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Development and evaluation of male-only strains of the Australian sheep blowfly, Lucilia cuprina.

Scott MJ - BMC Genet. (2014)

Bottom Line: 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.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.

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ABSTRACT
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.

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Tetracycline-repressible female lethal genetic systems for Lucilia cuprina. In the single component system [39], tTA expression is auto-regulated as the promoter contains 21 copies of the tTA binding site (tetO) upstream of the core promoter from the Lchsp70 gene [40]. The Chtra intron is inserted within the tTA coding region. As a consequence, only female overexpress tTA due to sex-specific splicing of tTA transcripts. Binding of tTA to tetO results in activation (+++) of tTA gene expression. High female lethality was observed in most transgenic lines when reared on diet that lacks tetracycline [40]. In the two-component system [41,42], which is under development for L. cuprina, tTA expression is controlled by a promoter from a cellularization gene (e.g. bnk), which is most active in early embryos. tTA activates (+++) expression of hid, but only the female hid transcript encodes HID protein due to sex-specific splicing of the Chtra first intron.
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Figure 4: Tetracycline-repressible female lethal genetic systems for Lucilia cuprina. In the single component system [39], tTA expression is auto-regulated as the promoter contains 21 copies of the tTA binding site (tetO) upstream of the core promoter from the Lchsp70 gene [40]. The Chtra intron is inserted within the tTA coding region. As a consequence, only female overexpress tTA due to sex-specific splicing of tTA transcripts. Binding of tTA to tetO results in activation (+++) of tTA gene expression. High female lethality was observed in most transgenic lines when reared on diet that lacks tetracycline [40]. In the two-component system [41,42], which is under development for L. cuprina, tTA expression is controlled by a promoter from a cellularization gene (e.g. bnk), which is most active in early embryos. tTA activates (+++) expression of hid, but only the female hid transcript encodes HID protein due to sex-specific splicing of the Chtra first intron.

Mentions: Moving to the U.S.A., the focus of the lab shifted to developing a transgenic male-only strain of the New World screwworm. However, since it was not possible to work with C. hominivorax in the U.S.A., we continued to work with L. cuprina (using a North American strain) as it is a close relative. Initially, we built a one-component system (Figure 4), based on the system developed by Luke Alphey and colleagues at Oxitec [39]. They found that overexpression of an auto-regulated tTA was lethal (usually pupal stage) but repressible by tetracycline. The lethality was thought to be due to a general interference with gene expression or "transcriptional squelching". The lethality can be made female-specific by incorporating the sex-specifically spliced tra intron into the tTA gene [39]. The appeal of making this system was that we had all of the necessary components, including the first intron from the C. hominivorax tra gene and an efficient transformation system. A disadvantage was that the late lethal period would not lead to any appreciable savings in the costs of diet for mass rearing. The initial system, FL3, used the core promoter from the D. melanogaster hsp70 gene and multiple copies of the tTA binding site (tetO) upstream of the core promoter. Two transgenic L. cuprina FL3 lines were made. In one line, over 99% of homozygous females died on diet that lacked tetracycline [40]. However, there was no decrease in female viability in heterozygous females. Further, homozygous females in the second line were fully viable on standard diet. Interestingly, in transgenic Drosophila FL3 lines, 100% of females died on diet lacking tetracycline. Thus the C. hominivorax tra intron was correctly sex-specifically spliced in D. melanogaster. However, it appeared that the system was not optimal for high levels of tTA expression in L. cuprina. We considered that expression could be improved by replacing the Drosophila core promoter with the core promoter from a L. cuprina hsp70 gene [30]. Two gene constructs were made, FL11 and FL12, which differ slightly in the length of the core promoter. In three of five FL11 lines and two of three FL12 lines, 100% of homozygous females die on diet that lacks tetracycline [40]. Moreover, for three of the lines female lethality is dominant, with one copy of the transgene sufficient for 100% female lethality. Thus these lines could potentially be used for a RIDL genetic control program [40]. Surprisingly, the sexes could be reliably sorted by fluorescence or color at larval stages as females that overexpress tTA also overexpress the linked marker gene. It would appear that tTA bound to tetO is either directly enhancing expression of the Lshsp83 promoter that drives the red fluorescent protein marker, or there is an indirect effect through changes to the structure of the local chromatin domain. Female larvae make so much of the marker protein that they show a crimson color under white light, easily distinguished from male larvae that have a very pale pink color.


