Source: NORTH CAROLINA STATE UNIV submitted to
AN INVESTIGATION INTO THE POTENTIAL RISKS OF RELEASE OF TRANSGENIC NEW WORLD SCREWWORM FLY COCHLIOMYIA HOMINIVORAX
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0226393
Grant No.
2011-33522-30730
Project No.
NC09229
Proposal No.
2011-02202
Multistate No.
(N/A)
Program Code
HX
Project Start Date
Sep 1, 2011
Project End Date
Aug 31, 2015
Grant Year
2011
Project Director
Scott, M.
Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695
Performing Department
Genetics
Non Technical Summary
Background: The New World screwworm (NWS) fly, Cochliomyia hominivorax, is a very serious pest of warm-blooded animals. Starting in the 1950s, the Sterile Insect Technique (SIT) was used to progressively eradicate the NWS from U.S.A., Mexico and Central America. The SIT involves rearing of millions of male and female NWS that are sterilized by exposure to radiation prior to being released into the wild. If a wild type female fly mates with a sterilized male fly, no offspring will be produced. By repeatedly releasing sterilized flies, the wild NWS population is gradually reduced in size until the fly has been eradicated from the targeted area. The economic benefits of this highly successful program have been substantial, with an estimated annual savings to the US livestock producers of $896 million. Currently, the USDA-APHIS and the Panamanian government jointly operate a NWS mass-rearing and sterilization facility in Pacora, Panama. Sterilized male and female NWS are released continually in a "buffer zone" in Southern Panama to prevent re-introduction from South America. APHIS has identified several advantages to incorporating transgenic NWS strains into the control program. Firstly, strains that carry stable fluorescent proteins will make it much easier to confirm that flies caught in the "buffer zone" were those released from the factory and not due re-invasion from Columbia. Secondly, strains that facilitate the release of only males would significantly increase the efficiency of SIT and thus reduce the costs of the program. Although it is only necessary to release sterilized males for SIT to work, both male and female flies are currently released as there is no easy way to separate out the female flies. We are currently developing transgenic NWS strains that carry genetic systems that cause female flies to die unless tetracycline is added to the diet. Project Goals. Obtain data for a future risk assessment of (1) Fluorescent protein-marked strains and (2) "male-only" strains. The data obtained from this project will be essential for regulatory agencies to evaluate the potential risks of incorporating engineered strains into the Screwworm Eradication Program. Methods. Various life parameters of transgenic NWS strains will be compared to the wild type strains. In particular we will evaluate longevity, potential to mate with a related fly species, stability (i.e. loss of the engineered gene), and efficacy of the female-killing gene in different genetic backgrounds and over time. Outcomes/Impacts. This project will provide: (1) Engineered strains of NWS that facilitate male-only releases. (2) Data on stability of the strains, mating behavior, fitness and other properties that will be essential for regulatory agencies to assess the risk of future field releases of these strains. Anticipated Benefits: Strains that increase the efficiency of SIT for control of NWS. This will reduce the costs of the ongoing NWS control program in Panama and facilitate any future eradication efforts in countries that are not free of NWS (e.g. some Caribbean islands). The latter pose a risk of re-invasion to screwworm-free countries.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
67%
Developmental
33%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3123110104030%
3123110108040%
3123110113030%
Knowledge Area
312 - External Parasites and Pests of Animals;

Subject Of Investigation
3110 - Insects;

