Figure 1.
Ovary structure of Laodelphax striatellus.
a: The ovariole of L. striatellus is composed of a terminal filament on the tip, the egg tube in the middle, and a pedicel at the bottom. The germarium is at the tip of the egg tube; ooecia, just below the germarium, are composed of oocytes and follicular cells. b: Ovarioles at the previtellogenesis stage were stained with DAPI for DNA (blue) and FITC-phalloidin for actin (green). The cell-free trophic core and several oocytes (white stars) are located in the middle and the base of the germarium, respectively. c: Schematic drawing of typical telotrophic meroistic ovariole. Nurse cells in the germarium radially arranged around the trophic core in the middle. The nutritive cord connects the trophic core and oocytes. ET: egg tube, Fc: follicular cell, Gr: germarium, NC: nurse cell, Nc: nutritive cord, O: oocyte, Pd: pedicel, TF: terminal filament, TC: trophic core.
Figure 2.
Analysis of interaction between RSV pc3 and different vitellogenin fragments in vitr.
a: The GST pull-down assay was used to detect any interaction between RSV pc3 and Vg. RSV pc3 was fused with GST to act as a bait protein with a single GST as a control. None of the prey proteins (VitN, DUF or vWD domains) interacted with GST. VitN had no binding ability, DUF had a weak interaction, but vWD strongly interacted with pc3-GST. b: Pull-down samples were stained with Coomassie brilliant blue to ensure the equal sample loading.
Figure 3.
Localization of RSV in ovaries of Laodelphax striatellus at different stages of ovarian development.
a: RSV in L. striatellus. In nymph and adult stage I, RSV was found only in the terminal filament and pedicel. In adult stage II, RSV invaded the tip of the gemarium; in adult stages III–IV, RSV was present throughout the germarium and in portions of the oocyte (anti-RSV monoclonal antibody is conjugated directly to Alexa Fluor 488, green;). Arrow: RSV antigens. Bar = 100 µm b: Vg was expressed at a background level in the nymph and Adult I (12 h postemergence) stages. Expression sharply increased in the Adult II stage (24 h postemergence) and continuously increased in Adult III (36 h postemergence) and IV (48 h postemergence) stages. c: RSV RNPs in the nutritive cord, which connects the germarium and oocytes. Ovarioles at Adult IV stage were treated with rhodamine-conjugated anti-RSV antibody (red) and stained with DAPI for DNA (blue), FITC- phalloidin for actin (green); bar = 100 µm. Gr: germarium, Nc: nutritive cord, O: oocyte, Pd: pedicel, TF: terminal filament.
Figure 4.
Colocalization of RSV and vitellogenin in Laodelphax striatellus ovaries.
a: Neither RSV nor Vg antigens were detected in the germarium at previtellogenic stage. b: RSV and Vg antigen colocalization spots at vitellogenic stage (Adult III). c: RSV and Vg arenaceous colocalization spots in germ cells at early stage of vitellogenesis. d: RSV and Vg antigens colocalization spots at later vitellogenic stage (Adult IV). Anti-RSV and anti-Vg monoclonal antibodies were conjugated to Alexa Fluor 488 (green) and Alexa Fluor 594 (red) separately. A, C, D: bar = 50 µm, B: bar = 30 µm. TF: terminal filament, Gr: germarium, O: oocyte, Pd: pedicel.
Figure 5.
Immunoelectron micrographs showing distribution of RSV and vitellogenin in the germarium.
a: Nurse cells radially arranged around the trophic core; Vg assembled in the highly electron-dense yolk granules in the nurse cells on the TC side. b: Both RSV and Vg antigens are found in the yolk granules. c: RSV (blue arrow) and Vg (yellow arrow) assembled in yolk granules. TC: trophic core; NC: nurse cells; y: yolk granule; blue arrow: 10-nm gold-conjugated goat-anti-mouse IgG against RSV used to detect virus; yellow arrow: 15-nm gold-conjugated goat-anti- rabbit IgG against Vg. A: bar = 10 µm, B: bar = 500 nm, C: bar = 100 nm.
Figure 6.
Quantitative assay of RSV in dsRNA-injected ovarioles.
RSV-infected nymphs were injected with Vg dsRNA, with gfp dsRNA as a control. a: After dsRNA injection, insects that had emerged for 48 hours were selected to detect Vg expression. b: The quantity of RSV pc3 RNA had decreased significantly by 79% in the ovarioles of viruliferous 48 h adult insects. c: RSV level in the other parts of the insect body did not decrease after injections with different dsRNAs.
Figure 7.
Injection of dsvg or dsgfp resulted in different RSV infection levels in the ovary.
Newly emerged adult insects were injected with dsvg or dsgfp, and ovaries were dissected to test RSV or Vg levels 48–c: Ovaries from dsvg-treated insects; d: ovaries from dsgfp-treated insects. a: Type A, ovariole had developed with more than two ooecia, but no Vg accumulated; RSV did not invade the ovariole. b: Type B, Vg had accumulated in the germarium and formed large spots in the tip; RSV began to invade the tip of the germerium of only a few ovarioles. c: Type C, a large amount of Vg had accumulated, forming large spots throughout the germarium, but not in the oocytes at the bottom (white arrows); RSV present in most of the germarium of nearly all ovarioles. d: Type C, Vg had accumulated in the oocyte at the bottom to form yolk globules (white arrows); RSV present throughout the germarium. RSV antibody was conjugated to Alexa Fluor 488. Bar = 100 µm. Gr: germarium, O: oocyte.
Table 1.
Percentage of RSV-infected ovaries with virus in the three location types from insects injected with dsgfp or dsvg dsRNAs.
Figure 8.
Model of RSV transovarial transmission.
RSV binds to Vg outside the ovary and is transported into the germ cells through VgR-mediated endocytosis. RSV first accumulates in yolk granules with Vg and is released into the cytoplasm. RSV finally enters the oocytes through the trophic core, then the nutritive cord. Gr: germarium, NC: nurse cell, Nc: nutritive cord, nc: nucleolus, TC: trophic core, O: oocyte, Y: yolk granule.