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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Insect Behavior and Biocontrol Research » Research » Research Project #438800

Research Project: An Assessment of Genetic Breakdown and Transposon Vector Horizontal Transfer as it Affects Transgenic Strain Stability in Insect Species

Location: Insect Behavior and Biocontrol Research

Project Number: 6036-22000-030-063-I
Project Type: Interagency Reimbursable Agreement

Start Date: Sep 1, 2020
End Date: Aug 31, 2024

Objective:
1. To test the frequency and genetic basis of lethality reversion resulting in survival under non-permissive conditions during large-scale rearing of i) a Anastrepha suspensa (Caribbean fruit fly) Tet-off uni-sex embryonic lethality strains (TELS) on Tet-free diet, and ii) an A. suspensa AsDTS7- pupal lethality strains reared at 29°C. 2. To create dual (stacked) TELS and AsDTS7- lethality strains in A. suspensa to test the frequency of adult survival under non-permissive conditions for both systems (Tet-free diet at 29°C), thereby evaluating the ability of AsDTS7- to mitigate the survival of TELS revertant lethals. 3. To test the ability of a transposon vector insertion in the A. suspensa genome to horizontally transfer into symbiotic bacterial populations in the presence and absence of vector transposase.

Approach:
Large-scale cage studies will be performed with an A. suspensa Tet-off embryonic conditional lethal system (TELS) strain and an A. suspensa Prosbeta2-1 (DTS7) dominant temperature-sensitive lethality system, where males are mated to wild type females and revertant lethal F1 surviving progeny, due to spontaneous mutations or inherent suppressors of lethality, are selected at non-permissive conditions of a tetracycline-free diet at 29°C. A determination of the frequency of heritable survival will be made and a structural analysis of the mutations and suppressors. Secondly, the TELS and DTS7 systems will be combined into a single dual lethality strain and mass-reared under permissive control and non-permissive experimental conditions to assess survival compared to use of the TELS system alone. Third, A. suspensa will be transformed with a piggyBac transposon vector marked with the Zeomycin-resistance gene and a fluorescent protein marker. Multiple generations of transformant strains will be screened at different life stages for Zeo-resistant bacterial populations (i.e. Enterobacter agglomerans and Klebsiella pneumoniae).