Field Of Science
1080 - Genetics; 1130 - Entomology and acarology; 1040 - Molecular biology;
Goals / Objectives
Goal 1: Evaluation of transgenic strains of the New World screwworm (NWS) fly, Cochliomyia hominivorax, carrying fluorescent marker genes Objectives a) Adult longevity at cooler temperatures: We will compare longevity at various temperatures of the transgenic strain to wild-type. b) Assessment of the potential for outcrossing: We will determine if transgenic males will mate with and transfer genetic material to females from a closely related species, the secondary screwworm fly Cochliomyia macellaria. c) Stability under field-cage conditions: Transgenic strains will be reared in large field cages for several generations and the presence of the transgene and green fluorescent protein (GFP) expression will be monitored. d) Development and assessment of "stabilized" lines: If the existing GFP-marked lines are unstable we will make "stabilized" GFP-marked lines where one piggyBac end has been excised. Transgene stability will then be assessed as described above in objective (c). Timeline: (a) and (b) Initiate and complete in year 1. (c) Initiate in year 1 and complete in year 2. (d) Initiate in year 2 and complete in year 3. Goal 2: Evaluation of male-only transgenic NWS strains Objectives a) Development of transgenic male-only strains. Transgenic strains will be made that carry tetracycline-repressible female-lethal genetic systems. b) Assessment of the fitness of the transgenic male-only strains. We will measure various fitness parameters such as fertility, fecundity, larval survival and pupal weight and compare to wild-type. Additionally longevity at various temperatures will be measured as described above. c) Assessment of the mating behavior of the transgenic male-only strains, potential for outcrossing and male-competitiveness. We will analyze the mating behavior of transgenic males compared to wild-type males and determine if transgenic males can compete effectively. We will also determine the potential for outcrossing with C. macellaria as described above. d) Assessment of the stability of the transgenic lines, including "stabilized" lines. Transgene stability in the male-only strains, including stabilized lines, will be assessed as described above for the GFP-marked strains. e) Assessment of the efficacy of "male-only" transgenic strains in different genetic backgrounds. Transgenic males will be crossed to females from wild-type strains that have been collected from various locations. f) Assessment of the potential for "male-only" lines to lose effectiveness over time. Transgenic lines reared for several generations in large cages and assessed for loss of female-specific lethality. Timeline: (a) Initiate in year 1 and complete in year 2. (b, c) Initiate in year 1 and complete in year 3. (d-f) Initiate in year 2 and complete in year 3. Expected Outputs: 1) Development of stable "male-only" transgenic New World Screwworm strains. 2) Data on the fitness, stability, outcrossing potential, efficacy in different genetic backgrounds and any loss of efficacy over time. Such data will be will be necessary for regulatory agencies to evaluate the potential risks of future field releases of the engineered strains.
Project Methods
Development of stable NWS strains carrying tetracycline-repressible female lethal genes. Several different DNA constructs containing a repressible female-lethal genetic system have been made. The key component of these systems is female-specific expression of the tetracycline-repressible transactivator or tTA. Female flies die when raised on diet that lacks tetracycline due to overproduction of tTA. Transgenic NWS strains carrying some of the DNA constructs have been developed and are being bred to homozygosity. The success of these experiments will be evaluated based on the % of the flies that develop as males when reared on diet that lacks tetracycline. The transgene contains the left and right ends of the piggyBac transposon. Both piggyBac ends and a source of piggyBac transposase are required for re-mobilization of the transgene. As the transgenic strains do not make piggyBac transposase, the integrated DNA should be stable. However, to eliminate the risk of re-mobilization, one of the piggyBac ends will be excised using a recombination system. The success of this step can be readily evaluated by molecular analyses and loss of marker genes. Evaluation of the fitness, mating behavior, stability and efficacy of the transgenic GFP and "male-only" strains: a) Fitness parameters that will measured for the male-only lines include average pupal weight, ratio of males to emerged adults, average fertility, fecundity, larval productivity and longevity. b) To provide data for a risk analysis, we will determine if transgenic males will mate with females from the closely related species Cochliomyia macellaria, the secondary screwworm fly. We will measure the egg production, % egg hatch rate (if any), % of larvae that develop to adults (if any) and adult fertility. c) To assess transgene stability, strains will be reared in large field cages for several generations and the presence and chromosomal location of the transgene in randomly selected flies (20 pools of 10 flies each pool) will be determined by molecular analysis of genomic DNA. d) The male mating competitiveness will be evaluated using cages that will contain 25 transgenic males, 25 wild-type males and 75 wild-type females. The offspring will be scored as either fluorescent (indicating presence of GFP transgene) or non-fluorescent. e) To evaluate if the efficacy of the female-killing system varies in different genetic backgrounds, mating cages will be set with virgin wild-type females and transgenic fertile males. Wild type NWS strains have been collected from several different geographical locations. If there is a genetic background effect, there will be a significant variation between crosses in the % of flies that develop as females on diet that lacks tetracycline. f) To assess the risk that a transgenic line could lose effectiveness over time, lines will be reared on medium supplemented with tetracycline for several generations in large cages. Several hundred embryos will be collected from each generation and raised on normal medium that lacks tetracycline. The % that develop as females will be calculated.