Development and evaluation of male-only strains of the Australian sheep blowfly, Lucilia cuprina.

Scott MJ - BMC Genet. (2014)

Tetracycline-repressible female lethal genetic systems for Lucilia cuprina. In the single component system [39], tTA expression is auto-regulated as the promoter contains 21 copies of the tTA binding site (tetO) upstream of the core promoter from the Lchsp70 gene [40]. The Chtra intron is inserted within the tTA coding region. As a consequence, only female overexpress tTA due to sex-specific splicing of tTA transcripts. Binding of tTA to tetO results in activation (+++) of tTA gene expression. High female lethality was observed in most transgenic lines when reared on diet that lacks tetracycline [40]. In the two-component system [41,42], which is under development for L. cuprina, tTA expression is controlled by a promoter from a cellularization gene (e.g. bnk), which is most active in early embryos. tTA activates (+++) expression of hid, but only the female hid transcript encodes HID protein due to sex-specific splicing of the Chtra first intron.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255793&req=5

Figure 4: Tetracycline-repressible female lethal genetic systems for Lucilia cuprina. In the single component system [39], tTA expression is auto-regulated as the promoter contains 21 copies of the tTA binding site (tetO) upstream of the core promoter from the Lchsp70 gene [40]. The Chtra intron is inserted within the tTA coding region. As a consequence, only female overexpress tTA due to sex-specific splicing of tTA transcripts. Binding of tTA to tetO results in activation (+++) of tTA gene expression. High female lethality was observed in most transgenic lines when reared on diet that lacks tetracycline [40]. In the two-component system [41,42], which is under development for L. cuprina, tTA expression is controlled by a promoter from a cellularization gene (e.g. bnk), which is most active in early embryos. tTA activates (+++) expression of hid, but only the female hid transcript encodes HID protein due to sex-specific splicing of the Chtra first intron.
Mentions: Moving to the U.S.A., the focus of the lab shifted to developing a transgenic male-only strain of the New World screwworm. However, since it was not possible to work with C. hominivorax in the U.S.A., we continued to work with L. cuprina (using a North American strain) as it is a close relative. Initially, we built a one-component system (Figure 4), based on the system developed by Luke Alphey and colleagues at Oxitec [39]. They found that overexpression of an auto-regulated tTA was lethal (usually pupal stage) but repressible by tetracycline. The lethality was thought to be due to a general interference with gene expression or "transcriptional squelching". The lethality can be made female-specific by incorporating the sex-specifically spliced tra intron into the tTA gene [39]. The appeal of making this system was that we had all of the necessary components, including the first intron from the C. hominivorax tra gene and an efficient transformation system. A disadvantage was that the late lethal period would not lead to any appreciable savings in the costs of diet for mass rearing. The initial system, FL3, used the core promoter from the D. melanogaster hsp70 gene and multiple copies of the tTA binding site (tetO) upstream of the core promoter. Two transgenic L. cuprina FL3 lines were made. In one line, over 99% of homozygous females died on diet that lacked tetracycline [40]. However, there was no decrease in female viability in heterozygous females. Further, homozygous females in the second line were fully viable on standard diet. Interestingly, in transgenic Drosophila FL3 lines, 100% of females died on diet lacking tetracycline. Thus the C. hominivorax tra intron was correctly sex-specifically spliced in D. melanogaster. However, it appeared that the system was not optimal for high levels of tTA expression in L. cuprina. We considered that expression could be improved by replacing the Drosophila core promoter with the core promoter from a L. cuprina hsp70 gene [30]. Two gene constructs were made, FL11 and FL12, which differ slightly in the length of the core promoter. In three of five FL11 lines and two of three FL12 lines, 100% of homozygous females die on diet that lacks tetracycline [40]. Moreover, for three of the lines female lethality is dominant, with one copy of the transgene sufficient for 100% female lethality. Thus these lines could potentially be used for a RIDL genetic control program [40]. Surprisingly, the sexes could be reliably sorted by fluorescence or color at larval stages as females that overexpress tTA also overexpress the linked marker gene. It would appear that tTA bound to tetO is either directly enhancing expression of the Lshsp83 promoter that drives the red fluorescent protein marker, or there is an indirect effect through changes to the structure of the local chromatin domain. Female larvae make so much of the marker protein that they show a crimson color under white light, easily distinguished from male larvae that have a very pale pink color.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus