2. 2 Scd is not required for P. berghei blood and mosquito stage development
To address the function of Scd, we generated Scd KO parasites by using double crossover homologous recombination (Fig. S2A). After recombination, the Scd ORF was replaced by the GFP and hDHFR:yFCU expression cassettes, and GFP-expressing KO parasites were confirmed using fluorescence microscopy (Fig. S2B). The correct gene replacement was confirmed by diagnostic PCR, and clonal lines were obtained by limiting dilution of the parasites. The clonal lines were further confirmed by diagnostic PCR, and the absence of the Scd ORF was determined by locus PCR, which amplified 4.33 kb in WT GFP and 7.16 kb in Scd KO parasites (Fig. S2C). Furthermore, we confirmed the insertion of the targeting cassette at the correct locus by Southern blotting. The genomic DNA was digested with EcoR V and probed with a 5’UTR probe, which revealed a band of 3.28 kb in KO compared to 8.4 kb in WT GFP due to the presence of an EcoR V site in the GFP cassette (Fig. S2D). The Scd-complemented parasite line was also generated to check the specificity of the phenotype (Fig. S2E). For this, a Scd expression cassette consisting of the 5’UTR, ORF, and 3’UTR was amplified and transfected into Scd KO parasites. After recombination, the WT locus was amplified in Scd comp parasites (Fig. S2F). Next, we checked the effect of gene deletion on parasite growth during asexual blood stage propagation and found a similar growth pattern of WT GFP and KO parasites (Fig. S2G). To further understand the role of Scd in mosquito and liver stages, we transmitted the parasites to the mosquitoes by allowing mosquitoes to feed on infected mice. Examination on day 14 post feeding demonstrated normal development of oocysts and oocyst-derived sporozoites in the mosquito midgut (Fig. S3A-D). On days 18-22 post feeding, we observed intact salivary glands that showed a normal sporozoite load and a comparable number of salivary gland sporozoites in KO and WT GFP parasites (Fig. S3E and F). These results indicate that Scd is dispensable for parasite blood and mosquito stage development.