Progress 09/01/11 to 08/31/15

Outputs
Target Audience:The principle target audience for this project is the Commission for the Eradication and Prevention of screwworm (COPEG), which is charged with running the New World screwworm (NWS) eradication and prevention campaign in Panama. COPEG is an international organization established by the United States and the Republic of Panama. The most promising male-only lines strains developed in this project will be transferred to COPEG for evaluation under mass rearing conditions. Additional target audiences include: - stakeholders/growers in the USA, Mexico and Central America that supported the NWS eradication campaign. -Administrators and scientists at USDA-APHIS and USDA-ARS that have supported or have an interest in using genetic modification to improve genetic control of insect pests. -the International Atomic Energy Agency (IAEA) that supports area-wide sterile insect technique programs in many countries. -scientists and academics that have an interest in using genetics for control of insect pests. -students (undergraduate and graduate) studying biology subjects including genetics, molecular biology and entomology. Changes/Problems:Goal 1. Marker-only line. Partially in response to feedback from the review panel it was decided to concentrate on the development of male-only strains (goal 2). Further, rather than concentrate on the existing CLAY line that contains a EGFP marker gene driven by a Drosophila gene promoter we decided that it would be better to make "marker-only" lines that contain the same bright ZsGreen and RFPex marker genes that were used for making the male-only strains. We developed several "marker-only" screwworm lines. The advantage of using these lines is that the data obtained from the marker-only strains can be more directly compared with the male-only strains as they contain the same marker. Goal 2 (d) Line stability. The transgenic lines have been stable over time with no loss of fluorescence or loss of effectiveness. Additionally, previous Southern blot DNA hybridization analysis of screwworm DNA found no evidence of the piggyBac transposon. Further, we did not find piggyBac in the recently completed L. cuprina genome sequence (the screwworm fly genome project is in progress). Thus we have seen no need to make "stabilized" lines by recombination/excision of one of the piggyBac ends. We had suggested in the proposal that this might be done if the lines appeared to be unstable. What opportunities for training and professional development has the project provided?Before moving to Panama, Dr. 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. Dr. Concha has successfully used the equipment to generate over 40 transgenic screwworm lines. Dr. Concha attended the annual BRAG PD meeting in 2013 and gave a poster presentation. Dr Concha attended the Entomology Society of America annual meeting in Austin, TX in 2013 and gave an oral presentation (joint talk with PD). Dr. Concha also attended the Xth European Congress of Entomology in York, England in 2014 and the IAEA Meeting on "Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-Based Technologies" held in Vienna in July, 2015. How have the results been disseminated to communities of interest?The results have been disseminated through primary publications in scientific journals, chapters in books, oral and poster presentations at scientific conferences and talks at stakeholder meetings (COPEG, growers, USDA-APHIS). These are listed under "products". What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT. To prevent the screwworm fly from returning to Central America, approximately 25 million sterilized male and female flies are released weekly along the Panama-Colombia border. Maintaining the barrier zone by constantly releasing sterilized flies is expensive. One way to significantly reduce costs and improve the efficiency of SIT is to release only sterile males. This is because females consume 50% of the diet but are ineffective as control agents and compete with females in the field for mating with sterile males. The major impact of this grant was the development and evaluation of transgenic screwworm strains that produce only males. Gene constructs were made and evaluated in Raleigh in Lucilia cuprina, a close relative of the screwworm. Dr. Concha (supported by this BRAG grant) then made 25 transgenic screwworm lines in Panama with the most effective gene constructs, FL11 and FL12. Ten of the strains were 100% female lethal on diet without tetracycline. 3 of these lines were found to have production characteristics and male mating behavior comparable to the wild type strain. One strain, FL12#56, is currently being evaluated under mass rearing conditions. An application for open field testing of sterilized transgenic males in Panama has been submitted to the Panamanian government. Goal 1. Marker-only lines. Lines were made that contain the same bright ZsGreen and RFPex marker genes that we have used for making male-only strains. These lines were bred to homozygosity and have been reared for over 20 generations. The general fitness parameters of these lines (e.g. pupal yield) is similar to the parental J06 wild type strain. Goal 2. a) Development of male-only strains Single-component late-acting. The genetic system outlined in the proposal consisted of an auto-regulated tetracycline transactivator (tTA) with the sex-specific intron from the C. hominivorax transformer (Chtra) gene. On diet without tetracycline, females should die due to high levels of tTA protein, which interferes with gene transcription. The system was first evaluated in L. cuprina at NCSU in Raleigh. High female lethality was observed in one line but not others. Modifications were made to achieve higher tTA levels. Two constructs, FL11 and FL12 were made that differed slightly in the length of the core promoter. Most transgenic L. cuprina FL11 or FL12 lines showed 100% female lethality without tetracycline in the diet. Females die at the late larval/pupal stage. These results have been published (Li et al, 2014). The FL11 and FL12 constructs were transferred to Panama. Dr. Concha made 9 FL11 lines and 16 FL12 lines. The lines were bred to homozygosity by selecting for brightly fluorescent larvae. They were tested for female lethality in the absence of tetracycline. Four FL11 lines and six FL12 lines showed 100% female lethality when reared in diet without tetracycline. Of these ten lines, three also show 100% female lethality as heterozygotes. Two-component early-acting. A two component early acting female lethal system was developed by Dr. Ying Yan for L. cuprina with funding provided by USDA-ARS and COPEG. The early lethal system comprised "Driver" lines that express tTA at the early embryo stage and "Effector" lines that contain a tTA-regulated hid cell death gene that is interrupted by the Chtra intron. Three L. cuprina lines homozygous for both driver and effector have been bred and evaluated. All lines produce only males if the parental generation is fed a diet with very low levels of tetracycline. Molecular analysis confirms females die at the embryo stage. A paper describing these results is currently under review. The gene constructs that were effective in L. cuprina were transferred to Panama and14 driver and 7 effector transgenic screwworm lines were made by Dr. Concha. These lines have been reared to homozygosity. Crosses between most driver and effector lines produce only male offspring with only one copy of each transgene. Five strains homozygous for both driver and effector have been made and are being evaluated. A female embryo lethal strain would have a major positive benefit for the program as there would be a considerable savings in diet costs. b) Fitness. The ten transgenic lines with the single component system that showed 100% female lethality on diet without tetracycline were evaluated