Mosquitoes strain used in the experiments
Mosquito strains used in this study consisted of a deltamethrin selected resistant colony and an unselected susceptible colony (hereafter referred to as resistant and susceptible mosquitoes) that were collected from Bungoma in western Kenya. These colonies were selected and maintained at the Centre for Global Health Research, Kenya Medical Research Institute (KEMRI) in Kisumu 21, Western Kenya, under standard rearing conditions of 27 ± 2 °C and relative humidity (RH) of 80 ± 10% °C under a L12: D12 h light: dark cycle. During the process, each colonized strain had three independent lineages that started with 200-250 females at every new generation to limit bottleneck effects. The progeny of F1 wild-caught mosquitoes from the same site were also used to undertake these experiments.
Resistant strain:
This colony underwent deltamethrin selection after each generation. The 6th generation used was highly resistant with 20% mortality according to the WHO criteria 22. Resistance in this colony was mainly mediated by cytochrome P450 detoxification enzyme. The two kdr mutations 1014S and 1014F were present and at high frequencies 21.
Susceptible strain:
This strain shares the same genetic background with the resistant colony, however, it was reared in the absence of insecticide selection pressure. After 9 generations without selection pressure, the population had almost lost resistance to deltamethrin (92%) and after 13 generations the population showed increased mortality (97.3%). The vgsc1014S was at a high frequency given that the allele was already fixed in the parent population21. The 14th generation was used in this study.
Wild population:
F1 progeny obtained from wild-caught An. gambiae female mosquitoes from the same area where the resistant and susceptible colonies were selected were used for validation. Each female(mother) was identified by PCR as An. gambiae s.s according to the methods of Scott, et al. 23. The wild population had 56% resistance to deltamethrin. The observed resistance is mediated by a mix of metabolic and kdr9,24-27.
Semi-field set up
The study was carried out in Western Kenya at the Centre for Global Health Research, Kenya Medical Research Institute, Kisumu. The release and recapture studies were conducted in a MalariaSphere a closed system 20m long x 8m wide28with slanted roofing (3m in the sides and 4.5m in the middle). The entire structure is covered with an insect-proof netting screen that prevents mosquitoes inside the system from escaping into the environment, or vice versa (Fig. 1 A). The system is also double-doored for the same reason. Inside the systema3m x 3m mud-walled hut is erected resembling a typical house in the study village in terms of size, structure and mosquito exit/entry points (eaves, window,and door) (Fig. 1B). The structure has local vegetation and grass floor to mimic the natural vegetation and provide shelter for mosquitoes in the outdoor environment (Fig. 1B). Two round clay pots are installed in the enclosure but outside the hut to act as outdoor resting sites (Fig 1C). Inside the hut either a treated LLIN inside a bed-net trap (Mbita trap)or an untreated net inside a bed-net trap as control was hanged (Fig 1D). Treated and untreated nets were used at different nights in the same hut. For each night, a consented and remunerated human volunteer slept under the bed-net trap in the hut. To offset any personal bias due to differential sleeping habits or relative attractiveness to mosquitoes, two sleepers were recruited for this experiment and took turns to sleep under the bed net. They were instructed not to consume alcohol or smoke and avoid deodorants during the study period. The volunteer who slept under the bed net served as a bait to attract the mosquitoes into the hut but was not bitten because of the net shield.
Bed net trap for the collection of host-seeking mosquitoes
The Mbita trap which is a bed-net trap described by Mathenge, et al. 29 . This is a modified conical bed-net made of light white cotton cloth instead of netting (Fig.2). The trap has two chambers; the upper trap chamber and the lower bait chamber. The upper chamber contained a netting panel fixed halfway (Fig. 2A) to prevent mosquitoes from reaching the human bait sleeping in the lower chamber (Fig. 2B). For this experiment, the netting panels were either treated or untreated. The treated netting panels were cut from DawaPlus 2.0 a long-lasting insecticidal net (LLIN) containing 80 mg/m2 deltamethrin. The nets were selected for this experiment based on the fact that they were distributed in the largest proportion in the study site by the National Malaria Control Programme in Kenya during the 2017 mass net campaign. The untreated bed net was obtained from the local market in Kisumu, Kenya.
