Volume 15 Supplement 2
Development and evaluation of male-only strains of the Australian sheep blowfly, Lucilia cuprina
© Scott; licensee BioMed Central Ltd. 2014
Published: 1 December 2014
The Australian sheep blowfly Lucilia cuprina (Wiedemann) is a major pest of sheep in Australia and New Zealand. From the 1960s to the 1980s there was a major effort to develop "field female killing" or FFK strains of L. cuprina that could be used for a cost-effective genetic control program. The FFK strains carried eye color mutations that were lethal to females in the field but not under conditions in the mass rearing facility. Males did not die in the field as normal copies of the eye color genes had been translocated to the Y chromosome and an autosome. Although the FFK strains showed some promise in field tests, a genetic control program in mainland Australia was never implemented for several reasons including instability of the FFK strains during mass rearing. A stable transgenic strain of L. cuprina that carried one or more dominant repressible female lethal genes offered the potential for efficient genetic control of blowfly populations. Here I review our research on tetracycline-repressible female lethal genetic systems, Lucilia germ-line transformation and sex determination genes that ultimately led to the successful development of transgenic "male-only" strains of L. cuprina. The technology developed for L. cuprina should be directly transferable to other blowfly livestock pests including L. sericata and the New World and Old World screwworm. 29
Female Lucilia cuprina lay eggs on the live animal and their larval offspring cause a cutaneous myiasis (flystrike) in sheep. L. cuprina is the major primary strike fly (fly that causes flystrike) in Australia  and an increasingly significant strike fly in New Zealand . Flystrike can lead to reduced wool quality and quantity and death of the animal if not treated, which largely involves the widespread use of insecticides and good farming practices. The annual economic cost of flystrike is estimated to be AUS $280 million in Australia  and NZ $30-40 million in New Zealand . For many years there was considerable interest in developing strains of L. cuprina that could be used for efficient genetic control programs. These efforts were inspired by the success of the sterile insect technique (SIT) in eradicating the related New World screwworm fly (Cochliomyia hominivorax) from the U.S.A. [4, 5]. Subsequently, New World screwworm was eradicated from all North and Central America . SIT involved mass rearing of the insect, sterilization by radiation and aerial release of sterile males and females over the targeted area. As the sterile males were in a large excess (at least 9:1) over fertile males in the field, the fertile females in the area were more likely to mate with the sterile males. For genetic control of L. cuprina to be cost-effective in Australia, strains were developed that were predicted to be more effective at lower release ratios than a bisexual release and that did not need to be sterilized by radiation [6, 7]. The latter would eliminate the need for a central large expensive mass rearing facility built around a radiation device. Instead several smaller facilities could be built which were expected to be more cost effective given the size of the Australian continent and distribution of sheep blowfly . Eliminating the radiation step could also improve the fitness of the released males, as radiation does reduce the fitness of C. hominivorax males .
"Field female killing (FFK)" strains were developed that were homozygous for two autosomal recessive eye color mutations that essentially made the females blind in the field . The FFK strains carried multiple chromosomal translocations involving the Y chromosome and the two autosomes that had normal copies of the eye color genes. Consequently, males were not blind and were semi-sterile, as most of their offspring would not develop due to aneuploidy. The combination of male semi-sterility and recessive female lethality was predicted to make the FFK strain more effective at population reduction than SIT, particularly at the more cost-effective low release ratios . Indeed, a small island (40 km2) trial in 1985-1986 was successful, achieving a very high genetic death and suppressing the L. cuprina population to undetectable levels . A subsequent larger island field trial initially had some success but ultimately failed. There were problems with mass rearing the strain, which was unstable and prone to breakdown due to recombination in males. With a decline in the wool price, the L. cuprina genetic control program was abandoned. Nevertheless, the concept of releasing flies that were not sterilized by radiation but carried female lethal genes had been established .
Development and evaluation of tetracycline-repressible female lethal genetic systems in Drosophila melanogaster
The successful germ-line transformation of D. melanogaster in 1982  opened the potential for making transgenic insect strains carrying female lethal genetic systems. As females would be needed for mass rearing, female-specific lethality needed to be repressible. In the mid 1990s, not long after establishing our laboratory at Massey University in New Zealand, we began building and testing such genetic systems in D. melanogaster since there were many genetic tools available for Drosophila engineering. Also a method for germ-line transformation of L. cuprina and not yet been developed.
Fortunately, the alternative approach of using a female-specific gene promoter was more successful. A 125 bp enhancer from the yolk protein 1 (yp1) gene had been shown to drive expression of a reporter gene in female fat body . This could have been used to control expression of hid to achieve female lethality but this system would not have been conditional. To achieve regulated expression of hid, we placed the tetracycline-dependent transactivator or tTA gene under the control of the yp1 enhancer (Figure 1). tTA is a fusion of the DNA binding domain from the Escherichia coli tet repressor (tetR) and the viral VP16 transcription activation domain . In the presence of tetracycline, tTA does not bind to the operator (tetO) from the E. coli tetracycline-resistance operon . We found that in transgenic Drosophila, yp1-tTA activated expression of a tetO-lacZ reporter gene in female fat bodies but only in the absence of tetracycline . Thus we had a made a female-specific conditional expression system. The last step was to make tetO-hid "effector" lines and cross with yp1-tTA "drivers" (Figure 1). We found that females carrying both components died unless tetracycline was added to the diet . Thus we had developed a transgenic FFK system. Unknown to us, a group in Oxford led by Luke Alphey had developed an almost identical system, which they called RIDL for "release of insect carrying dominant lethals" . Modeling suggested that the RIDL system would be more efficient than SIT , particularly if the strains carried multiple repressible female-lethal genes that had low fitness costs . It should be noted that, although there are several studies on fitness costs in transgenic RIDL strains (e.g. [21, 22] and references therein), this remains to be evaluated under mass rearing and under field conditions.
