Location: Insect Behavior and Biocontrol Research
Project Number: 6036-22000-034-001-A
Project Type: Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Jun 30, 2025
Objective:
The objective of this cooperative research project is to develop genetically modified strains for the Caribbean fruit fly (Anastrepha suspensa) and spotted-wing drosophilid (Drosophila suzukii) for improved biological control of these important pests of fruit crops. Specific aim-1 of this project is to create strains in both species for male sterility that, when hybridized to a female-lethal strain, results in a sterile males-only population for field release in a Sterile Insect Technique (SIT) population control program. Specific aim-2 of this project is to evaluate the sterile male strains for fitness and mating competitiveness for SIT field release, compared to wild type males sterilized by irradiation.
Approach:
Spermatocyte-specific gene cognates of the Drosophila genes, Prosalpha6T (proteasome subunit) and wampa (dynein subunit), will be isolated by degenerate PCR protocols in A. suspensa and D. suzukii based on homologous sequences in Drosophila and other dipteran species. Tissue-specific expression of these genes during adult development will assayed by quantitative reverse-transcriptase PCR (qPCR). Once expression is verified, functional analysis based on the necessity of these genes for male fertility will assessed by dsRNA feeding for RNAi analysis and CRISPR-Cas9-mediated gene-edited null mutations. Conditional male sterile strains will then be created by the use of transposon-mediated transformation to introduce single guide RNA (sgRNA) constructs that target the spermatocyte-specific genes and a Cas9 gene under tetracycline-suppressible regulation. These strains will be tested for sgRNA and Cas9 expression on Tet-diet, which is expected to suppress CRISPR targeting resulting in male fertility, and Tet-free diet that is expected to allow Cas9 gene expression and CRISPR targeting resulting in male sterility. Sterile male strains will be evaluated for life fitness and mating competitiveness for SIT field release, compared to wild type males sterilized by irradiation.
