but comparisons were made between all treatment groups. In all analyses, individual huts and sleepers were included in the models as random effects.