MiRNAs and hormonal pathways regulating postembryonic development in insects

This thesis focusses on the study of the physiological role and mode-of-action of hormonal and small RNA pathways in the control of post-embryonic development and reproduction in locusts, which are globally considered as the most devastating migratory pest insects. Juvenile hormones (JH) are key endocrine regulators produced by the corpora allata (CA) of insects. Together with ecdysteroids, as well as nutritional cues, JH coordinates different aspects of insect post-embryonic development and reproduction. The JH signalling pathway was only recently described with the characterization of a genuine JH receptor, Methoprene-tolerant (Met), a transcription factor belonging to the basic-helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) family. Binding of JH stimulates Met to form a complex with other bHLH-PAS proteins, which in turn induces the expression of JH response genes, such as krüppel-homolog 1 (Kr-h1), the main effector of the anti-metamorphic action of JH. The ecdysteroid early response gene, E93, is a key determinant promoting adult morphogenesis. The ancestral regulatory mechanism by which JH inhibits metamorphosis is defined as the MEKRE93 pathway. The function of Met appears to be required in a wide variety of processes regulated by JH. However, its functional interactions with other hormonal signalling pathways seem highly dependent on the feeding habits and on the developmental and reproductive strategies employed by the insect species investigated. In this thesis, we report on the effects of RNA interference (RNAi) mediated SgMet knockdown during the first gonadotrophic cycle in female desert locusts (Schistocerca gregaria). We show that the JH receptor Met is essential for ovarian maturation, vitellogenesis and associated ecdysteroid biosynthesis in adult female S. gregaria. Interestingly, knockdown of SgMet also resulted in a significant decrease of insulin-related peptide (SgIRP) and increase of neuroparsin (SgNP) 3 and 4 transcript levels in the fat body, illustrating the existence of an intricate regulatory interplay between different hormonal factors. In addition, SgMet knockdown in females resulted in delayed display of copulation behaviour with virgin males, when compared with dsGFP injected control animals. Moreover, we observed an incapacity of adult dsSgMet injected female locusts to oviposit during the time of the experimental setup. Kr-h1 is a zinc-finger transcription factor maintaining the status quo in immature insect stages and promoting reproduction in adult insects through transduction of the JH signal. Knockdown studies have shown that precocious silencing of Kr-h1 in the immature stages results in premature development of adult features. However, the molecular characteristics and reproductive potential of these premature adult insect stages are still poorly understood. In this thesis, we report on an adult-like or 'adultoid' phenotype of the migratory locust, Locusta migratoria, obtained after a premature metamorphosis induced by silencing of Kr-h1 in the penultimate instar. The freshly moulted adultoid showed precocious development of adult features, coinciding with increased LmE93 transcript levels. Furthermore, accelerated ovarian maturation and vitellogenesis were observed in female adultoids, coinciding with elevated expression of LmCYP15A1 in corpora allata (CA) and LmKr-h1 and vitellogenin genes (LmVg) in fat body, whereas LmE93 and Methoprene-tolerant (LmMet) transcript levels decreased in fat body. Expression of several Halloween genes in the ovaries was elevated as well. In addition, the processes of copulation and oviposition were severely disturbed in these females. Where Kr-h1 is maintaining the status quo in immature insect stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. In this thesis, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria. We describe a supernumerary nymphal phenotype which still displayed juvenile morphological features, such as a nymphal colour scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary moult. Interestingly, when compared to control locusts, the total duration of the fifth, and normally final, nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in the haemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, also the levels of the Ecdysone receptor (SgEcR) and Retinoid-X-Receptor (SgRXR) transcripts were altered, suggesting that the silencing of SgE93 had affected both ecdysteroid synthesis and signalling. A very potent upregulation of the SgKr-h1 transcript levels was also observed upon knockdown of SgE93. Moreover, the process of moulting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50 % of the N6 and all N7 nymphal instars eventually died. Recently, the importance of microRNAs (miRNAs) in regulating key insect processes was established as well. Especially the study of extracellular miRNAs (ex-miRNAs) is a new, still underexplored, but very intriguing research area. In the present study, we demonstrated the presence of ex-miRNAs in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates (EAgo-1), obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed these qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects. Furthermore, we have also demonstrated the presence of extracellular miRNAs in haemolymph of fifth nymphal L. migratoria. More specifically, we analyzed small RNA sequencing reads of of fifth nymphal L. migratoria haemolymph characterized by low (early fifth nymphal) versus high (late fifth nymphal) ecdysteroid levels. Differential expression analysis revealed that the relative abundances of 43 miRNAs significantly differed between the tested conditions. To our knowledge, this is the first report on the differential extracellular miRNA occurrence throughout the moulting cycle of an insect. Although the transcriptional regulation of ecdysteroid and MEKRE93 pathway components has been extensively studied, little is known about their regulation at the post-transcriptional level. In this study, we have reported the importance of specific miRNAs in the metamorphic transition into adult L. migratoria. Systemic RNAi mediated silencing of LmDicer-1, an enzyme that is required for miRNA biogenesis, during the fifth nymph stage disrupted the nymphal-to-adult moult. Moreover, mimicking Lmi-miR-13b-3p (agomiR) in late instar L. migratoria caused a prolonged duration of the fifth nymphal stage, whereas the majority (60 %) of the Lmi-bantam-3p agomiR- and 20 % of Lmi-miR-306-5p agomiR-injected fifth nymphal locusts showed severe moulting defects and eventually died. Altogether, these findings support a role for miRNAs as important players in the metamorphic transition in L. migratoria. L. migratoria and S. gregaria are well-known, swarm-forming pest insects that can destroy crops and harvests in different continents of the world, including some of the world's poorest countries. Even today, locusts are threatening food supply and livelihoods of millions of people. As such, components of pathways regulating crucial insect processes, as investigated in this thesis, are very promising candidate targets for future development of novel, more selective locust control strategies.status: accepte