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Schistocerca gregaria or the desert locust is a ravenous swarm-forming phytophagous pest insect, which can form a real threat to agricultural production in some of the world’s poorest countries. Most of the time, these locusts live a solitary life, which is relatively harmless. However, under certain circumstances they can undergo a phase transition to the swarm-forming gregarious phase. Swarms may contain millions of individuals eating everything on their path. Plagues can cover an area of more than 30 million square kilometres between the west of Northern Africa and the eastern border of India. Unfortunately, non-specific chemical insecticides are still the preferred method to combat locust plagues, thereby having a negative impact on the environment and non-target organisms. Hence, the search for new, more biorational strategies to control locusts is crucial.An important reason why insects have been so undeniably successful on this planet is the protection provided to them by the presence of a rigid exoskeleton or cuticle. Although the rigidity of this cuticle protects them, it renders the apparently ‘simple’ process of growing extremely complex. For allowing the insect to increase in volume, it must be shed periodically and a larger one must be synthesized in a process called moulting. Failure to successfully moult will in most cases result in death, making this process an excellent target in the search for new insect pest management strategies. Another important feature in the life cycle of insects is reproduction. In the case of S. gregaria the high reproductive rate is one of the reasons why it is so difficult to effectively control this pest insect. Consequently, reproduction is another excellent target process to combat this harmful pest insect.In this thesis, we investigated several signalling pathways involved in the regulation of moulting and reproduction. The common theme running through all subprojects was the arthropod-specific hormone family of ecdysteroids. Ecdysteroids play an important role in regulating diverse physiological processes, such as moulting and metamorphosis, reproduction, diapause and innate immunity. Cytochrome P450 enzymes encoded by the Halloween genes, Spook, Phantom, Disembodied, Shadow and Shade, catalyse the final steps in ecdysteroid biosynthesis. Ecdysteroids mediate their response by binding to a heterodimeric complex of two nuclear receptors, the ecdysone receptor (EcR) and the retinoid-X-receptor/ultraspiracle (RXR/USP). This receptor complex binds to ecdysone response elements (EcRE) associated with the target genes. Upon binding of its ligand a cascade of transcription factors will be transcribed, which in turn mediate specific developmental and reproductive responses.First, we examined the role of EcR and RXR in metamorphosis and development of S. gregaria. We performed an in-depth profiling study of the transcript levels of SchgrEcR and SchgrRXR, as well as its downstream response genes, in different tissues isolated throughout the last instar stage, showing a clear correlation with circulating ecdysteroid titres. Using RNA interference (RNAi), we have proven the importance of the receptor components, SchgrEcR and SchgrRXR, for successful moulting of locust nymphs into the adult stage. Some SchgrEcR/SchgrRXR knockdown females were arrested in the last instar stage, and 65 % of them initiated vitellogenesis and oocyte maturation, which normally only occurs in adults. Furthermore, our results clearly indicate that at the peak of ecdysteroid synthesis, on day six of the last instar stage, knockdown of SchgrEcR/SchgrRXR is affecting the transcript levels of the Halloween genes, Spook, Shadow and Shade.Second, we investigated a key factor in the neuropeptidergic cascade regulating moulting. In Holometabola, a pulse in ecdysteroids activates a peptide-mediated signalling cascade, in which ecdysis triggering hormone (ETH) acts as the key factor. We identified the ETH precursor and pharmacologically and functionally characterized the ETH receptor in S. gregaria. Activation of SchgrETHR by SchgrETH results in an increase of both Ca2+ and cyclic AMP, suggesting that SchgrETHR displays dual coupling properties in an in vitro cell-based assay. Using qRT-PCR, an in-depth profiling study of SchgrETH and SchgrETHR transcripts was performed. RNAi-mediated silencing of SchgrETH and SchgrETHR resulted in lethality at the expected time of ecdysis, thereby showing their crucial role in moulting. Silencing of SchgrEcR/SchgrRXR resulted in significantly lower transcript levels of SchgrETH and SchgrETHR, indicating that the ETH system acts downstream of ecdysteroids.Third, we functionally characterised the ecdysone receptor complex, SchgrEcR/SchgrRXR, and the Halloween genes, Spook, Phantom, Disembodied and Shade, in the female reproductive physiology. Tissue and temporal distribution profiles were analysed during the first gonadotrophic cycle of adult female locusts. Using RNAi, we have proven the crucial role of ecdysteroid synthesis and signalling in ovulation and oviposition in S. gregaria. Silencing of the Halloween genes affected ovarian maturation by preventing the proper formation of interfollicular tissue, while silencing the ecdysone receptor complex resulted in impaired chorion formation. We also found evidence for the regulation of JH biosynthesis by ecdysteroids.Finally, we investigated the role of a venus kinase receptor (VKR) in the female reproductive physiology of S. gregaria. We performed an in-depth profiling study of the SchgrVKR transcript levels in different tissues throughout the female adult stage. RNAi-mediated silencing of SchgrVKR had significant effects on ovarian ecdysteroid levels and on the size of oocytes during the vitellogenic stage. This receptor is probably involved in the complex cross-talk between several important hormonal pathways, such as ecdysteroids, regulating female reproductive physiology.

