Molecular screening of colony and wild caught mosquitoes
Locked-nucleic acid (LNA) probe-based PCR diagnostics were designed for all three mutations (Supplementary materials Appendix 1).
Genotype:phenotype association testing was performed using two colonies of An. gambiae s.s. , BusiaUG (resistant) and Mbita (susceptible). The BusiaUG strain was established in the lab in November 2018 from Busia, eastern Uganda and exhibits high resistance to pyrethroids and organochlorines (<10% mortality following WHO exposure test), but full susceptibility to organophosphates and carbamates (Oruniet al. unpublished). The Mbita strain was first colonised from Mbita Point, Kenya in 1999, and is fully susceptible to pyrethroids. Colonies were reared in insectaries targeted to 25-27oC and 70-80% relative humidity.
Freshly emerged females from the Mbita line were mated with 3-5 day-old BusiaUG males and then blood fed. The offspring from this cross were then crossed back to the parental BusiaUG line. This design was chosen as resistance variants are often recessive. The resultant backcrossed 3-5-day-old females were exposed for one hour to deltamethrin, permethrin or α-cypermethrin (the three insecticides most commonly used on LLINs) or DDT (a non-pyrethroid sodium channel antagonist), following WHO standard procedures. Mosquitoes were maintained on a 10% sugar solution after exposure and mortality was recorded 24 hours post exposure.
Democratic Republic of Congo. Anopheles gambiae s.s. were obtained from the President’s Malaria Initiative supported entomological surveillance project [29] and from collections conducted by Lyndet al [30]. Mosquitoes were collected by human landing catch or pyrethrum spray collection from 15 locations between 2013 and 2018. Resistance-phenotyped individuals were also obtained from Pwamba, Bassa and Fiwa in Nord Ubangi Province in 2016. These mosquitoes were assessed for susceptibility to deltamethrin or permethrin using a standard WHO tube assay or a cone bioassay where Permanet 3.0 (deltamethrin plus PBO) or Olyset Plus (permethrin plus PBO) were the test nets [23].Kenya and Uganda. Collection details for specimens from contiguous areas of western Kenya and eastern Uganda have been published previously [20, 24, 25].Tanzania. Mosquito collections were conducted in Geita, Bagamoyo and Muleba districts of Tanzania in 2018.
Mosquitoes from all collections were genotyped at the ZZB-TE,Cyp6p4-236M and Cyp6aap-Dup1 loci and genotype:phenotype association testing was performed by Fisher’s exact tests.