Abstract
The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2ts) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2ts SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.
Highlights
The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating
A single guide RNA and a 140 nt single-stranded oligodeoxynucleotide repair template were designed to introduce the amino acid exchanges corresponding to the Dmel ts[1] or ts[2] mutations, to create temperature-sensitive versions of the CcTRA2 protein
The D. melanogaster tra2ts temperature-dependent sex-conversion phenotype promises great advantages for creating male-only populations needed for SIT programs, as it doubles the amount of male offspring per parental egg capacity, and only heat is needed for induction
Summary
The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Can be switched off for strain maintenance by adding the antibiotic tetracycline to the fly food (Tet-off system) Compared to these systems, a sexing system based on temperature-inducible female-to-male conversion would have two advantages: (1) doubling or, compared to semi-sterile GSS, even quadrupling the number of males for the release and (2) abolishing the use of antibiotics. A sexing system based on temperature-inducible female-to-male conversion would have two advantages: (1) doubling or, compared to semi-sterile GSS, even quadrupling the number of males for the release and (2) abolishing the use of antibiotics Both factors would considerably reduce costs and increase the efficiency of a medfly SIT program. Based on this fact and the promising results from the previous transient knock-down of tra[2] in C. capitata[23], Cctra[2] is a good candidate for the construction of a temperature-based sex-conversion system in medfly
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