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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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Conditional female lethal genetic systems evaluated in Drosophila melanogaster. (Top) Heat inducible female lethal system. The hid open reading frame was interrupted with the female-specific intron from the dsx gene, which contains a weak splice acceptor site. Immediately 3' of hid was the dsxRE, which includes several TRA/TRA2 binding sites. Binding of TRA/TRA2 to the dsxRE enhances (+++) splicing of the dsx intron in females. Further downstream was the intron-exon from the Dror2 gene, which has an optimal splice acceptor site. Males were predicted to use the stronger Dror2 splice site whereas females would use the dsx acceptor site. Thus only the female hid transcript was predicted to encode full-length protein. (Bottom) Tetracycline-repressible female lethal system. On normal diet, expression of tTA in the female fat body of third instar larvae led to activation (+++) of hid gene expression and female-specific lethality. Lethality was repressed by the addition of tetracycline to the diet.
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Figure 1: Conditional female lethal genetic systems evaluated in Drosophila melanogaster. (Top) Heat inducible female lethal system. The hid open reading frame was interrupted with the female-specific intron from the dsx gene, which contains a weak splice acceptor site. Immediately 3' of hid was the dsxRE, which includes several TRA/TRA2 binding sites. Binding of TRA/TRA2 to the dsxRE enhances (+++) splicing of the dsx intron in females. Further downstream was the intron-exon from the Dror2 gene, which has an optimal splice acceptor site. Males were predicted to use the stronger Dror2 splice site whereas females would use the dsx acceptor site. Thus only the female hid transcript was predicted to encode full-length protein. (Bottom) Tetracycline-repressible female lethal system. On normal diet, expression of tTA in the female fat body of third instar larvae led to activation (+++) of hid gene expression and female-specific lethality. Lethality was repressed by the addition of tetracycline to the diet.

Mentions: To achieve female-specific gene expression, it was apparent that this required either a gene promoter that was female-specific or an intron that was sex-specifically spliced. For the latter, we turned to the genes that are part of the well-characterized Drosophila sex determination regulatory pathway [12]. Transcripts from the master gene Sex lethal, from the transformer (tra) gene and from the doublesex (dsx) gene were all known at that time to be sex-specifically spliced. Wilkins had suggested that the genes at the bottom of the regulatory pathway, dsx and fruitless, would be more highly conserved than the master gene at the top of the pathway [13]. This hypothesis has largely proven to be correct. Consequently, we focused on using the sex-specifically spliced intron from the D. melanogaster dsx (Dmdsx) gene as we reasoned that dsx transcripts were also likely to be sex-specifically spliced in L. cuprina. Steller and colleagues had shown that widespread expression of the proapoptotic gene head involution defective (hid), also known as Wrinkled (W), led to organismal death [14]. Thus we decided to insert the Dmdsx intron within the hid gene to obtain a female-specific lethal gene. Expression was controlled with the heat inducible hsp70 gene promoter [15] (Figure 1). The expectation was that after a heat shock, D. melanogaster females would die, as only the female hid transcript would code for fully functional HID protein. Unfortunately, after heat shock both males and females died as in both sexes hid transcripts were spliced using the weak female-specific Dmdsx splice acceptor site. It appeared that the hid transcript contained a nucleotide sequence that enhanced the use of the weak female acceptor site in both sexes [15].


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

Scott MJ - BMC Genet. (2014)

Conditional female lethal genetic systems evaluated in Drosophila melanogaster. (Top) Heat inducible female lethal system. The hid open reading frame was interrupted with the female-specific intron from the dsx gene, which contains a weak splice acceptor site. Immediately 3' of hid was the dsxRE, which includes several TRA/TRA2 binding sites. Binding of TRA/TRA2 to the dsxRE enhances (+++) splicing of the dsx intron in females. Further downstream was the intron-exon from the Dror2 gene, which has an optimal splice acceptor site. Males were predicted to use the stronger Dror2 splice site whereas females would use the dsx acceptor site. Thus only the female hid transcript was predicted to encode full-length protein. (Bottom) Tetracycline-repressible female lethal system. On normal diet, expression of tTA in the female fat body of third instar larvae led to activation (+++) of hid gene expression and female-specific lethality. Lethality was repressed by the addition of tetracycline to the diet.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Conditional female lethal genetic systems evaluated in Drosophila melanogaster. (Top) Heat inducible female lethal system. The hid open reading frame was interrupted with the female-specific intron from the dsx gene, which contains a weak splice acceptor site. Immediately 3' of hid was the dsxRE, which includes several TRA/TRA2 binding sites. Binding of TRA/TRA2 to the dsxRE enhances (+++) splicing of the dsx intron in females. Further downstream was the intron-exon from the Dror2 gene, which has an optimal splice acceptor site. Males were predicted to use the stronger Dror2 splice site whereas females would use the dsx acceptor site. Thus only the female hid transcript was predicted to encode full-length protein. (Bottom) Tetracycline-repressible female lethal system. On normal diet, expression of tTA in the female fat body of third instar larvae led to activation (+++) of hid gene expression and female-specific lethality. Lethality was repressed by the addition of tetracycline to the diet.
Mentions: To achieve female-specific gene expression, it was apparent that this required either a gene promoter that was female-specific or an intron that was sex-specifically spliced. For the latter, we turned to the genes that are part of the well-characterized Drosophila sex determination regulatory pathway [12]. Transcripts from the master gene Sex lethal, from the transformer (tra) gene and from the doublesex (dsx) gene were all known at that time to be sex-specifically spliced. Wilkins had suggested that the genes at the bottom of the regulatory pathway, dsx and fruitless, would be more highly conserved than the master gene at the top of the pathway [13]. This hypothesis has largely proven to be correct. Consequently, we focused on using the sex-specifically spliced intron from the D. melanogaster dsx (Dmdsx) gene as we reasoned that dsx transcripts were also likely to be sex-specifically spliced in L. cuprina. Steller and colleagues had shown that widespread expression of the proapoptotic gene head involution defective (hid), also known as Wrinkled (W), led to organismal death [14]. Thus we decided to insert the Dmdsx intron within the hid gene to obtain a female-specific lethal gene. Expression was controlled with the heat inducible hsp70 gene promoter [15] (Figure 1). The expectation was that after a heat shock, D. melanogaster females would die, as only the female hid transcript would code for fully functional HID protein. Unfortunately, after heat shock both males and females died as in both sexes hid transcripts were spliced using the weak female-specific Dmdsx splice acceptor site. It appeared that the hid transcript contained a nucleotide sequence that enhanced the use of the weak female acceptor site in both sexes [15].

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