for various parameters that are important for production. These assays include "biological yield" (number pupae that develop from 100mg eggs), pupal weight, egg hatch, egg production and percent pupal eclosion. In general, most of the transgenic lines are comparable to the wild type parental J06 strain. Longevity, particularly at more temperate temperatures, is a consideration when evaluating the risk of an open field release of transgenic strains. Male longevity for most of the transgenic lines was less than the control J06 strain. Only two lines were comparable to J06. We subsequently found that male longevity increased dramatically if males were separated from females (i.e. male only cage). The studies are now being repeated at 25°C and 19°C with J06 males and transgenic males. Until more replicate experiments are performed, we cannot yet conclude if any of the transgenic males live longer than the J06 males at the cooler temperature. c) Mating behavior. Outcrossing potential. The species most closely related to C. hominivorax in Panama is the secondary screwworm C. macellaria. To test for outcrossing potential, 5 transgenic C. hominivorax males were placed in a cage with 15 C. macellaria females. No inseminated females were found in any of experiments. In control experiments with C. macellaria males, 73-93% of the C. macellaria females were inseminated. Thus we conclude the transgenic C. hominivorax males do not have an increased potential for outcrossing. Male aggression and male-competition. To measure male sexual aggression, 5 transgenic males were placed in a cage with 15 wild type J06 virgin females for 8 hours. The females were then examined for the presence of sperm. In all but one of the lines, transgenic males showed a similar level of aggression as the control J06 males. To measure male mating competitiveness, ten transgenic males, ten J06 males and ten virgin J06 females were placed in a cage for 20 hours. The offspring of each female from each cage was screened for fluorescence. Males from three of the transgenic lines (e.g. FL12#56) were fully competitive with J06 males. d) Stability. The FL11 and FL12 screwworm lines have now been maintained for over 40 generations. The strains are checked every few generations and have shown no evidence of instability. That is, fluorescence intensity is maintained and non-fluorescent larvae have not been found. Further, there has been no loss of female lethality over time (see below). e) Genetic backgrounds. Female lethality in different genetic backgrounds was tested by crossing FL11#18B line males with females from 6 different wild type strains. Female lethality was between 98-100% in all crosses, thus genetic background appears to have a only a small influence on female lethality due to overexpression of tTA. f) Effectiveness over time. Female lethality on diet without tetracycline was measured for the FL11#2B line at generations 6, 9, 13, 27 and 33. High female lethality (98-100%) was found at each generation. Similarly, FL12#56 has shown no loss of general fitness or female lethality after 40 generations in culture. Thus we find no evidence for loss of effectiveness over time.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Li, F., Wantuch, H.A., Linger, R.J., Belikoff, E.J. and Scott, M.J. (2014) Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina. Insect Biochemistry and Molecular Biology, 51: 80-88. doi: 10.1016/j.ibmb.2014.06.001
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Sandeman, R. M., Levot, G., Heath, A. G., James, P. J., Greeff, J. C., Scott, M. J., Batterham, P. and Bowles, V. M. (2014) Control of the Sheep Blowfly - are we there yet? Int. J. Parasitol., 44:879-891 pii: S0020-7519(14)00231-8. doi: 10.1016/j.ijpara.2014.08.009
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Scott, M.J. (2014) Development and evaluation of male-only strains of the Australian sheep blowfly, Lucilia cuprina. BMC Genetics, 15(Suppl 2):S3. doi:10.1186/1471-2156-15-S2-S3
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Scott, M.J., Morrison, N.I., Simmons, G.S. (2014) Transgenic approaches for sterile insect control of dipteran livestock pests and lepidopteran crop pests. In: Transgenic Insects: Techniques and Applications (Benedict, M.Q., ed.) pp 152-167, CABI, Wallingford, UK.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2015 Citation: Scott, M.J. and Benedict, M.Q. (2015). Concept and history of genetic control. In: "Genetic Control of Malaria and Dengue", (Adelman, Z. N. ed.) Elsevier, in press.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: COPEG Stakeholders Meeting, Panama City, Republic of Panama May 6, 2015. Presentation: " Prospectus of Genetically Modified Male only Screwworm Strain", with Steve Skoda, USDA-ARS.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: XIV COPEG commissioners meeting, Washington DC, October 9, 2014. Presentation "Status of Developing the Genetically Modified Screwworm Strain(s)".
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: American Association of Veterinary Parasitologists (AAVP) annual meeting, Boston, MA, July 11-14, 2015. Invited plenary presentation " Genetic control of ectoparasite dipteran livestock pests".
  • Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: First Research Co ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project Comparing Rearing Efficiency and Competitiveness of Sterile Male Strains Produced by Genetic, Transgenic or Symbiont-based Technologies in Vienna, Austria from 6 to 10 July, 2015. Presentation: "Transgenic sexing systems for genetic control of the New World screwworm and the Australian sheep blowfly".
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: 10th European Congress of Entomology in York, England, August 3-8, 2014. Presentation: Transgenic sexing systems for genetic control of the New World screwworm Cochliomyia hominivorax.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Fourth Research Co ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project Development and Evaluation of Improved Strains of Insect Pests for SIT in Capri, Italy from April 7-11, 2014. Presentation: " Transgenic sexing systems for genetic control of the New World Screwworm and the Australian sheep blow fly ".
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: "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".
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Annual meeting of the Entomological Society of America, Austin, TX, November 10-13, 2013. Developing male-only strains of the screwworm and the Australian sheep blowfly.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Annual meeting of the Entomological Society of America, Knoxville, TN, November 11-14, 2012. Two presentations: "Characterization of blowfly gene promoters in transgenic Lucilia cuprina" and "Development of "male-only" strains of the New World screwworm fly, Cochliomyia hominivorax"
  • Type: Conference Papers and Presentations Status: Other Year Published: 2012 Citation: Project Directors meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (June 5-6, 2012), Riverdale, MD. "Development and evaluation of male-only transgenic strains of the New World screwworm fly"
  • Type: Journal Articles Status: Other Year Published: 2016 Citation: "Development and evaluation of male-only strains of the New World screwworm" to be submitted.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Project Directors meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (June 5, 2014), Riverdale, MD. "Development and evaluation of male-only transgenic strains of the New World screwworm"
  • Type: Conference Papers and Presentations Status: Other Year Published: 2013 Citation: Project Directors meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (June 14, 2013), Riverdale, MD. "Development and evaluation of male-only transgenic strains of the New World screwworm"


