2013 Annual Report
1a.Objectives (from AD-416):
The objective of the overall APHIS-ARS project is the design of a gene system which, if used to stably transform a higher dipteran, would result in a transgenic strain of flies in which embryonic female lethality could be chemically induced. In this new specific cooperative agreement, the horn fly, Haematobia irritans, would be used as a model insect to develop a transgenic insect to both demonstrate feasibility of the embryonic lethal gene system in a higher dipteran and also provide the capability to investigate horn fly control through genetic means.
1b.Approach (from AD-416):
1. In previous research, our laboratory has developed a DNA plasmid that contains the piggybac transposable element, the green fluorescent protein marker, and the necessary components to induce embryonic female lethality regulated by the presence or absence of tetracycline. The evaluation of this DNA plasmid by microinjection/transformation protocols developed in our laboratory is underway in Drosophila melanogaster and will soon be underway in the New World screwworm, Cochliomyia hominivorax. Transgenics in these two species will be created by microinjection of early stage dechorionated embryos.
2. The horn fly was to be transformed by microinjection as part of the APHIS-ARS project. However, the extreme fragility of the early embryonic stages of the horn fly prevented the successful microinjection of the DNA plasmid into the embryos. We will utilize the technique of electroporation to attempt to create a transgenic horn fly.
3. Published electroporation protocols exist that have been used for the successful transformation of early stage embryos of D. melanogaster. These protocols will be used, and modified as necessary, to attempt to electroporate early stage horn fly embryos to facilitate the uptake and integration of the recombinant DNA plasmid containing the embryonic female lethal system.
4. The electroporation protocol must be preceded by dechorionation, the extent of which must be determined as part of this agreement. Dechorionation by bleach or enzymatic treatment will be evaluated, assaying for % survival, in conjunction with electroporation. The specific parameters of the electroporator settings will have to be determined empirically, with guidance from published protocols.
The objective is to determine the conditions necessary for creating a transgenic horn fly and to conduct the experiments at ARS. We have successfully created a transgenic horn fly, and a manuscript describing this research for publication is being revised pending the outcome of experiments required by journal reviewers.