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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICALLY BASED TECHNIQUES TO LIMIT THE DISPERSAL OF INVASIVE PESTS

Location: Insect Behavior and Biocontrol Research Unit

Title: A DsRed fluorescent protein marker under polyubiquitin promoter regulation allows visual and amplified gene detection of transgenic Caribbean fruit flies in liquid traps

Authors
item Nirmala, Xavier -
item Olson, Shelley -
item Holler, Timothy -
item Cho, Keun Ho -
item HANDLER, ALFRED

Submitted to: Biocontrol
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 28, 2011
Publication Date: March 11, 2011
Citation: Nirmala, X., Olson, S., Holler, T., Cho, K., Handler, A.M. 2011. A DsRed fluorescent protein marker under polyubiquitin promoter regulation allows visual and amplified gene detection of transgenic Caribbean fruit flies in liquid traps. Biocontrol. 56(3):333-340.

Interpretive Summary: Tephritid fruit flies cause significant damage to agriculture forcing major quarantine regulations on agricultural trade. The population size of these flies is reduced by release of sterile males that mate with fertile females in the field resulting in sterile eggs that fail to hatch. These flies are marked with fluorescent powders before release to distinguish them from fertile flies in the field that is critical in pest monitoring programs. Scientists at the USDA-ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida, in collaboration with colleagues from USDA-APHIS and the University of Florida examined whether such temporary markers could be replaced by more permanent and environmentally friendly markers. A red fluorescent protein marker gene that is stably integrated in the genome of the Caribbean fruit fly could be visually detected under a microscope for at least three weeks after being in the torula yeast/borax and propylene glycol solutions, that are commonly used in fly traps to monitor pests. This marker could also be detected at the molecular level. A comparison with the enhanced green fluorescent protein showed the reliability of red compared to green. This study shows that transgenic flies can be effectively and unambiguously identified after field release.

Technical Abstract: Field population surveillance of a targeted insect pest species is critical to the evaluation of management programs such as the sterile insect technique. Use of markers to distinguish released insects from the field population is essential, but fluorescent powder dyes used on tephritid species are less than optimal in terms of reliability and human health issues. The ability to genetically transform several tephritid species presents the possibility of using fluorescent protein transgenes for marking. Here we tested the ability to detect fluorescent flies maintained in two commonly used liquid trap solutions, aqueous torula yeast borax and propylene glycol. DsRed fluorescence in transgenic flies was stable in both solutions for up to three weeks by visual microscopic observations and could unambiguously distinguish them from non-fluorescent wild type flies. To compensate for any potential ambiguity in visual identification a diagnostic PCR method was developed that could specifically amplify the exotic marker protein gene in transgenic flies. The use of sterile transgenic insect strains carrying stably integrated fluorescent marker genes in biologically-based control programs would greatly improve released fly identification, and thereby pest management programs.

Last Modified: 9/10/2014
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