Extended Data Fig. 9: IgM promotes Leishmania backcross hybrids in the gut of sand flies.
From: Leishmania genetic exchange is mediated by IgM natural antibodies
(a) Diagram outlining the generation of backcrosses. Sand flies were fed on mice lesions composed of a parental and a hybrid line. L. major Parental 1 (WR-SSU-HYG) and Parental 2 (FVI-FKP40-BSD) resistant to hygromycin (HYG) or blasticidin (BSD), respectively, were crossed to produce F1 hybrids. HYG/BSD double resistant F1 hybrids were backcrossed to L. major Parental 2′ (FV1-FKP40-SAT) or 2″ (FV1-SSU-SAT), both resistant to Nourseothricin (SAT) inserted at loci in chromosome 16 and 27, respectively. This resulted in the recovery of backcross hybrids resistant to HYG/BSD/SAT. (b) Leishmania major backcross hybrid genotyping by PCR targeting parental selectable drug markers HYG, BSD and SAT. (1×2) F1 hybrid (a cross between L. major Parental 1 and Parental 2); Parental 2′, FV1-FKP40-SAT; Parental 2″, FV1-SSU-SAT.; ntc, no template control; L, 1kb-plus ladder. Triple drug resistant hybrid lines were cloned before genotyping. Only sand flies provided a second bloodmeal containing IgM produced backcross hybrids. A single backcross hybrid exemplar is shown for positive events from each group. “n” detailed on Fig. 5c. Parasite number (c,d) and percentage of metacyclic promastigotes (e,f) 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. 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)×2′ (c,e) n = 3; (1×2)x2′ (d,f) n = 2.
