Building early-larval sexing systems for genetic control of the Australian sheep blow fly Lucilia cuprina using two constitutive promoters

Abstract

Transgenic sexing strains (TSS) that carry conditional female lethal genes are advantageous for genetic control programs based on the sterile insect technique (SIT). It is desirable if females die early in development as larval diet is a major cost for mass production facilities. This can be achieved by using a gene promoter that is only active in embryos to drive expression of the tetracycline transactivator (tTA), the transcription factor commonly used in two-component TSS. While an embryo-specific promoter is ideal it may not be essential for assembling an effective TSS as tTA can be repressed by addition of tetracycline to the diet at larval and/or adult stages. Here we have investigated this idea by isolating and employing the promoters from the Lucilia spitting image and actin 5C genes to drive tTA expression in embryos and later stages. L. cuprina TSS with the tTA drivers and tTA-regulated tetO-Lshid effectors produced only females when raised on a limited tetracycline diet. The Lshid transgene contains a sex-specific intron and as a consequence only females produce LsHID protein. TSS females died at early larval stages, which makes the lines advantageous for an SIT program.

Figure 1

Schematic illustration of the driver constructs. All constructs contain the ZsGreen marker driven by the Lchsp83 gene promoter, a phiC31 attP site and 5′ and 3′ piggyBac ends. Expression of tTA is controlled by the Lsspt promoter in DR3 (A) and the Lcact5C promoter in DR5 (B). The Lcact5C promoter was also used to drive expression of the dtTomato (RFPto) reporter gene (C).

Figure 2

The Lcact5C gene promoter directs expression of the tdTomato (RFPto) marker in most cells but predominantly in gut. Whole body (A) and dissected gut tissues (B) from L. cuprina 3^rd instar marked with constitutively expressed fluorescent protein marker genes. The larvae in (A) were from pBac-Lch83 promoter-ZsGreen -Lcactin5C promoter-RFPto transformed homozygous line (left), pBac-Lch83 promoter-ZsGreen transformed homozygous line (middle) and wild type (right), respectively, and the photos were taken under white light (a), GFP filter (b) and DsRed filter (c). The dissected gut tissues in (B) were from wild type (left), pBac-Lch83 promoter-ZsGreen transformed homozygous line (middle) and pBac-Lch83 promoter-ZsGreen -Lcactin5C promoter-RFPto transformed homozygous line (right), and the images were taken under GFP filter (a) and DsRed filter (b).

Figure 3

tTA expression at different developmental stages in transgenic driver lines. RNA was isolated from the lines DR3#2, DR3#4 and DR5#4 and analyzed by RT-PCR. The sizes of RT-PCR products are: 206bp for tTAv, 165bp for tTAo, 515bp for Lcspt, 495bp for Lcact5C and 121 bp for LcGST1.

Figure 4

Female-specific lethality of double heterozygous lines. Homozygous DR3#2 (A) and DR3#4 (B) males were crossed with virgin females from each of the indicated homozygous effector lines. For the X-linked DR5#4 line (C), virgin females were crossed with males from each homozygous effector line. The offspring of the crosses were raised on diet without tetracycline. Error bars show the standard error of the mean (n = 3).

Figure 5

Female-specific lethality of double homozygous strains. DH3 (A), DH4 (B) and DH5 (C) were raised under different tetracycline feeding conditions. +W: adults given water with 100 μg/mL tetracycline from day 1 (D1) to D8 after pupal eclosion; +−W: water with limited tetracycline (3 μg/mL D1 to D2, then switched to water without tetracycline from D3 to D8); −W: water without tetracycline from D1 to D8; +M: larval diet (93% ground meat) with 100 μg/mL tetracycline;-M: meat without tetracycline. Eggs collected from 8 pairs of adults from each DH strain and error bars show the standard error of the mean (n = 3).

Figure 6

Staged lethality of double-homozygous transgenic sexing strains. DH3, DH4, DH5 and WT flies reared on diet with a high concentration of tetracycline (A) and without (WT, DH5) or limited tetracycline (+−W, DH3, DH4) (B). 1000 embryos were collected and the number of L1, L3, pupae, adult males and adult females were recorded. Error bars show the standard error of the mean (n = 3). The relative proportion of male and female larvae at different stages was determined by RT-PCR (C). With the Lctra primer pair used, 636 bp and 325 bp products were obtained from male and female transcripts respectively. RNA was isolated from WT, DH4 and DH5 strains. cDNA prepared from RNA from adult WT male and female RNA was mixed at ratios of 1:1 (lanes 1,8), 10:1 (lanes 2,9) and 100:1 (lanes 3,10) prior to amplification. Samples from DH4 were: 4) 100 L1 under ++W condition; 5) 100 L1 under +−W condition; 6) 100 L2 under ++W/+M condition; 7) 100 L2 under +−W/−M condition. Samples from DH5 were: 11) 100 L3 under ++W/+M condition; 12) 100 L1 under −W condition; 13) 100 L2 under −W/−M condition; 14) 100 L3 under −W/−M condition. L, DNA ladder.

Figure 7

tTA expression in gut and ovary tissue from adult females. Relative expression of LcSer6 (left panel), Lcvasa (center panel), tTAv (DR3) and tTAo (DR5) (right panel) transcripts in total RNA isolated from adult female gut and ovary tissue dissected from DR3#4 and DR5#4 lines. Transcript levels were normalized to Lcalpha-tubulin. Mean relative expression +/− standard error from three biologically independent replicate qRT-PCR experiments are shown.