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. 2020 Dec 18;21(Suppl 2):141.
doi: 10.1186/s12863-020-00947-y.

Building a transgenic sexing strain for genetic control of the Australian sheep blow fly Lucilia cuprina using two lethal effectors

Ying Yan  1   2 , Maxwell J Scott  3
Affiliations

Building a transgenic sexing strain for genetic control of the Australian sheep blow fly Lucilia cuprina using two lethal effectors

Ying Yan et al. BMC Genet. .

Abstract

Background: The sterile insect technique (SIT) has been successfully used in many pest management programs worldwide. Some SIT programs release both sexes due to the lack of genetic sexing strains or efficient sex separation methods but sterile females are ineffective control agents. Transgenic sexing strains (TSS) using the tetracycline-off control system have been developed in a variety of insect pests, from which females die by either of two commonly used lethal effectors: overexpression of the transcription factor tetracycline transactivator (tTA) or ectopic expression of a proapoptotic gene, such as head involution defective (hid). The lethality from tTA overexpression is thought to be due to "transcriptional squelching", while hid causes lethality by induction of apoptosis. This study aims to create and characterize a TSS of Lucilia cuprina, which is a major pest of sheep, by combining both lethal effectors in a single transgenic strain.

Results: Here a stable TSS of L. cuprina (DH6) that carries two lethal effectors was successfully generated, by crossing FL3#2 which carries a female-specific tTA overexpression cassette, with EF1#12 which carries a tTA-regulated LshidAla2 cassette. Females with one copy of the FL3#2 transgene are viable but up to 99.8% of homozygous females die at the pupal stage when raised on diet that lacks tetracycline. Additionally, the female lethality of FL3#2 was partially repressed by supplying tetracycline to the parental generation. With an additional LshidAla2 effector, the female lethality of DH6 is 100% dominant and cannot be repressed by maternal tetracycline. DH6 females die at the late-larval stage. Several fitness parameters important for mass rearing such as hatching rate, adult emergence and sex ratio were comparable to those of the wild type strain.

Conclusions: Compared to the parental FL3#2 strain, the DH6 strain shows stronger female lethality and lethality occurs at an earlier stage of development. The combination of two tTA-dependent lethal effectors could improve strain stability under mass rearing and could reduce the risk of resistance in the field if fertile males are released. Our approach could be easily adapted for other pest species for an efficient, safe and sustainable genetic control program.

Keywords: Genetic pest management; Head involution defective (hid); Sterile insect technique (SIT); Tetracycline transactivator (tTA).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
L. cuprina transgenic sexing strain DH6 carrying the two lethal effectors. a Schematic illustration of the two lethal effectors strategy. The FL3 piggyBac construct contains a ZsGreen marker gene driven by Lchsp83 promoter and a sex-specific tTA overexpression cassette (tetO21-Dmhsp70 core-Chtra intron-tTA-SV40 polyA). The EF1 piggyBac construct contains a DsRed marker gene driven by the Lchsp83 promoter and a sex-specific Lshid Ala2 effector cassette (tetO21-Lchsp70 core-Chtra intron-Lshid Ala2-SV40 polyA). In the absence of tetracycline, tTA is overexpressed from the FL3 transgene causing female lethality at the pupal stage. However, in the two lethal effectors strategy tTA would also activate expression of Lshid Ala2, which acts as the second lethal effector. Consequently, females die at an earlier late-larval stage because of activation of apoptosis. b DH6 (FL3#2; EF1#12) shows both green and red fluorescence in third instar larvae and young adults
Fig. 2
Fig. 2
Female-specific lethality of DH6 with one copy of each transgene. Eight homozygous DH6 males were crossed with eight WT virgin females and their offspring raised on diet without tetracycline. The number of wandering third instar larvae (L3), pupae and adult male and female offspring from each cross were counted. Each experiment was performed three times. Mean ± standard deviation are shown
Fig. 3
Fig. 3
Female-specific lethality of FL3#2 (a) and DH6 (b) under different tetracycline feeding regimens. Containers were set with eight pairs of adults and the number of third instar (L3), pupae and adult male and female offspring were counted. +W: parental generation fed water with 100 μg/mL tetracycline from day 1 (D1) to D8; −W: parental generation fed water without tetracycline from D1 to D8; +M: ground meat (larval diet) with 100 μg/g tetracycline; −M: meat without tetracycline. Each experiment was performed three times. Mean ± standard deviation are shown
Fig. 4
Fig. 4
Staged lethality of FL3#2 and DH6 under different tetracycline feeding regimens. 1000 embryos were collected and the numbers of first instar (L1), third instar larvae (L3), pupae, adult males and adult females were recorded. Each experiment was performed three times. Mean ± standard deviation are shown
Fig. 5
Fig. 5
Fitness parameters of L. cuprina TSS. Homozygous FL3#2 and DH6 were raised in diet containing tetracycline (100 μg/mL), while WT and effector line EF1#12 were raised in diet without tetracycline. a percentage of first instars that hatch from embryos, b percentage of embryos that develop into pupae, c percentage of adults that emerge from pupae, and d sex ratio of emerged adults. Each experiment was performed three times. Mean ± standard deviation are shown
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