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Title: Inter- and Intraspecific Identification of the New World Screwworm Using Random Amplified Polymorphic DNA-Polymerase Chain Reaction

Author
item Skoda, Steven
item FIGAROLA, JAMES - Beckman Research Institute
item PORNKULWAT, SAOWALUCK - University Of Nebraska
item FOSTER, JOHN - University Of Nebraska

Submitted to: Journal of Insect Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/9/2012
Publication Date: 8/14/2013
Citation: Skoda, S.R., Figarola, J.L., Pornkulwat, S., Foster, J.E. 2013. Inter- and Intraspecific identification of the New World screwworm using random amplified polymorphic DNA-polymerase chain reaction. Journal of Insect Science. 13:76.

Interpretive Summary: Screwworms were one of the most important pests of livestock and other animals, including humans, before they were eradicated from the U.S., Mexico, and Central America with the sterile insect technique. A barrier against screwworms is now maintained at the Panama to Colombia border. When outbreaks of this devastating pest are suspected, quick and accurate identification of samples is necessary to develop the appropriate response. Early stages of screwworms are nearly impossible to accurately distinguish from early stages of other blow flies that may inhabit animal wounds. We developed random amplified polymorphic DNA – polymerase chain reaction (RAPD), a molecular genetic technique, to accurately and rapidly distinguish all life stages of screwworms from several other common flies that may inhabit animal wounds. Further analysis indicated that RAPD may also be useful in distinguishing the geographic origin of screwworm samples. RAPD was used with suspicious samples of early life stage flies collected from cattle in Nicaragua, which has been free of screwworms for many years. Results from morphological identification and RAPD concurred that the samples were not screwworm; this saved millions of dollars because emergency response efforts were not required. Samples from a suspicious outbreak of screwworms in Mexico were shown, using RAPD, not to have originated from the screwworm rearing facility, alleviating the suspicion of sabotage. Further development of RAPD wherein the geographic origin of an outbreak could be determined would be a valuable addition to the eradication and barrier maintenance efforts against screwworms.

Technical Abstract: New World screwworms (NWS), Cochliomyia hominivorax (Coquerel), are one of the most important arthropod pests of livestock in the Western Hemisphere. Early instars are very difficult to distinguish morphologically from several closely related blow fly species. Random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) markers were developed for discrimination of NWS from other wound inhabiting species. Forty decameric primers were screened; nine showed clear reproducible RAPD profiles suitable for distinguishing all life stages of C. hominivorax from C. macellaria (Fabr.), Phormia regina (Meigen), Lucilia sericata (Meigen), Calliphora vicina Robineau Desvoidy, Chrysomya rufifacies (Macquart), Sarcophaga sp., and Musca domestica L. Results from RAPD-PCR with field collected samples of unknown first instars agreed with morphological identification that the samples were not NWS. Three different primers showed DNA polymorphisms (intraspecific) for NWS populations originating from Mexico, Costa Rica, Panama, Jamaica, and Brazil; RAPD-PCR may be useful for determining the geographic origin of NWS samples. These primers, used with screwworm samples from an outbreak in Mexico, clearly showed that the outbreak did not originate from the mass rearing facility. Accurate identification of suspected NWS samples is possible using RAPD-PCR; further development to identify the geographic origin of samples would benefit the ongoing surveillance programs against NWS and the decision process during suspected outbreaks of this important pest.