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The fundamental processes of feeding and digestion are necessary to acquire sufficient building blocks and energy to fuel anabolic processes, such as growth, development and reproduction. Animals require the ability to respond to both environmental and internal cues and relate these to their nutritional state. A rigid regulatory system is necessary in all animals to ensure that feeding and digestion are dynamically controlled. This regulation of food uptake and digestion is coordinated by the nervous and endocrine system. Neuropeptides are ubiquitous and essential messengers that can serve as neurotransmitters, neuromodulators or circulating neurohormones and intervene in many processes that are influenced by the nervous and endocrine system. One of the many neuropeptides involved in the regulation of feeding and digestion in insects is sulfakinin (SK). SK is a sulfated neuropeptide that is homologous to vertebrate cholecystokinin and gastrin.SK acts as an inhibitor of food uptake and regulator of intestinal contractions in various insects. However, multiple effects of SK on the regulation of feeding and digestion have only been reported in the fruit fly, Drosophila melanogaster, so far. To expand the knowledge of the sulfakinin signaling system and its control of feeding and digestion, this study utilized two complementary insect models. The red flour beetle, Tribolium castaneum, provided complete and annotated sequence information that could be used to clone and characterize the different components of the SK signaling pathway. In the migratory locust, Locusta migratoria, the SK peptide has been isolated and can be used to assess multiple in vivo actions. The relative size of L. migratoria allows for straightforward physiological testing by means of peptide injections and microdissection. In addition, both models are categorized as important pest insects with a huge economical and humanitarian impact on cultivated and stored human food sources.An important step in elucidating the function of a neuropeptidergic system is the identification of its (G protein-coupled) receptor and peptide precursor and evidencing their functional coupling. In the current study, we managed to clone two putative SK receptors fromT. castaneum and were able to activate both of them using the endogenous SKs as ligands in an in vitro cell-based screening system. Stimulation of either receptor demonstrated positive coupling to cAMP and Ca2+ second messenger pathways in heterologous cell systems. Furthermore, the sulfated tyrosine residue and the C-terminal HMRFamide tetrapeptide were identified as the essential ligand core necessary for high-affinity receptor activation.A qRT-PCR study in T. castaneum mainly localized SK receptor transcripts in the brain and optic lobes, with little expression in the peripheral tissues. In L. migratoria, high expression of SK receptor was apparent in the brain and along the alimentary tract. Relative transcript levels of the components of the SK signaling system were influenced by starvation: inT. castaneum, significant upregulation was quantified upon starvation, while SK receptor transcripts were depleted in starved L. migratoria.Multiple experiments were performed to gain insight into the regulation of feeding and digestion by SK in L. migratoria. Injection of SK provided confirmation that it can inhibit food uptake. Furthermore, SK injections caused a significant downregulation of digestive enzyme activity in midgut and gastric caeca secretions and were able to mediate the removal of partially digested material and digestive enzymes from the gastric caeca. The sulfated tyrosine residue was revealed as an indispensable structural requirement for biological activity of SK in L. migratoria. SK-induced effects on digestive enzyme secretion and gastric caeca emptying were abolished by RNAi-mediated knockdown of the putative SK receptor inL. migratoria.In conclusion, this study describes the first in-depth characterization of SK receptor pharmacology and signaling properties in insects. The SK signaling system is clearly influenced by starvation in both L. migratoria and T. castaneum. Furthermore, SK negatively influences food uptake and digestive enzyme secretion and mediates the transport of partially digested food through the gut in L. migratoria. These observations greatly contribute to the understanding of the SK signalling system and its functions in insects.