Fig. 3: Transmission-associated genes from quiescent sporozoites have daily rhythms.
From: Parasite and vector circadian clocks mediate efficient malaria transmission
a, The experimental design from two independent experiments for the sequencing of P. berghei (P.b.) sporozoites transcripts from A. stephensi mosquito salivary glands. Salivary glands were collected every 4 h over 3 days for light/dark (LD) and dark/dark (DD) conditions. b, Representation of six cycling genes selected from the top 20 cycling genes (based on significance) for each condition (LD and DD) and the circadian-algorithm-fitted lines. c, The daytime distribution of the peak expression of cycling genes showing that cycling transcription peaks between 0 h and 4 h after lights on (beginning of daytime, light phase). d, The phase (time of peak of expression) of the 409 common genes (that cycle in LD and DD) is maintained in both conditions. Red dashed lines denote linear correlation between the phases of 409 common genes in each condition. Solid grey lines denote 0, 24 or 48 h timepoints on each axis. e, Circadian fold change of the common cycling genes in both conditions. The violin plot width depicts distributions of fold-change values. f, Heat map of sporozoite cycling genes from mosquitos in LD and DD conditions. Each row represents a gene. The gene expression is z-scored. g, Representative immunofluorescence micrographs from purified salivary-gland sporozoites at days 22–24 after mosquito’s bloodmeal at different timepoints ZT3 (zeitgeber time, 3 h after lights on), ZT9, ZT15 and ZT21. DNA in blue and CSP in red. Scale bar, 5 µm. n = 20 dissected mosquitos per timepoint; two independent experiments. h, Representative immunofluorescence micrographs of Anopheles mosquito midguts with oocysts from P. berghei-TK-GFP and GFP (parental) line after EdU incorporation. The white circles delineate three oocysts. The arrowheads show oocysts that in the 48 h before fixation, replicated their DNA as demonstrated by the incorporation of EdU. The asterisk shows replicating mosquito midgut cells. Scale bar, 10 µm. n = 20 dissected mosquitos per genotype; two independent experiments. i, Representative immunofluorescence micrographs of salivary-gland sporozoites from P. berghei-TK-GFP and GFP (parental) parasite line upon feeding of mosquitos with EdU. The white line delineates a nucleus of a sporozoite. Sporozoites show no DNA replication as observed by the absence of EdU (red) at DNA (blue). Scale bar, 5 µm. n = 20 dissected mosquitos per genotype (repl.); two independent experiments. Mosquito icon in panel a designed by Freepik.
