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Keywords:

  • Sarcophagidae;
  • Wohlfahrtia magnifica;
  • cytochrome b;
  • Mediterranean basin;
  • mitochondrial DNA;
  • post-glacial dispersal;
  • Greece;
  • Hungary;
  • Morocco;
  • Spain

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Abstract 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 (≤ 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The flesh fly, Wohlfahrtia magnifica, is an important cause of traumatic myiasis in livestock throughout the Mediterranean basin and eastwards into China, including the steppe regions of continental Europe (Hall & Farkas, 2000). It occurs along much of what is known as the Eurasian ruminant street or road (Slingenbergh et al., 2004), an avenue of trade which runs from Europe through Turkey, Iraq and Iran into Asia, along which a considerable amount of livestock trade occurs. Its distribution in Iran and Iraq partly overlaps with that of the Old World screwworm (OWS) fly, Chrysomya bezziana Villeneuve, with which it can potentially be confused because of similarities in larval morphology and wound characteristics (Hall, 2004).

Female W. magnifica are attracted to wounds or natural body orifices of their hosts, where they deposit first instars. Sheep are a major host, but other livestock including poultry (Farkas et al., 2001), wildlife (Rosen et al., 1998) and even humans (Iori et al., 1999; Lmimouni et al., 2004) can be affected. In Europe, W. magnifica has been a particular problem for breeds of sheep introduced in Romania and Hungary, such as Merino, Romney and Corriedale, which are very susceptible to infestation (Lehrer & Verstraeten, 1991; Farkas et al., 1997). Newly deposited larvae immediately start to feed on the host’s cutaneous and underlying tissues, causing serious damage. These wounds become increasingly attractive to females (Hall et al., 1995) and, therefore, more flies arrive and deposit their larvae, which enlarge the wound still further. Infestations of livestock can lead to death if left untreated. Even if death does not result, wohlfahrtiosis is a major animal welfare problem, causing pain and suffering. The genitalia of both sexes are a major site of infestations, which can lead to problems in the reproductive ability of hosts (e.g. sheep [Farkas & Hall, 1998] and camels [Hadani et al., 1989; Valentin et al., 1997]).

Recent molecular studies have shown that there are at least two genetic lineages of W. magnifica, one in Spain and France and the other in all countries studied to the east of these (Hall et al., 2009). The distribution of the species is dynamic, possibly in part because of movements of infested animals. Since 1999, W. magnifica has been recorded on the island of Crete, Greece, where it has caused significant health and welfare problems in livestock, particularly sheep and goats, which form the basis of the local dairy industry (Sotiraki et al., 2003, 2005a, 2005b). Its status as a pest in Morocco has also increased since 2001, when outbreaks were first reported in the northern province of Al-Hoceima, which borders the Mediterranean Sea (El Abrak et al., 2002).

Most cases in Morocco were found in livestock, but human cases have also been reported (Lmimouni et al., 2004; Tligui et al., 2007). It was initially thought that cases may have been caused by larvae of OWS, Ch. bezziana, but they were confirmed as W. magnifica (M.J.R. Hall, unpublished report to the Food and Agriculture Organization of the United Nations, GF AGAHP RA213A6622010, 2002). It is probable that a similar misidentification occurred in Algeria, where a case of myiasis caused by Ch. bezziana was reported in a young shepherd who had not travelled outside the region (Abed-Benamara et al., 1997). Such potential confusion confirms the need for a thorough taxonomic study of all fly species that can produce obligate traumatic myiasis in humans and livestock.

At least two publications have claimed that the distribution of W. magnifica included Morocco (James, 1947; Verves, 1986). James (1947) wrote that the records in his review were prepared either from published records or from specimens present in the collection of the United States National Museum, or were determined by the author from other sources. He did not specifically state where his information on the distribution of W. magnifica came from. At least four other species of Wohlfahrtia are reported to occur in Morocco: Wohlfahrtia bella (Macquart), Wohlfahrtia nuba (Wiedemann), Wohlfahrtia indigens Villeneuve and Wohlfahrtia trina (Wiedemann) (Verves, 1986). Wohlfahrtia magnifica is known to occur in mainland Greece (Verves, 1986), but until 1999 there are no records, to our knowledge, of its presence in Crete. In addition, neither farmers nor veterinarians on Crete were familiar with wohlfahrtiosis until 1999 (Sotiraki et al, 2003).

