Progress 10/01/12 to 09/30/13
Outputs
Target Audience: North Carolina State University: Seminars given by the PD to the departments of Genetics and Plant Pathology on October 1 2012 and November 5 2012 respectively. The seminars included progress on developing and evaluating male-only strains of C. hominivorax. Efforts. The PD co-taught the graduate level course "Principles of Genetic Pest Management" in the fall of 2012. Thirteen graduate students (mix PhD and MS) from a wide range of disciplines from both sciences and humanities took the course (eg Entomology, Economics, Communication). The course is required for the six doctoral students supported by the new NSF IGERT program on "Genetic engineering and society: the case of transgenic pests" and will be taught again in fall 2013. Entomology Society of America Annual Meeting. On 14 November 2012 the PD gave a talk entitled “Development of "male-only" strains of the New World screwworm fly, Cochliomyia hominivorax” in a symposia on “Genetic Pest Management” at the conference. Changes/Problems: The initial version of the single-component tetracycline-repressible female genetic lethal system was not very effective in L. cuprina and C. hominivorax. We found that replacing the Drosophila core promoter with a core promoter from a Lucilia hsp70 gene was sufficient to achieve the high levels of transgene expression needed for 100% female lethality in L. cuprina and C. hominivorax. What opportunities for training and professional development has the project provided? Before moving to Panama, Dr. Carolina Concha was trained at NCSU by Ms Esther Belikoff (manager insect transgenesis facility) on how to use the advanced microinjection equipment from Sutter to microinject blow fly embryos. The same equipment is housed within the screwworm research unit in the biosecure facility in Pacora, Panama. How have the results been disseminated to communities of interest? The PD attended and gave seminar presentations at the ESA annual meeting in November 2012 and at a conference on “Sex determination in insects” in June 2013. Dr. Concha attended the annual BRAG PD meeting in Washington DC in June and gave a poster presentation. What do you plan to do during the next reporting period to accomplish the goals? C. hominivorax. Complete evaluation of female viability of transgenic lines on diet with and without tetracycline. Each line to be tested in triplicate. The most promising lines that show 100% female lethality on diet that lacks tetracycline will be further evaluated. For example, we will measure fertility, fecundity and male competitiveness. L. cuprina. Transgenic lines carrying the two component female embryo lethal system will be made and evaluated. The expectation is that 100% of females will die on diet that lacks tetracycline and that lethality will be predominately in the embryo stage. If successful, the system will be transferred to C. hominivorax.
Impacts
What was accomplished under these goals?
Our main aim has been on developing and evaluating repressible female-lethal genetic systems for the New World screwworm fly, Cochliomyia hominivorax. C. hominivorax was eradicated from North and Central America using the sterile insect technique or SIT. As SIT is 3-10 times more efficient if only sterile males are released, a male-only transgenic gene would be of significant benefit for the ongoing genetic control program. As we cannot work with C. hominivorax in Raleigh, we have tested genetic systems in Lucilia cuprina, a close relative of C. hominivorax. L. cuprina. We completed evaluation of 8 transgenic L. cuprina lines carrying single component tetracycline-repressible female lethal genetic systems. Most of the lines showed 100% female lethality when homozygous for the transgene and three lines were 100% effective as heterozygotes. Fitness testing suggests the transgenic males were competitive with wild type, particularly when in excess. A manuscript describing the research was being drafted for publication in September 2013. We have also been isolating the genetic components needed to make a two-component female lethal genetic system that dies at the embryo stage. To that end we have isolated and functionally characterized Lucilia gene promoters that are active in the early embryo and Lucilia cell death genes. C. hominivorax. As noted earlier, we have developed 22 transgenic lines of the New World Screwworm fly at the USDA facility in Panama. The transgenic lines carry a single component tetracycline-repressible female lethal genetic system. Preliminary analysis suggests that some of the lines are 100% female lethal when raised on diet that lacks tetracycline.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
" Li, F., Vensko II, S.P., Belikoff, E.J. and Scott, M.J. (2013) Conservation and sex-specific splicing of the transformer gene in the calliphorids Cochliomyia hominivorax, Cochliomyia macellaria and Lucilia sericata. PLOS ONE, 8(2):e56303. doi: 10.1371/journal.pone.0056303
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Activities. Most of the experiments conducted in the laboratory were aimed at developing a male-only strain of the New World screwworm fly, Cochliomyia hominivorax, a devastating pest of livestock. To that end we isolated and characterized the transformer gene from three blowflies, C. hominivorax, C. macellaria and Lucilia sericata. Several single component tetracycline repressible female lethal (FL) gene constructs were made and evaluated in transgenic Drosophila melanogaster and the blowfly Lucilia cuprina. Sex-specific splicing of lethal gene transcripts was achieved by using the first intron from the C. hominivorax transformer gene. D. melanogaster is a model genetic organism and is an excellent system for testing gene constructs and we use L. cuprina as a model blowfly species. The most promising FL gene constructs were also tested in C. hominivorax in Panama by our USDA-ARS collaborators, including Dr. Felix Guerrero. We have also been isolating and testing the genetic components required to make an embryo female lethal system (e.g. cell death genes, early embryo gene promoters) as the one component systems are lethal at late stages of development. The classes taught by the PD (Scott) that are relevant to this project include a graduate class in "Epigenetics" in spring 2012 and a graduate class in "Principles of Genetic Pest Management" in fall 2012. With regard to mentoring, the PD met regularly with the postdoctoral fellow (Dr. Li), graduate student (Ms. Rebecca Edman) and visiting scientist (Mr. Ying Yan) to discuss progress with their research. Events. Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (June 5-6, 2012). Presentation. "Development and evaluation of male-only transgenic strains of the New World screwworm fly." "XXIV International Congress of Entomology" in Daegu, South Korea, August 19-25, 2012. Presentation: "Development of "male-only" strains of the New World screwworm fly, Cochliomyia hominivorax". Third Research Co ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project (CRP) "Development and Evaluation of Improved Strains of Insect Pests for SIT" in Daegu, South Korea, August 19-25, 2012. Contributed to writing a report for the IAEA/FAO on progress made by members of the CRP since the previous meeting in Nanjing, China 2011. Dissemination The PD is one of the twenty "most critical" faculty listed in the recently funded NSF Integrative Graduate Education and Research Traineeship (IGERT) program on "Genetic engineering and society: the case of transgenic pests." The program involves faculty from the sciences and social sciences to train graduate students in "technologies used for manipulation of pest genomes as well as methods needed to assess the environmental and socio-cultural appropriateness of specific products of these manipulations." In addition to co-teaching one of the core courses for the program on "Principles of genetic pest management", the PD has participated in weekly sessions that have involved discussions of the research interests of the faculty involved in the program. Science communication is a major component of the IGERT training. PARTICIPANTS: Individuals. Dr. Maxwell J. Scott (PD). Designed experiments, analyzed data, wrote manuscripts, mentored staff and students and provided overall guidance for the project. Dr. Fang Li. Supported 100% by a NIFA Biotechnology Risk Assessment grant to work on the New World Screwworm fly. Dr. Li developed the female lethal gene constructs and evaluated in D. melanogaster and L. cuprina. Ms. Rebecca Edman. A Masters student supported in part by start-up funds to the PD from the College of Agriculture and Life Sciences and my a teaching assistantship. Ms Edman isolated cell death genes and early gene promoters from L. sericata. Mr. Ying Yan is a visiting doctoral student from the Chinese Academy of Agricultural Sciences in Beijing, China and was supported 100% by funds from China. Mr Yan isolated and characterized L. cuprina heat shock and constitutive gene promoters. Ms. Holly Wantuch. A research technician supported in part by start-up funds to the PD from the College of Agriculture and Life Sciences and by a specific cooperative agreement with the USDA-ARS. Ms. Wantuch maintains the blowfly stocks, bred the transgenic lines to homozygosity and performed lethality tests on normal diet and diet supplemented with tetracycline. Partner Organizations USDA-ARS. The USDA-ARS provide financial support through a specific cooperative agreement to develop a male-only strain of the New World screwworm fly. Our USDA-ARS collaborators (Dr. Felix Guerrero and technical staff at the screwworm research unit) test the female lethal (FL) gene constructs developed in the PDs laboratory in the screwworm research unit in Panama. Collaborators and contacts Dr. Nambi Palavesam (Uni of Maryland) made transgenic New World screwworm lines with our FL gene constructs. He worked with our USDA-ARS collaborators in Panama. Training or professional development The postdoctoral fellow and graduate students who worked on this project are listed above under "individuals". TARGET AUDIENCES: Target Audiences. The New World screwworm (NWS) fly is a devastating pest of livestock. The pest was eradicated from the USA using the sterile insect technique. A male-only strain would improve the efficiency of genetic control of NWS, lowering current costs and thus helping to ensure that livestock producers will not have to deal with stock losses due to NWS. Efforts. Formal instruction. The PD developed a new graduate class in "Epigenetics" in spring 2012 and co-developed a new graduate class in "Principles of Genetic Pest Management" in fall 2012. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
change in knowledge. Three of our activities have produced sufficient new information that we anticipate they will lead to future publications. Firstly, the transformer genes from three blowfly species, C. hominivorax, C. macellaria and L. sericata were fully characterized and shown to be essential for female development. A bioinformatics analysis identified conserved motifs in the first intron that could play an important role in sex-specific splicing of this intron. Secondly, a single component female lethal genetic system based on the sex-specific intron from the C. hominivorax transformer gene was very effective in D. melanogaster. 100% of females died when raised on diet that lacked tetracycline. However, the same system was less effective in L. cuprina and C. hominivorax. The system was modified slightly to be more effective in blowflies. Initial results with the modified system in L. cuprina and C. hominivorax are promising. Thirdly, we completed an analysis of the L. sericata cell death genes hid and reaper. Initial functional assays in D. melanogaster indicates that expression of either gene leads to cell death. change in actions. The bioinformatic analysis of the blowfly transformer genes highlighted the likely importance of specific nucleotides at the exon/intron junctions for correct sex-specific splicing. This knowledge was used in building the single and two component female lethal genetic systems. Indeed, the lethal gene transcript was correctly spliced in female D. melanogaster and L. cuprina transgenic lines. Our observation that the initial female lethal genetic system was more effective in D. melanogaster than in blowflies, led to the realization that the gene construct needed to be modified to achieve higher expression levels in blowflies.
