VPS38/UVRAG and ATG14, the variant regulatory subunits of the ATG6/Beclin1-PI3K complexes, are crucial for the biogenesis of the yolk organelles and are transcriptionally regulated in the oocytes of the vector Rhodnius prolixus.

In insects the reserve proteins are stored in the oocytes into endocytic-originated vesicles named yolk organelles. VPS38/UVRAG and ATG14 are the variant regulatory subunits of two class-III ATG6/Beclin1 PI3K complexes that regulate the recruitment of the endocytic (complex II) and autophagic (complex I) machineries. In a previous work from our group, we found that the silencing of ATG6/Beclin1 resulted in the formation of yolk-deficient oocytes due to defects in the endocytosis of the yolk proteins. Because ATG6/Beclin1 is present in the two above-described PI3K complexes, we could not identify the contributions of each complex to the yolk defective phenotypes. To address this, here we investigated the role of the variant subunits VPS38/UVRAG (complex II, endocytosis) and ATG14 (complex I, autophagy) in the biogenesis of the yolk organelles in the insect vector of Chagas Disease Rhodnius prolixus. Interestingly, the silencing of both genes phenocopied the silencing of ATG6/Beclin1, generating 1) accumulation of yolk proteins in the hemolymph; 2) white, smaller, and yolk-deficient oocytes; 3) abnormal yolk organelles in the oocyte cortex; and 4) unviable F1 embryos. However, we found that the similar phenotypes were the result of a specific cross-silencing effect among the PI3K subunits where the silencing of VPS38/UVRAG and ATG6/Beclin1 resulted in the specific silencing of each other, whereas the silencing of ATG14 triggered the silencing of all three PI3K components. Because the silencing of VPS38/UVRAG and ATG6/Beclin1 reproduced the yolk-deficiency phenotypes without the cross silencing of ATG14, we concluded that the VPS38/UVRAG PI3K complex II was the major contributor to the previously observed phenotypes in silenced insects. Altogether, we found that class-III ATG6/Beclin1 PI3K complex II (VPS38/UVRAG) is essential for the yolk endocytosis and that the subunits of both complexes are under an unknown transcriptional regulatory system.

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

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 de novo 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 Rhodnius prolixus. 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.