A drug-inducible sex-separation technique for insects

Abstract

Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.

Multiplexed drug-based selection and counterselection genetic manipulations in Drosophila

“…Of the sixteen possible two-input binary Boolean logic gates, four commonly used gates can be directly represented using combinations of markers (AND, NOR, and both versions of NOT), whereas the other four commonly used gates (OR, NAND, XOR, and XNOR) can be generated indirectly by combining differentially selected and counterselected populations ( Figure S6 ). Our experiments show that combining selection and/or counterselection drugs can generate a diverse set of genotypically pure populations in one or two generations, going beyond sex-specific selection of flies with a genetically engineered circuit coupled to G418 or puromycin resistance ( Kandul et al, 2020 ). This combinatorial drug flexibility opens up opportunities for sophisticated genetic manipulations, crossing schemes, as well as population-control approaches.…”

“…To eliminate the workload of screening, a drug-based selection approach can be used instead ( Figure S1B ; Steller and Pirrotta, 1985 ; Handler and O’Brochta, 1991 ; Semple et al, 2010 ; Giordano-Santini and Dupuy, 2011 ; Kandul et al, 2020 ). Selection markers confer resistance to universally toxic antibiotics, killing non-transgenic progeny but allowing transgenic, drug-resistant animals to survive ( Steller and Pirrotta, 1985 ).…”

“…Drug-sensitized animals will not survive, whereas non-sensitive progeny will be unaffected by drug exposure. In vivo animal counterselection, akin to counterselection in bacteria and cell culture, has been primarily used for efforts to control disease vector and agricultural pest insect populations by biasing genetic sexing (i.e., generation of purely male populations; Thomas et al, 2000 ; Flores and O’Neill, 2018 ; Kandul et al, 2020 ). Of the three most notable examples, two of the approaches use a two-component tetracycline-repressible expression system to drive female-specific expression of the proapoptotic gene head involution defective ( hid ) ( Heinrich and Scott, 2000 ; Thomas et al, 2000 ).…”

Determining effective drug concentrations for selection and counterselection genetics in Drosophila melanogaster

“…A minor limitation of this protocol is that the resulting transgenic flies may not be physically distinct from wild type animals as drug-based selection/counterselection does not rely on physical markers, requiring molecular analysis to confirm successful transgenesis. This can be remedied by the addition of a physical marker into the design of a selection marker containing transgenic construct for simple, visual confirmation of accurate drug selection/counterselection ( Kandul et al., 2020 ; Matinyan et al, 2021 ; Steller and Pirrotta, 1985 ).…”

Temperature-Inducible Precision Guided Sterile Insect Technique

“…Even the accidental release of a small fraction of Wolbachia-infected fertile females could lead to the wide-scale spread of Wolbachia, which would immunize populations against the particular IIT program, underscoring the importance of effective sex separation. However, with a few species-specific exceptions 10,11 , insect sex-sorting can be time consuming, labor intensive, errorprone, and species-dependent [12][13][14] .…”