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

Abstract

In insects, the last stage of oogenesis is the process where the chorion layers (eggshell) are synthesized and deposited on the surface of the oocytes by the follicle cells. Protein homeostasis is determined by the fine‐tuning of translation and degradation pathways, and the ubiquitin–proteasome system is one of the major degradative routes in eukaryotic cells. The conjugation of ubiquitin to targeted substrates is mediated by the ordered action of E1‐activating, E2‐conjugating, and E3‐ligase enzymes, which covalently link ubiquitin to degradation‐targeted proteins delivering them to the proteolytic complex proteasome. Here, we found that the mRNAs encoding polyubiquitin (pUbq), E1, and E2 enzymes are highly expressed in the ovaries of the insect vector of Chagas Disease Rhodnius prolixus. RNAi silencing of pUbq was lethal whereas the silencing of E1 and E2 enzymes resulted in drastic decreases in oviposition and embryo viability. Eggs produced by the E1‐ and E2‐silenced insects presented particular phenotypes of altered chorion ultrastructure observed by high‐resolution scanning electron microscopy as well as readings for dityrosine cross‐linking and X‐ray elemental microanalysis, suggesting a disruption in the secretory routes responsible for the chorion biogenesis. In addition, the ovaries from silenced insects presented altered levels of autophagy‐related genes as well as a tendency of upregulation in ER chaperones, indicating a disturbance in the general biosynthetic‐secretory pathway. Altogether, we found that E1 and E2 enzymes are essential for chorion biogenesis and that their silencing triggers the modulation of autophagy genes suggesting a coordinated function of both pathways for the progression of choriogenesis.

Functional characterization of maternally accumulated hydrolases in the mature oocytes of the vector Rhodnius prolixus reveals a new protein phosphatase essential for the activation of the yolk mobilization and embryo development

“…Within the ovary, all mRNAs tended to be more expressed in the tropharium, suggesting that this site is indeed the main source of maternal mRNA synthesis in the ovary and that cytoplasmic bridges are used to deliver the mRNAs to the transcriptionally inactive developing oocytes (Kunkel, 1991). This pattern, of highest expression levels in the tropharium, has been observed before in R. prolixus for genes of the autophagy pathway (Vieira et al, 2018;Bomfim and Ramos, 2020;Pereira et al, 2020), the ubiquitin-proteasome system (Pereira et al, 2022) and the unfolded protein response (Rios et al, 2022). It is important to notice, however, that although the temperature, humidity, light/dark cycles, and blood feeding intervals for each insect in this study were strictly controlled, it is expected that adult insects develop certain phenotypes during their early life.…”

Silencing of the 20S proteasomal subunit-α6 triggers full oogenesis arrest and increased mRNA levels of the selective autophagy adaptor protein p62/SQSTM1 in the ovary of the vector Rhodnius prolixus

“…While proteasome activity is regarded as widely conserved and ubiquitous, functional investigations have shown that proteasomes have a specific role in the reproduction biology of several models [ 35 , 42 , 47 , 49 ], particularly in the signals that trigger oocyte maturation [ 36 – 40 , 43 ]. In this study, we discovered that the vitellogenic ovaries exhibit significant proteasomal activity, and that the silencing of the essential 20S proteasome subunit Prosα6 causes reduced proteasomal activity, which ultimately leads to complete oogenesis arrest and reproduction ablation in the Chagas disease vector R .…”

“…In the kissing bug R . prolixus , RNAi knockdown of the ubiquitin enzymes E1 and E2 resulted in impaired oogenesis and embryo lethality [ 49 ].…”

Vector Control: Insights Arising from the Post-Genomics Findings on Insects’ Reproductive Biology

“…Accordingly, electron microscopy evidenced the FC's typical secretory cell morphology with a high content of vesiculated rough endoplasmic reticulum [57,58]. Systemic RNAi experiments targeting the autophagy-related genes ULK1/ATG1, the autophagy-dedicated E2-conjugating enzyme ATG3 [57,59], and E1-activating and E2-conjugating ubiquitin enzymes [60] generated particular phenotypes of chorion malformations due to alterations in the general protein homeostasis of the FCs during choriogenesis, resulting in extremely lower rates of embryo viability. Taken together, the data points to a high degree of complexity in the chorion biogenesis program in R. prolixus, rendering the process extremely sensitive to changes in proteostasis of the FCs, and, thus, an interesting target for slight but effective interventions.…”