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Thesis Format

Monograph

Degree

Master of Science

Program

Biology

Supervisor

Grbic, Vojislava

Abstract

Tetranychus urticae, commonly known as the two-spotted spider mite, poses a significant threat to agriculture due to its ability to feed on a diverse range of plant hosts and its strong detoxification abilities in overcoming xenobiotic response. With global warming projected to increase spider mite infestations, it is vital to study the detoxification genes that enable the mite to adapt and survive. The spider mite genome sequence reveals a unique set of detoxification genes that can be studied using RNAi as a promising reverse genetic tool. However, the current genetic toolkit requires improvement. This study examined the effectiveness of three precursor molecules (dsRNA, shRNA, and amiRNA) in inducing RNAi response in spider mites. While shRNA and amiRNA demonstrated variable responses, dsRNA significantly reduced transcript levels of all three target genes. The establishment of primary cell cultures from T. urticae embryos provides a valuable tool for investigating the uptake mechanism of different precursor RNAi molecules through 'RNAi of RNAi' experiments. Ultimately, this research could contribute to the development of RNAi-based pesticides that selectively target spider mites detoxification pathways, leading to more effective pest control in agriculture. Future research should aim to improve our understanding of the spider mite RNAi machinery, including the uptake mechanism and precursor molecule stability, to facilitate functional gene analysis and the development of new pest control strategies.

Summary for Lay Audience

The two-spotted spider mite, Tetranychus urticae, is a pest that can feed many different types of plants, causing significant economic losses in agriculture. One of the main challenges in controlling this pest is its ability to develop resistance to pesticides, making it crucial to explore alternative approaches for pest management. The two-spotted spider mite has developed high resistance to pesticides due to its ability to detoxify harmful compounds, which is controlled by a large number of genes. To understand the function of these genes, a tool called RNAi can be used to turn off their expression. The RNAi process begins by introducing precursor molecules into the system. These precursors are designed to interfere with the normal activity of the target gene, resulting in what is referred to as "knockdown" or suppression of its expression. However, the most commonly used precursor molecule, dsRNA, can have off-target effects, which means other genes may also be affected. To overcome this problem, my thesis investigated the use of alternative precursor molecules like shRNA and amiRNA for RNAi in spider mites. Additionally, primary cell cultures from spider mites were established, which are cells grown in a laboratory setting and are capable of mimicking the behavior of whole mites. This allowed us to study RNAi responses at the cellular level. The findings from this study suggest that dsRNA-induced RNAi is the most highly effective method, and that spider mite primary cell cultures are suitable for performing cellular assays such as RNAi experiments. Overall, this study establishes primary cell cultures as a valuable tool for performing RNAi, which can be used to further investigate the reasons for the lower efficiency of other precursor molecules compared to dsRNA in the future, by studying the enzymes involved in the RNAi machinery at the cellular level.

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