Fig. 1: A reporter parasite strain to assess sporozoite viability.

a Schematic of the gene targeting strategy to insert a GFP-Luc reporter gene under the control of the csp promoter in the pfs47 locus of P. falciparum NF54 parasites. The expression cassette was integrated through homologous recombination. Subsequently, the hdhfr selection cassette was removed through FLP-mediated recombination. X (XcmI), N (NcoI): restriction sites; pf47: pf47 locus; hrp: histidine rich protein; cam: calmodulin; hsp: heat shock protein; hdhfr: human dihydrodrofolate reductase coding region; GFP-Luc: GFP-luciferase fusion protein; fcu: cytosine deaminase/uracil phosphoribosyl transferase; pbdt: P. berghei dhfr transcription termination region. b Microscope images in phase contrast (upper row) and fluorescence (lower row) channels of mosquito midguts at day 10 postinfection (left panels) and isolated sporozoites at day 17 postinfection (right panels) mounted in PBS on a glass slide. c Luciferase activity in infected mosquitoes in time. Mosquitoes were collected 6, 8, 9, 12, and 14 days after infection with a blood meal containing NF54-CGL gametocytes. Luciferase activity was determined in whole mosquito homogenates. Symbols indicate luminescence in individual mosquitoes with 5–6 mosquitoes per timepoint. d Luminescence signal of a serial dilution of asexual blood-stage parasites (blue squares) and sporozoites (red dots) of the NF54-CGL parasite strain. The dashed line represents the detection limit in the luminescence assay. Symbols indicate values from 4 to 12 replicates per dilution. e Luminescence activity and assay Z′ as a function of time. Isolated salivary gland sporozoites were incubated with vehicle control (0.1% DMSO, red dots) or 1 µM gramicidin (blue squares), and luminescence activity was measured in time. The graph shows data from 12 replicates. These were also used to calculate Z′ values (light blue open triangles). The dotted line indicates Z′ = 0.5.