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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 #444629

Research Project: Enabling Sustainable Control Of Fruit-fly Agricultural Pests Using CRISPR-Editing Of High-throughput Selectable Traits Of Target Insect Species

Location: Insect Behavior and Biocontrol Research

Project Number: 6036-22000-034-225-R
Project Type: Reimbursable Cooperative Agreement

Start Date: Jul 1, 2023
End Date: Apr 30, 2026

The overall goal of this project is to create strains in Bactrocera zonata (Bz) and Anastrepha suspensa (As) for a genetic sexing strategy to improve the Sterile Insect Technique. Following a similar strategy used in the Medfly, an autosomal temperature-sensitive lethal mutation (tsl-) will be created by gene-editing, and once temperature-dependent lethality is verified, its wild type functional allele (tsl+) will be inserted into the male Y-chromosome. Thus, at restrictive temperatures female embryos carrying only the mutant alleles will be expected to die while males carrying the functional allele should survive. Visible mutations will be created for strain and chromosomal linkage identification. Objective 1: To create databases for whole genomic sequences and expressed genes for Bactrocera zonata (Bz) and Anastrepha suspensa (As) for identifying genes that are potential targets for temperature-sensitive conditional lethality and visible mutation markers; and sequencing data for mapping genes encoding fruit fly selectable traits and Y-chromosome insertion sites for male-specific expression in Bz and As. Objective 2: To generate, characterize and genetically map visible mutant markers in Bz and As by CRISPR-Cas9 gene-edited knock-out mutations such as white eye (we) and white pupae (wp). Objective 3: To generate, characterize and genetically map temperature-sensitive lethal (tsl) mutations in Bz and As. Temperature sensitivity and temperature sensitive periods will be established for mutant rearing and selection, and eventual use for sexing during rearing. The tsl+ wild type gene will be inserted into the male-specific Y-chromosome by gene-editing or site-specific recombination.

Genetic pest control is a form of biological control in which modified, laboratory-reared male insects are released into the environment that mate with wild females. In doing so these males transmit a suppression trait (e.g. sterility) to the wild population. Genetic control is species-specific, environmentally non-polluting and has a strong track record in controlling insects of agricultural importance, including in Israel and the USA. A well known example is the Sterile Insect Technique (SIT) that entails mass-release of sterile males. The efficient separation of males and females before field-release is a high priority enhancement for genetic control, but is difficult to technically achieve in a manner that is scalable. Sex separation remains arguably the most challenging technical step, when considering the transfer of such technologies to a new target species. In the medfly, sex separation is achieved using so-called genetic sexing strains (GSSs) that use sex-linked traits, selectable visible markers and temperature-sensitive lethal (tsl) mutations, to distinguish and separate males from females. This project will leverage recent advances in insect CRISPR-based gene editing to begin developing genetic sexing strains (GSSs) in two important fruit fly pests, B. zonata (aka peach fruit fly) in Israel and A. suspensa (aka Caribbean fruit fly) in the USA, for which efficient control measures are not available. Our strategy benefits directly from recent discoveries that we and partners have made in the medfly Tephritid pest. These include identification of genes residing on the Y chromosome acting as master-regulators of sex determination and more recently, the elucidation of ancillary genes underlying medfly GSS characteristics.