2013 Annual Report
1a.Objectives (from AD-416):
Develop molecular genetic techniques for identifying the geographic origin of screwworm samples. The successful exclusion of screwworms from eradicated, as well as from historically non-endemic, areas relies on the accurate and timely identification of suspicious samples and would be greatly enhanced with identification of the origin of outbreaks. In previous research by the collaborator from UN-L, molecular genetic techniques including random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLP) have shown usefulness in identifying ambiguous samples and promise for determining their geographic origin: these techniques need further optimization and standardization. Additionally, genetic variability identified from the molecular data may then be analyzed in a Geographical Information System (GIS) for landscape distribution of genetic variation. If geographic barriers are shown to inhibit movements of screwworms, as inferred by genetic differences, then these barriers would be useful to establishing new eradication programs. Full success of this project will provide primers to identify the species and the geographic area of origin for all life stages of screwworms, providing a valuable new tool for eradication and exclusion personnel responding to suspected outbreaks.
1b.Approach (from AD-416):
Standard Protocols, developed in the Genetics Laboratory of the Department of Entomology at UN-L, for RAPD and AFLP have shown potential for both species identification and determination of geographic origin. They will be used to further explore and catalog genetic variability of screwworm samples collected from different geographic regions (more than 5 areas) of South America; more than two sub-samples, separated by several weeks, will be collected. We will use Landsat TM imagery of the study sites to select areas of favorable screwworm habitat: this will streamline the field collections for each of the areas. Banding patterns for each technique will be evaluated for uniqueness of 'DNA fingerprints'. Band(s), from either or both techniques, that are unique (for identifying the geographic origin of screwworms) and consistently produced on gels will be excised from the gel, sequenced, and primers developed and tested for unique amplification from screwworm samples from different geographic origins. Resultant analyses of molecular data, particularly distance matrices and dendrograms that summarize genetic variability, will be used in GIS analysis for visualizing spatial population structures for the remaining areas of native screwworm populations. We will analyze differences in DNA 'fingerprints', and possibly limited sequence differences, of geographically different populations of screwworm. All spatial and geographical analysis will be performed using ESRI ARCGIS 8. Kridging, a spatial modeling technique, will be used on these structures to identify geographic areas of high genetic variability.
The manuscript "Inter- and Intraspecific Identification of the Screwworm using Random Amplified Polymorphic DNA-Polymerase Chain Reaction" submitted to the Jornal of Insect Science has been accepted. This manuscript reports that RAPD-PCR can be used to distinguish species that may co-inhabit a wound and showed promise in distinguishing the geographic origin of samples of screwworm.
Based on, and an extension of, the above work, DNA from excised markers generated with RAPD-PCR was used as the basis to generate two species-specific Sequence Characterized Amplified Regions (SCAR). The SCARs are specific to, or only recognize, screwwworms. We are developing a scientific manuscript to report these results. We also have communicated, along with you, for initiation of technology transfer to customers of the screwworm eradication program.
We are awaiting samples from South America and the Caribbean, for use with techniques developed as part of this agreement, to determine if we can identify the geographic origin of screwworm samples. We intend to distinguish the 'most reliable' identification technique and determine the genetic variability of screwworms across their current range.