Author: Paula Cristina Walger de Camargo Lima
Walger de Camargo Lima, Paula Cristina, 2013 The application of RNA interference to study the biology of the Neoparamoeba genus, Flinders University, School of Biological Sciences
This electronic version is made publicly available by Flinders University in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material and/or you believe that any material has been made available without permission of the copyright owner please contact firstname.lastname@example.org with the details.
RNA interference (RNAi) is a natural regulatory mechanism of most eukaryotic cells that uses small double-stranded RNA (dsRNA) molecules as triggers to direct homology-dependent control of gene activity. This technique has emerged as a powerful tool for rapid analysis of gene function in non-model organisms and has the potential to identify candidate targets for intervention against diseases of economic importance to aquaculture. With regards to amoebic gill disease (AGD) of farmed Atlantic salmon, RNAi could become an invaluable research instrument to unravel the role of proteins involved in amoeba attachment and pathogenicity, as well as to validate important treatment targets by investigating the effect of specific gene knockdown on amoeba survival and physiology. Additionally, RNAi technology could greatly assist in the elucidation of possible factors associated with the loss of virulence in certain species from the Neoparamoeba genus. However, before RNAi technology can be employed in Neoparamoeba, it is important to consider whether members of this genus possess the required set of proteins involved in the RNAi pathway. As a result, the main purpose of the present study was to use functional and comparative genomics approaches to investigate whether functional RNAi machinery has been retained or lost in species from the Neoparamoeba genus. As the in vitro culture of the causative agent of AGD (Neoparamoeba perurans) has been successfully achieved only recently, most of the gene regulation assays were performed using the closely-related Neoparamoeba pemaquidensis, which is readily amenable to culture. Using a N. perurans and N. pemaquidensis transcriptome database we were able to identify putative proteins containing conserved domains of RNAi-related genes, such as Dicer and Argonaute. For N. pemaquidensis, the candidates' involvement in the RNAi pathway was validated by assessing their levels of expression followed the administration of dsRNA and small interference RNA (siRNA), respectively. The presence of an active Dicer in both species was also corroborated by utilizing an RNAse III assay, which showed complete degradation of dsRNA following incubation in amoeba lysate. Further evidence for the presence of an active RNAi machinery was also supported by gene silencing experiments, where N. pemaquidensis specific genes (β-actin and EF1α) were successfully downregulated by the administration of RNAi-trigger molecules. However, knockdown efficiency was dependent on dose, administration frequency, target gene, delivery method and RNAi molecule. Additionally, trophozoites soaked with bacterially expressed dsRNA targeting β-actin unexpectedly transformed into a cyst-like stage, which has not been previously reported in this species. Unfortunately, the attempts to employ the Entamoeba histolytica U6 promoter to confirm the existence of a functional RNAi pathway in N. perurans haven't succeeded yet. The results altogether provide strong evidence for the presence of functional RNAi machinery in Neoparamoeba spp. Despite being promising, these findings are still preliminary and the reality of applying RNAi technology to develop new treatment strategies against AGD still needs further effort. Therefore, more work needs to be undertaken in order to fully elucidate the RNAi mechanisms in Neoparamoeba perurans.
Keywords: aquaculture,amoebic gill disease,salmon,RNA interference
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Dr James Harris