*2.3. Mosquitoes*

Pyrethroid-resistant *Anopheles arabiensis* (Kingani strain, established 2017) and pyrethroid susceptible *Anopheles gambiae* (Ifakara strain, established 1996) were used in this study. *An. arabiensis* (Kingani) is metabolic-resistant and expresses the upregulation of cytochrome p450s, with 14% mortality upon exposure to WHO 1x discriminating dose of alphacypermethrin that was reversed by piperonyl butoxide (PBO) pre-exposure, reconfirmed before this study was initiated. *An. gambiae* s.s. (Ifakara) is fully susceptible to all insecticide classes at 1x WHO discriminating doses, reconfirmed before this study was initiated. The mosquito colony was maintained according to MR4 Guidelines [36] at 27 ± 2 ◦C and 40%–100% relative humidity, with an ambient (approximately 12:12) light–dark cycle. The colony was maintained on a Tetramin fish food for larvae, 10% glucose for adults. Females were offered cattle blood in a membrane feeder or were offered a human arm as a blood source. Mosquitoes were 5–8 days old, nulliparous, sugar starved for eight hours, and acclimatized to the test room for an hour before the experiment (Table 1). As VCPTU do not have resistant *An. gambiae* in the colony, we used metabolic resistant *An. arabiensis* instead. Since the bioassay measured contact toxicity, it was deemed that the mechanism for resistance was more critical than the species used for the evaluation.

#### *2.4. The Standard WHO Tunnel Test Procedure*

WHO tunnel tests were conducted following WHO guidelines [20] (Figure 1A). The tunnel was divided into two chambers separated by a netting sample that were deliberately holed with 9 small (1 cm) holes through which the mosquitoes had to pass to reach the bait. The bait was placed in the short chamber. In the long section, 100 unfed female mosquitoes aged 5–8 days were released at 19:00 h. The tunnel was covered with a black cloth and left overnight. The following morning, between 07:00 and 09:00 h, mosquitoes were removed from the tunnel using an aspirator. Mosquitoes were scored as alive fed, alive unfed, dead fed, or dead unfed in each chamber and put into a separate paper cup for post exposure mortality monitoring. Mosquitoes were supplied with access to 10% sugar solution *ad libitum* and were then scored for post-exposure delayed mortality at 24-h and 72-h. The experiment and post exposure holding was conducted at a temperature of 27 ± 2 ◦C and a relative humidity of 80% ± 10. For the experiment to be considered valid, the following thresholds were used: control 24-h mortality ≤10% in all experiments and blood-feeding success ≥50% with experiments using the rabbit bait.

**Figure 1.** WHO tunnels for comparison of baits: (**A**) Conduct of standard WHO Tunnel with the bait chamber to the left of the picture and mosquitoes being placed into the longer end of the chamber; (**B**) Rabbit—in Experiments 1–4; (**C**) Hemotek® membrane—in Experiment 1 and 4; and (**D**) Human arm—in Experiment 1.

#### *2.5. Bait Used and Preparation*

**Rabbit:** three groups of five healthy rabbits were used. Rabbits were maintained under veterinary supervision. The rabbit was shaved on its back to allow the mosquitoes to feed. The rabbit was gently restrained in a mesh tube that was suspended in the short section of the WHO tunnel throughout the 12-h experiment (Figure 1B). **Membrane feeding:** A Hemotek® membrane feeder (SP-6 System, Hemotek Ltd., Blackburn BB6 7FD, UK) was used. Two membrane feeders were placed on top of the "bait chamber" of each tunnel (Figure 1C). Each feeder reservoir contained 3 mL of cow blood covered by a stretched parafilm membrane and tightened with an o-ring to prevent any leakage. Cow blood was obtained from cattle maintained under veterinary supervision at VCPTU and was stored for up to two weeks at 4–8 ◦C in heparinized tubes. Socks worn by the investigator (DK)

for 8 h on the day of testing were stretched across the surface of the membrane feeder reservoir to provide host kairomones and increase mosquito attraction to the feeder. The Hemotek® was switched on 10 min before the experiment. The temperature of the feeder was set at 37–39 ◦C throughout the 12-h experiment. **Human arm:** Five healthy male volunteers conducted arm feeding by inserting their arms into the bait short section of the tunnel (Figure 1D). Before testing, their arms were washed with water. The volunteers were non-smokers and did not drink alcohol or use perfumed lotions during the experimental period. The experimental time for arm feeding was 1 h to allow for standardized evaluation and to minimize volunteer discomfort. Previous work has shown that 30 min of exposure resulted in high blood feeding [35]. To protect human participants, several procedures are routinely undertaken in the laboratory. Anybody who works in the insectary and bloodfeeds mosquitoes (including the participants) are screened weekly for malaria parasites using malaria rapid tests (SD bioline). Colony mosquitoes are not kept beyond 10 days, as it takes 12–14 days for mosquitoes to develop sporozoites. Mosquitoes used in the experiments were nulliparous. Therefore, participants were not at risk of malaria infection as a result of the experiments.
