Artificial membrane feeder

Aedes aegypti were maintained on raisins at 27 °C and 80% humidity according to standard rearing procedures. Mosquitos were propagated by feeding defibrinated sheep blood using a Hemotek artificial membrane feeder^{23 (link)}. Female mosquitoes were infected by intrathoracic microinjection and inoculated with approximately 10³ genome equivalents of virus in a volume of 200nL. Whole mosquitoes were harvested 5 days post-infection by placing individual mosquitoes in 100 μL phosphate-buffered saline (PBS) and storing samples at −80 °C. Prior to RT-LAMP, thawed mosquito samples were homogenized 10 times with a P10 pipet tip in the PBS and the 2 µL crude lysate was used in RT-LAMP reactions.

Missouri isolate (MO) D. immitis microfilariae in dog blood (NR-48907, provided by the NIH/NIAID Filariasis Research Reagent Resource Center, FR3, for distribution through BEI Resources) were fed to female Aedes aegypti mosquitoes (Liverpool strain) at a density of 5,000 mf/ml through an artificial membrane feeder (Hemotek, UK). Blood-fed mosquitoes were reared for 15 days with daily sugar-water feeding to allow development to the L3 stage. At day 15, DiL3 were collected from infected mosquitoes by crushing and concentration using a Baermann apparatus and Roswell Park Memorial Institute (RPMI) 1640 with 1% penicillin–streptomycin (both Sigma-Aldrich, UK). For validation studies at TRS Labs, US, an in-house Georgia III (GAIII) isolate of D. immitis was utilized. Dirofilaria immitis mf were used to infect female A. aegypti mosquitoes (Liverpool strain) in dog blood using a glass feeder at a density of 1,000–2,500 mf/ml. At day 14, DiL3 were collected from infected mosquitoes using crushing and straining with RPMI 1640 and 1% penicillin–streptomycin.

The life cycle of Brugia malayi (Bm) was maintained in mosquitoes and Mongolian gerbils. For Bm larvae (BmL3) generation, microfilariae (mf) were collected from infected gerbils via catheterisation. For this, Mongolian gerbils were anaesthetized with isoflurane and subjected to peritoneal washes with RPMI 1640 media (ThermoFisher Scientific) to harvest mf. Mf were then purified using PD10 column size exclusion chromatography (Amersham), enumerated by microscopy and mixed with human blood to a final concentration of 15–20,000 mf/ml. Mf were then fed to female Aedes aegypti mosquitoes through an artificial membrane feeder (Hemotek). Blood fed mosquitoes were reared for 14 days with daily sugar-water feeding to allow development to BmL3 stage. At day 14, BmL3 were collected from infected mosquitoes by crushing and concentration using a Baermann’s apparatus and RPMI media.