Ctenocephalides felis is a major pest of companion animals worldwide. This project aimed to generate novel genetic resources for C. felis and develop tools to aid drug-target identification and validation. Sample handling methods were assessed and candidate reference genes validated, to ensure quality of RNA samples and reliable gene expression normalisation. Piercing C. felis samples prior to storage in RNAlater ensured RNA integrity was maintained over time. Glyceraldehyde 3-phosphate dehydrogenase , 60S ribosomal protein L19 and elongation factor-1α were demonstrated as stable reference genes across all comparisons tested. A C. felis transcriptome encompassing multiple developmental stages, sexes and tissues was sequenced and de novo assemblies produced with two assemblers, Trinity and Oases. Each assembly contained >100000 contigs. Annotation of the assemblies generated functional insight, such as top BLAST hits, GO annotations and signal peptide predictions. The Trinity assembly was deemed the highest quality and searched for genes of interest, involved in development. Expression analysis of selected transcripts across stadia gave insight into developmental processes, and demonstrated the utility of the transcriptome. This study was the first to demonstrate that C. felis can mount an RNAi response upon exposure to dsRNA. Knockdown of glutathione S-transferase σ (GSTσ), was demonstrated in adult C. felis: ≈80 % knockdown following microinjection of dsGSTσ; ≈64 % knockdown after soaking in dsGSTσ; ≈96 % knockdown after continuous feeding on dsGSTσ. RNAi machinery was identified in C. felis. siRNAi pathway components, Dicer 2 and Argonaute 2, were upregulated following dsRNA exposure. Dicer 2 was knocked-down by soaking in dsDicer2, although results of an “RNAi of RNAi” experiment were inconclusive. Transcripts encoding machinery putatively involved in dsRNA uptake and breakdown were also identified. Through these studies, this project has generated novel insights into C. felis biology and opened up new avenues for research.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:742385 |
| Date | January 2018 |
| Creators | Edwards, Catriona Helen |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=236461 |
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