title: Improved method for screening mitochondrial cytochrome b markers to identify regional populations of the Old World screwworm fly and other myiasis agents.
abstract: A new protocol was developed to overcome obstacles to the high-throughput sequence analysis of the 716-717 nucleotides at the carboxyl terminal of the mitochondrial gene cytochrome b (cyt b) of the myiasis flies Chrysomya bezziana and Wohlfahrtia magnifica. For both of these obligate parasites, cyt b haplotypes provide diagnostic markers for phylogeographic populations, markers that identify the origins of emerging populations causing economically important myiasis in livestock and, in the case of C. bezziana (Old World screwworm fly), could help select reproductively-compatible populations for use in the Sterile insect technique as part of area wide integrated pest management. High sequence quality is important for unambiguously detecting the few mutations that are diagnostic for regional cyt b haplotypes and their lineages. A key innovation is the design of a new forward primer for the specific PCR amplification and high-quality sequencing of cyt b. The improved protocol will facilitate the use of this established comparative cyt b sequence analysis, not only by teams lacking the resources for whole genome sequencing (WGS) but also by those requiring reference sequences for developing comparative mitogenomics based on WGS.
authors.affiliation: Life Sciences Department, Natural History Museum, London SW7 5BD, UK; Disease Control Department, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK. Electronic address: P.Ready@nhm.ac.uk.
authors.forename: P D
authors.initials: PD
authors.lastname: Ready
authors.affiliation: Life Sciences Department, Natural History Museum, London SW7 5BD, UK; Parasitology Department, Indonesian Research Centre for Veterinary Science (Balai Besar Penelitian Veteriner), Bogor, Indonesia.
authors.forename: A H
authors.initials: AH
authors.lastname: Wardhana
authors.affiliation: Life Sciences Department, Natural History Museum, London SW7 5BD, UK.
authors.forename: Z J O
authors.initials: ZJ
authors.lastname: Adams
authors.affiliation: VRI, HAO-Demeter, Thessaloniki, Greece.
authors.forename: S
authors.initials: S
authors.lastname: Sotiraki
authors.affiliation: Life Sciences Department, Natural History Museum, London SW7 5BD, UK. Electronic address: M.Hall@nhm.ac.uk.
authors.forename: M J R
authors.initials: MJ
authors.lastname: Hall
mesh_terms: D000818:Animals; D045303:Cytochromes b / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics*; D004175:Diptera / Q000145:classification* / Q000235:genetics*; D004772:Entomology / Q000379:methods; D005820:Genetic Testing / Q000379:methods; D006239:Haplotypes; D009198:Myiasis / Q000209:etiology; D058974:Phylogeography; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D004272:DNA, Mitochondrial; D045303:Cytochromes b
keywords: Chrysomya bezziana; Integrated pest management; Mitochondrial haplotypes; Regional haplotype identification; Wohlfahrtia magnifica