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Title: Amplified fragment length polymorphism used for inter- and intraspecific differentiation of screwworms (Diptera: Calliphoridae)

item Skoda, Steven

Submitted to: Entomological Research Bulletin of
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/12/2008
Publication Date: 4/9/2009
Citation: Alamalakala, L., Skoda, S.R., Foster, J.E. 2009. Amplified fragment length polymorphism used for inter- and intraspecific differentiation of screwworms (Diptera: Calliphoridae). Bulletin of Entomological Research. 99(2):139-149.

Interpretive Summary: The successful eradication of screwworms, severe and debilitating pests of animals, from the U.S., Mexico and Central America to the Darien Province of Panama using the sterile insect technique has very positively impacted animal and human health and welfare as well as the animal husbandry industries. Protecting this accomplishment by continued exclusion of screwworms will rely on rapid, accurate identification of suspected outbreaks. Because the early stages of maggots that infest wounds look very similar, we used the molecular genetic technique of amplified fragment length polymorphism (AFLP) to develop genetic markers that distinguish the early, immature stages of screwworm from the secondary screwworm, a closely related and morphologically very similar fly. Results indicate that the two species are readily distinguished based on their AFLP fingerprint patterns. Further investigation showed potential of the use of AFLP markers for distinguishing strains of screwworms from different geographic origins. The further development of diagnostic AFLP markers could provide tools that not only distinguish species but may also distinguish the general geographic origin of a sample; this would be useful for determining from where outbreaks had originated and may aide the process of responding to and eradicating the screwworm outbreak.

Technical Abstract: Morphologically, early immature stages of screwworms, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), and secondary screwworms, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), are nearly indistinguishable. Correct identification is crucial to the ongoing eradication and exclusion program against screwworms, economically important pests now eradicated from the United States, Mexico, and most of Central America but that persist in South America and the Caribbean; secondary screwworms are not economically important pests. The use of amplified fragment length polymorphism (AFLP) polymerase chain reaction was explored to differentiate and identify populations of C. hominivorax as well as to discriminate them from C. macellaria. Ten AFLP primer pairs screened for interspecific discrimination studies with four populations of C. hominivorax and one population of C. macellaria generated 52 discrete bands used in analyses. Results indicate that the two calliphorid species are readily distinguished based on their AFLP fingerprint patterns. The two species clustered at 16% similarity level with 100% bootstrap support. C. macellaria populations grouped together at the 92% level; C. hominivorax populations grouped together at the 68% level. Seven of the 52 bands were monomorphic for both species, 22 bands were specific to C. macellaria, 10 bands were present only in C. hominivorax, and the remaining 13 bands differentiated C. hominivorax populations. Separate studies using 10 strains of C. hominivorax showed a higher level of genetic similarity within populations than between populations. Analyses were performed using 72 bands (19 bands were monomorphic and 53 bands grouped all 10 strains at the 58% similarity level). At the 85% similarity level, seven clusters representing seven mutant strains from Mexico were resolved; at the 72% similarity level, all 10 clusters representing the 10 strains were resolved. Diagnostic bands were identified for species and strain identification. Although the technique has some limitations (such as dominant expression and requisite assumptions for interpreting the AFLP data), AFLP fingerprinting can be a valuable tool for studies of interspecific and intraspecific genetic variation in screwworm populations.