ATG3 Is Important for the Chorion Ultrastructure During Oogenesis in the Insect Vector Rhodnius prolixus

Abstract

In insects, the last stage of the oogenesis is the choriogenesis, a process where the multiple layers of the chorion are synthesized, secreted, and deposited in the surface of the oocytes by the follicle cells. The chorion is an extracellular matrix that serves as a highly specialized protective shield for the embryo, being crucial to impair water loss and to allow gas exchange throughout development. The E2-like enzyme ATG3 (autophagy related gene 3) is known for its canonical function in the autophagy pathway, in the conjugation of the ubiquitin-like ATG8/LC3 to the membranes of autophagosomes. Although the ATGs were originally described and annotated as genes related to autophagy, additional functions have been attributed to various of these genes. Here, we found that Rhodnius prolixus ATG3 is highly expressed in the ovaries of the adult vitellogenic females. Parental RNAi depletion of ATG3 resulted in a 15% decrease in the oviposition rates of depleted females and in the generation of unviable eggs. ATG3-depleted eggs are small and present one specific phenotype of altered chorion ultrastructure, observed by high resolution scanning electron microscopy. The amounts of the major chorion proteins Rp30, Rp45, Rp100, and Rp200 were decreased in the ATG3-depleted chorions, as well as the readings for dityrosine cross-linking and sulfur, detected by fluorescence emission under ultraviolet excitation and X-ray elemental detection and mapping. Altogether, we found that ATG3 is important for the proper chorion biogenesis and, therefore, crucial for this vector reproduction.

Knockdown of E1‐ and E2‐ubiquitin enzymes triggers defective chorion biogenesis and modulation of autophagy‐related genes in the follicle cells of the vector Rhodnius prolixus

“…Such regulations were observed before in mammalian cells, where proteasome inhibition triggered the induction of p62/SQSTM and ATG8/GABARAP (Sha et al, 2018). This hypothesis is further supported by the fact that similar results of chorion malformation were observed in R. prolixus in experiments of RNAi targeting ULK1/ATG1 and the autophagydedicated E2-conjugating enzyme ATG3 Santos & Ramos, 2021). Nevertheless, it is important to consider that although all dsRNAs we tested in silico for potential offtargets with no significant predictions, it is not possible to completely rule out off-target effects, as well as pleiotropic effects contributing to the observed phenotypes.…”

Dynamics of maternal gene expression in Rhodnius prolixus

“…Early oogenesis (genes that affect germ cell survival or results in atresic follicles): Rp-ATG-8 112 ; Rp-cactus 46 ; Rp-Piwi-2 , Rp-Piwi-3 , Rp-Argonauta-3 48 ; 2. Late oogenesis (genes that results in eggs with incomplete yolk load or impaired choriogenesis): Rp-Bicaudal C 50 ; ULK/Rp-ATG-1 113 ; Rp-ATG-3 114 ; Rp-ATG-6 115 ; 3. Embryonic patterning genes: Rp-dorsal 46 ; Rp-Bicaudal D (this work).…”

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

“…This is compatible with the fact those genes are highly expressed in the ovaries and accumulated in the oocytes. Also, it is interesting to note that the elevated expressions of VPS38/UVRAG and ATG14 in the tropharium suggest that the nurse cells (located in the tropharium and connected to the oocytes by cytoplasmic bridges) are indeed an important site of maternal mRNAs transcription and delivery to the transcriptionally inactive developing oocytes [75], and this pattern has been previously observed for other ATGs in R. prolixus [76][77][78].…”