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Development of a biotechnologically enhanced sterile insect technique to fight coleopteran pests

dc.contributor.advisorWimmer, Ernst A. Prof. Dr.
dc.contributor.authorIsah, Musa Dan'azumi
dc.titleDevelopment of a biotechnologically enhanced sterile insect technique to fight coleopteran pestsde
dc.contributor.refereeWimmer, Ernst A. Prof. Dr.
dc.description.abstractengTribolium castaneum commonly referred to as the Red Flour Beetle (RFB) belongs to the class Insecta and order Coleoptera, and it’s the best model organism for coleopterans. About 380,000 coleopteran species, constituting approximately 25% of all the animal species described in the world have been identified, thus making them the most taxonomically described species in the animal kingdom, and they are found occupying different ecosystems due to their extraordinary diversities. Approximately 75% of the beetle described are polyphagous as larvae and adult, and are found to live on plants, wood products and stored products and are of economic importance for agriculture, as well as forestry and other household products. By such activities they cause several and significant damage both in direct and indirect losses. The current predominant control measure employed for controlling coleopteran pests is the injurious application of chemicals which results in the development of resistance over time, and also have negative effect to the environment and human health. Hence, there is a need for more efficient and environmentally friendly control measures that is devoid of the drawbacks identified, hence the development and usage of biotechnological approaches. Transgenic approaches use recombinant DNA via genetic engineering to mimic classical genetic approaches, which is also less expensive, and addresses the drawbacks of the classical genetic approach. However, while these practices have been employed in various insect pests mostly in the order of dipteran, none has been employed to any coleopteran insect pest so far. Biotechnological approaches could improve techniques such as sexing for male alone releases, sperm marking, and female/embryonic specific lethality strategies, where classical genetic approaches use for example radiation for causing sterility, fluorescent dyes for monitoring and phenotypic strategies such as pupal sizes or colour for sexing which have several disadvantages. Tribolium castaneum as a model insect pest has a comprehensive genome database, which can be utilized for both evolutionary and population genetics approaches. The organism is also the most utilized insect for development biology studies aside from Drosophila melanogaster due to the ease of handling the insect in the laboratory, short generation time, high fecundity, etc. Here I provide the first attempt and proof of principle of transgenic approaches for sterility, sperm marking for monitoring, and sexing using recombinant DNA. Firstly, for monitoring, Iused the Promoter/Enhancer (P/E) region of the Tribolium spermatogenesis specific ß2 -tubulin gene to drive DsRed and/or EGFP fluorescent protein in the sperm of male individuals by first integrating the constructs by piggyBac germline transformation to generate transgenic lines. I observed the marked sperm in the testis of the male, and in the spermatheca of the female when a wildtype female is mated with a transgenically sperm-marked male individual. This can solve the drawback that is observed in the bodily application of fluorescent dyes on the released individuals, which will result in either false negative or false positive in the classical SIT in capture and recapture experiments. Also, the generated sperm marked individuals provided us with a tool to answer some reproductive biology question on sperm storage and usage by twice mated females. The result showed that the female store the sperm for a long period, and use the sperm of the second inseminated males first, with the first male inseminated sperms used subsequently. Moreover, we discovered a new early embryonically expressed gene (Tc007675) which was hitherto not reported and used it for functional studies. I used the amplified 1.4Kb or 2.1Kb of the Promoter/Enhancer (P/E) upstream region of the gene to drive the artificial heterologous transactivator tTA in order to generate several driver strains by piggyBac germline transformation. By Whole Mount In Situ Hybridization (WMISH), two driver lines expressing tTA at cellularization stage were identified. In addition, using CRISPR-Cas9 genome editing, I effectively generated a driver line targeting the Tc007675 gene locus by Homology Directed Repair (HDR) strategy to generate a bi-cistronic locus. The effectiveness of the driver line was verified using WMISH, where we detected a transient expression of tTA under the control of the Tc007675 gene. Furthermore, using the spermatogenesis specific ß2t enhancer/promoter to drive tTA, we generated a driver line that expresses tTA in testis of one of the lines generated. With this line, we intend to express Cas9 and respective guide RNAs in the testis as a new approach to cause reproductive sterility. At the same time, we generated a potential Cas9 expressing effector line which we crossed with our driver line but for now we were not able to drive Cas9 in the testis. This idea will be used to mimic the use of ionizing radiation used in classical approach, where it could take care of the drawback arising from fitness loss due to the mutation effect on somatic cells of the treated organism. The CRISPR-Cas9 mediated reproductive sterility system will provide a novel and efficient way, in which males will have no fitness or competitiveness disadvantages to wildtype when
dc.contributor.coRefereeBucher, Gregor Prof. Dr.
dc.subject.engTribolium casteneumde
dc.subject.engSterile Insect techniquede
dc.subject.engTransgenic Sperm markingde
dc.subject.engEmbryonic sexing systemde
dc.subject.engFlour beetlede
dc.affiliation.instituteBiologische Fakultät für Biologie und Psychologiede
dc.subject.gokfullBiologie (PPN619462639)de

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