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Molecular-based identification of the New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae)

Joan A Christen, University of Nebraska - Lincoln

Abstract

Since the beginning of civilization, man and insects have struggled to gain superiority over the other. Such is the case with the New World Screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). Although eradicated using the Sterile Insect Technique (SIT) in the United States (1982), Mexico (1991), and Central America to Panama (2000) the obligate ectoparasite screwworm remains an important economic pest in the Western Hemisphere. ^ Morphological differentiation between larvae of the Calliphoridae (blowflies) is difficult until at least the third instar. This study was conducted in part to confirm that random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) markers could differentiate, with no intraspecific differences, between the adult and larval stages of the NWS and C. macellaria (Fabricius) (Diptera: Calliphoridae) and provides the basis for future sequencing of informational RAPD-PCR generated bands. Twelve 10-mer primers were initially screened and used to confirm previous work with NWS and C. macellaria . One 10-mer primer (OPA-12) was selected to generate an amplicon resolved on agarose gel that revealed distinct banding patterns differentiating the two species with no difference in banding between larval and adult stages. ^ Bands were then excised from agarose gel and purified. Invitrogen's pCR ®4-TOPO® TA Cloning Kit was used to transform and clone the ∼860 bp amplicon in C. hominivorax and the ∼292 bp amplicon generated in C. macellaria. Restriction digests were performed to verify the presence of the insert prior to sequencing. Genomic sequencing of the 852 and the 848 base pair sequences of C. hominivorax revealed no statistical genomic differences between adult and larval specimens of each strain examined. Primers were constructed and used to develop sequence characterized amplified regions (SCAR)-Polymerase Chain Reaction (PCR) amplicons that amplify only C. hominivorax and not other flies. The results showed 100% support for the ability of the two primers, named CR92A1 and J1A2, to discriminate between C. hominivorax and all other flies tested. The disadvantage of RAPD-PCR reproducibility is eliminated with this technique. ^

Subject Area

Biology, Entomology|Biology, Genetics

Recommended Citation

Christen, Joan A, "Molecular-based identification of the New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae)" (2008). ETD collection for University of Nebraska - Lincoln. AAI3297477.
http://digitalcommons.unl.edu/dissertations/AAI3297477

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