A project undertaken at The University of Melbourne, and supervised by Dr Simon Baxter.
Sterile insect technique (SIT) is a species-specific and environment-friendly genetic pest control approach, whereby overwhelming numbers of sterile male insects are released into a targeted location. They mate with wild females which then produce infertile embryos, causing a localised population collapse. This approach is currently helping control outbreaks of the horticultural pest Queensland fruit fly (Bactrocera tryoni, Figure 1.), however, we can’t separate males from females, so need to use less efficient mixed-sex releases. Opportunities exist to improve the efficacy of SIT programs, by development of genetic sexing strains for conditional separation of males and females, and to establish factory reared strains that look physically different from field populations, to help tell them apart in monitoring traps.
Figure 1. Life stages of the Queensland fruit fly, Bactrocera tryoni. Females can lay eggs directly into ripening fruit which the larvae consume. Their pupae are brown in colour.
This project applied CRISPR/Cas9 gene editing techniques to modify specific Queensland fruit fly genes and develop strains for potential use in SIT applications. As only male insects are required for SIT releases, we aimed to introduce conditional temperature sensitive mutations or sex-linked pupae colour mutations that could be used to remove females from cultures. We also aimed to generate changes to the fly’s body colour, to distinguish between SIT flies and wild flies with the naked eye.
Figure 2. CRISPR/Cas9 mutagenesis of the Black Pupae gene.
Genes involved in female sex determination and development were extensively targeted with gene editing techniques. We hoped to introduce temperature sensitive mutations to the genome of Queensland fruit fly in the laboratory by changing just one DNA residue. Although these results were largely unsuccessful, our efforts helped to improve many aspects of fruit fly gene editing techniques. We found greater success in manipulating genes involved with pigmentation and colour development, creating two strains with dramatic pupal phenotypes: Black Pupae and Greyscale Pupae.
Figure 3. CRISPR/Cas9 mutagenesis of the Greyscale gene creates pupae colour variation. Ongoing research is creating a genetic sexing strain to separate male and female pupae based on colour differences.
Black Pupae: Here we created mutations in a B. tryoni gene we called “black pupae” by using CRISPR/Cas9 editing to delete a small region of DNA (Figure 2). Both the pupae and flies themselves were black in colour and clearly different to regular flies. However, there were some fitness costs associated with the strain, as they were quite difficult to maintain in the laboratory.
Figure 4. Comparison of Bactrocera tryoni wild type flies with a “Yellow mutant” created through a CRISPR/Cas9 knockout of the gene yellow-y. The yellow mutant strain shows reduced melanisation in the wings and regions on their three pairs of legs.
Greyscale Pupae: Introducing mutations to a gene called MFS disrupted normal pigmentation of the pupae case (Figure 3). This mutation has become a highly promising visible marker for developing a genetic sexing strain and this research is ongoing. Greyscale and normal colour pupae can be separated using automated seed sorting machines and we are now assessing their fitness and rigour in the laboratory setting.
Irradiated Queensland fruit flies used in SIT programmes are marked with detectable fluorescent dyes to distinguish them from wild flies when recaptured in monitoring traps. Although dyes enable differential monitoring of sterile and wild flies in the field, coating sterile pupae with powdered dyes can reduce emergence rates or fly quality, can be flawed on a small number of occasions because of inadequate coating or loss of dye, and the dye can be passed onto wild flies through contact. Here we created a phenotypically distinct B. tryoni strain that lacks typical melanisation patterns, again using CRISPR/Cas9. Disrupting the gene yellow-y caused flies to develop with pale coloured wing structures, which are normally dark. Minimal fitness costs were associated with the visible trait and fitness assessments are continuing to determine if these “yellow” flies are suitable for field trials (Figure 4).
The production of improved Qfly SIT strains will increase efficiency of pest control and help reduce reliance on chemical pesticides. Here we created two promising strains for potential use in SIT: a Greyscale strain for sorting males from females at the pupal stage, and a yellow strain, with markings that distinguish them from flies found in the wild.