Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters.

For genetic approaches for controlling insect pests such as the sterile insect technique (SIT), it is advantageous to release only males as females are ineffective as control agents and they consume about 50% of the diet. Here we developed tetracycline-repressible Lucilia cuprina transgenic strains in which adult females were fully fertile and viable on a diet that lacked tetracycline and all of their female offspring died at the embryo stage. The transgenic strains are an improvement over the strains we developed previously, which had the disadvantage that adult females on diet without tetracycline were sterile and died prematurely. This was possibly due to the low level expression of the effector gene in ovaries. In the strains developed in this study, the early promoters from L. cuprina nullo or Cochliomyia macellaria CG14427 genes were used to drive the tetracycline transactivator (tTA) expression in the early embryo. In the absence of tetracycline, tTA activates expression of the proapoptotic gene Lshid which contains a female-specific intron. Consequently, only females produce active HID protein and die at the embryo stage. Crossing the tTA-expressing driver lines with an RFPex reporter line confirmed that there was no expression of the effector gene in the ovary. These new embryonic L. cuprina transgenic sexing strains hold great promise for genetic control programs and the system reported here might also be transferable to other major calliphorid livestock pests such as the New World screwworm, Cochliomyia hominivorax.

Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos.

The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making 'male-only' strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.