Abstract

Genetic approaches, including the sterile insect technique (SIT), have previously been considered for control of the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. In an SIT program, females consume 50% of the diet but are ineffective as control agents and compete with females in the field for mating with sterile males, thereby decreasing the efficiency of the program. Consequently, transgenic sexing strains of L. cuprina were developed that produce 100% males when raised on diet that lacks tetracycline. However, as females die mostly at the pupal stage, rearing costs would not be significantly reduced. Here we report the development of transgenic embryonic sexing strains of L. cuprina. In these strains, the Lsbnk cellularization gene promoter drives high levels of expression of the tetracycline transactivator (tTA) in the early embryo. In the absence of tetracycline, tTA activates expression of the Lshid proapoptotic gene, leading to death of the embryo. Sex-specific RNA splicing of Lshid transcripts ensures that only female embryos die. Embryonic sexing strains were also made by combining the Lsbnk-tTA and tetO-Lshid components into a single gene construct, which will facilitate transfer of the technology to other major calliphorid livestock pests.

Highlights

  • Be reconsidered for control of L. cuprina, with an initial emphasis on the island state of Tasmania[8]

  • The L. sericata bottleneck (Lsbnk)-tTAo gene cassette was in a piggyBac transformation vector containing a constitutively expressed ZsGreen marker gene

  • Nine L. cuprina transgenic lines were obtained by piggyBac-mediated germ-line transformation and bred to homozygosity

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Summary

Introduction

Be reconsidered for control of L. cuprina, with an initial emphasis on the island state of Tasmania[8]. A yp promoter was shown to direct high levels of a reporter gene in transgenic adult female L. cuprina, expression was not detected until the adult stage and only after a protein-meal This was too late a stage to be useful for a building a TSS. Two groups have developed transgenic embryonic sexing systems (TESS) for C. capitata and the Caribbean fruit fly Anastrepha suspensa[22,23] In these systems, tTA expression is driven by a promoter from a cellularization gene that is expressed in the early embryo. We report development of TESS where both Lsbnk-tTA and tetO-Lshid components are contained within a single gene construct This is advantageous for transferring the technology to other calliphorid livestock pests and for “stabilizing” the transgene. The latter involves a two-step recombination/transposition procedure to effectively remove one of the piggyBac ends[26,27]

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