DEVELOPMENT OF NOVEL SYSTEMS FOR ENHANCED GENETIC CONTROL OF THE NEW WORLD SCREWWORM

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1020092
• Project No.: NC02772
• Project Start Date: Oct 1, 2019
• Project End Date: Sep 30, 2024

Project Director
Scott, MA.

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Entomology and Plant Pathology

Non Technical Summary
One the greatest accomplishments of the USDA in the 20th century was the eradication of the New World screwworm (NWS) fly from all of North and Central America. NWS females lay their eggs in open wounds or a natural orifice. The hatched larvae then feed on the animal's living tissue. Animals with severe screwworm infestations may die if untreated. NWS was eradicated using the sterile insect technique, which involved the mass rearing of NWS, sterilization by irradiation and distribution of sterile flies over the targeted area. Currently, sterile NWS are released in a " buffer zone" in along the Panama-Colombia border to prevent re-infestation from South America.Transgenic "male-only" NWS offer the potential of more efficient genetic suppression, reduced diet costs, higher plant capacity, improved monitoring and reduced risk should (in the very unlikely event) any NWS that escape the plant. Transgenic NWS lines have been obtained carrying a single-component tetracycline-repressible female-lethal system. Females die at the pupal stage, which does not reduce diet costs. Further, the transgenic males appear to be less fit than the current production strain males.The aim of this project is produce new male-only strains that could be an improvement on the previously generated strains. We propose to make and evaluate 3 novel genetic systems. With the first system, females should die at the embryo stage due to expression of a cell death gene. This would lead to considerable savings as the larval diet is a major cost for NWS production. However, any low level expression of the cell death gene in males could lead to reduced fitness. With the second system, females do not die but instead develop as XX males. This approach could produce males with improved fitness. With the third system, the CRISPR/Cas9 system is used to degrade the X chromosome during sperm development. As a consequence only eggs fertilized by Y-bearing sperm will develop. This system also has the advantage of being embryo-specific but does not rely on tight control of expression of a cell death gene. Thus it could produce competitive males. All strains will be further evaluated for characteristics that influence production and male fitness in the field. We will measure parameters such as fertility, fecundity and male competitiveness.

Animal Health Component

0%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
312	3110	1040	100%

Knowledge Area
312 - External Parasites and Pests of Animals;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology;

Keywords

new world screwworm

sterile insect technique

crispr/cas9

transformer

male-only strain

rnai

Goals / Objectives
Improve conditional female embryo lethal systems. Make and evaluate new gene constructs that use embryo-specific promoters to drive expression of tTA. Evaluate if tetracycline derivatives that bind to tTA, but have no antibiotic activity, can be used for rearing strains and if so improve male fitness.Develop conditional female transformation systems. Without transformer, females develop as males. Develop tetracycline-repressible systems that target the transformer gene. For example, constructs that express double-stranded RNA to trigger use RNA interference (RNAi) degradation of transformer gene RNA.Develop Cas9-based systems that target the X chromosome during sperm development. This is the so-called "X shredding" approach for making male-only strains. Using a bioinformatics approach to identify repetitive sequences on the screwworm X chromosome. Make transgenic lines that express Cas9 and gRNAs in the testes. The gRNAs target Cas9 to the X-specific repeats.

Project Methods
Efforts.Gene constructs and transgenic screwworm will be made using established procedures. Fitness tests of transgenic lines (e.g. egg hatch, pupal weight) and male behavior tests (e.g. sexual aggression, male competition) will be done using our previously developed procedures (Concha et al [2016] BMC Biology).Evaluation.Transgenic strains will be evaluated for effectiveness by counting the number of phenotypic males and females on standard diet that lacks tetracycline.The milestones for this project include:Female embryo lethalMake new DR7 and DR6 lines and breed to homozygosityMake new female embryo lethal strains by combining DR7 and DR6 lines with existing effector lines and determine the level of female lethality in diet that lacks tetracyclineFemale to male transformationMake transgenic Lucilia cuprina and screwworm strains that carry tra RNAi constructsCombine RNAi lines with tTA driver lines and determine the level of female to male transformation on diet that lacks tetracyclineX shredderIdentify X-linked repetitive sequencesDevelop strains that express Cas9 and gRNA in the testes and evaluate effectiveness by crossing with wild type femalesAll strainsDetermine the fitness levels of the new transgenic strains by measuring several important biological parameters including male competitiveness

