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.

Fig. 1. Development of sex-sorter cassette in Drosophila.

a Chemical structures of puromycin and geneticin (G418). b Supplementing fly food with puromycin or geneticin to a final concentration of 0.4 mg/ml completely arrests development of wildtype (wt) D. melanogaster. Both drugs are also toxic to wt larvae at 0.2 mg/ml, but a few adult flies do emerge. c Fly survival in two independent transgenic lines harboring one copy of either Opie2-PuroR or Opie2-NeoR mixed with wt flies on food supplemented with 0.4 mg/mL of puromycin or geneticin. The PuroR and NeoR genes expressed under the Opie2 promoter rescued transgenic flies harboring one copy of a transgene on the corresponding drug, while all wt flies perished. Bar plots show the average  ± one standard deviation (s.d.) over five biological replicates. Statistical significance was estimated using a t-test with equal variance. (***P < 0.001). d Schematic of genetic constructs engineered and tested in the study. The expression of antibiotic-resistance genes (PuroR and NeoR) throughout Drosophila development confers resistance to puromycin and geneticin, respectively, supplemented on fly food. To ensure that functional antibiotic-resistance proteins will be produced only in one or the other sex, sex-specific introns from two sex-determination genes (tra and dsx) were inserted into coding sequences of PuroR and NeoR. The entire sequences of female-specific traF and male-specific dsxM introns (highlighted in pink) are spliced out in the corresponding sex, but some sequences carrying a stop codon (TGA) are retained in the opposite sex (Supplementary Fig. 1). The transgenic flies harboring one copy of a genetic construct were identified by the strong ubiquitous expression of dsRed (highlighted in purple). e Survival of females and/or males carrying the respective constructs when supplemented with the indicated antibiotic. Source data available in Supplementary Data 1–3.

Fig. 2. Positive selection of a specific sex.

Sex-specific drug resistance is achieved by inserting the female-specific traF or male-specific dsxM intron into the coding sequences of PuroR and NeoR. a The efficiency of drug-induced sex sorting was assessed for a few independent transgenic lines of the same genetic construct. Transgenic flies harboring one copy of the antibiotic-resistance genes were raised on drug-supplemented food. When sex sorting was not 100% efficient, the higher drug concetration of 1.0 mg/ml was used. b Expression of Opie2-PuroR^dsxM or Opie2-PuroR^traF transgene rescues only transgenic males or females (red fluorescence) raised on the food supplemented with puromycin, while all wildtype (wt) flies (no red fluorescence) and the transgenic flies of the selected-out sex die during early development. c Both antibiotic-resistance genes expressed in the two sexes were combined into one sex-sorter cassette, Opie2-NeoR^traF + Opie2-PuroR^dsxM. Three independent transgenic lines harboring one copy of the sex-sorter cassette were tested on food supplemented with either puromycin or geneticin at 0.4 and 1.0 mg/ml. We found two transgenic lines that can produce 100% males or 100% females when raised on food supplemented with 1.0 mg/ml of geneticin or puromycin, respectively. Bar plots show the average ± one standard deviation (s.d.) over at least three biological replicates. Statistical significance was estimated using a t test with equal variance. (^nsP ≥ 0.05 , *P < 0.05, **P < 0.01, and ***P < 0.001). Source data available in Supplementary Data 1–3.

Fig. 3. Sex selection and fitness of flies carrying two copies of sex-sorter cassette.

The sex-sorter cassette includes NeoR^traF and PuroR^dsxM genes that confer resistance to the antibiotics puromycin and geneticin, respectively, in a sex-specific manner (Fig. 2a). The PuroR^dsxM gene is properly spliced, which results in the expression of the functional PuroR protein only in males, while NeoR^traF expresses the functional NeoR protein only in females. To estimate the lowest concentration of an antibiotic at which male or female selection is enforced at 100%, the homozygous sex-sorter flies were raised on various concentrations of puromycin or geneticin. a Only male flies emerged from the food supplemented with 0.4 mg/mL or more of puromycin. b Raising the same flies on food containing 0.2 mg/ml or more of geneticin resulted in the emergence of only female flies. To compare the fitness of homozygous sex-sorter flies to that of wildtype (wt) flies, the embryo-to-adult survival of both fly types were compared under normal and selective conditions. c Embryos of both sex-sorter (gray bars) and wt flies (white bars) survived to the adulthood equally well on food without any antibiotics and died on the food supplemented with both puromycin and geneticin at concentrations of 0.4 and 1.2 mg/ml. d The survival of male or female sex-sorter flies under selection treatments was statistically identical to that of the corresponding sex from wt flies raised under normal conditions. Bar plots show the average ± one standard deviation (s.d.) over at least three biological replicates. Statistical significance was estimated using a t test with equal variance. (^nsP ≥ 0.05 , *P < 0.05, **P < 0.01, and ***P < 0.001). Source data available in Supplementary Data 1–3.