Germ-line transformation of L. cuprina
Drosophila transformation was achieved by using vectors based on the P transposable element. However, plasmid-based mobility assays showed that the P element was not functional outside of Drosophilidae . Thus the first step in developing a L. cuprina transformation system was to identify transposable elements that were functional in L. cuprina embryos. Peter Atkinson and colleagues used interplasmid transposition assays to show that the mariner transposable element was functional in L. cuprina . We used similar assays to show that the Minos and piggyBac transposable elements were active in L. cuprina embryos . The next step was to identify a marker gene that could be used to identify transgenic L. cuprina. Initially we used eye color genes as several L. cuprina strains carrying eye color mutations were available, including white eye. However, our attempts at making transgenic L. cuprina using Minos or piggyBac vectors carrying the medfly white gene as a marker were unsuccessful . An alternative to eye color genes were fluorescent protein marker genes . An EGFP gene driven by the D. melanogaster polyubiquitin promoter (PUbnlsEGFP) had been successfully used to identify transgenic D. melanogaster and Anastrepha suspensa [27, 28]. Using a piggyBac vector containing this EGFP marker gene we were successful in transforming L. cuprina . The same vector was subsequently used to make transgenic C. hominivorax .
Genes that are sex-specifically expressed in L. cuprina
In order to develop transgenic L. cuprina carrying the tetracycline-repressible female lethal system that was successful in D. melanogaster, we isolated a L. cuprina yolk protein gene promoter (LcypA) . L. cuprina that carried an LcypA-lacZ transgene showed high levels of β-galactosidase in adult females, but only after a protein meal. Thus, although the LcypA promoter was female-specific, it was active at too late a stage for a FK system .
Development of transgenic "male-only" L. cuprinalines
To achieve female lethality at an earlier stage of development, we have also been working on a two-component genetic system (Figure 4). A similar system was used to develop A. suspensa and C. capitata transgenic embryonic sexing strains [41, 42]. In this system a promoter from a cellularization gene (e.g. sry-α bnk, slam, nullo) that is most active in early embryos is used to drive expression of tTA. The effector is a tetO-hid construct that contains the sex-specifically spliced tra intron inserted in the hid gene. Thus in the absence of tetracycline, tTA is expressed in the early embryo and activates transcription of hid, but only female embryos die as only the female transcript codes for HID protein. To make transgenic embryonic sexing strains of L. cuprina, promoters from Lucilia cellularization genes and Lucilia cell death genes were required. Transcripts for the L. sericata bnk, rpr and hid genes were identified in RNA-seq libraries . The L. sericata bnk promoter was isolated and shown to be active in transgenic L. cuprina embryos using a GFP reporter gene . Expression of the L. sericata rpr and hid genes in transgenic Drosophila induced widespread apoptosis, with Lshid being particularly effective at inducing organismal death . Thus with the required components on hand, we can proceed with assembling the gene constructs necessary to make transgenic sexing strains of L. cuprina where females die during embryogenesis.
Transgenic sexing strains of L. cuprina have been developed that carry a tetracycline -repressible female lethal genetic system. The successful development of these strains built upon prior research on female-lethal genetic systems in D. melanogaster, Lucilia germ-line transformation and sex determination genes in Lucilia. In the near future development of transgenic L. cuprina embryonic sexing strains should be possible as the required components have been isolated from Lucilia and shown to be functional. If the transgenic sexing strains were to be used for a genetic control program in Australia (or New Zealand), the next step would be to introgress the transgene into a more appropriate genetic background by crossing with a field strain from Australia. Cage studies would then be performed to evaluate the ability of the strains to suppress a population. The strains could be used for conventional SIT or a fertile release program. However, if fertile males are released, the male larval offspring will feed on tissue of live sheep, causing damage that may not be acceptable to growers. The systems that have been developed for L. cuprina should be directly portable to other blowfly livestock pests including the New World and Old World screwworm. We are actively working on making and evaluating transgenic sexing strains of New World screwworm (C. hominivorax).
We have been fortunate to receive funding from several sources over the past 15 years including from Wools of New Zealand, Australian Wool Innovation, Massey University, North Carolina State University, USDA-ARS and the NIFA Biotechnology Risk Assessment Grant Program. 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".
This article has been published as part of BMC Genetics Volume 15 Supplement 2, 2014: Development and evaluation of improved strains of insect pests for SIT. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcgenet/supplements/15/S2.
Publication of this supplement was funded by the International Atomic Energy Agency. The peer review process for articles published in this supplement was overseen by the Supplement Editors in accordance with BioMed Central's peer review guidelines for supplements. The Supplement Editors declare that they have no competing interests.
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