RNA Interference-Mediated Suppression of Ecdysone Signaling Inhibits Choriogenesis in Two Coleoptera Species.

During choriogenesis in insects, chorion (eggshell) is formed by surrounding follicular epithelial cells in ovarioles. However, the regulatory endocrine factor(s) activating choriogenesis and the effect of chemical components on eggshell deserve further exploration. In two representative coleopterans, a coccinellid Henosepilachna vigintioctopunctata and a chrysomelid Leptinotarsa decemlineata, genes encoding the 20-hydroxyecdysone (20E) receptor heterodimer, ecdysone receptor (EcR) and ultraspiracle (USP), and two chitin biosynthesis enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP) and chitin synthase (ChS1), were highly expressed in ovaries of the young females. RNA interference (RNAi)-aided knockdown of either HvEcR or Hvusp in H. vigintioctopunctata inhibited oviposition, suppressed the expression of HvChS1, and lessened the positive signal of Calcofluor staining on the chorions, which suggests the reduction of a chitin-like substance (CLS) deposited on eggshells. Similarly, RNAi of LdEcR or Ldusp in L. decemlineata constrained oviposition, decreased the expression of LdUAP1 and LdChS1, and reduced CLS contents in the resultant ovaries. Knockdown of LdUAP1 or LdChS1 caused similar defective phenotypes, i.e., reduced oviposition and CLS contents in the L. decemlineata ovaries. These results, for the first time, indicate that 20E signaling activates choriogenesis in two coleopteran species. Moreover, our findings suggest the deposition of a CLS on the chorions.

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.

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.

The transition from vitellogenesis to choriogenesis triggers the downregulation of the UPR sensors IRE1 and PERK and alterations in the ER architecture in the follicle cells of the vector Rhodnius prolixus.

In insects, the follicle cells (FCs) give rise to a single-layered tissue of binucleated professional secretory cells that surround the oocytes during oogenesis. In the latest stage of oocyte development, the FCs rapidly synthesize and secrete the chorion (eggshell) immediately before degenerating through apoptosis. Here, we used RT-qPCR, electron microscopy, and RNAi silencing to explore the role of the main unfolded protein response (UPR) receptors IRE1 and PERK, as well as the ultrastructure dynamics of the FCs during oogenesis of the insect vector of Chagas disease Rhodnius prolixus. We found that IRE1 and PERK mRNAs are highly expressed in the ovaries of vitellogenic females. Interestingly, we observed that IRE1 and PERK, as well as different isoforms of the chaperones Bip and PDI, have their FCs gene expression levels decreased during the vitellogenesis to choriogenesis transition. Using transmission electron microscopy, we observed that the downregulation of the UPR gene expression is accompanied by dramatic changes in the FCs ultrastructure, with an 80% reduction in the mean area of the ER tubules, and circularization and enlargement of the mitochondria. Additionally, we found that parental RNAi silencing of both IRE1 and PERK resulted in minor changes in the chorion protein composition and ultrastructure, accessed by urea extraction of the chorion proteins and scanning electron microscopy, respectively, but did not impact the overall levels of oviposition and F1 embryo development.

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

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 parental RNAi 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.

An ovary-specific mucin is associated with choriogenesis mediated by prostaglandin signaling in Spodoptera exigua.

Polytrophic ovarioles of Spodoptera exigua, a lepidopteran insect, begins with the development of oocytes and differentiation of nurse cells followed by vitellogenesis and choriogenesis. Compared with previtellogenic and vitellogenic developments, choriogenesis has not been clearly understood yet in endocrine control. This study investigated the expression and function of a mucin-like structural protein of S. exigua called Se-Mucin1 in choriogenesis. It was highly expressed in ovarioles containing chorionated oocytes. The expression level of Se-Mucin1 was increased during adult stage as early as 18 h after adult emergence, reaching the maximal level at 24 h and later. Interestingly, DNA amount of Se-Mucin1 was increased by almost four folds during early adult stage while other genes (hexokinase and glyceraldehyde-3-phosphate dehydrogenase) not directly associated with chorion formation did not show genomic DNA increase, suggesting specific gene amplification of Se-Mucin1. RNA interference (RNAi) suppressed Se-Mucin1 expression by injecting 1 µg of double-strand RNA to teneral females (<5 h after emergence), which exhibited significantly impaired fecundity and egg hatching rate. Eggs laid by RNAi-treated females were malformed in eggshell structures with loss of mesh-like fibers. Treatment with aspirin, a prostaglandin (PG) biosynthesis inhibitor, suppressed the induction of Se-Mucin1 expression during early adult stage and impaired egg development. An addition of PGE2 significantly rescued such impairment in Se-Mucin1 expression and subsequent egg development. These results suggest that PGs mediate choriogenesis of S. exigua by activating the expression of chorion-associated genes including Se-Mucin1.

