Production of Plasmodium vivax sporozoites in laboratory-bred Anopheles darlingi mosquitoes

Obtaining Plasmodium vivax sporozoites for screening and development of vaccines and drugs is challenging since production of P. vivax gametocyte culturesin vitrois not feasible. We report P. vivax sporozoite production by membrane feeding of laboratory-bred Anopheles darlingionfield collected P. vivax-infected blood samplesand potential sporozoite production enhancement by providing a supplementary, non-infectious blood meal to infected mosquitoes. Plasmodium vivax-infected blood samples collected from 65 patients in Iquitos, Peru,from July 2014 to December 2015, were used for membrane feeding assaysof lab-bred (F18-F36) An. darlingi. Oocyst and sporozoite assessments were performed 5-8 and 10-19 days post infection, respectively. A second, non-infectious bloodmeal was provided to oocyst-positive mosquitoes 5 days after the infectious bloodmeal in assays conducted from August to December 2015. Blood samples from 50 patients (77%) led to sporozoite production. Mosquito feeding rate was on average 63%; mosquito survival14 days post infection was on average 51%. Oocyst intensity and oocyst prevalence were on average 18 ± 4.2 (1.3 -132.6) oocysts/midgut and 71.9 ± 3.2 % (6.3 –100%) infected midguts / dissected midguts, respectively. Sporozoite intensity was on average 7,647± 3,373(4-199,810). Mosquitoes given a second, non-infectious bloodmeal had a sporozoite intensity 2.9 times higher than mosquitoes given a single, infectious bloodmeal (26,007 ± 15,635 vs. 9,116 ± 5,029 sporozoites / salivary gland pair), yet further replication is needed to confirm a significant effect onsporozoite production enhancement. Our sporozoite production system represents a valuable resource for anti-malaria vaccine and drug research.