|Handler, Alfred - Al|
Submitted to: Fruit Flies of Economic Importance International Symposium
Publication Type: Book / chapter
Publication Acceptance Date: 11/20/2009
Publication Date: 1/16/2009
Citation: Schetelig, M.F., Scolari, F., Handler, A.M., Gasperi, G., Wimmer, E.A. 2009. New genetic tools for improving SIT in Ceratitis capitata: embryonic lethality and sperm marking. Fruit Flies of Economic Importance International Symposium. 299-305. Interpretive Summary: The ability to create transgenic strains of economically and medically important insect species has the potential to greatly enhance our ability to improve existing biological control methods and develop more novel means of control, which is a major goal of our laboratory at the Center for Medical, Agricultural and Veterinary Entomology, USDA, Agricultural Research Service, Gainesville, FL. The aim of the studies reported here is to establish and evaluate embryonic lethality and sperm marking systems in Mediterranean fruit fly, Ceratitis capitata. A sperm marking system for medfly was developed by isolating the testis-specific Beta 2tubulin gene, and using its promoter to drive the gene expression of a fluorescent marker protein. Transgenic medfly strains were then created having the Beta 2tubulin::DsRed transgene that were evaluated for fitness, marker gene accuracy, efficiency of the sexing procedure, and strain stability. Possible applications for these transgenic strains in operational SIT programs are the genetic sexing of larvae and the identification of mated females in the field. The testes and sperm-marking systems will also facilitate studies on the reproductive biology of C. capitata by sperm precedence analysis. Functional large-scale SIT activities, like those established for Ceratitis capitata, are ideal for comparing the effectiveness and usefulness of novel transgenic SIT approaches.
Technical Abstract: The aim of this research is the development of a sperm marking system for Ceratitis capitata, which is based on the use of the Ceratitis capitata spermatogenesis-specific Beta 2t promoter driving a fluorescent marker. The testes-specific Beta 2-tubulin gene was isolated by a degenerate PCR approach, with the promoter regulatory sequence identified after sequencing. The promoter was linked to the DsRed-Express fluorescent protein gene and inserted into a piggyBac vector having GFP linked to a polyubiquitin promoter used as a transgenic promoter. After thorough strain evaluation and a test phase for fitness, accuracy and stability of the sexing procedure as well as the stability of these strains, they could be used for different purposes. A possible application might be the use as a transgenic sexing strain in combination with the ability for an easy monitoring in an operational SIT program. The system will also help in providing more detailed information on reproductive biology of Ceratitis capitata. The aim of the studies reported here was to establish and evaluate such embryonic lethality and sperm marking systems in Ceratitis capitata. Functional large-scale SIT activities, like those established for Ceratitis capitata, are ideal for comparing the effectiveness and usefulness of novel transgenic SIT approaches.