RpATG6 is highly expressed in the ovary of <i>Rhodnius prolixus</i>.

<p><b>A.</b> 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>B.</b> qPCR showing the relative expression of RpATG6 in different organs. MG, Midgut; FB, Fat body; Ov, Ovary. <b>C.</b> 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).</p

Silencing of RpATG6 resulted in abnormal morphology of the oocytes.

<p><b>A.</b> Ovary of females previously injected with dsMal or dsRpATG6 dissected 7 days after the blood meal. <b>B.</b> Detail of the ovariole of females previously injected with dsMaL or dsRpATG6, 7 days after the blood meal.</p

Silencing of RpATG6 impaired the yolk accumulation and the biogenesis of the yolk organelles in the insect vector <i>R</i>. <i>prolixus</i>

<div><p>In oviparous animals, the egg yolk is synthesized by the mother in a major metabolic challenge, where the different yolk components are secreted to the hemolymph and delivered to the oocytes mostly by endocytosis. The yolk macromolecules are then stored in a wide range of endocytic-originated vesicles which are collectively referred to as yolk organelles and occupy most of the mature oocytes cytoplasm. After fertilization, the contents of these organelles are degraded in a regulated manner to supply the embryo cells with fundamental molecules for <i>de novo</i> synthesis. Yolk accumulation and its regulated degradation are therefore crucial for successful development, however, most of the molecular mechanisms involved in the biogenesis, sorting and degradation of targeted yolk organelles are still poorly understood. ATG6 is part of two PI3P-kinase complexes that can regulate the recruitment of the endocytic or the autophagy machineries. Here, we investigate the role of RpATG6 in the endocytosis of the yolk macromolecules and in the biogenesis of the yolk organelles in the insect vector <i>Rhodnius prolixus</i>. We found that vitellogenic females express high levels of RpATG6 in the ovaries, when compared to the levels detected in the midgut and fat body. RNAi silencing of RpATG6 resulted in yolk proteins accumulated in the vitellogenic hemolymph, as a consequence of poor uptake by the oocytes. Accordingly, the silenced oocytes are unviable, white (contrasting to the control pink oocytes), smaller (62% of the control oocyte volume) and accumulate only 40% of the yolk proteins, 80% of the TAG and 50% of the polymer polyphosphate quantified in control oocytes. The cortex of silenced oocytes present atypical smaller vesicles indicating that the yolk organelles were not properly formed and/or sorted, which was supported by the lack of endocytic vesicles near the plasma membrane of silenced oocytes as seen by TEM. Altogether, we found that RpATG6 is central for the mechanisms of yolk accumulation, emerging as an important target for further investigations on oogenesis and, therefore, reproduction of this vector.</p></div

RNAi knockdown of RpATG6 does not affect major physiological processes of <i>R</i>. <i>prolixus</i>.

<p><b>A.</b> PCR showing the knockdown efficiency of RpATG6 in the ovary and fat body at different days after the blood meal. <b>B.</b> qPCR showing the relative expression of RpATG6 in the ovary (7 days after the blood meal). <b>C.</b> PI3P detection by TLC and densitometric quantification (n = 2). <b>D.</b> Effect of RpATG6 knockdown in the blood protein digestion in the midgut. Graph shows mean ± SEM (n = 3). <b>E.</b> Survival curve of silenced females (n = 3). *p<0.05, t-Tests.</p

Knockdown of RpATG6 leads to a decrease in the accumulation of TAG and PolyP but not in the glycogen content of the eggs.

<p><b>A.</b> 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>B.</b> Glycogen content of the eggs of females previously injected with dsMal or dsATG6. <b>C.</b> 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.</p

Knockdown of RpATG6 results in abnormal distribution of the yolk organelles and less endocytic vesicles in the oocytes.

<p><b>Left panel:</b> 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. <b>Right panel:</b> 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.</p

<i>In vivo</i> FITC-uptake by the oocyte yolk organelles.

<p>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.</p

Silencing of RpATG6 increases oviposition but decreases embryo viability.

<p>The oviposition and hatching were monitored after the blood meal. <b>A.</b> Number of eggs laid per female over 4 weeks. Graph shows mean ± SEM (n = 4). <b>B.</b> Total of eggs laid by silenced and control females. Graph shows mean ± SEM (n = 25). **p<0.01, t-Test. <b>C.</b> Hatching rates after silencing of RpATG6. Graph shows mean ± SEM (n = 3). ***p<0.001, t-Test. <b>D.</b> Phenotypic distribution observed after knockdown of RpATG6. Percentage (%) of hatching per phenotype is also showed. <b>E.</b> Representative image of the phenotypes observed in the eggs. (N), normal (control) eggs; (C), collapsed eggs, (W), white eggs.</p

Effects of molecules of distinct redox status on epimastigote proliferation <i>in vitro</i>.

<p><i>T</i>. <i>cruzi</i> epimastigotes (2.5 x 10⁶cells/mL) were incubated in BHI medium supplemented with 10% FCS in the absence (control) or in the presence of 30 μM heme, <b>(A)</b> 30 μM β-hematin; <b>(B)</b> different concentrations of GSH (30 μM or 1 mM) in the absence or presence of 30 μM heme; or with <b>(C)</b> different concentrations of NAC (30 μM or 1 mM) in the absence or presence of 30 μM heme. All data are presented as the means ± standard deviation. Statistical analysis was performed for the 12^th day of treatment, ^*p<0.05 compared with the control group and ^#p<0.05 compared with heme treatment by one-way ANOVA and Tukey’s test.</p

Real-time PCR analysis of parasite loads during R. prolixus infection: the effect of antioxidants in vivo 5 dpi.

<p>Fifth instar <i>R</i>. <i>prolixus</i> nymphs were fed serum-inactivated rabbit blood or blood supplemented with 1 mM NAC or 1 mM urate and 5 x 10⁷ epimastigotes/mL (at least ten insects per group in each experiment). Five days post infection, the bugs were dissected, and the total RNA of the <b>(A)</b> anterior midgut, <b>(B)</b> posterior midgut or <b>(C)</b> the rectum was extracted in TRIZOL reagent. A cDNA strand was synthetized and used as a template for amplification with TCZ primers. RpMIP was used as an endogenous control, ^*p<0.05 compared with the blood group by one-way ANOVA and Tukey’s test.</p