2.3.1. Mosquito Rearing

Mosquito colonies were maintained as described by Williams et al., in the LITE facility at the Liverpool School of Tropical Medicine (LSTM) [12]. Insectary conditions were maintained at 26 ± 2 ◦C and 70 ± 10% relative humidity (RH), with a L12:D12 h light: dark cycle anda1h dawn and dusk. Larvae were reared in purified water and fed ground TetraMin® tropical flakes (Tetra U.S., Blacksburg, VA, USA), adults were provided continuous access to a 10% sucrose solution, and adult females were given access to blood using a Hemotek membrane feeding system (Hemotek Ltd., Blackburn, UK). Two wellcharacterized laboratory strains of mosquito were used as representative populations, one susceptible and one resistant to commonly used insecticides. *A. gambiae* Kisumu is a reference insecticide-susceptible strain originally from Kisumu, Kenya, reared at LSTM since 1975, and *A. gambiae* Tiassalé 13 is a resistant *Anopheles* strain which was colonized from Tiassalé, Côte d'Ivoire and has been reared at LSTM since 2013. Kisumu has no selection procedure and so is susceptible, whereas Tiassalé 13 is selected witha1h exposure to 0.05% deltamethrin and shows high resistance to pyrethroids, which is mediated by both target sites 1014F *kdr* and *ace-1*, and metabolic resistance, which is mediated by several cytochrome P450s.

#### 2.3.2. WHO Tube Bioassay Testing

A WHO holding-tube and its exposure tube pair are referred to as a 'test unit'. Test units using mosquitoes from the same cohort are technical replicates of each other. Test units using mosquitoes from different cohorts are referred to as biological replicates. Three biological replicates, each made up of 2 negative control test units and 12 insecticide test units, were carried out for each experiment. There were 2 test units per treatment within a biological replicate, which were technical replicates of each other. The WHO tube bioassay 4 was used with some adaptation to allow investigation of 2 individual parameters:


Other factors, such as orientation of the test unit, sample size required per treatment, and sample size required per control, were either already clearly defined in the current test procedures or supported by previously published literature.

Grade 1 Whatman filter papers of size 15 × 12 cm were coated with 0.043% permethrin dissolved in silicone oil for Kisumu testing (an LC50 determined from previous work in the department by WHO tube bioassay for the Kisumu strain) and 0.75% (WHO recommended discriminating concentration for *Anopheles* [1]) permethrin for Tiassalé 13 testing. The 0.043% papers were made in the LITE laboratories, whereas the 0.75% papers were purchased from WHO Malaysia (Universiti Sains Malaysia, Penang, Malaysia). Permethrin was chosen as it is a heavily used insecticide for profiling, with a well understood mode of action and established resistance mechanisms in the Tiassalé 13 strain.

To investigate the effect of the number of mosquitoes per test unit and compare the results from covered and uncovered tubes in parallel, the experimental layout for a single replicate is outlined in Table 1. Additional technical replicates of the tubes containing fewer mosquitoes were conducted to ensure equivalent numbers of mosquitoes were screened per treatment and to nullify the potential bias of a smaller sample size influencing the mortality estimate.

**Table 1.** Experimental outline of a single biological replicate to investigate the effect of covered vs. uncovered exposure tubes and number of mosquitoes per test unit. The test concentration was 0.043% for *Anopheles gambiae* (Kisumu, susceptible) testing and 0.75% for *An. gambiae* (Tiassalé 13, resistant) testing. Mosquito age was 2–5 days for both strains and all treatments.


Mosquitoes were exposed in test units for 1 h at 26 ± 2 ◦C and 70 ± 10% RH and then transferred back to holding-tubes post-exposure and held in the same conditions for 24 h at which point their mortality was scored. Data from three biological replicates, each prepared independently, were used to generate the data.

To investigate the effect of mosquito age, a second experiment was completed using a single cohort of each strain, from which a subsample of 150 mosquitoes was taken at the WHO recommended testing age [3] (2–5 days), 2 days after testing age (overlapping with the recommended age range, 4–7 days), and 4 days after testing age (outside the recommended age range, 6–9 days). Mosquitoes were tested with two negative control test units and four insecticide test units. The test concentrations were 0.043% for Kisumu testing and 0.75% for Tiassalé 13 testing, as in the previous experiment. This experiment was repeated three times, each with independently reared cohorts of mosquitoes.
