Eggshell ultrastructure and delivery of pharmacological inhibitors to the early embryo of R. prolixus by ethanol permeabilization of the extraembryonic layers

Abstract

Most vectors of arthropod-borne diseases produce large eggs with hard and opaque eggshells. In several species, it is still not possible to induce molecular perturbations to the embryo by delivery of molecules using microinjections or eggshell permeabilization without losing embryo viability, which impairs basic studies regarding development and population control. Here we tested the properties and permeability of the eggshell of R. prolixus, a Chagas disease vector, with the aim to deliver pharmacological inhibitors to the egg cytoplasm and allow controlled molecular changes to the embryo. Using field emission scanning and transmission electron microscopy we found that R. prolixus egg is coated by three main layers: exochorion, vitelline layer and the plasma membrane, and that the pores that allow gas exchange (aeropiles) have an average diameter of 10 μm and are found in the rim of the operculum at the anterior pole of the egg. We tested if different solvents could permeate through the aeropiles and reach the egg cytoplasm/embryo and found that immersions of the eggs in ethanol lead to its prompt penetration through the aeropiles. A single five minute-immersion of the eggs/embryos in pharmacological inhibitors, such as azide, cyanide and cycloheximide, solubilized in ethanol resulted in impairment of embryogenesis in a dose dependent manner and DAPI-ethanol solutions were also able to label the embryo cells, showing that ethanol penetration was able to deliver those molecules to the embryo cells. Multiple immersions of the embryo in the same solutions increased the effect and tests using bafilomycin A1 and Pepstatin A, known inhibitors of the yolk proteolysis, were also able to impair embryogenesis and the yolk protein degradation. Additionally, we found that ethanol pre-treatments of the egg make the aeropiles more permeable to aqueous solutions, so drugs diluted in water can be carried after the eggs are pre-treated with ethanol. Thus, we found that delivery of pharmacological inhibitors to the embryo of R. prolixus can be performed simply by submersing the fertilized eggs in ethanol with no need for additional methods such as microinjections or electroporation. We discuss the potential importance of this methodology to the study of this vector developmental biology and population control.

Deficiency of ULK1/ATG1 in the follicle cells disturbs ER homeostasis and causes defective chorion deposition in the vectorRhodnius prolixus

“…While dissecting the insects under the stereomicroscope, we noticed that some of the silenced eggs were penetrable to saline solution, contrasting the chorion properties of control eggs, which are highly impermeable to polar solvents ( Figure 2D, Video S1). 28 To test if the observed differences in permeability were due to ultrastructure alterations, we processed the chorion of freshly laid eggs for scanning electron microscopy (SEM) (Figure 2E-G). ULK1-silenced small eggs presented a milder phenotype, where the typical hexagonal patterns of the depressions in the chorion (where the FCs used to occupy) ( Figure 2E) are not well defined and spaces among the depressions can be observed (arrows) ( Figure 2F).…”

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

“…After choriogenesis the mature oocyte is ready to be fertilized and laid in the environment. In species that colonize land, the chorion layers allow gas exchange and serve as a shielding barrier to impair water loss during embryogenesis ( Kerkut and Gilbert, 1985 ; Bomfim et al, 2017 ). The process of chorion formation and its complex ultrastructure represent a remarkable model for in vivo studies of biogenesis and assembly of the network of macromolecules in an extracellular matrix.…”

“…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 also described ( Bouts et al, 2007 ).…”

The Chorion Proteome of Diaphorina citri, the Vector of Huanglongbing Disease in Citrus

“…During incubation, the chorion (eggshell) protects insect embryos from the environment [38]. During the later stages of oogenesis, the chorion matrix is secreted between the oocyte and overlaying somatic follicle cells [39]. Its multilayered structure performs a variety of specialized functions such as providing substrate attachment, resistance to acid or alkalis, preventing water loss, and allowing gas exchange throughout development [39,40].…”

“…During the later stages of oogenesis, the chorion matrix is secreted between the oocyte and overlaying somatic follicle cells [39]. Its multilayered structure performs a variety of specialized functions such as providing substrate attachment, resistance to acid or alkalis, preventing water loss, and allowing gas exchange throughout development [39,40]. The differences in ecological environment and, specifically, oviposition substrates have determined through evolution the chorion morphology, organization, and composition among different species [41].…”