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Many pathogen infections in nature are accompanied by injury and subsequent coagulation. Despite the contribution of hemolymph coagulation to wound sealing, little is known about its immune function. Based on the molecular knowledge of Drosophila innate immunity, this thesis investigated the immune function of clot both in vitro and in vivo, the immune relevant genes involved in a natural infection model, involving entomopathgenic nematodes (EPN) and the factors leading to crystal cell activation. Transglutaminase (TG) and its substrate Fondue (Fon) have been identified as bona fide clot components in Drosophila larvae. By knocking down TG or Fon via RNAi, we observed an increased susceptibility to EPN in larvae. In addition, this increased susceptibility was associated with an impaired ability of hemolymph clots to entrap bacteria. Immunostaining revealed that both clot components (Fon and TG) were able to target microbial surfaces. All these data suggest an immune function for the Drosophila hemolymph clot. Strikingly, similar results were obtained when we ran parallel experiments with human FXIIIa, an ortholog of Drosophila TG, indicating a functional conservation. We also found evidence for the regulation on both clot and immunity by eicosanoids in Drosophila larvae. The combination of EPN infection with the Drosophila model system allowed us to discover an immune function for TEP3 and Glutactin. However the molecular mechanism underlying the involvement of these two proteins in this particular host-pathogen interaction remains to be elucidated. Prophenoloxidase, the proform of enzyme involved in hardening the clot matrix, has been shown to be released by rupture of crystal cells. This cell rupture is dependent on activation of the JNK pathway, Rho GTPases and Eiger. Our work further identified the cytoskeletal component, Moesin, and the cytoskeletal regulator Rac2 as mediators of cell rupture. Despite the possible role of caspases in crystal cell activation, such cell rupture was turned out to be different from apoptosis. The implication of Rab5 in this process indicated that proper endocytosis is required for cell activation and subsequent melanization. Our findings furthered not only our understanding of the release of proPO via cell rupture but also our knowledge on different paths of immune cell activation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In press. Paper 4: Manuscript.<strong></strong></p>

How are neuromodulatory networks organised to adapt sensory discrimination for different contexts? I hypothesised that neurons within a sensory circuit express different neuromodulatory receptors for differential modulation. Here I aimed to use the simple and genetically amenable Drosophila larval Mushroom Body (MB) calyx, a higher order processing area involved in learned odour discrimination, as a model to map octopamine (OA) neuromodulatory circuitry. I first identified olfactory projection neurons (PNs), a GABAergic feedback neuron and cholinergic extrinsic neurons as putative postsynaptic partners to OA neurons in the MB calyx using GFP reconstitution across synaptic partners. Next, I used novel EGFP-tagged OA receptors generated from recombination-mediated cassette exchange with MiMIC insertions in receptor genes to visualise endogenous expression patterns of OA receptors. Most notably, this is the first report of α2-adrenergic-like OA receptor localisation in any insect. For the first time, I showed that the α1-adrenergic-like OAMB localised to PN presynaptic terminals in the calyx; while Octβ1R localised diffusely in the calyx, resembling the innervation pattern of MB neuron dendrites. I detected EGFP-tagged Octα2R and Octβ2R in some PN cell bodies but not in neuron terminals - suggesting that Octα2R and Octβ2R may be expressed in some PNs, provided the misfolded fusion proteins are retained in the cell bodies of the neurons they are normally expressed in. Furthermore, I found that Octα2R and GABAAR fusion proteins localised to OA cell bodies but not to neuronal terminals, suggesting that OA neurons are subjected to inhibition, again given that these are not artefacts of the fusion proteins. To obtain tools to study OA modulation in the larval calyx, I then confirmed the expression patterns of driver lines that more specifically labelled calyx-innervating OA and extrinsic neurons, and tested the efficacy of three OAMB receptor knockdown lines. This initial attempt of mapping OA receptors, while subjected to further verification and development, is consistent with my hypothesis that a single neuromodulatory source can regulate multiple neuronal types in the same circuit through the distribution of different types of neuromodulatory receptors. This provides a new perspective in how the anatomical organisation of neuromodulation within a sensory network may translate to flexible outputs.