Location: Insect Behavior and Biocontrol Research
Project Number: 6036-22000-034-061-N
Project Type: Non-Funded Cooperative Agreement
Start Date: Jun 1, 2019
End Date: Mar 28, 2024
Objective:
The research objective is to develop new transgenic strains for the fruit fly pest species Anastrepha (A.) fraterculus, A. obliqua, and A. suspensa for tetracycline-suppressible (Tet-off) and dominant temperature sensitive (DTS) female-specific conditional lethality strategies for genetic-sexing. Notably, both systems will use highly conserved genetic systems for regulatory and sex-specific control and lethal effectors that, with minor modification, can be used as generic systems in other related species. DTS lethality (using the AsProsbeta2 mutation) has already been demonstrated in A. suspensa, and temperature-dependent female-specific lethality will be tested for the first time in additional Anastrepha species. Tet-off female-specific embryonic lethality has been achieved in tephritid species, but in A. ludens, maternal pre-zygotic lethality resulting in female-sterility also occurred. Thus, new promoter systems that are strictly embryo-specific will be identified and tested for use in A. ludens, in addition to A. fraterculus and A. obliqua and other species where the serendipity-alpha promoter exhibits pre-zygotic activity. The development of two independent female-specific lethality systems for genetic sexing, that are highly conserved, will broaden the capabilities for achieving efficient sexing in a large number of related insect pest species.
Approach:
Year 1:
- evaluate promoter expression from candidate embryonic genes in ovaries (pre-zygotic oocytes) and early stage post-zygotic embryos by quantitative Polymerase Chain Reaction (qPCR) in Anastrepha (A.) ludens, A. fraterculus and A. obliqua - candidate embryonic genes will include cognates to the Drosophila genes, serendipty alpha (sry-alpha), spitting image (spt), slam, and CG2186.
- isolate the Prosbeta2 genes from A. fraterculus and A. obliqua and perform in vitro single nucleotide mutagenesis on each gene to create the DTS-7 point mutant allele.
Year 2:
- create transgenic tetracycline-Transcriptional Activator (tTA) driver lines for A. fraterculus and A. obliqua with vectors inserted with optimal embryonic-specific promoters linked to tTA; and assay embryonic-specific tTA expression by qPCR in transgenic lines.
- create transgenic Tet-off female-specific lethal effector lines for A. fraterculus and A. obliqua using the previously created TRE-Cctra-Intron1-Alhid-Ala2 transformation vector (that includes a Tet-Response Element linked to the A. ludens hid-Ala2 cell death gene having an insertion of the Ceratitis (C.) capitata transformer gene intron 1 immediately 3’ to the ATG initiation codon).
- create transgenic DTS-7 female-specific temperature-dependent lethality lines for A. suspensa, A. fraterculus and A. obliqua using vectors having the sex-specific Cctra-Intron1 linked to the Prosbeta2 DTS-7 mutant allele.
Year 3:
- create homozygous transgenic lines for the Tet-off and DTS-7 female-specific lethality systems and evaluate them under small-scale rearing (2,000 adults) for male and female viability when reared under permissive (Tet-diet or 25°C or below) and restrictive conditions (Tet-free diet or 29°C).
