Silencing of RpATG8 impairs the biogenesis of maternal autophagosomes in vitellogenic oocytes, but does not interrupt follicular atresia in the insect vector Rhodnius prolixus

Abstract

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 PLOS Neglected Tropical Diseases | https://doi.

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

“…The unrelated bacterial MalE gene was used as a control dsRNA, as previously described. 24,26 Knockdown efficiency was confirmed by RT-qPCR at different days after blood meal.…”

“…Alignments of the ULK1/ATG1 protein sequence to the ULK1/ATG1 sequences from different species were performed using Clustal Omega (https://www.ebi.ac.uk/Tools/ msa/clust alo/). The ATG8, 24 IRE, PERK, and SEC16A sequences are listed in Table S1.…”

“…For LC3/ATG8, the dsRNA fragment was previously reported in. 24 Unfed adult females were injected with 1 µg of a mix of the two dsRNAs (0.5 µg of each) for ULK1 and SEC16A, and 1 µg of dsRNA for IRE, PERK and ATG8/LC3, and fed 2 days later. The unrelated bacterial MalE gene was used as a control dsRNA, as previously described.…”

Bicaudal Cis required for the function of the follicular epithelium during oogenesis inRhodnius prolixus

“…The oogenesis of R. prolixus is one of the best studied among insects, from the morphological work of Huebner [22,24–26,34,63] to the biochemistry studies of Masuda [64–70]. Although studies on the cellular biology of oogenesis have recently emerged [71,72], we still lack enough information on genetic to understand the molecular basis of egg formation and patterning in R. prolixus . This is the first report on the function of Rp-BicC in R. prolixus to provide a bona fide mechanism for egg formation.…”

“…The physiology and biochemistry of yolk metabolism is well known in R. prolixus [41], however, the molecular and cellular mechanisms of egg formation and yolk accumulation are still scarce. It has been recently demonstrated that Rp-ATG6 and Rp-ATG8 , part of PI3P-kinase complexes that regulate the endocytic and autophagy machinery, are essential for yolk accumulation [71,72]. The phenotype of silenced Rp-ATG6 females shares similarities with the one of Rp-BicC : unviable, small and white eggs that accumulate a minor fraction of yolk.…”