Progress 09/01/13 to 08/31/14

Outputs
Target Audience: North Carolina State University: The PD co-taught the graduate level course "Principles of Genetic Pest Management" in the fall of 2013. Seventeen 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 NSF IGERT program on "Genetic engineering and society: the case of transgenic pests". Entomology Society of America Annual Meeting Austin, TX, November 10-13, 2013. The PD gave a talk entitled “Developing male-only strains of the screwworm and the Australian sheep blowfly”. Fourth Research Co?ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project “Development and Evaluation of Improved Strains of Insect Pests for SIT” in Capri, Italy from April 7-11, 2014. The PD gave a talk entitled "Transgenic sexing systems for genetic control of the New World Screwworm and the Australian sheep blow fly ". BRAG PD annual meeting, Program (June 5, 2014), Riverdale, MD. The PD presented a Poster entitled: "Development and evaluation of male-only transgenic strains of the New World screwworm". “10th European Congress of Entomology” in York, England, August 3-8, 2014. The PD gave a talk entitled: “Transgenic sexing systems for genetic control of the New World screwworm Cochliomyia hominivorax”. Changes/Problems: Personnel. The USDA-ARS laboratory has dealt with the resignation of one technician and the secretary during the course of the last year. Both of these positions take several months to fill. As a result of the absence of these two people there is more limited availability to the technicians that are in ARS as they must also complete the tasks of the absent two colleagues. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Conference Presentations Entomology Society of America Annual Meeting Austin, TX, November 10-13, 2013. The PD gave a talk entitled “Developing male-only strains of the screwworm and the Australian sheep blowfly”. Fourth Research Co?ordination Meeting (RCM) on the FAO/IAEA Co-ordinated Research Project “Development and Evaluation of Improved Strains of Insect Pests for SIT” in Capri, Italy from April 7-11, 2014. The PD gave a talk entitled "Transgenic sexing systems for genetic control of the New World Screwworm and the Australian sheep blow fly ". BRAG PD annual meeting, Program (June 5, 2014), Riverdale, MD. The PD presented a Poster entitled: "Development and evaluation of male-only transgenic strains of the New World screwworm". “10th European Congress of Entomology” in York, England, August 3-8, 2014. The PD gave a talk entitled: “Transgenic sexing systems for genetic control of the New World screwworm Cochliomyia hominivorax”. What do you plan to do during the next reporting period to accomplish the goals? Plans to achieve goals Goal 1: The general fitness parameters of marker-only strains have been evaluated and compared to the parental wild type strain. In addition, in the following months we will measure longevity at various temperatures (a) and outcrossing potential (b) and strain stability (c,d). However, the strain stability experiments have a lower priority than similar experiments with the selected male-only strains (Goal 2). Goal 2: Ten FL11 and FL12 lines of C. hominivorax have been evaluated for female lethality and general fitness parameters. Longevity assays will be completed during the following months and the most promising lines will be tested for longevity at cooler temperatures. Male aggression and male competition assays will be performed to evaluate the performance of transgenic males in comparison to the J06 plant production strain. Stability assays will also be performed and the study of the potential for outcrossing to C. macellaria is in progress.

