Location: Insect Behavior and Biocontrol Research
Project Number: 6036-22000-030-58-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Apr 1, 2018
End Date: Mar 31, 2020
The objective of this cooperative research project is to develop transgenic strains in the Caribbean fruit fly (Anastrepha (A.) suspensa) and spotted-wing drosophilid (Drosophila (D.) suzukii) for improved biological control of these important pests of fruit crops. Specific aim-1 of this project is to create A. suspensa strains genetically modified with additional antioxidant genes for superoxide dismutase I, superoxide dismutase II, and catalase in order to improve the viability, fitness, and mating competitiveness of males sterilized by irradiation for use in the sterile insect technique (SIT). Fitness and reproductive competitiveness parameters will be evaluated in adult transgenic males to assess whether ectopic expression of these genes have a beneficial effect on viability and mating behavior in males sterilized by irradiation for the sterile insect technique. Specific aim-2 of this project is to test the viability, fitness, and mating competitiveness of A. suspensa and D. suzukii chromosomal females that develop as phenotypic males as a result of modifying their genomes with a temperature-sensitive allele of the transformer-2 (tra-2) sex determination gene. These tests will determine if mutant sterile males created by the sex reversal of chromosomal females will have the physiological parameters, compared to sterilized wild type males, for effective use in SIT.
Antioxidant genes, including those for superoxide dismutase I, superoxide dismutase II , and catalase, will be isolated by polymerase chain reaction (PCR) protocols in Anastrepha (A.) suspensa based on homologous sequences in dipteran species. Complete genomic sequences placed under various modes of conditional regulation will be introduced by germline transformation into wild type A. suspensa strains. With assistance from the Cooperator, lines homozygous for the transgenes will be created and adult males will be assayed for antioxidant gene expression and enzymatic activity. Untreated males and males sterilized by gamma-irradiation will be tested for life fitness and reproductive competitiveness parameters. The tra-2ts2 mutation will be created in A. suspensa and Drosophila (D.) suzukii by CRISPR/Cas9 homology-directed repair gene-editing, and sex reversal of XX; tra-2ts2 chromosomal mutant females to phenotypic males at restrictive temperatures (i.e. 26°C to 29°C) will be tested for both species. With assistance from the Cooperator, mating competitiveness and reproduction of XX; tra-2ts2 phenotypic males will be compared to XY; tra-2ts2 and wild type males.