Chorion genes: a landscape of their evolution, structure, and regulation.

Differential regulation at the level of transcription provides a means for controlling gene expression in eukaryotes, especially during development. Insect model systems have been extensively used to decipher the molecular basis of such regulatory cascades, and one of the oldest such model systems is the regulation of chorion gene expression during ovarian follicle maturation. Recent experimental and technological advances have shed new light onto the system, allowing us to revisit it. Thus, in this review we try to summarize almost 40 years' worth of studies on chorion gene regulation while-by comparing Bombyx mori and Drosophila melanogaster models-attempting to present a comprehensive, unified model of the various regulatory aspects of choriogenesis that takes into account the evolutionary conservation and divergence of the underlying mechanisms.

Analysis of developmentally regulated chorion gene promoter architecture via electroporation of silk moth follicles.

In the silk moth Bombyx mori, chorion genes of the same developmental specificity are organized in divergently transcribed α/ß gene pairs, sharing a common 5' flanking promoter region. This bidirectional promoter contains a complete set of cis-elements responsible for developmentally accurate gene expression. In the present paper, based on the observation that Bombyx chorion gene promoters contain cis-elements for the same transcription factors without concrete evidence on which of them are essential, we address the question as to how promoter architecture (number, orientation and position of common factor binding sites) facilitates developmentally accurate chorion gene regulation. To this end, we constructed several mutated promoter regions of an early-middle gene pair and cloned them upstream of a reporter gene to introduce these plasmid constructs into silk moth follicle epithelial cells via electroporation as an efficient and quick method for transient expression. This is the first time that an ex vivo method had been applied to test the impact of systematic cis-element mutations on a chorion gene promoter. Our results confirmed the importance of the HMGA factor and the role of the GATA factor as an early repressor, and led to a more detailed understanding of which C/EBP sites participate in the regulation of early-middle chorion gene expression.

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

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.

Gene expression profiling of ovary identified eggshell proteins regulated by 20-hydroxyecdysone in Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive pests worldwide. The frequent use of chemical insecticides has led B. dorsalis to develop resistance to many insecticides in recent decades. New high-throughput-sequenced transcriptomes, as well as genomes, have revealed a large number of reference genes for functional target identification. Here, we performed digital gene expression profiling of ovary and testis of B. dorsalis adults. Various genes were identified to be highly expressed in B. dorsalis ovary. The genes encoding components of eggshell, vitelline membrane proteins (Vmps) and chorion-related proteins, were identified to be tissue-specifically expressed in ovary. Five cytochrome P450 genes were also identified to be highly expressed in ovary. Three of them were ecdysone synthesis pathway genes indicating the ovary as a potential synthesis site of female. The up-regulated expression of Vmps by exogenous 20-hydroxyecdysone implied the hormonal regulation of eggshell formation during ovarian development. Many other genes with potential functions in ovarian development were also identified, including vitellogenin receptor, insulin receptor, NASP protein, and odorant binding protein. These findings should promote our understanding of the regulation of vitellogenesis and eggshell formation and enable exploration of potentially novel pest control targets.

Inhibition of prostaglandin biosynthesis leads to suppressed ovarian development in Spodoptera exigua.

Prostaglandins (PGs) are a group of eicosanoids that are C20 oxygenated polyunsaturated fatty acids. PGs can mediate various physiological processes such as immunity, salivary secretion, excretion, and reproduction in insects. The objective of this study was to determine the effect of PG on oocyte development in Spodoptera exigua, a lepidopteran insect known to biosynthesize PGs. Polytrophic ovarioles of S. exigua females exhibited follicle development in germarium, in which oocytes were distinct from nurse cells. During vitellogenesis, nurse cells degenerated by losing cytoplasm called "nurse cell dumping" while oocytes showed increase in cell volume. When PG biosynthesis inhibitors such as ibuprofen or aspirin were applied, nurse cell dumping was not complete and no chorion was formed, thus preventing egg formation. However, addition of PGE2 significantly rescued such inhibition and resumed oocyte development and choriogenesis. To support the observation with genetic factor, RNA interference (RNAi) specific to peroxynectins (Pxts: Se-Pxt1 and Se-Pxt2) known to act as insect cyclooxygenase was performed to suppress PG biosynthesis. Both Se-Pxt1 and Se-Pxt2 were highly expressed in the ovary of control female. RNAi treatment against Se-Pxt1 or Se-Pxt2 specifically suppressed target genes and inhibited oocyte development. Addition of PGE2 to adults treated with RNAi rescued the suppressed development of oocytes. Results of this study suggest that PGs can stimulate oocyte development as autocrine/paracrine mediators of vitellogenesis and choriogenesis in insects.