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

Anna Santos,Isabela Ramos

doi:10.3389/fphys.2021.638026

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.

Highlights

The ability of insects to produce a large number of eggs in a short period of time is one of the characteristics that contributes to their remarkable adaptive success in inhabiting many different habitats and acting as vectors of human diseases
We found that ATG3 is important for the proper chorion biogenesis and, crucial for this vector reproduction
Using quantitative PCR (RT-qPCR) we found that the ovary of vitellogenic females express an average of 3× more ATG3 than the midgut and fat body (Figure 1A)

Summary

Introduction

The ability of insects to produce a large number of eggs in a short period of time is one of the characteristics that contributes to their remarkable adaptive success in inhabiting many different habitats and acting as vectors of human diseases. Exploring the specific mechanisms of egg formation and embryo development should be of great importance to further understand the reproductive biology of various vectors and to elaborate new strategies for vector population control (Pereira et al, 2020). This is especially important for the neglected, vector borne tropical diseases, which are endemic to developing countries such as dengue fever and Chagas Disease. In Drosophila, studies about choriogenesis focus mostly on the programed gene-specific transcriptional activation of chorion genes (Tootle et al, 2011; Velentzas et al, 2018) and on the biochemical characterization of chorion proteins, mainly by mass spectrometry (Fakhouri et al, 2006). In Rhodnius prolixus, the chorion ultrastructure and permeability properties were previously explored (Beament, 1948; Dias et al, 2013; Bomfim et al, 2017; Bomfim and Ramos, 2020) and the identification of the specific chorion proteins Rp30 and Rp45, the latter associated to an antifungal activity, were described (Bouts et al, 2007)

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Conclusion