Dscam gene expression in invertebrate immunity : alternative splicing in response to diverse pathogens
Smith, Paul Hugh
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Invertebrates show enhanced immunity and even specific primed immunity in response to repeat infections, analogous to vertebrate adaptive immunity. Little is known of the mechanism for this phenomenon, or which molecules are involved. A candidate gene for the underlying mechanism for a pathogen-specific response in invertebrate immunity is Down syndrome cell adhesion molecule (Dscam). Dscam can produce thousands of different protein isoforms through the mutually exclusive splicing of many exon variants contained within variable regions of the gene. It is an important receptor of the invertebrate nervous system but has been implicated in having a role in immunity. Dscam has been shown to be involved in phagocytosis across members of the Pancrustacea, and it has been reported to respond in a pathogen-specific manner in mosquitoes and crayfish. In this thesis, I have investigated the splicing of Dscam in response to diverse pathogens in different host species. In the Anopheles mosquito, I cloned and sequenced a fragment of Dscam spanning across two of its variable exon regions to enable me to detect mutually exclusively splice variants and their associations in different treatments (Chapter 2). I discovered that the expression diversity of the hypervariable Dscam is higher in parasite-exposed mosquitoes. In Chapter 3, I extended the study to the more experimentally amenable Drosophila fruit fly. A new Illumina-based sequencing assay was developed and implemented to examine more closely Dscam expression in response to diverse pathogens. The new method successfully quantified non-random expression of Dscam variable exons 4 and 6. I also describe a small but detectable effect of pathogen-exposure on the expression of Dscam exon 4 variants. In Chapter 4, I expanded the work of Chapter 3 to study tissue-specific Dscam expression in response to well-characterised immune elicitors of Drosophila. I describe how exon 4 variants were expressed in a tissue-specific manner, but not exon 6 variants. I also found a small exon 4-by-tissue-by-pathogen effect, which although detectable, did not dominate over the tissue effects. Finally, in Chapter 5, I turned to the crustacean, Daphnia, to study Dscam expression in a natural host-parasite interaction and in a clonal organism. I describe the non-random expression of exons 4 and 6, and another small effect of pathogen-exposure on the expression of Dscam exon 4. My work aimed to further investigate the putative pathogen-specific alternative splicing of the hypervariable Dscam receptor. The data presented quantified the constitutive expression of Dscam exons 4 and 6 in different pancrustacean species. The data also suggest that infection-responsive splicing of Dscam may occur but that effects are small, and may be diluted within the background of the highly important Dscam expression of the nervous system if they exist at all. The study supports the high-throughput sequencing method for future studies of alternative splicing and Dscam expression.