Extended Data Fig. 4: IgM promotes Leishmania mating clump formation and hybridization inside the mating clump.

(a) SEM of L. major (upper panel) and L. tropica (lower panel) mating clump showing the 3D organization of promastigote forms. Scale bar, 15 µm. (b) TEM of a L. major mating clump showing a cell with 3 nuclei. Scale bar, 3 µm. (c) TEM images of a L. tropica mating clump showing cells with 3 nuclei. Scale bars: 5 µm. Arrows point to individual nuclei within a parasite cell body. Three-nucleated cells are suggestive of an earlier step that precedes the merging of nuclei. The cell cycle progression follows a strict pattern were Nucleus, Kinetoplast, Flagellum (NKF) division is always constant and goes from 1N1K1F to 2N2K2F culminating with cytokinesis. Note the presence of 3 nuclei without changes to either the kinetoplast or flagellum suggestive of a potential early fusion event inside the LMC. (d, e) Leishmania mating clump formation within the sand fly midgut. Twelve days post-infection with L. major; Lu. longipalpis females were provided with a second reconstituted blood meal containing IgM (500 µg/mL). (d) Initiation of Leishmania mating clump (LMC) formation 30 min after imbibing a second blood meal (blue circles). (e) Parasites merge to form larger LMCs within 24 h after imbibing a second blood meal. Scale bars = 50 μm. LMCs formation in sand flies can also be observed in Supplementary Videos 11 and 12. (f) Confocal immunofluorescence of a 5 µm transversal section from L. tropica mating clump. Promastigotes were grown to stationary phase in the presence of EdU or BrdU then extensively washed and used at a 1:1 ratio to assemble the LMC with IgMn. EdU, green; BrdU, red. White arrows point to yellow nuclei indicative of fusion and exchanged genetic material. Scale bars, 3 µm.