Extended Data Fig. 5: Hybrid genotyping and infection status in sand flies after a naturally acquired Leishmania infection from mice lesions composed of two parental lines.
From: Leishmania genetic exchange is mediated by IgM natural antibodies
(a) Leishmania major hybrids formed in sand flies given one infectious blood meal (BM1) or provided 6 days later with an additional uninfected bloodmeal (BM2) in the presence (+IgM) or absence (−IgM) of IgM were genotyped by PCR targeting parental selectable drug markers HYG (Hygromycin), BSD (Blasticidin) and SAT (Nourseothricin). Parental 1, WR-SSU-HYG; Parental 2, FVI-FKP40-BSD; Parental 3, FVI-FTL-SAT; ntc, no template control; L, 1kb-plus ladder. Double drug resistant hybrid lines were cloned before genotyping. A single hybrid exemplar is shown for positive events from each group. “n” detailed on Fig. 4d. Parasite number (b,c,d) and percentage of metacyclic promastigotes (e,f,g) in L. major-infected Lu. longipalpis. At 6 days post-infection, a proportion of the sand flies were provided a second uninfected blood meal via a membrane feeder composed of rabbit red blood cells reconstituted with fetal bovine serum with (+IgM) or without (−IgM) 500 µg/ml adult bovine IgM or allowed to feed on uninfected mice as a blood source for natural IgM. Infection status of individual sand flies was assessed at 14 days after the first blood meal (8 days after the second bloodmeal). Parental line combination 1×2 (b,e) n = 4, 1×3 (c,f) n = 3, 2×3 (d,g) n = 3. data represented by individual values scatter plots. Dashed bars, medians. (h) An antibiotic cocktail (ABTs) was used to control contamination with sand fly gut microbiota when isolating parasites from sand fly midguts. ABTs has no effect on parasite growth and viability. ABTs: Penicillin-Streptomycin (100 U/mL); Gentamicin (50 µg/mL); Caspofungin (15 µg/mL); 5-fluorocytosine (30 µg/mL). Growth curve represented by median ± interquartile range. n = 3.
