*2.6. Study Design*

Experiments were comparative bioassays with a minimum of 5 replicates per net type, per permutation (Table 1). A total of sixty one experimental nights were run between March 2021 and February 2022. All procedures for preparation, release, collection, and mosquito scoring were performed as per the standard WHO tunnel test procedure [20] (Figure 1A) outlined above with the factors of interest (bait, exposure time, and density) varied (Table 1). The endpoints measured were blood feeding success (BFS) or blood feeding inhibition (BFI), mortality at 24-h (M24), and mortality at 72-h (M72).

#### 2.6.1. Experiment 1: The Impact of Bait/Host

The bio-efficacy of unwashed and 20 times washed Interceptor® G2 and Interceptor® ITNs was tested using 100 pyrethroid-resistant *An. arabiensis* per replicate with membrane, human arm, and rabbit bait (Figure 2A). Mosquitoes were left in the tunnel for 12 h overnight and BFS, M24, and M72 endpoints were evaluated. Five samples for each ITN type and condition (Interceptor® G2 unwashed and 20× washed and Interceptor® unwashed and 20× washed) for each host type were evaluated using five tunnels. One control and four treatments—i.e., one per net type and condition—were conducted each night for 15 nights with each bait (membrane, human, and rabbit) and were evaluated for five nights each. Each bait type was tested on different nights to allow for an independent comparison of each bait in the absence of competing host kairomones.

#### 2.6.2. Experiment 2: The Impact of Exposure Time

The bio-efficacy of unwashed and 20× washed Interceptor® G2 and Interceptor® was tested using 100 pyrethroid-resistant *An. arabiensis* per replicate with either a human arm or membrane bait (Figure 2B). When investigating 1 h exposure, mosquitoes were exposed to ITNs for only 1 h with a human arm or membrane and were then removed from the tunnel and placed in holding cups with access to sugar for 11 h overnight. For the 12-h exposure, the human arm was only available for 1 h, but the mosquitoes were left in the tunnel for the remaining 11 h of the experiment. In the membrane assay, mosquitoes interacted with membrane in the tunnel throughout the 12 h of exposure. In both tests, the BFS, M24, and M72 endpoints were evaluated. Five samples for each ITN type (Interceptor® G2 unwashed and 20× washed and Interceptor® unwashed and 20× washed) plus a negative control were tested using five tunnels. Five replicates per treatment arm for each bait and exposure time were conducted over 10 nights. The 1 h and 12 h of exposure were conducted on the same night for either the membrane or the human arm. The 1 h exposure was performed and then a 12-h exposure was conducted on the same net using a fresh batch of mosquitoes.

Each bait type was tested on different nights to allow for an independent comparison of each bait in the absence of competing host kairomones.

**Figure 2.** Flow chart of experimental procedure, experiment 1 (**A**—impact of baits) and experiment 2, (**B**—effects of exposure time 12-h vs. 1-h) on WHO tunnel test outcomes.

2.6.3. Experiment 3: Effects of Mosquito Density on the Bio-Efficacy Measurement of Blood-Feeding Inhibition and Mortality at 24-h or 72-h

The effect of mosquito density on bio-efficacy measurements of BFS, M24, and M72 endpoints was evaluated in the WHO tunnel using 50-mosquitoes compared to the standard 100-mosquitoes (Figure 3A). Experiments were conducted following standard procedures with 12 h of exposure and continuous access to a restrained rabbit. For this, two species were used: pyrethroid-resistant *An. arabiensis* tested for the pyrethroid and chlorfenapyr Interceptor® G2 (unwashed or 20× washed) and pyrethroid-susceptible *An. gambiae* for the pyrethroid only Interceptor® ITN (unwashed or 20× washed). A total of seven tunnels (one control, 3 with unwashed, and 3 with washed ITNs) per night were run with 15 replicates conducted per net condition for each mosquito density. Each strain and density (Table 1) were evaluated in a separate 5-night block. This was done to ensure the fitness of mosquitoes used, as the experiments were conducted at a time when the mosquito colony was under pressure from multiple evaluations.

2.6.4. Experiment 4: Possibility to Replace Standard Bait (Rabbit) with the Membrane Assay

To determine whether the rabbit can be replaced with the membrane assay as the bait, the bio-efficacy measurements of BFS, M24, and M72 endpoints were evaluated in the WHO tunnel with 12 h of exposure using 50-membrane and 100-rabbit (gold standard) with resistant *An. arabiensis* mosquitoes (Figure 3B). The same procedure was used for all five arms: a negative control and four treatment arms of Interceptor® unwashed or 20× washed and Interceptor® G2 unwashed or 20× washed (Table 1). For the membrane, a total of 5 tunnels (1 per arm) were run per night, and for the rabbit, 9 tunnels (1 control and 2 replicates per treatment arm) were run per night, with a total 15 replicates per arm for each assay. Different baits were run on separate nights to allow for an independent comparison of each bait in the absence of competing host kairomones.

**Figure 3.** Flow chart of experimental procedure: Experiment 3 (**A**—effects of mosquito density 100 vs. 50) and Experiment 4 (**B**—possibility to replace 100-rabbit bioassay with 50-Hemotek membrane).
