Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni.

BACKGROUND: Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. RESULTS: Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. CONCLUSIONS: We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

Identification of Y-chromosome scaffolds of the Queensland fruit fly reveals a duplicated gyf gene paralogue common to many Bactrocera pest species.

Bactrocera tryoni (Queensland fruit fly) are polyphagous horticultural pests of eastern Australia. Heterogametic males contain a sex-determining Y-chromosome thought to be gene poor and repetitive. Here, we report 39 Y-chromosome scaffolds (~700 kb) from B. tryoni identified using genotype-by-sequencing data and whole-genome resequencing. Male diagnostic PCR assays validated eight Y-scaffolds, and one (Btry4096) contained a novel gene with five exons that encode a predicted 575 amino acid protein. The Y-gene, referred to as typo-gyf, is a truncated Y-chromosome paralogue of X-chromosome gene gyf (1773 aa). The Y-chromosome contained ~41 copies of typo-gyf, and expression occurred in male flies and embryos. Analysis of 13 tephritid transcriptomes confirmed typo-gyf expression in six additional Bactrocera species, including Bactrocera latifrons, Bactrocera dorsalis and Bactrocera zonata. Molecular dating estimated typo-gyf evolved within the past 8.02 million years (95% highest posterior density 10.56-5.52 million years), after the split with Bactrocera oleae. Phylogenetic analysis also highlighted complex evolutionary histories among several Bactrocera species, as discordant nuclear (116 genes) and mitochondrial (13 genes) topologies were observed. B. tryoni Y-sequences may provide useful sites for future transgene insertions, and typo-gyf could act as a Y-chromosome diagnostic marker for many Bactrocera species, although its function is unknown.