Impacts
What was accomplished under these goals? Goal 1. Marker–only transgenic C. hominivorax lines. We have developed “marker-only” lines that contain the same bright ZsGreen and RFPex marker genes that we have used for making male-only strains. The advantage of using these lines is that the data obtained from the marker-only strains can be more directly compared with the male-only strains as they contain the same marker. During the course of 2013, we obtained five transgenic lines containing the Lchsp83-ZsGreen marker and four lines containing the Lchsp83-RFPex marker. These lines were bred to homozygosity and have been reared in the USDA-ARS laboratory in Panama for 22 generations. We have also evaluated general fitness parameters of these lines such as pupal weight, biological yield, hatching and egg laying, showing that they are similar to J06, the strain of origin from which the transgenic lines were derived. Goal 2. Male-only transgenic C. hominivorax lines. Single component late-acting system. In 2013 we developed 7 new FL11 and 14 new FL12 lines containing a construct that was optimized for expression in C. hominivorax. All of these lines were raised to homozygosity and tested for female lethality in the absence of tetracycline on a small-scale preliminary assay. The lines that showed significant female lethality in this assay were further studied in larger assays involving three or more replicates. We obtained four FL11 lines and six FL12 lines that show 100% female lethality when reared in diet without tetracycline. Of these ten lines, three also show 100% female lethality as heterozygotes. During the course of 2014 we have evaluated fitness parameters for the four FL11 lines and six FL12 lines that are 100% homozygous female lethal. Biological yield, pupal weight, hatching and egg laying parameters have been completed and longevity studies are in progress. We have also started a set of assays to measure male competition under different controlled conditions in order to determine which is the best assay to measure and compare male performance for the different transgenic male-only lines. We have selected two types of assays to measure male aggression and male competition and these are in progress. Female lethality in different genetic backgrounds has also been tested for the FL11#18B line, which is dominant female lethal. For this assay, the FL11#18B line was crossed to five different strains of C. hominivorax obtained from different locations in South America and regularly reared in the USDA-ARS laboratory in Panama. The results show small differences between the crosses, where heterozygotes of strains from Colombia (Valledupar) and Jamaica (J03) show small levels of female survival. Two-component early acting system. A two component early acting female lethal system was developed for L. cuprina by making “Driver” piggyBac vectors containing an early developmental promoter (either L. sericata bottleneck or serendipity alpha) controlling expression of tTA and “Effector” piggyBac vectors containing a L. sericata cell death gene (Hid or reaper) under the control of a tTA regulated promoter and interrupted by the Lctra intron. Transgenic lines of L. cuprina were made for the different Drivers and Effectors and these lines were reared to homozygosity. Several crosses were made between Driver and Effector lines in order to put in place a system where early expression of tTA drives expression of a cell death gene only in females. The crosses were tested for female lethality in heterozygotes containing one copy of each component of the system. Six different Driver-Effector lines of L. cuprina showed 100% dominant female lethality. Based on these results, one Driver and one Effector construct were sent to Panama for injection into C. hominivorax. During the course of this year, 14 Driver lines and 7 Effector lines of C. hominivorax were made in the USDA-ARS laboratory in Panama. These lines have been reared to homozygosity and are ready to be tested for female lethality in the following months.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: " Li, F., Wantuch, H.A., Linger, R.J., Belikoff, E.J. and Scott, M.J. (2014) Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina. Insect Biochemistry and Molecular Biology, 51: 80-88. doi: 10.1016/j.ibmb.2014.06.001