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:1. COPEG. The Commission for the Eradication and Prevention of Screwworms [Comisión Panama - Estados Unidos para la Erradicación y Prevención del Gusano Barrenador de Ganado (COPEG)], cooperatively managed by the USDA and the Panama Ministry of Agriculture and Livestock (MIDA), operates the mass rearing facility in Panama. We participated in monthly calls with COPEG, USDA-APHIS and USDA-ARS to report on progress on the development of male-only strains. We also traveled to Panama in February 2020 to report on progress on male-only strains. 2. Scientists. We participated in relevant conferences, giving talks on the screwworm male-only strains. We also published our results in peer-reviewed journals. Changes/Problems:Progress over the reporting period was limited due to covid restrictions on the number of people allowed at any one time in my lab in Raleigh and in the USDA screwworm research laboratory in Panama What opportunities for training and professional development has the project provided?Due to covid restrictions in 2020, there were few opportunities for training and professional development. How have the results been disseminated to communities of interest?Dr. Scott was on "scholarly reassignment" (sabbatical) in the fall semester of 2019 at the University of Zurich in Switzerland. Consequently, several talks were given at European venues early in the reporting period. Few talks were given in 2020 due to covid restrictions. Presentations: Groningen Institute for Evolutionary Life Sciences (GELIFES) in Groningen, Netherlands, October 3, 2019. Invited talk on "Development of genetic systems for control of insect pests". Max Planck Institute of Immunology and Epigenetics, Freiburg im Breisgau, Germany, October 18, 2019. Invited talk on "Development of genetic systems for control of insect pests". Fourth and final 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 Adelaide, Australia from December 2-6, 2019. Presentation: "Development of a simple gene drive system in Drosophila and an update on screwworm transgenic embryo sexing strains". Developmental Biology at the Johann Friedrich Blumenbach Institute of Zoology and Anthropology, Georg August University, Göttingen, Germany, (May 28, 2020), invited talk (zoom) "Development of genetic/genomic resources for basicinvestigations and for genetic control of blow flies". Annual meeting of the American Veterinary Medical Association (AVMA), virtual convention, August 21 2020. Invited talk (zoom) on "Genetic control of the New World screwworm". What do you plan to do during the next reporting period to accomplish the goals?1. Improve conditional female embryo lethal systems for the New World screwworm. Make EF3 transgenic lines and evaluate their fitness and effectiveness when crossed with existing tTA driver lines. 2. Develop conditional female transformation systems. Complete evaluation of Lucilia cuprina tTA driver, traIR double homozygous strains. Make traIR gene constructs for screwworm transformer and transfer to Panama. 3. Develop Cas9-based system that target the X chromosome during sperm development. Bioinformatic analysis of screwworm and L. cuprina X chromosomes, including identification of any X-specific repetitive sequences. Make and evaluate screwworm gene promoters for expression of tTA and Cas9 in testes.

