The Tephritid Problem
The global fruit industry is burdened by the Tephritidae family of invasive fruit flies, which cause significant agricultural yield and economic losses. As pictured on the right, females of this family lay eggs in ripe fruits/vegetables, where the larvae hatch and feed on the flesh.
The current method of population suppression for tephritids is the sterile insect technique (SIT). Here, mass-produced sterile males are released into the wild to increase infertile matings, which results in the decline of the wild population. This technique has been widely and successfully used since its proposal in 1955. However, it is necessary that these releases only consist of males this adds extra costs and labour in sex sorting flies.
Sterile Insect Technique
This technique involves frequent releases of sterile males into wild populations that lead to infertile matings and consequently population suppression.
Males are sterilised at large scales using ionizing radiation that induces chromosomal rearrangements, affecting their overall fitness, and mating competitiveness.
The widespread continued use of SIT is restricted by the requirement for male-only releases as bisexual ones are found to be less efficient (Rendón et al., 2004).
Since released females can still contribute to crop damage and mate with co-released males, mass-reared populations are sex-sorted, a labour-intensive process requiring large facilities.
Furthermore, the female embryos need to be reared till adulthood when they are sorted and removed from release. Wild-type females have been previously reported to favour matings with fellow wild-type males over the released sterile males (McInnis et al., 1996).
With this method, a single release of 1 million sterile males costs an estimated US$150 million and given that repeated releases are required to suppress the wild population significantly, this method is inefficient.
Precision-guided Sterile Insect Technique
A newly suggested alternative to SIT utilising CRISPR/Cas9 constructs is known as precision-guided SIT (pgSIT) (Kandul et al., 2022, 2021, 2019).
Here, transgenic strains are maintained separately, harbouring Cas 9 or double guide RNAs (dgRNAs) simultaneously targeting essential genes for male fertility and female sex determination (see Fig 2A).
The genetic crosses between these two strains would, theoretically, generate a progeny of all sterile males for mass release (Kandul et al., 2019). Sterile males are generated for mass release by eliminating the need for large-scale sex sorting, and the cost of female rearing. Compared to SIT, CRISPR/Cas9 systems efficiently knock out target genes, i.e. pgSIT causes fewer off-target mutations that would have otherwise led to compromised fitness and mating competitiveness. Finally, pgSIT male releases into the wild population require less sterile males when compared to SIT.
Fig 2A: When targeted embryonic silencing of genes transformer and B2-tubulin leads to male development and sterility.
Fig 2B: In a pgSIT Test cross a Cass9 harboring female mates with a dgRNA harboring male.
pgSIT has been shown to eliminate 100% of females and sterilize 100% of males in a proof-of-concept study in Drosophila melanogaster (Kandul et al., 2019).
To establish pgSIT in the medfly, the Meccariello lab previously modelled several promoters and generated two dsRED tagged Cas9 lines under the control of Drosophila melanogaster polyubiquitin promoter (Dmel pUb). The Dmel pUb mediated Cas9 transgenic strains, OAM_S14 and OAM_S16, were established by PhD student Serafima Davydova. When crossed with a C. capitata U6.1 promoted-gRNA strain targeting the white eye (we) gene, homologous to its orthologue in Drosophila melanogaster, the lines were characterised to be 100% efficient in generating a nonred eye phenotype. This suggested the CRISPR/Cas9 system successfully knocked out the we gene (unpublished data).
Aims of my study
01
Homozygous lines
Establish and charcterise homozygous transgenic lines that harbour Cas 9 and dgRNA targeting tra and b2tub, under the control of endogenous pUb and U6.1 promoters respectively.
02
Perform a test cross
Demonstrate pgSIT in the medfly by crossing newly established dgRNA lines with Dmel pUb-Cas9 strains
03
Analyse the offsprings
Assess the fecundity and fertility of pgSIT F1 progeny.
Methods
Plasmid Constructs
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Results
