Improved method for screening mitochondrial cytochrome b markers to identify regional populations of the Old World screwworm fly and other myiasis agents.

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.

Genetic diversity of populations of Old World screwworm fly, Chrysomya bezziana, causing traumatic myiasis of livestock in the Gulf region and implications for control by sterile insect technique.

Fly larvae were collected from 181 cases of traumatic myiasis in livestock in 10 regions of four countries in the Middle East Gulf region: Iran, Iraq, Saudi Arabia and Oman. The predominant fly species responsible for cases was the Old World screwworm (OWS) fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae). In cases from Iran and Oman, which included non-OWS fly species, OWS fly was found solely responsible for 67.6% of cases and jointly with other fly species for a further 12.7% of cases. The major hosts were sheep and goats, together comprising 84.6% of the total, which reflects their predominance among the livestock of these Gulf countries. The major site of wounding on sheep and goats was the tail (40.3%), followed by female genitalia (14.0%). The 3' terminal 715 nucleotides of the mitochondrial cytochrome b gene were sequenced for 178 larvae of OWS. Five haplotypes were identified: three had been recorded previously in the region (two were common throughout and one was unique to Oman), and two were newly identified, one from southern Iraq and the other from Saudi Arabia, both in regions sampled for the first time. The haplotypes varied from one another only at one or two nucleotide sites, equivalent to an intraspecific difference of 0.14-0.28% across the entire 715-bp fragment. There was a single statistically significant association between host species and haplotype in Saudi Arabia, a first such record for OWS fly. The small degree of genetic diversity between geographical populations of OWS fly within the Gulf region suggests that a single Gulf colony could be used to implement the sterile insect technique within an integrated control programme.

Morphological and mitochondrial DNA characters for identification and phylogenetic analysis of the myiasis-causing flesh fly Wohlfahrtia magnifica and its relatives, with a description of Wohlfahrtia monegrosensis sp. n. Wyatt & Hall.

Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is a major cause of traumatic myiasis in livestock in Central and Eastern Europe and in countries bordering the Mediterranean. The present study explored the utility of external body characters, genitalia characters and mitochondrial DNA characters for identification of this and related species in the subfamily Paramacronychiinae. Sequence analyses of the 3' terminal 273 bp of the mitochondrial cytochrome b gene revealed two lineages of W. magnifica, one from Spain and France and the other from the rest of Eurasia, differing by only two base pairs. Phylogenetic analysis of cytochrome b showed that W. magnifica and Wohlfahrtia vigil Walker were sister species; this conclusion was not contradicted by a phylogenetic analysis of the morphological characters. Based on cytochrome b, the genetic distance between specimens of W. vigil from Europe and North America was sufficiently large to justify the recognition of more than one species. A new species, Wohlfahrtia monegrosensis, from northern Spain, was described, based on morphology and cytochrome b. A unique combination of external body characters of males or females were diagnostic for W. magnifica, the W. vigil group and Wohlfahrtia bella, but only the genitalia characters were diagnostic for all nine species studied.

Molecular genetic analysis of populations of Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica, in outbreak populations from Greece and Morocco.

Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae), is the most important cause of traumatic myiasis in the southern Palaearctic region. Larval stages are obligate parasites and the wounds caused by infestations are very similar to those caused by Old and New World screwworm flies. During the last decade, W. magnifica appears to have expanded its range to parts of northern and central Morocco, and to Crete, Greece. Specimens of W. magnifica were collected in Morocco and Crete either as larvae (preserved in 80% ethanol) or as adults (dry-pinned). Comparison specimens were collected in Spain, Hungary and mainland Greece. A DNA fragment containing the 3' 715 base pairs of the mitochondrial cytochrome b gene was amplified by polymerase chain reaction from each of 132 larvae or adults of W. magnifica and the amplicons were directly sequenced and analysed phylogeographically. Twelve cytochrome b haplotypes were detected. All haplotypes from Morocco belonged to a lineage that included specimens from the Iberian peninsula, and restricted mixing of central and northern populations in Morocco was demonstrated. Cytochrome b haplotyping combined with an analysis of larval size provided clear evidence of multiple infestations of hosts in all geographical areas, with one quarter of wounds containing larvae from two to at least four females. More than 80% of specimens from Crete contained a haplotype predominating in mainland Greece and Hungary. Our survey indicated that wohlfahrtiosis was more widespread in northern and central Morocco than previously recorded by government veterinarians. However, the prevalence of wohlfahrtiosis was low (< 1%). The high genetic diversity of Moroccan populations is consistent with longterm endemicity, rather than recent introduction. Crete showed a higher prevalence of wohlfahrtiosis (< or = 15%) and less genetic diversity of W. magnifica, which is consistent with a recent introduction. The western and eastern Mediterranean lineages may have been isolated in different Pleistocene ice-age refugia, from which there has been limited post-glacial dispersal.

A comparison of Lucitraps and sticky targets for sampling the blowfly Lucilia sericata.

The Lucitrap (Miazma Pty Ltd, Queensland, Australia) combined with a synthetic odour bait, Lucilure (Miazma Pty Ltd, Queensland, Australia), is a commercially available trap for sampling and control of Lucilia cuprina (Wiedemann) in Australia. It was tested in Hungary against Lucilia sericata (Meigen) (Diptera: Calliphoridae), a cause of sheep strike throughout temperate Europe. The standard Lucitrap was tested against black or yellow sticky target traps. Both trap types were baited with either Lucilure or liver and 10% w/v sodium sulphide solution. With Lucilure as bait, L. sericata were caught on sticky traps but not in Lucitraps. With liver and sodium sulphide as bait, sticky traps caught 500-1500 times more L. sericata than Lucitraps. An adhesive sheet fitted to the top of a Lucitrap captured 30-300 times more L. sericata then were captured inside an unaltered Lucitrap. Direct observation of metallic green calliphorids (92.1% L. sericata) alighting on Lucitraps indicated that most flies stayed for a short while (modal class 2-4 s) and only a few stayed longer, to an observed maximum of 28 s. Flies explored a mean of 1.5 entry holes (range 0-7) during a visit but only 6% entered the trap. Size of L. sericata was not a physical barrier to Lucitrap entry, because many larger species were captured. However, L. sericata captured inside Lucitraps were significantly smaller than those captured on sticky traps, demonstrating that size was of behavioural importance. The data demonstrate that the Lucitrap is not effective as a trap for L. sericata in Hungary, due mainly to a failure of flies to enter the trap in large numbers. In Australia and South Africa, L. sericata is commonly caught in Lucitraps baited with Lucilure, although L. cuprina is more numerous. Our study highlights the potential for diversity of fly behaviour between different geographical populations of the same species. Such diversity can have a significant effect on the functioning of systems for fly sampling and control, when these systems depend for their success on certain behavioural responses of the target species.