Abstract
Simple SummaryThe sterile insect technique is a pest control strategy used to suppress or eliminate regional populations of insects that pose significant threats to agriculture or human health. The process involves mass-rearing, sterilization and release of male insects who fail to produce viable offspring when they mate with wild females, which leads to a population decline. Females are essential for colony propagation in rearing facilities and their selective removal prior to sterile releases remains an ongoing challenge. Developing genetic sexing strains with conditional temperature sensitive lethal mutations offers one strategy to eliminate female embryos through heat treatment, while males carry a wild type allele translocated to the Y-chromosome (or sex determination locus) to maintain their fitness. Here we review point mutations in Drosophila melanogaster genes that cause temperature sensitive phenotypes with the potential or ability to cause embryonic lethality. Re-engineering these known temperature sensitive mutations in other insects using CRISPR/Cas9 technology presents new opportunities to engineer genetic sexing strains for the sterile insect technique.A major obstacle of sterile insect technique (SIT) programs is the availability of robust sex-separation systems for conditional removal of females. Sterilized male-only releases improve SIT efficiency and cost-effectiveness for agricultural pests, whereas it is critical to remove female disease-vector pests prior to release as they maintain the capacity to transmit disease. Some of the most successful Genetic Sexing Strains (GSS) reared and released for SIT control were developed for Mediterranean fruit fly (Medfly), Ceratitis capitata, and carry a temperature sensitive lethal (tsl) mutation that eliminates female but not male embryos when heat treated. The Medfly tsl mutation was generated by random mutagenesis and the genetic mechanism causing this valuable heat sensitive phenotype remains unknown. Conditional temperature sensitive lethal mutations have also been developed using random mutagenesis in the insect model, Drosophila melanogaster, and were used for some of the founding genetic research published in the fields of neuro- and developmental biology. Here we review mutations in select D. melanogaster genes shibire, Notch, RNA polymerase II 215kDa, pale, transformer-2, Dsor1 and CK2α that cause temperature sensitive phenotypes. Precise introduction of orthologous point mutations in pest insect species with CRISPR/Cas9 genome editing technology holds potential to establish GSSs with embryonic lethality to improve and advance SIT pest control.
Highlights
IntroductionThe sterile insect technique (SIT) is an area-wide, environmentally friendly and speciesspecific biocontrol method aimed at suppressing or eliminating insect pest populations to reduce damage to crops, livestock or transmission of insect-vectored diseases
Initial reports showed no evidence of the shits2 mutation resulting in embryonic lethality, we have found that heat treatment of shits2 embryos at 29 ◦C resulted in lethality of 88% of embryos compared to 26% observed for wild type Canton S flies (Figure 2A), indicating that the shits2 mutation has a moderate effect on D. melanogaster embryonic viability
A temperature sensitive lethal mutation generated by random mutagenesis has been the key to success for the Mediterranean fruit fly (Medfly) Genetic Sexing Strains (GSS), but strain development and optimisation has taken considerable time
Summary
The sterile insect technique (SIT) is an area-wide, environmentally friendly and speciesspecific biocontrol method aimed at suppressing or eliminating insect pest populations to reduce damage to crops, livestock or transmission of insect-vectored diseases. Generation of TSSs requires insertion of foreign DNA from other species into the insect genome, which can result in social and legislative implications regarding their acceptance and adoption [60] To potentially avoid these issues, Subtractive Transgene Sex Sorting has recently been developed in D. melanogaster as an innovative strategy to mass produce non-transgenic males by crossing specialised stocks of transgenic insects that carry tetracycline repressible lethal circuits on sex chromosomes [61]. Several advantages of developing strains with dominant W chromosome conditional lethal temperature sensitive mutations include: (i) elimination of females using a restrictive temperature is inexpensive (ii) insect crosses could transfer the W chromosome (carrying the dominant allele) into a genetic background that best suited a particular country or region (iii) and the released sterile males will not carry transgenic constructs or factors associated with the sexing selecting process [66]. This would involve identification of suitable candidates among known D. melanogaster temperature sensitive mutations, introducing the orthologous mutation into the genome of the pest species using CRISPR/Cas, and subsequently, translocation of a dominant wild type allele to the male-specific Y chromosome to rescue males, or translocation of a dominant mutant allele to the female-specific W chromosome to cause female conditional lethality
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