Progress 09/01/12 to 08/31/13

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. 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. BRAG PD annual meeting, 14 June 2013. Dr. Carolina Concha, the postdoctoral fellow currently supported by the grant, presented a poster on her work on male-only strains of C. hominivorax in Panama at the meeting. Changes/Problems: Personnel. The two postdoctoral fellows who worked on this project in year 1 (Dr Li at NCSU, Dr. Palavesam at USDA-ARS Panama[subcontract]) both returned to their home countries in 2012. Dr. Li left NCSU in July 2012 and Dr. Palavesam in May 2012. Consequently there was a delay in making new transgenic C. hominivorax lines until a new postdoctoral fellow was recruited and trained on the equipment. Dr. Concha was appointed to the position and moved to Panama in January 2013. Goal 1. As described under accomplishments, rather than using the existing CLAY transgenic line we have made several new “marker-only” lines that carry the very bright highly expressed red and green markers that are currently being used to identify transgenic screwworm. It is these new marker-only lines that will be evaluated in year 3. Goal 2. As described under accomplishments, as the initial single-component gene constructs outlined in the proposal were not effective, modified gene constructs further optimized for blow flies were made. These new constructs appear to be very effective but the time frame for evaluating these lines has been shifted to later in the grant period due to the additional time required to optimize the gene constructs. What opportunities for training and professional development has the project provided? Before moving to Panama, Dr. 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. 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 and gave a poster presentation. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Goal 1: The general fitness parameters of the new “marker-only” lines will be evaluated and compared to the parental wild type strain. In addition we will measure longevity at various temperatures (a) and outcrossing potential (b) and strain stabilization (c,d). However, the strain stability experiments have a lower priority than similar experiments with the selected male-only strains (Goal 2). Goal 2: After measuring female lethality on normal and tetracycline supplemented diets and measuring various fitness parameters, selected FL11 and FL12 lines will be evaluated for male behavior and stability as previously outlined. The two-component systems will continue to be evaluated at NCSU in Lucilia species. The most effective gene constructs will be transferred to Panama for evaluation in C. hominivorax.

Impacts
What was accomplished under these goals? Goal 1. Marker-only transgenic C. hominivorax line. Partially in response to feedback from the review panel it was decided to concentrate on the development of male-only strains (goal 2) in the first year. Further, rather than concentrate on the existing CLAY line that contains a EGFP marker gene driven by a Drosophila gene promoter we decided that it would be better to make “marker-only” lines that contain the same bright ZsGreen and RFPex marker genes that were used for making the male-only strains. In this way the data obtained from the marker-only strains could be more directly compared with the male-only strains as they contained the same marker. Thus, C. hominivorax embryos were injected with piggyBac vectors containing either the Lchsp83-ZsGreen or Lchsp83-RFPex marker genes. We have identified five independent transgenic lines with each marker gene. They are currently being bred to homozygosity. Goal 2. “Male-only” C. hominivorax lines. (a) Development. Single-component late-acting. As described in the previous report, although transgenic C. hominivorax lines carrying the gene constructs described in the proposal were made, the lines showed little female-specific lethality on diet lacking tetracycline. The gene construct was subsequently optimized for expression in C. hominivorax and one line carrying the first optimized construct (FL11) was obtained in June 2012. Initial testing suggested this line (FL11NA) had a high female lethality on diet lacking tetracycline (98-100% female lethality) but males and females were equally viable on diet containing tetracycline. This line continues to show high lethality (see f). Development of additional lines with the optimized gene constructs (FL11 and FL12) was delayed for 8 months while a new postdoctoral fellow was recruited and trained. In 2013 Dr. Concha has succeeded in isolating 7 new FL11 lines and 14 FL12 lines. Preliminary analysis suggests that some of these lines are more effective (i.e. higher female lethality in heterozygotes) than the FL11NA line. Two-component early-acting. We have isolated the promoters from three blowfly genes, bnk, slam and sry-alpha that are expressed early in development. The slam and bnk gene promoters have been evaluated in transgenic Lucilia (green blowfly) at NCSU. Both were active in early embryos, with the bnk gene promoter having the highest activity. The Lucilia hid and reaper cell death genes were evaluated in transgenic Drosophila using the GAL4/UAS expression system. Both blow fly genes were effective at inducing cell death in Drosophila with hid being the most active. Three “Driver” gene constructs in piggyBac vectors have been made using the early gene promoters to control expression of tTA. Two additional driver gene constructs are being assembled. Four “effector” gene constructs have been made in piggyBac vectors that contain the hid or reaper cell death gene under the control of a tTA-regulated promoter. Transgenic Lucilia lines have been made with one driver and one effector gene construct. The lines are being bred to homozygosity. (b) Fitness. Some basic fitness properties have been measured for the FL11NA line. The line produces a consistent volume of pupae per 100 mg of embryos and the a consistent pupal weight. These parameters have been measured on at least 10 occasions over the past 6 months. Moreover the pupal volume and pupal weight are comparable to the wild type parental strain. (f) Transgenerational effects. The FL11NA line has now been maintained for 17 generations. Lethality tests have ben performed at each generation. On diet lacking tetracycline, 96-100% of the flies that develop are male, whereas an equal number of males and females develop on diet containing tetracycline. Thus we see no loss of effectiveness of the female-lethal system over time (i.e. no generational effect). Work on objectives c, d, and e will be undertaken after new FL11 and FL12 lines have been made homozygous and fully evaluated for female lethality. The best performing lines will be further analyzed for fitness, stability etc.