The objectives of the work reported here were to characterize genetically the populations of W. magnifica sampled in outbreak situations in Greece and Morocco, using mitochondrial DNA markers, in order to assess their geographical ranges and discover any evidence for exchange of flies within the region.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Surveys in Crete and mainland Greece

Field sampling was undertaken on Crete in August 2001, May and July 2002, and July and September 2003 during studies of fly behaviour and control (Sotiraki et al., 2003, 2005a, 2005b). Samples from mainland Greece were either collected by the authors during the summers of 2005 and 2006 or were submitted by local veterinarians (Fig. 1).

image

Figure 1. Outline map of Greece showing approximate origins of specimens of each cytochrome b haplotype (•, CB_magn01; ▴, CB_magn10; ♦, CB_magn11;⋆, CB_magn12).

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Survey in Morocco

Field visits to Morocco were made in December 2001, July and September 2002 and May 2003 (Fig. 2). During each field trip, many smallholder farms and communal wells, used for watering of livestock, were visited. Local farmers and animal owners were questioned about myiasis and encouraged to present active cases, either on the day of first visit or on a revisit. All larvae were collected from each active infestation presented.

image

Figure 2. Outline map of central and northern Morocco showing approximate origins of specimens of each cytochrome b haplotype (•, CB_magn02; ▴, CB_magn03; ▪, CB_magn04;⋆, CB_magn05/06/07).

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In July 2002 M.J.R. Hall and R. Farkas visited the Diptera collections of the Institut Scientifique, University Mohammed V, in Rabat, to look for any historical evidence of W. magnifica in Morocco. Eight specimens labelled as W. magnifica were studied, two of which were identified as W. magnifica (Verves, 1986). These did not have clear date labels, but their localities were indicated as Volubilis (possibly in 1978), the site of an old Roman town about 19 km north of Meknes, and Mazagan, the old name for the sea port of El-Jadida, about 90 km southwest of Casablanca.

In July 2002 sampling was concentrated on Al Hoceima Province in northern Morocco, but samples were also collected at Volubilis in central Morocco. In September 2002, sampling concentrated on the Maaziz-Meknes-Fes (including Volubilis) and Middle Atlas regions, with a 1-day visit to Al Hoceima Province. In May 2003 sampling was undertaken around Maaziz and in the High Atlas region.

Samples from Spain and Hungary

Samples of larvae from Spain (Zaragoza) and Hungary (Harkakotony) were collected during ongoing studies of wohlfahrtiosis.

Sampling, preservation methods and morphological identifications

Larvae were kept alive on moist tissue paper within plastic containers for 1–8 h and were then killed by immersion in boiling water for 15–30 s. This extended the larvae, enabled their preservation in 80% ethanol without tissue blackening (Adams & Hall, 2003) and had no negative impact on DNA extraction. At each site hand-netting was undertaken of adult flies settled on rocks, walls and other prominent objects in the environment. Most of these flies were males at ‘waiting stations’ or ‘mating stations’ (Guillot et al., 1978; Hall et al., 1995). Larvae were identified according to Zumpt (1965) and Spradbery (1991), and adults were identified according to Verves (1986) and Hall et al. (2009), following examination under a dissecting microscope at up to 50× magnification.

DNA extraction, PCR amplification, sequencing and analysis

All steps used the protocols reported by Ready et al. (2009). DNA was extracted from either an abdominal tissue block of larvae (approximately from body segments 6–9) or from the thoracic muscle of adult flies. DNA was extracted from the tissues using the DNAzol® kit (Invitrogen Corp., Carlsbad, CA, U.S.A.; Chomczynski et al., 1997), dissolved in 1× Tris-EDTA solution and stored short-term at 4 °C and long-term at − 20 °C. The 3′ terminal 715 bp of the mitochondrial cytochrome b gene (cyt b) was amplified by polymerase chain reaction (PCR), directly sequenced and analysed.

