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ARS Home » Southeast Area » Gainesville, Florida » Center for Medical, Agricultural and Veterinary Entomology » Insect Behavior and Biocontrol Research » Research » Publications at this Location » Publication #305487
Title: Fitness cost implications of phiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae)

Author
item MEZA, JOSE - Chiapas Health Services
item DIAZ-FLEISCHER, FRANCISCO - Ecology Institute
item SANCHEZ-VELASQUEZ, LAZARO - Ecology Institute
item ZEPEDA-CISNEROS, CRISTINA SILVIA - Chiapas Health Services
item Handler, Alfred - Al
item SCHETELIG, MARC - Justus-Liebig University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2014
Publication Date: 10/1/2014
Citation: Meza, J.S., Diaz-Fleischer, F., Sanchez-Velasquez, L.R., Zepeda-Cisneros, C., Handler, A.M., Schetelig, M.F. 2014. Fitness cost implications of phiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae). PLoS One. 10(9):e109690. DOI: 10.1371/journal.pone.0109690.

Interpretive Summary: The creation of transgenic strains of economically important insects for the development of more effective biological control programs is a major goal of the USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida. This article describes the development and evaluation of a new genomic targeting system in the Mexican fruit fly, Anastrepha ludens, which is a highly significant agricultural pest species in Mexico and the southern United States. Genomic targeting is an important method for the efficient development of new transgenic strains that high highly fit and can be stabilized for increased ecological safety. Here, a phiC31-integrase mediated recombination vector system was integrated at several genomic positions within the mexfly genome. These target site strains were then evalautaed for optimal transgene expression and host strain viability, fitness and male mating competitiveness which are attributes that could be compromised by the initial transgene genomic integration site. Two strains were found to have normal attributes similar to non-transgenic wild type flies, and were then used for the first time in the mexfly for targeted transgene insertion, to determine if a secondary integration could compromise the host strain fitness and transgene expression. Fitness tests for one strain showed no effects after targeted integration, while the second strain exhibited significantly lower fitness relative to the original target-site host strain. These results indicated that at some genomic positions, insertion of additional DNA into a previously integrated transgene can have a negative effect that fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.

Technical Abstract: Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its affect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.