Fig. 3

Sperm-mediated paternal transmission of RGDV from V⁺ males to offspring. a–c Immunofluorescence microscopy showing RGDV infection in V⁻ a or V⁺ male reproductive systems b, c. c is the enlargement of boxed area in b. The male reproductive systems were stained with virus-rhodamine (red) and actin dye Phalloidin-FITC (green). Bars: a, b, 200 μm; c, 50 μm. d Immunofluorescence microscopy showing association of RGDV virions with sperm heads. Sperms from V⁺ male were stained with virus-rhodamine (red), actin dye Phalloidin-FITC (green) and DAPI (blue). Bar, 10 μm. e, f Electron micrograph showing association of RGDV virions (red arrows) with the plasma membrane of sperm heads. f is the enlargement of boxed area in e. Bars, 200 nm. g–o Immunofluorescence microscopy showing infection route of RGDV in V⁻ female reproductive system after mating with V⁺ males. RGDV accumulated in the spermatheca g and i but not in other parts g and h at 3 days post mating. h and i are the enlargements of boxed areas in g. j–l RGDV accumulation in the opening of the spermatheca k where it connected to the oviduct l at 6 days post mating. k and l are the enlargements of boxed areas in j. m–o RGDV accumulation in the spermatheca o but not in female ovaries n at 10 days post mating. n and o are the enlargements of boxed areas in m. Bars, 200 μm. g–o The female reproductive systems were stained with virus-rhodamine (red) and actin dye Phalloidin-FITC (green). p Electron microscopy showing association of RGDV virions (red arrows) with the plasma membrane of sperm heads in the spermatheca of V⁺ females at 10 days post mating. Bar, 100 nm. q Proposed model for sperm-mediated paternal transmission of RGDV. During mating, virus-associated sperms accumulate first in the female spermatheca, then move to the oviduct to fertilize eggs. All immunofluorescence images are representative of at least three replicates. AG, accessary gland; BC, bursa copulatrix; Od, oviduct; Oo, oocyte; Ovl, ovariole; Pd, pedicel; PM, plasma membrane; Sh, sperm head; Sp, spermatheca; SpG, spermatheca gland; Sv, seminal vesicle; T, testis; Tf, terminal filament; Vd, vas deferens