MOLECULAR OOGENESIS OF INSECT VECTORS
RESEARCH/PESQUISA
The vector of Chagas Disease R. prolixus/ Mature oocyte seen under the stereoscope/ Scanning electron microscopy high resolution image the organelles inside the oocyte.
The lab's primary research interest is to understand the essential cellular and molecular mechanisms that control oogenesis with particular emphasis on basic model systems and in insect vectors of neglected tropical diseases (NTDs) and to determine how these mechanisms can be explored for the management of vector population control.
Our short term goal is to build a biochemical and genetic framework to elucidate the machinery and the molecular mechanisms underlying the calcium-dependent pathways that allow the fine-regulated mechanisms of secretion, meiotic progression, and intracellular yolk protein degradation in NTD insect vectors. Our projects employ the techniques of RNAi and CRISPR to disturb the expression of selected genes and interpret the phenotypes to test hypotheses regarding the biological function of selected cell pathways.
PI3K complexes signal to recruit the yolk endocytic machinery:
Dissected ovaries from control and RNAi silenced insects.
In 2005, the WHO created the department of Neglected Tropical Diseases (NTDs), recognizing their importance and aiming to manage their incidence mostly in Africa and Latin America (http://www.who.int/neglected_diseases/en/). Rhodnius prolixus is one of the main vectors of Chagas disease, which is one of the several NTDs that are important in Latin America. Currently 6 to million people are estimated to be infected by Chagas Disease, and vector control is still the most useful method to prevent this illness. Because embryo development relies entirely on the buildup of the yolk nutritional reserves, the proper biogenesis of the yolk organelles is indispensable for vector reproduction representing a promising target for interference in vector’s reproduction.
In this project we investigate 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.
Knockdown of ULK1 and Sec16 trigger changes in the ER and mitochondria architectures and impair chorion biogenesis:
Nucleus
Endoplasmic Reticulum
Lipid Droplets
Mitochondria
3D reconstruction of the perinuclear region of a follicle cell designed using the technique of Focused Ion Beam Scanning electron micorscopy (FIB-SEM)
In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parentalRNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to
the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.
The autophagy machinery and follicular atresia triggered under immune stress:
Yolk organelles from control and silenced oocytes stained with acidic marker acridine orange and observed under the fluoresecence microsocpe.
Follicular atresia is the mechanism by which the oocyte contents are degraded during oogenesis in response to stress conditions, allowing the energetic resources stored in the developing oocytes to be reallocated to optimize female fitness. Autophagy is a conserved intracellular degradation pathway where double-membrane vesicles are formed around target
organelles leading to their degradation after lysosome fusion. The autophagy-related protein 8 (ATG8) is conjugated to the autophagic membrane and has a key role in the elongation and closure of the autophagosome. Here we identified one single isoform of ATG8 in the genome of the insect vector of Chagas Disease Rhodnius prolixus (RpATG8) and found that it is highly expressed in the ovary during vitellogenesis. Accordingly, autophagosomes were detected in the vitellogenic oocytes, as seen by immunoblotting and electron microscopy. To test if autophagosomes were important for follicular atresia, we silenced RpATG8 and elicited atresia in vitellogenic females by Zymosan-A injections. We found that silenced females were still able to trigger the same levels of follicle atresia, and that their atretic oocytes presented a characteristic morphology, with accumulated brown aggregates. Regardless of the difference in morphology, RpATG8-silenced atretic oocytes presented the same levels of protein, TAG and PolyP, as detected in control atretic oocytes, as well as the same levels of acidification of the yolk organelles. Because follicular atresia has the ultimate goal of restoring female fitness, we tested if RpATG8-silenced atresia would result in female physiology and behavior changes. Under insectarium conditions, we found that atresia- induced control and RpATG8-silenced females present no changes in blood meal digestion, survival, oviposition, TAG content in the fat body, haemolymph amino acid levels and overall locomotor activity. Altogether, we found that autophagosomes are formed during oogenesis and that the silencing of RpATG8 impairs autophagosome biogenesis in the oocytes. Nevertheless, regarding major macromolecule degradation and adaptations to the fitness costs imposed by triggering an immune response, we found that autophagic organelles are not essential for follicle atresia in R. prolixus.
The autophagy-related gene 3 is essential for the chorion biogenesis
Scanning electron micrographs of the chorion outer surface in eggs laid by control and silenced insects/ X-ray elemental mapping of the main elements in the chorion outer surface.
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.
FINANCIAMENTO
