Fig 1.
RpATG6 is highly expressed in the ovary of Rhodnius prolixus.
A. Schematic diagram of the predicted conserved functional domains of RpAtg6 and HsAtg6/Beclin1 (Gene ID: 8678). BH3, BCL-2 homologous domain; CCD, coiled-coil domain; ECD, evolutionarily conserved domain. B. qPCR showing the relative expression of RpATG6 in different organs. MG, Midgut; FB, Fat body; Ov, Ovary. C. qPCR showing the relative expression of RpATG6 throughout oogenesis. Troph, tropharium; PreVit, pre-vitellogenic oocytes; Vit, vitellogenic oocytes; EpVit, follicular epithelium of the vitellogenic oocyte; Chor, chorionated oocytes. The relative expression was quantified using the ΔCT method with Rp18S as endogenous control. Graphs show mean ± SEM (n = 3).
Fig 2.
RNAi knockdown of RpATG6 does not affect major physiological processes of R. prolixus.
A. PCR showing the knockdown efficiency of RpATG6 in the ovary and fat body at different days after the blood meal. B. qPCR showing the relative expression of RpATG6 in the ovary (7 days after the blood meal). C. PI3P detection by TLC and densitometric quantification (n = 2). D. Effect of RpATG6 knockdown in the blood protein digestion in the midgut. Graph shows mean ± SEM (n = 3). E. Survival curve of silenced females (n = 3). *p<0.05, t-Tests.
Fig 3.
Silencing of RpATG6 resulted in abnormal morphology of the oocytes.
A. Ovary of females previously injected with dsMal or dsRpATG6 dissected 7 days after the blood meal. B. Detail of the ovariole of females previously injected with dsMaL or dsRpATG6, 7 days after the blood meal.
Fig 4.
Silencing of RpATG6 leads to accumulation of yolk proteins the hemolymph.
A. Representative images of hemolymphs extracted from females previously injected with dsMal or dsRpATG6. B. Total amount of protein in the hemolymph from silenced and control females. Graph shows mean ± SEM (n = 3). *p<0.05, t-Test. C. 13% SDS-PAGE showing the protein profile of the hemolymph from silenced and control females. All experiments were done at 7 days after the blood meal. VG, vitellogenin subunits; RHPB, Rhodnius heme binding protein. D. RHBP and vitellogenin/vitellin quantifications in the fat bodies (secreted samples), hemolymphs and chorionated oocytes from silenced and control females. Graphs show mean ± SEM (n = 3). *p<0.05, **p<0.01, ***p<0.001, t-Test.
Fig 5.
Silencing of RpATG6 increases oviposition but decreases embryo viability.
The oviposition and hatching were monitored after the blood meal. A. Number of eggs laid per female over 4 weeks. Graph shows mean ± SEM (n = 4). B. Total of eggs laid by silenced and control females. Graph shows mean ± SEM (n = 25). **p<0.01, t-Test. C. Hatching rates after silencing of RpATG6. Graph shows mean ± SEM (n = 3). ***p<0.001, t-Test. D. Phenotypic distribution observed after knockdown of RpATG6. Percentage (%) of hatching per phenotype is also showed. E. Representative image of the phenotypes observed in the eggs. (N), normal (control) eggs; (C), collapsed eggs, (W), white eggs.
Fig 6.
Silencing of RpATG6 affects the yolk protein content of the eggs.
A. Representative images of the gradual phenotype observed in the eggs of females previously injected with dsRpATG6. P, pink egg (control); LP, light pink egg; W, white egg. B. Total amount of protein in the white eggs of females previously injected with dsMal or dsRpATG6 measured by the Lowry (Folin) method. Graph shows mean ± SEM (n = 3). ***p<0.001, t-Test. C. 13% SDS-PAGE showing the protein profile of 24h eggs from females injected with dsRNAs. dsMal (control pink eggs), dsRpATG6 light pink eggs (dsRpATG6LP) and dsRpATG6 white eggs (dsRpATG6W). Arrowheads, Vitellin subunits. Arrow, RHPB–Rhodnius Heme Binding Protein.
Fig 7.
Knockdown of RpATG6 leads to a decrease in the accumulation of TAG and PolyP but not in the glycogen content of the eggs.
A. Densitometry measurements of the TAG content in the silenced eggs after neutral lipid separation by TLC. Upper panel: inset of representative TAG spots in the TLC. B. Glycogen content of the eggs of females previously injected with dsMal or dsATG6. C. Total PolyP content of the eggs of females previously injected with dsMal or dsATG6. All graphs show mean ± SEM (n = 3). *p<0.05, t-Tests.
Fig 8.
Knockdown of RpATG6 results in abnormal distribution of the yolk organelles and less endocytic vesicles in the oocytes.
Left panel: Representative images of cross-sections of early vitellogenic oocytes and 24h-eggs from control and silenced females were observed in the light microscope. The images show an accumulation of larger yolk organelles in the core cytoplasm (*) and an irregular distribution of these organelles in the periphery of dsRpATG6 oocytes and eggs (white arrowheads). Bars: 200 μm. Right panel: Representative images of cross-sections of cortex and plasma membrane of vitellogenic oocytes from control and silenced females observed under the TEM. Silenced oocytes do not show projected microvilli or endocytic vesicles as seen in the control oocytes. Black arrowheads: endocytic vesicles. Bars: 0.5 μm.
Fig 9.
In vivo FITC-uptake by the oocyte yolk organelles.
Silenced and vitellogenic females were injected with 2 μg of FITC and their ovaries were dissected 18 h later for observation under the stereomicroscope. Note the abnormal morphology of the endocytic-originated yolk organelles in the early vitellogenic oocytes from silenced animals (dsRpATG6) when compared to control females (dsMal). Upper panel: brightfield images. Lower panel: fluorescence images. The exposure time (in milliseconds) for the fluorescence images are indicated.