Chapter 2. Functional characterization and fitness cost of spinosad resistant alleles in Ceratitis capitata…..33 2.1. Rationale for the study…..35 2.2. Materials and methods…..36 2.2.1. C. capitata strains and generation of isolines…..36 2.2.2. Genomic DNA extraction, PCR of intronic/exonic boundaries and sequencing of Ccα6…..40 2.2.3. RNA extraction, RT-PCR, qPCR of Ccα6 exons, PCR and sequencing of codifying regions…..40 2.2.4. GAL4>UAS targeted expression of Ccα6 isoforms in D. melanogaster…..42 2.2.4.1. Cloning of wild-type and mutant mRNA isoforms of Ccα6 …..42 2.2.4.2. Construction of vectors for transformation of D. melanogaster…..43 2.2.4.3. Microinjection of Ccα6 constructs to generate transgenic lines…..44 2.2.5. Susceptibility bioassays with spinosad…..44 2.2.5.1. Susceptibility of C. capitata…..44 2.2.5.2. Susceptibility of D. melanogaster…..45 2.2.6. Stability of Ccα6 3aQ68*Δ3b-4 and Ccα6 3aQ68*-K352* alleles in C. capitata…..45 2.2.7. Fitness of resistant isolines and the susceptible strain of C. capitata…..46 2.2.7.1. Developmental time to pupation and adults' weight…..46 2.2.7.2. Attraction to a parapheromone in a wind tunnel…..46 2.2.7.3. Electrophysiological Antennal Activity…..47 2.2.7.4. Male competition in mating…..47 2.2.8. Statistics…..48 2.3. Results…..48 2.3.1. Expression of Ccα6 3aQ68* , Ccα6 3a8b and Ccα6 3b8a mRNA isoforms in D. melanogaster…..48 2.3.2. Characterization of Q68* and Q68*-K352* isolines of C. capitata…..50 2.3.3. Stability of spinosad resistant alleles Ccα6 3aQ68*-K352* and Ccα6 3aQ68*Δ3b-4 in C. capitata…..53 2.3.4. Fitness of Q68* and Q68*-K352* isolines of C. capitata…..57 2.3.4.1. Biological traits…..57 2.3.4.2. Behavioral traits…..57 2.4. Discussion…..61 Chapter 3. Inheritance of resistance to lambda-cyhalothrin and search of molecular markers associated with resistance…..65 3.1. Rationale for the study…..67 3.2. Materials and methods…..68 3.2.1. C. capitata strains…..68 3.2.2. Study of the inheritance of lambda-cyhalothrin resistance…..69 3.2.3. Bioassays with the synergist PBO…..70 3.2.4. Expression and CNV analysis of P450 genes from C. capitata…..70 3.2.5. Search of markers involved in lambda-cyhalothrin resistance by variant calling analysis…..72 3.2.6. Statistics…..73 3.3. Results…..73 3.3.1. Study of the inheritance of lambda-cyhalothrin resistance…..73 3.3.2. Effect of PBO treatment on lambda-cyhalothrin resistance…..74 3.3.3. Analysis of P450 expression and CNV…..77 3.3.4. Search of markers involved in lambda-cyhalothrin resistance by variant calling analysis…..81 3.4. Discussion…..85 Chapter 4. Detection and management of insecticide resistance in Ceratitis capitata…..89 4.1. Rationale for the study…..91 4.2. Materials and methods…..92 4.2.1. Laboratory strains, field populations and generation of a multiresistant strain of C. capitata…..92 4.2.2. Bioassays…..95 4.2.3. F1-screen assays…..96 4.2.4. Detection of mutations in laboratory strains and field populations of C. capitata…..97 4.2.5. Evolutionary model of spinosad resistance…..99 4.2.6. Statistics….....
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