Culex quinquefasciatus, Say, 1823

Moura, Aires Januário Fernandes da, Tomaz, Filipe, Melo, Tiago, Seixas, Gonçalo, Sousa, Carla A. & Pinto, João, 2024, Vector competence of CUlex QUInQUefaSCIaTUS from Santiago Island, Cape Verde, to West Nile Virus: exploring the potential effect of the vector native WolbaCHIa, Parasites & Vectors (536) 17 (1), pp. 1-12 : 4

Culex quinquefasciatus artificial infection with WNV

Te infective blood meal was prepared by mixing blood and viral stock produced in the previous step to a final titer of 7.0 log 10 PFU/ml. An aliquot of the infectious blood meal was stored at − 80 °C for subsequent backtitration by plaque assay.

Mosquito females between 7 and 10 days old from both thewPip− and wPip + colonies were kept in groups of 100 individuals in paper cups and exposed to the infective blood meal using a Hemotek ® membrane feeding system for 1 h. After feeding, engorged females were selected following sedation with CO 2, transferred to new paper cups and maintained inside a climate chamber (Percival Scientific, Inc., Perry, IA, USA) maintained at 28 °C and 70% relative humidity RH, under 12:12-h light:dark cycle, with access to a 10% sucrose solution.

At days 7, 14 and 21 post-infection (dpi), 30 mosquitoes per group were anesthetized using CO 2,; the legs and wings were then removed and saliva was collected by inserting the mosquito proboscis into pipette tips filled with 5 µl of DMEM with 10% FBS for 60 min. Te collected saliva was then transferred to a microtube containing 200 µl of DMEM supplemented with 10% FBS. Te dissected body and legs plus wings were stored separately in microtubes containing 300 µl of complete DMEM with 10% FBS, 1% amphotericin B and three 2-mm-diameter glass beads for subsequent homogenization during the RNA extraction. All samples were kept at − 80 °C until further use in the RNA extraction.