Publications


    Progress 09/01/11 to 08/31/12

    Outputs
    OUTPUTS: Activities: Several Transgenic New World screwworm lines were made and evaluated. The initial lines showed either partial or no decrease in female viability when raised on standard diet. Based on data obtained at NCSU from transgenic Drosophila melanogaster and Lucilia cuprina, the single component system was modified from that described in the grant application. To date, one transgenic New World screwworm line has been obtained with the modified system and this has been bred to homozygosity. On standard diet 98-100% of the females die. On diet supplemented with tetracycline equal numbers of males and females develop. Events: Dr Scott gave a presentation on this project at the XXIV International Congress of Entomology" in Daegu, South Korea, August 19-25, 2012. The title of the presentation was "Development of "male-only" strains of the New World screwworm fly, Cochliomyia hominivorax". Dr Scott also gave a talk at the Project Director's meeting for the Biotechnology Risk Assessment Grants (BRAG) Program (June 5-6, 2012) in Riverdale, MD. The title of the talk was "Development and evaluation of male-only transgenic strains of the New World screwworm fly" Dissemination: In May (21-25) 2012, Dr. Scott visited the New World screwworm mass rearing facility in Pacora, Panama and met with several staff from USDA-APHIS and USDA-ARS. Discussions focused on the future development of male-only strains and the potential advantages of transgenic strains for the genetic control program. PARTICIPANTS: Dr. Maxwell J. Scott (PD). Designed gene constructs, analyzed data and supervised Dr. Li. Dr. Felix Guerrero (co-PD). Supervised Dr. Palavesam and analyzed data. Dr. Fang Li. (postdoctoral fellow) Made all of the FL series of single component gene constructs. Generated transgenic Drosophila melanogaster and Lucilia cuprina carrying the FL transgenes and evaluated female viability on standard diet and diet supplemented with tetracycline. Analyzed data. Dr. Azhahianambi Palavesam (postdoctoral fellow). Generated transgenic New World screwworm lines with the FL gene constructs. Supervised the Panamanian technicians who bred lines to homozygosity and performed female lethality tests on diet +/- tetracycline. Analyzed data. Partner Organization: USDA-ARS. The co-PD (Guerrero) works for the USDA-ARS as do the technicians in Panama (not supported by this grant) who rear the transgenic New World screwworm lines. TARGET AUDIENCES: Target Audiences: The development of male-only strain of the New World screwworm would provide several advantages for the ongoing sterile insect program that is run by USDA-APHIS. For example, diet costs should be lowered if female lethality occurs early in development (ie diet is only consumed by males). Further, sterile male-only releases would improve the efficiency of population suppression. In the long term, cattle ranchers and other livestock owners would benefit from a more efficient genetic control program. Efforts. Dr. Scott is a co-instructor for a new graduate level course offered at NCSU called " Principles of Genetic Pest Management". The course is being offered in the fall semester 2012, beginning on August 16, 2012. Thirteen graduate students (mix PhD and MS) from a wide range of disciplines from both sciences and humanities are enrolled in 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". PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

    Impacts
    Change in knowledge: One of the initial single component gene constructs (FL3) was more effective at inducing female lethality in Drosophila than in blowfly species (New World screwworm and Lucilia cuprina). This was at first surprising as most of the parts of the gene construct were of blowfly origin. However, one part of the gene construct was of Drosophila origin, providing a possible explanation for our observations. Change in action: The FL3 single component gene construct was modified taking into account the knowledge gained from our initial experiments. Specifically the part from Drosophila was replaced with a functionally equivalent part from Lucilia. The modified single component system is significantly more effective at reducing female viability in New World screwworm and Lucilia cuprina.

    Publications

    • No publications reported this period