Improving the sex-specificity of a conditional female lethal system for genetic biocontrol of the New World screwworm, Cochliomyia hominivorax

Abstract

The New World screwworm is an obligate parasitic fly and a significant economic pest of livestock in the Americas. Although eradicated from the USA using the Sterile Insect Technique (SIT), enhancing SIT efficiency remains a priority. A promising approach involves conditional female-lethal genetic strains that produce only males in the absence of tetracycline, ideally eliminating females early in development to reduce larval diet costs. However, while some strains match wild-type production levels, lower male fitness reduces the net benefit of replacing the current wild-type strain with one of these genetic-sexing strains. This study aimed to improve strain performance through female-specific expression of both the driver and effector components of the lethality system. We tested four transgenic strains using early embryo-specific promoters from the Chhalo and g6451 genes. Strains with the Chhalo promoter driving tTA expression exhibited early-stage female lethality under a modified doxycycline regimen but suffered from reduced male fitness. In contrast, one strain with the g6451 promoter produced males with excellent fitness but female lethality occurred at the late pupal stage. Despite imperfect female lethality timing, the overall fitness characteristics of this strain makes it a good candidate for future sterile or fertile male release genetic control programs.

Keywords: Cochliomyia hominivorax; Screwworm; Sterile insect technique; Tetracycline transactivator; Transformer; fsRIDL.

Fig. 1

Developmental gene expression in the DR9 and DR10 lines. (a) Schematic of the gene constructs. In the DR9 construct, tTA is driven by the promoter from the Chhalo gene, while for DR10 construct the promoter from the g6451 gene was used. The tTA coding region is interrupted by the sex specific intron from the Chtra gene in both constructs (schematized with the small triangle in yellow). ZsGreen is constitutively driving by Lchsp83. In the effector construct EF1, there are 21 copies of tetO upstream of the core promoter from the L. cuprina hsp70 gene. The promoter is driving expression of the proapoptotic gene hid from L. sericata (b-e) RT-PCR analyses of RNA isolated from 0–1 (E1H), 2–3 (E2H) and 4–6 h (E6H) embryos, wandering third instar larvae (L3) and pupae (P2 stage 48 h). (b,c) Chhalo and tTA expression in the DR9 lines. (d,e) g6451 and tTA expression in the DR10 lines. The ChGAPDH gene was used as a positive internal control. For images of the complete agarose gels refer to Figures S1 and S2 in the supplementary material.

Fig. 2

Sex specific splicing of tTAv transcripts in the DR9#3-2B line. (a) Excision of the Chtra intron in females is expected to produce a 639 bp RT-PCR product. (b) Retention of the male exon should produce a 933 bp product in males. (c) DNA fragments obtained after amplification of RNA from mixed sex embryos at 2 h. Green, blue and yellow arrowheads indicate the 639 and 933 bp fragments, respectively. The full gel image is shown. Alignments of DNA sequences of cloned RT-PCR products supported these assignments (a,b).

Fig. 3

Sex specific splicing of tTAv transcripts in the DR10#5A line. Alignment of transcript sequences identified an intron in the 5’UTR of the g6451 gene promoter fragment that was not annotated in the assembly (a) Excision of the Chtra intron in females is expected to produce a 526 bp RT-PCR product. (b) Retention of the male exon should produce a 820 bp product in males. (c) DNA fragments obtained after amplification of RNA from mixed sex embryos. Green and yellow arrowheads indicate the 526 and 820 bp fragments, respectively. The full gel image is shown. Alignments of DNA sequences of cloned RT-PCR products supported these assignments (a,b).

Fig. 4

Comparison of two doxycycline feeding schedules on the lethality of two component strains. Percentage survival at 8, 24, 48 and 72 h after hatching is shown. The comparative analysis of average survival was carried out between each stage of development. (a) Standard doxycycline feeding protocol for embryo lethal strains. + D = 25 μg/mL doxycycline added to adult and larval diets. − D = Doxycycline is omitted from the parental adult diet and larval diet of offspring. Only the DR10#5AxEF1#16 strain shows significantly reduced viability on the − D regimen. Although DR9#3xEF#1 at − D regimen shows significant differences at 8, 24 and 72 h after hatching, the percentage of mortality does not correspond to the expected 50% if females had died. (b). Survival of embryos and larvae in the modified feeding protocol. Δ + D 25 μg/mL doxycycline added to adult and larval diets. Δ-D Doxycycline is omitted from the parental third larval and adult diets and the larval diet of offspring. The DR9#3xEF1#16 strain shows significantly reduced viability on the Δ-D regimen. J-06 is the parental control colony (black bar). The solid bars correspond to the DR9 and DR10 double component strains with doxycycline and the striped bars without doxycycline. Statistical differences vs J-06 are indicated with letters.

Fig. 5

Female-specific lethality of DR9 and DR10 double component strains. The percentage of pupae that emerged as males, females or did not emerge (NE) are shown. + D and Δ + D, diet with 25 μg/mL doxycycline at all stages. − D, doxycycline omitted from parental adult diet and larval offspring diet. Δ-D, doxycycline omitted from parental L3 and adult diets and larval offspring diet. Strains reared in the absence of doxycycline show significant differences between sexes (p-value < 0.001, Wilcoxon test).

Fig. 6

Mating success, competitiveness, and longevity of double component males. Two component strains were reared without doxycycline in the parental adult diet and larval offspring diets (D) or in addition doxycycline was omitted from the parental L3 diet (Δ-D). (a) Male mating success. Percentage females that mated when 5 transgenic males were placed with 15 J-06 females for 4 h. DR9#4-1xEF1#6 and DR10#5AxEF1#6 were less competent when compared to J-06 (p-value < 0.05, T-test). (b) Male competitiveness. Mating success of 10 transgenic lines males and 10 J-06 males for 10 J-06 females over 20 h. The male competitiveness index (MCI) values are shown. An MCI of 0.5 indicates that the transgenic strain males are equally competitive with the control strain. Asterisks indicate the MCI was significantly less than the null-hypothesized value of 0.5. With males from the DR9#4-1xEF1#6 strain, the preference for J-06 males was borderline statistically significant (MCI = 0.37, 95% confidence interval of 0.249–0.496). The DR10#4-3xEF1#6 males were equally competitive with the J-06 control line on the either antibiotic feeding regimen [(-D) or (Δ-D)]. (c) Male survival measured as days post adult emergence. The mean values of three replicate experiments are shown. There were no significant differences in the longevity of any of the strains compared to J-06 regardless on the diet regimen, except for DR9#3-2BxEF1#6 on the modified feeding protocol Δ-D (p < 0.001, Multiple Comparisons of Means, Tukey Contrasts)).