This PDF file includes: 640 Supplementary text: Cyp6 expression analysis using the ΔΔCt method 641 Figs. S1 to S4 642 Tables S1 and S2 643 29 Supplementary Text 644 Cyp6 expression analysis using the ΔΔCt method 645 Threshold cycle (Ct) values were corrected for primer efficiency applying the formula Corrected 646 Ct = Ct (Logx / Log2), where x = 2 if efficiency is >100% and x = 1+Efficiency if efficiency is 647 <100%. 648 Ct values obtained for target genes were normalised against those of the housekeeping genes 649 RPS7 and Ubiquitin using the formula ΔCt = Ct target -Ct housekeeping. 650Mean ΔCt values of GAL4/+ mosquitoes were then compared to those of GAL4/UAS mosquitoes 651 using the formula ΔΔCt = ΔCt GAL4/+ -ΔCt GAL4/UAS. Fold change (FC, x) expression 652 between the two populations was calculated with the formula FC = (2ᶺ GAL4/UAS ΔΔCt) / (2ᶺ 653 GAL4/+ ΔΔCt). 654Statistical differences in fold change expression between GAL4/UAS and GAL4/+ were 655 determined using t test (unpaired, two-tails, assuming equal variance). 656

24 44(0)1517053137 25 Gareth.Lycett@lstmed.ac.uk 26

Pyrethroid-impregnated nets have contributed significantly to halving the burden of malaria but resistance threatens their future efficacy and the pipeline of new insecticides is short. Here we report that an invertebrate automated phenotyping platform (INVAPP), combined with the algorithm Paragon, provides a robust system for measuring larval motility in Anopheles gambiae (and An. coluzzi) as well as Aedes aegypti with the capacity for high-throughput screening for new larvicides. By this means, we reliably quantified both time- and concentration-dependent actions of chemical insecticides faster than using the WHO standard larval assay. We illustrate the effectiveness of the system using an established larvicide (temephos) and demonstrate its capacity for library-scale chemical screening using the Medicines for Malaria Venture (MMV) Pathogen Box library. As a proof-of-principle, this library screen identified a compound, subsequently confirmed to be tolfenpyrad, as an effective larvicide. We have also used the INVAPP / Paragon system to compare responses in larvae derived from WHO classified deltamethrin resistant and sensitive mosquitoes. We show how this approach to monitoring larval response to insecticides can be adapted for use with a smartphone camera application and therefore has potential for further development as a simple portable field-assay with associated real-time, geo-located information to identify hotspots.

No abstract