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

Abstract

3 RESULTS: ...……………………………………………………………………………...16 3.1 Transgenic sperm marking to enhance monitoring of coleopteran insect pests: ...…………………………………………………..………………………………….17 3.2 Identification and characterization of early embryonic gene(s) for the development of transgenic embryonic lethality systems in Tribolium castaneum: .………………………………………………..………………….………………...58 3.3 Novel genetic engineering approach for the management of coleopteran insect pests using CRISPR-Cas9: ………………….…………………………………….97 3.4 Genetic improvement and use of CRISPR/Cas9 to generate an early embryonic driver line in Tribolium castaneum: ……………………………...………………119 4 General discussion: .…………………………………………………………………….145 4.1 Transgenic sperm marking in Tribolium castaneum: .………………………145 4.2 Identification and characterization of potential cellularization gene(s) in Tribolium castenuem: .……………………………………………………….....…146 4.3 Female Specific Embryonic Lethality systems (FSELs):.……………………147 4.4 Genetic Improvement and Use of CRISPR/Cas9 in Tribolium castaneum for genome editing: .……………………………………………………………...……148 4.5 Novel genetic engineering approach for the management of coleopteran (Tribolium castaneum) insect pests using CRISPR-Cas9 system for reproductive sterility: .…………………………………………………………………………...149 5 Outlook: .………………………………………………………………………………...151 6. References:………………………………………………………………………………152 6 Curriculum Vitae:………………………………………………………………………..170 2 GENERAL INTRODUCTION: 2.1 Red Flour Beetle (RFB) Tribolium castaneum (Tenebrionidae): Coleopteran model insect pest:The class Insecta is the largest group of organisms 1 in the animal kingdom, and their successes can be attributed to their ability to survive in different niches such as water bodies 2 , tropical rain forest 3 , several land areas such as mountains, caves, hot spring, polar zones and their staggering diversity 4 . Coleopterans insect also known as beetles belong to the class Insecta, and comprises about 380,000 described species 5,6 of the class, thereby making them about ~40% of all described species 7 , and known to occupy different ecological niches 8 .Tribolium casteneum otherwise called The Red Flour Beetle, 9 belongs to the order Coleoptera., and it is the coleopteran model organism 10 , and its second only to Drosophila melanogaster (Order: Diptera), for various genomic studies due to: 1) Easy of handling 2) High fecundity and 3) Easy to maintain in a laboratory 17 . The organism is helping in answering several developmental and evolutionary biology questions 11 which hitherto cannot be investigated.Female T. casteneum have the capacity to lay about 200 -500 eggs within a total lifespan of about 3 years 11 . T. casteneum is found to develop favorably at 27 o C 11 , with the first instar larvae looking slender and wormlike in appearance 12 . The larvae are covered with fine hairs, and also possess a spine like structure called urogomphi 11 found at the last abdominal segment, and sexing of individuals are mostly done at the pupal stage 13 by the possession of lobe like structure notices at th...