Impacts
What was accomplished under these goals? 1. Improve conditional female embryo lethal systems for the New World screwworm. Conditional female lethal strains are made by combining a line that expresses the tetracycline transactivator (tTA) in embryos with a tTA-activated effector gene. The effector gene, EF1, promotes widespread cell death or apoptosis leading to death of females. Females survive it tetracycline is added to the diet. We proposed to evaluate new DR6 and DR7 driver lines as prior research in Raleigh indicated these drivers expressed very high levels of tTA in embryos. Five DR7 and five DR6 transgenic lines were obtained and bred to homozygosity in the USDA-ARS facility in Panama. Two DR6 lines (#2, #5) were selected to combine with two of the EF1 lines. The two component strains did not show 100% dominant female lethality on diet without tetracycline and are thus are not an improvement on the existing strains made with other tTA driver lines. Attempts to combine the new DR7 lines with existing EF1 lines have not been successful. The new lines appear to make too much tTA, thereby reducing fitness. We will continue to evaluate the DR6 lines and plan to make and test a new effector, EF3. In male competition tests in Raleigh with transgenic Lucilia cuprina (a close relative of screwworm) we have found that males carrying only the EF1 effector have reduced fitness, possibly due to leaky expression of the potent cell death gene. In contrast, some EF3 lines are as fit as wild type, while producing only males in combination with DR6 driver lines. 2. Develop conditional female transformation systems. Transgenic lines were made that contain tTA regulated transformer RNAi (traIR) genes. The traIR lines were crossed with established tTA driver lines; DR2, DR3, DR6 and DR7 on diet without tetracycline. Expression of tTA would lead to induction of the traIR transgenes, production of transformer double-stranded RNA that would trigger an RNAi response and lead to degradation of tra mRNA. If there is a significant decrease in TRA protein expression, XX flies would develop as males. While most driver/traIR combinations were ineffective, several combinations were found that produced partial female transformation. The best combinations were interbred on diet with tetracycline and made homozygous for both tTA driver and traIR effector transgenes. In the absence of tetracycline the strongest double homozygous lines produced 100% males. These straisn will be further evaluated.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Concha, C., Wallbank, R.W.R., Hanly, J.J., Fenner, J., Livraghi, L., Rivera, E.S., Paulo, D.F., Arias, C., Vargas, M., Sanjeev, M., Morrison, C., Tian, D., Aguirre, P., Ferrara, S., Foley, J., Pardo-Diaz, C., Salazar, C., Linares, M., Massardo, D., Counterman, B.A., Scott, M.J., Jiggins, C.D., Papa, R., Martin., A. and McMillan, W.O. (2019). "Interplay between Developmental Flexibility and Determinism in the Evolution of Mimetic Heliconius Wing Patterns." Current Biology. 29 (23): 3996-4004. https://doi.org/10.1016/j.cub.2019.10.010
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yan, Y., Williamson, M.E., Davis, R.J., Andere, A.A., Picard, C.J. and Scott, M.J. (2020) Improved transgenic sexing strains for genetic control of the Australian sheep blow fly Lucilia cuprina using embryo-specific gene promoters. Molecular Genetics and Genomics, 295(2): 287-298. https://doi.org/10.1007/s00438-019-01622-3
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Webster S.H., Vella, M.R. and Scott, M.J. (2020) Development and testing of a novel Killer-Rescue self-limiting gene drive system in Drosophila melanogaster. Proceedings of the Royal Society B, 287: 20192994. http://doi.org/10.1098/rspb.2019.2994.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Knudsen, K.E., Reid, W.R., Barbour, T.M. Bowes, L.M., Duncan, J., Philpott, E., Potter, S. and Scott, M.J. (2020) Genetic variation and potential for resistance development to the tTA overexpression lethal system in insects. G3,10: 1271-1281. https://doi.org/10.1534/g3.120.400990.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Scott, M.J., Benoit, J.B., Davis, R.J., Bailey, S., Varga, V., Martinson, E.O, Hickner, P.V., Syed, Z., Cardoso, G.A., Torres, T.T., Weirauch, M.T., Scholl, E.H., Phillippy, A.M., Sagel, A., Vasquez, M., Quintero, G. and Skoda, S.R. (2020) Genomic analyses of a livestock pest, the New World screwworm, find potential targets for genetic control programs. Communications Biology, 3: 424. https://doi.org/10.1038/s42003-020-01152-4.