Fig 1.
VPS38/UVRAG and ATG14 are conserved and highly expressed in the ovaries.
A-B. Diagram of the predicted conserved functional domains of human, yeast and Rhodnius prolixus VPS38/UVRAG and ATG4. C2, a protein structural domain involved in targeting PI3K to the cell membrane, CC, coiled coil domain. C-D. RT-qPCR showing the relative expression of VPS38 and ATG4 in different organs and during oogenesis of vitellogenic females. MG, Midgut; FB, Fat body; FM, flight muscle; Ov, Ovary. Troph, tropharium; PreVit, pre-vitellogenic follicles; Vit, vitellogenic follicles; Chor, chorionated oocytes. The relative expression was quantified using the ΔCT method with Rp18S as endogenous control. Graphs show mean ± SEM (n = 6). * p<0.05, **p<0.01, One Way ANOVA.
Fig 2.
RNAi knockdown of VPS38/UVRAG and ATG14 results in reduced levels of PI3P in the ovary.
A-B. RT-qPCR showing the relative expression levels of VPS38/UVRAG and ATG14 in the ovary and fat body of control (dsMal) and silenced females. The organs were dissected 7 days after the blood meal (n = 8). The expression levels of VPS38/UVRAG were reduced by 70% in the ovary and by 50% in the fat body. The expression levels of ATG14 were reduced by 80% in the ovary and by 90% in the fat body. C. Densitometric measurements of PI3P detection by TLC in the chorionated oocytes of control and silenced insects (n = 5). * p<0.05, **p<0.01, One Way ANOVA.
Fig 3.
Silencing of VPS38/UVRAG and ATG14 results in small and white oocytes.
A. Representative images of the ovaries of vitellogenic females injected with dsMal, dsVPS38/UVRAG and dsATG14. B. Representative image of ovarioles dissected from females previously injected with dsMal, dsVPS38/UVRAG and dsATG14. C. Image: representative image of the hemolymph samples extracted from females previously injected with dsMal, dsVPS38/UVRAG and dsATG14. Graph: total amount of protein in the hemolymph from silenced and control females. Graph shows mean ± SEM (n = 8). D. 13% SDS-PAGE showing the protein profile of the hemolymph from silenced and control females. All experiments were performed 7 days after the blood meal. Arrowheads, Vg, vitellogenin subunits; RHPB, Rhodnius heme binding protein (n = 4).
Fig 4.
Silencing of VPS38/UVRAG and ATG14 does not alter oviposition but decreases embryo viability.
A. Number of eggs laid per female over 4 weeks after the blood meal. Graph shows mean ± SEM (n = 52). B. Total of eggs laid by silenced and control females. Graph shows mean ± SEM (n = 52). C. Phenotypic distribution of the F1 eggs observed after knockdown of VPS38/UVRAG and ATG14. Percentage (%) of hatching per phenotype is also showed inside the bars. D. Representative image of the phenotypes observed in the F1 eggs laid by control and silenced females. E. Total amount of protein from control and VPS38/UVRAG and ATG14 silenced eggs, measured by the Lowry method. Graph shows mean ± SEM (n = 6). ***p<0.001, One Way ANOVA. F. 13% SDS-PAGE showing the protein profile of freshly laid eggs from control and silenced females. Arrowheads, VT, Vitellin subunits; RHPB–Rhodnius Heme Binding Protein (n = 3).
Fig 5.
Knockdown of VPS38/UVRAG and ATG14 results in abnormal distribution of the yolk organelles.
A. Representative images of cross-sections from chorionated oocytes were observed in the light microscope. The images show an accumulation of larger yolk organelles in the core cytoplasm (red asterisk) and an irregular distribution of smaller/abnormal organelles in the periphery of silenced oocytes (white arrowheads). Bars: 200 μm. (n = 3). B. Flow cytometry FSC x SSC dot-plots of the yolk organelles extracted from chorionated oocytes obtained from control and silenced females. The plots are representative of five experiments (n = 5). C. Quantification of the organelles (events) frequency in each quadrant of the plots shown in B (RT, Right top; LT, left top; RB, right bottom; LB, left bottom). (n = 5) *p<0.05,***p<0.001, One Way ANOVA.
Fig 6.
VPS38/UVRAG, ATG14 and ATG6/Beclin1 are transcriptionally regulated in the oocytes of Rhodnius prolixus.
Oocytes dissected from silenced females were tested for the expression levels of the other class III PI3K components, and for the control genes ATG8/LC3, ATG1/ULK1, EF1 and PERK (non-related to the class III PI3K complexes). A. Expression levels of different genes in the ovaries of females silenced for VPS38/UVRAG (dsVPS38). B. Expression levels of different genes in the ovaries of females silenced for ATG14 (dsATG14). C. Expression levels of different genes in the ovaries of females silenced for ATG6/Beclin1 (dsATG6/Beclin1). Graphs show mean ± SEM. (n = 8) **p<0.01,***p<0.001. One Way ANOVA. D. Model for the transcriptional regulations between ATG6/Beclin1, VPS38/UVRAG and ATG14.