Development of Transgenic Sterile Insect Technique Strains for the Invasive Fruit Pest Drosophila suzukii
by Hassan Mutasim Mohammed Ahmed
Date of Examination:2019-12-20
Date of issue:2020-12-03
Advisor:Prof. Dr. Ernst A. Wimmer
Referee:Prof. Dr. Ernst A. Wimmer
Referee:Prof. Dr. Gregor Bucher
Referee:Prof. Dr. Reinhard Schuh
Referee:Prof. Dr. Ralf Heinrich
Referee:Prof. Dr. Daniel John Jackson
Referee:PD Dr. Roland Dosch
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Description:Dissertation
Abstract
English
Globalization has contributed enormously to emergence and expansion of invasive pests. A recent example is the invasion in Europe and the USA by the cherry vinegar fly Drosophila suzukii. The pest has high potential to invade and establish itself in different climatic zones, the fact that is made clear by looking at the current global distribution map of the fly. The use of pesticide chemicals is so far the first line of defense against it due to lack of reliable alternatives. The use of pesticides against this fly has major problems including possible emergence of insecticide resistance due to the short generation time and number of generations per year. Secondly, the fly lays eggs inside the fruit which make the egg and larvae not exposed to insecticide. Thirdly and most importantly is the time of invasion with regard to ripening and harvest of the fruits. This makes the pesticide choice a very hard one. To be able to fight this onerous pest, the Sterile Insect Technique (SIT) offers an effective, environment friendly genetic pest management that does not interfere with the natural enemies and pollinators. Here, I propose the use of biotechnological approaches to develop transgenic strains suitable for SIT against D. suzukii. In the first part of this study, the use of a CRISPR/Cas9 genome editing system to engineer a sex conversion suppression gene drive in Drosophila melanogaster is presented. We found that current designs of homing-based gene drive induce development of resistance against them. We proposed possible solutions to avoid rapid development of resistance and the application of such drive in Tephritid flies where targeting the sex determination gene transformer leads to fertile XX males. Second, the hyperactive version of the piggyBac transposase was successfully used to improve insect transgenesis with demonstration in three species belonging to two different orders, the genetic model Drosophila melanogaster, the global fruit pest Ceratitis capitata and the new beetle model for development and evolution Tribolium castaneum. In the third chapter improvement in CRISPR/Cas9 genome editing of the invasive fruit pest Drosophila suzukii is presented. Different endogenous regulatory elements were isolated and used to drive the expression of Cas9 and the guide RNAs. In addition, the application of CRISPR/Cas9 to engineer the first sperm marking strain for D. suzukii is presented, which represents an important contribution to the establishment of the SIT for this pest. In the fourth part, the φC31 integrase based site-specific germline transformation of the fruit pest D. suzukii was established both by integration in one attP landing site and by recombinase mediated cassette exchange (RMCE). This provides a platform for testing several enhancers and/or complete system in the same genome context. We also present the development of embryonic and spermatogenesis specific driver lines that can be used for different biotechnological improvements of SIT against D. suzukii. The last three chapters present a perspective paper describing a new reproductive sterility system and the first steps toward its development. It is based on CRISPR/Cas9 chromosome shredding during spermatogenesis that should lead to the same results commonly achieved by ionizing radiation but with no deleterious effect on the males’ fitness. This should culminate in embryonic lethality due to chromosomal aberration causing aneuploidy. We first generated several spermatogenesis specific driver lines and Cas9 responder lines to test the suitability of the tet-off binary system to control gene expression during spermatogenesis. Suitable genomic targets for chromosome shredding were identified and gRNAs to target them were designed. Genome editing and insect transgenesis tools developed in this study will facilitate further biotechnological improvements of the SIT and its transfer to the invasive agricultural pest D. suzukii.
Keywords: Gene drive; Molecular entomology; Insect pests; Genome editing; CRISPR/Cas; Genetic engineering; piggyBac; Transgenesis; Drosophila suzukii