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.
KeywordsSterile insect technique Transgenic Lucilia cuprina Genetic control Cellularization gene
We thank Amy Berger and Scott Harrison for assistance with fly rearing and Jason Osborne for statistical analysis. The authors acknowledge the use of the Cellular and Molecular Imaging Facility (CMIF) at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation. The project benefitted from discussions at International Atomic Energy Agency funded meetings for the Coordinated Research Project: “The Use of Molecular Tools to Improve the Effectiveness of SIT”.
Y.Y. did qRT-PCR analysis for cellularization genes, carried out the driver plasmid construction, made the transgenic lines, performed the lethality and longevity tests and drafted the manuscript. M.W. made the tetO-RFPex strain and did the confocal microscopy analysis, performed some of the lethality and longevity testing and carried out the RT-PCR analysis for female embryo lethality testing. R.J.L. isolated RNA and performed qRT-PCR analysis for tTA expression of transgenic lines. A.A.A. and C.J.P. assembled and annotated a draft genome of C. macellaria. M.J.S. conceived of the study, participated in its design and coordination and co-wrote the manuscript. All authors read and approved the final manuscript.
Funding is gratefully acknowledged from specific cooperative agreements (59-6205-3-001 and 58-3094-7-013) between the USDA-ARS and NCSU, a grant (01-15) from the Panama-United States Commission for the Eradication and Prevention of Screwworm (COPEG) (MJS) and National Institute of Justice Grant 2012-DN-BX-K024 (CJP).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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