Viral pathogens hitchhike with insect sperm for paternal transmission

Abstract

Arthropod-borne viruses (arboviruses) can be maternally transmitted by female insects to their offspring, however, it is unknown whether male sperm can directly interact with the arbovirus and mediate its paternal transmission. Here we report that an important rice arbovirus is paternally transmitted by the male leafhoppers by hitchhiking with the sperm. The virus-sperm binding is mediated by the interaction of viral capsid protein and heparan sulfate proteoglycan on the sperm head surfaces. Mating experiments reveal that paternal virus transmission is more efficient than maternal transmission. Such paternal virus transmission scarcely affects the fitness of adult males or their offspring, and plays a pivotal role in maintenance of viral population during seasons unfavorable for rice hosts in the field. Our findings reveal that a preferred mode of vertical arbovirus transmission has been evolved by hitchhiking with insect sperm without disturbing sperm functioning, facilitating the long-term viral epidemic and persistence in nature.

Fig. 1

Vertical transmission of RGDV by viruliferous R. dorsalis. a Experimental design for vertical virus transmission assays. b, c Vertical transmission of RGDV by the laboratory reared b and the field caught c V⁺ and V⁻ leafhoppers via mating. Eggs were collected for tracing RGDV. d Maintenance and epidemic cycle of rice viral disease caused by RGDV in the field in Guangdong. e Viruliferous rates of R. dorsalis populations in different generations in Guangdong during overwintering seasons from 2013 to 2016. f Viruliferous rates in overwintering population of male or female R. dorsalis in Guangdong in 2015 and 2016. g Successive paternal and maternal transmission rates of RGDV by R. dorsalis for three generations. Data are presented as mean ± SE of three independent experiments of four mating combinations in b, c, and f, ^*P < 0.05, ^**P < 0.01; ANOVA followed by Tukey’s HSD test. Values are means ± SD of three independent experiments of four mating combinations in e and g (Student’s t test, two tailed). V⁺, viruliferous. V⁻, nonviruliferous. e, f O, the original overwintering insects collected in November, 1st, the first generation of overwintering insects collected around January, 2nd, the second generation of overwintering insects collected in late February. g 1st, the first generation, 2nd, the second generation, 3rd, the third generation

Fig. 2

Comparison of fitness variables between male and female vectors. a Mean number of V⁻ females that copulated with single V⁺ or V⁻ male in 24, 48, or 72 h. b Effects of RGDV infection on the longevity of male and female adult R. dorsalis. c Sex ratio of offspring produced by parental insects from different mating combinations (V⁻♀ × V⁻♂, V⁺♀ × V⁻♂, V⁻♀ × V⁺♂ or V⁺♀ × V⁺♂). d Progeny egg number, size and hatching rate of female adults from different mating combinations (V⁻♀ × V⁻♂, V⁺♀ × V⁻♂, V⁻♀ × V⁺♂ or V⁺♀ × V⁺♂). Data are presented as mean ± SE of three independent experiments of four mating combinations. The significance of any differences was tested using Student’s t test a–b or Tukey’s HSD test c–d. ^*P < 0.05. Different letters after means in the same column a or line (d) indicate a significant difference at P = 0.05, and the means do not differ significantly if they are indicated with the same letter. V⁺, viruliferous. V⁻, nonviruliferous

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

Fig. 4

Interaction of RGDV P8 with HSPG. a Live sperms were incubated with purified virions for 5, 30, or 90 min, then stained with virus-rhodamine (red) and DAPI (blue). b Structure of RGDV virion. c Live sperms were incubated with purified virions, P8 or P2, then stained with virus-, P8-, or P2-rhodamine (red) and DAPI (blue). d Live sperms were incubated with purified virions and RGDV-, P8-, or P2-specific antibodies, then stained with virus-rhodamine (red) and DAPI (blue). e Schematic representation of R. dorsalis HSPG gene. f Interactions between different domains of HSPG and RGDV P8 in the yeast two-hybrid system. SD-2: SD-Trp-Leu, SD-4: SD-Trp-Leu-His-Ade. g Pull-down analysis of RGDV P8 interaction with HSPG DoIII. HSPG DoIII was fused with GST as a bait protein. P8 was fused with His as a prey protein. P2 and GFP were fused with His as controls. h, i The transcript h or protein i levels of HSPG in reproductive systems of V⁻ or V⁺ male adults. j Confocal micrographs showing colocalization (white triangles) of RGDV (red) and HSPG (green) on sperm head (blue) surface. k Live sperms were incubated with purified virions and 3% BSA, pre-immune serum, purified HSPG DoIII, or HSPG-specific antibodies, then stained with virus-rhodamine (red) and DAPI (blue). l The reduced colocalization (white triangles) of RGDV (red) and HSPG (green) on the surface of sperm heads (blue) after dsHSPG treatment. m, n The transcript m or protein n levels of HSPG and RGDV P8 in male reproductive systems after dsGFP or dsHSPG treatments. o Paternal transmission rates of RGDV from crosses between dsHSPG- or dsGFP-treated V⁺ males with V⁻ females. Eggs were collected for tracing RGDV. p Proposed model for interaction of RGDV P8 with HSPG mediating viral attachment on the surface of sperm heads and subsequent paternal transmission. All images are representative of at least tree replicates. h, m, and o Data are presented as mean ± SD from three independent experiments (Student’s t test, two tailed); ^*P < 0.05, ^**P < 0.01. V⁺, viruliferous. V⁻, nonviruliferous. Ab, antibodies. PM, plasma membrane. N, nucleus. Bars, 5 μm

Fig. 5

Paternal transmission of RGDV by minor vector, the rice leafhopper N. cincticeps. a The viruliferous rates of R. dorsalis and N. cincticeps (25 males and 25 females) collected from the fields in Guangdong, China. b The acquisition efficiencies of RGDV by R. dorsalis and N. cincticeps collected from the fields. c Vertical transmission of RGDV by the field-caught V⁺ and V⁻ N. cincticeps via mating. Eggs were collected for tracing RGDV. d Association of RGDV particles with sperm heads of N. cincticeps and the non-vector wheat leafhopper P. alienus. The sperms dissected from the male leafhoppers N. cincticeps or P. alienus were microinjected with RGDV virions, stained with virus-rhodamine (red) and DAPI (blue), and examined by immunofluorescence microscopy. All images are representative of at least three replicates. Bars, 10 μm. e Comparison of amino-acid sequences of HSPG DoIII of R. dorsalis, N. cincticeps, and P. alienus. f A yeast two-hybrid assay was used to detect the interaction between RGDV P8 and HSPG DoIII from R. dorsalis (R. d), N. cincticeps (N. c) and P. alienus (P. a). a–c Data are presented as mean ± SD from three independent experiments (Student’s t test, two tailed), **P < 0.01