Mosquito release and recapture
Batches of 200 uninfected and unfed female mosquitoes aged 3-5 days from the resistant or susceptible colonies were gently mouth-aspirated into a clean paper cup. The mosquitoes were sugar-starved for 6 hours before releases and color-marked with green (for the susceptible colony) and pink (for resistant colony) fluorescent powder to distinguish them after simultaneous release in the semi-field environment. Mosquito releases were done outside the hut and at the same time of day (1840hrs) to avoid circadian cycle effects. The volunteer entered the bed net 30 mins after the release of the mosquitoes. Fifteen (15) tests were conducted with each net (treated or untreated net). The release was done every 3 days to allow for the wash-out period. Windows of huts were fitted with exit traps to catch exiting mosquitoes (Fig. 1E). The floor of the hut was covered by white sheets to ease the collection of knocked-down mosquitoes. Host-seeking mosquitoes trapped in the bed net trap were collected and recorded (Fig. 1F). Validation of these behaviors was done using the F1 progeny obtained from An. gambiae s.s caught from the field.
Indoor and outdoor resting mosquito recapture
Mosquitoes that were not caught in the bed net trap or window exit trap were collected from inside the hut and outside at 0700HRS using Prokopack aspirators (John W Hock, Gainesville, FL, USA). For mosquitoes resting indoors, walls and ceilings were systematically aspirated using progressive down and upward movements along its entire length. For outdoor resting mosquitoes, the collection was done from the clay pots (Fig. 1 F) by placing a white mesh from a mosquito cage over the mouth and agitating the mosquitoes inside the pot, causing them to fly and move to the cage 30. The corners of the screen house and the vegetation cover were also scanned for resting mosquitoes using the Prokopack aspirator.
WHO cone bio-assay to determine bed-net efficacy
The treated net insecticidal efficacy was confirmed by exposing mosquitoes for 3 mins according to the standard WHO cone bioassay procedure. This was done with 4–5day old, non-blood fed, An. gambiae s.s. The bioassays included 5 replicates from both the resistant and susceptible colony with an average of five mosquitoes per tube. The cone bioassays were conducted using DawaPlus 2.0 long-lasting insecticidal net treated with deltamethrin. The F1 progeny of wild-caught mosquitoes were also used for this experiment for validation. After exposure, the groups of mosquitoes were placed in a single 1 L paper cup and provided with cotton wool soaked with 10% sugar solution for 24 hrs. Their knock-down status was measured 60 min post-exposure and mortality were recorded after 24 h. An untreated net was used as a negative control for the assay.
Scientific and Ethical clearance
This study was approved by the Ethical Review Board of the Kenya Medical Research Institute (KEMRI) under the scientific steering committee (SSC 3434). Prior to commencement of the study, volunteers were given an information sheet describing the aims, study procedures, risks and benefits of their participation in this study. Written informed consent was obtained from individual volunteers before the experiments. The experiments were performed in accordance with the institution guidelines and regulations.
Statistical analysis
The proportions of mosquitoes caught in the bed net trap were interpreted as host-seeking mosquitoes. The proportions were calculated by dividing the number of mosquitoes caught in the bed net trap/exit trap/resting with the total number of mosquitoes recaptured for each phenotype (resistant and susceptible mosquitoes) respectively. Observations of host-seeking behavior and exit behavior of resistant and susceptible phenotypes were compared between treatments using generalized linear model (GLM) with binomial distribution and logit link function. The LLINs were considered bio-effective when the percentage of mosquitoes knocked down after 60 min post-exposure was above 95% or mortality after 24 h was above 80% in the WHO cone bioassays31. Statistical analysis was done using the statistical program Stata (Version 14, StataCorp, College Station, Texas).