White pupae phenotype of tephritids is caused by parallel mutations of a MFS transporter

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp− mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.


nature research | reporting summary
April 2020

Data analysis
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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. -RNAseq was carried out on 3rd instar and pre-pupa libraries in triplicates for C. capitata, B. dorsalis, and Z. cucurbitae. Sequencing six libraries per species, three at each stage, is sufficient to confirm and identify deletions. As the white pupa strains were founded by single individuals, the mutation causing the phenotype would be identical between all individuals allowing a smaller sample size to be used.
in situ hybridizations -In situ hybridizations were done at least in duplicate and at least ten nuclei were analyzed per sample.
CRISPR/Cas9 injections into B. tryoni embryos were carried out over multiple times days. In total, 591 embryos were injected with two RNA guides targeting the first protein coding exon of the MFS gene. In total, 19 (3.2%) individuals survived and completed development, and presented with either wild type brown puparium (n = 12) or were mosaic with a somatic white-brown puparium (n = 7).

No data was excluded
Replication was carried out by generating multiple mutant lines in both, B. tryoni and C. capitata. Multiple individuals containing different mutations were used to generate pure breeding white pupa lines: Bactrocera tryoni CRIPSR/Cas9: Five crosses involving G0 survivors were fertile and produced G1 progeny. After crossing G1 siblings, three of these lines produced individuals with white pupae. Four different mutations were identified among the white pupa individuals through Sanger sequencing. C. capitata: six G0 survivors were crossed individually, the remaining G0 flies were crossed in seven groups of seven to ten flies. Five out of 13 crosses produced white pupae phenotype G1 offspring (reciprocal crosses). Eight different mutations were identified through Sanger sequencing.
RNAseq libraries used to identify and confirm mutations were sequenced from replicate libraries containing different individuals.
In situ hybridizations were done at least in duplicate and at least ten nuclei were analyzed per sample.
PCRs on the D53 inversion breakpoints (C. capitata) were done at least twice, PCRs for non-lethal genotyping (knock-out experiment in C. capitata) were done once per individual, wp-CRISPR alleles were verified via sequencing.
All attempts at replication were successful.
Individuals for sequencing, larvae for in situ, and embryos for injections were sampled from laboratory stocks in a randomized way.
Comparisons between species in both WGS, whole genome comparative and RNAseq comparisons carried out in this paper were a posteriori due to the nature of the experiments and comparisons being made. No other randomization was necessary due to the experiments requiring the sequencing and in situ analysis of targeted species and strains.
Blinding was carried out when WGS samples were mapped to the reference, sample names were changed before genotyping. Blinding was not necessary for the experiments carried out to identify the introgressed region as populations needed to be known ahead of time to carry out tests.