Publications
- Concha, C., Edman, R.M., Belikoff, E.J., Schiemann, A.H., Carey, B., and Scott, M.J. (2012) Organization and expression of the Australian sheep blowfly (Lucilia cuprina) hsp23, hsp24, hsp70 and hsp83 genes. Insect Molecular Biology, 21: 161-180. doi: 10.1111/j.1365-2583.2011.01123.x
- Sze, S-H., Dunham, J.P., Carey, B., Chang, P.L., Li, F., Edman, R.M., Fjeldsted, C., Scott, M.J., Nuzhdin, S.V. and Tarone, A.M. (2012) A de novo transcriptome assembly of Lucilia sericata (Diptera: Calliphoridae) with predicted alternative splices, single nucleotide polymorphisms and transcription expression estimates. Insect Molecular Biology, 21: 205-221. doi: 10.1111/j.1365-2583.2011.01127.x
- Stasiuk, S.J., Scott, M.J. and Grant, W.N. (2012) Developmental plasticity and the evolution of parasitism in an unusual nematode, Parastrongyloides trichosuri. EvoDevo, 3:1 doi:10.1186/2041-9139-3-1
- Fitzsimons, H.L. and Scott, M.J. (2011) Genetic modulation of Rpd3 expression impairs long-term courtship memory in Drosophila. PLoS ONE, 6(12): e29171. doi: 10.1371/journal.pone.0029171
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Activities: isolated a number of genes from the blowfly Lucilia sericata that will be useful for developing a male-only strain of the primary screwworm Cochliomyia hominivorax. Isolated sex-determining genes from C. hominvorax that can be used to make tetracycline-repressible female lethal strains. Events: Attended ARS-APHIS Screwworm Project Update Meeting on November 16 at the ARS facility in Kerrville, Texas. Gave a presentation on "Development of molecular approaches for genetic control of insect pests". Participated in planning the development of a male-only strain to be made at USDA-APHIS facility in Panama.Some of the preliminary work will be performed at NCSU in 2011. PARTICIPANTS: Dr Fang Li: Has worked on this project. She started at NCSU on 1 July 2010. Dr. Li has isolated blowfly genes that are highly expressed in embryos, genes that control cell death and genes that determine sex. All of these genes are needed for developing early-acting tetracycline repressible female lethal strains of Cochliomyia hominivorax. Ms. Esther Belikoff: Has worked on this project. She started in mid-August 2010. Ms. Belikoff has established cultures and rearing conditions for the blowfly species Lucilia sericata and Cochliomyia macellaria. These will be used as models for evaluating female killing systems for eventual use in Cochliomyia hominivorax. Ms. Belikoff is an expert in insect transgenesis and has been setting up a core facility at NCSU. Collaborators: Dr. Felix D. Guerrero, Project Leader, USDA-ARS Knipling-Bushland US Livestock Insects Research Laboratory, Kerrville, Texas Dr. Aaron Tarone, Department of Entomology, Texas A&M University, College Station, Texas TARGET AUDIENCES: Target audiences: the primary screwworm Cochliomyia hominivorax was a major livestock pest in the USA prior to its eradication by use of the sterile insect technique. The livestock industry continues to benefit from this eradication program. The strains developed with this project could eventually replace the strain currently used in the buffer zone in Panama and be used to suppress screwworm populations in currently infested areas. Efforts: co-taught a graduate class on "genetic pest management" in fall 2010. 8 students took the class. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge: Learned how sex determining genes are alternatively spliced in C. hominvorax that will form the basis for a future publication. Learned how cell death is regulated in blowfly embryos, which can be published in the future and be applied to developing early-lethal genetic systems.
Publications
- Schiemann, A.H., Li, F., Weake, V.M., Belikoff, E.J., Klemmer, K.C., Moore, S.A. and Scott, M.J. (2010) Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila. BMC Molecular Biology, 11:80. doi:10.1186/1471-2199-11-80
- Concha C., Li F. and Scott M. J. (2010) Conservation and sex-specific splicing of the doublesex gene in the economically important pest species Lucilia cuprina. J. Genet. 89: 279-285
- Moore, S.A., Ferhatoglu, Y., Jia, Y., Al-Jiab, R.A. and Scott, M.J. (2010) Structural and biochemical studies on the chromo-barrel domain of male specific lethal 3 (MSL3) reveal a binding preference for mono or dimethyl lysine 20 on histone H4. J. Biol. Chem. 285: 40879-90. doi:10.1074/jbc.M110.134312
- Schiemann, A.H., Weake, V.M., Li, F., Laverty, C., Belikoff, E.J., and Scott, M.J. (2010) The importance of location and orientation of male specific lethal complex binding sites of differing affinities on reporter gene dosage compensation in Drosophila. Biochemical and Biophysical Research Communications, 402: 699-704. doi:10.1016/j.bbrc.2010.10.088.
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