The PCR amplification was carried out using a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems, Inc., Foster City, CA, U.S.A.). Positive (voucher sample of the cyt b region) and negative (no DNA) controls were used. The size and quantity of the amplified DNA fragments were determined by fractionating the PCR products on 1.5–2.0% agarose gels along with DNA fragments of known sizes and quantities (Promega PCR Markers G316A; Promega Corp., Madison, WI, U.S.A.). The DNA in the excised gel fragments was purified with glassmilk (Geneclean II®; MP Biomedicals, Solon, OH, U.S.A.) and each strand directly sequenced using one of the PCR primers (3.2 pmoles) and the ABI Prism® Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit (Version 2.0; PE Applied Biosystems, Inc.). Purification of the extension products was carried out using the ethanol precipitation method. Sequences were resolved using the ABI 377 system. Sequences were edited and aligned with database sequences using Sequencher™ (Gene Codes Corp., Ann Arbor, MI, U.S.A.), all according to ABI protocols (PE Applied Biosystems, Inc.), and were identified and their relationships analysed using paup* software (Swofford, 2002).

The term ‘adult equivalents’ (AEs) was used when assessing haplotype frequency to indicate an individual adult or the offspring of one female. The use of AEs gives a minimum number of adults. This is because all larvae in a single wound with the same haplotype may derive from a single adult, but they may also derive from more than one. However, all specimens of the same haplotype in a wound were considered to be from just one adult unless they were obviously the product of two larvipositions, such as when mature third instars and second instars were found in the same wound, clearly indicating at least two larvipositions.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

Morphological identifications of larvae causing myiasis infestations and of adult flies

Of nine active cases sampled in Crete, all were exclusively infested by larvae of W. magnifica. Hosts sampled were sheep (8/9, 88.9%) and a dog (1/9, 11.1%).

Of 19 active cases of traumatic myiasis sampled in Morocco, 17 (89.5%) were infested exclusively by W. magnifica, one (5.3%) was infested jointly by W. magnifica and an unknown sarcophagid species and one (5.3%) was infested jointly by a Lucilia species and Chrysomya albiceps (Wiedemann), but had previously been infested by W. magnifica, based on the deeply penetrating wounds. Thus, W. magnifica was involved in all cases seen. Hosts encountered were sheep (8/19, 42.1%), dogs (8/19, 42.1%), goats (2/19, 10.5%) and a horse (1/19, 5.3%). All Wohlfahrtia adults collected in Greece were of W. magnifica. In Morocco, adults of W. bella were also collected, but these were not sequenced.

For comparison with the outbreak populations, four larvae from Spain (Zaragoza; hosts: horse [1], unknown [1]), two larvae and three adults from Hungary (Harkakotony; host: pig [1]) and 45 larvae from the northern half of mainland Greece (north of 38°50′ N; hosts: sheep [9], goat [1], horse [1], dog [5], unknown [4]) were sequenced.

mtDNA haplotype diversity

In total, 12 mtDNA haplotypes (= unique sequence types) were identified based on analyses of the 3′ terminal 715 nucleotides of cyt b (Tables 1 and 2; GenBank accession numbers: FJ379605–FJ379616). The 16 specimens (all larvae) of W. magnifica from Crete belonged to one of two mtDNA haplotypes of the eastern Mediterranean lineage. Larvae of the rarer haplotype (CB_magn10) were recovered from two wounds, always in association with larvae of the common haplotype (CB_magn01). The 62 specimens (54 larvae, eight adults) of W. magnifica from Morocco were found to contain one of six mtDNA haplotypes (Tables 1 and 2) belonging to the cyt b lineage of the western Mediterranean (Spain, France) (Hall et al., 2009) and they shared one haplotype with the sample from Spain (CB_magn02).

Table 1.  Numbers of individuals of Wohlfahrtia magnifica with each cytochrome b gene haplotype in different geographical locations and regions. Numbers in brackets are adult equivalents.
Geographical locationWohlfahrtia magnifica cyt b haplotype number (CB_magn01–CB_magn12)Total of all haplotypes
Western Palaearctic regionCentral and eastern Palaearctic region
020304050607080901101112
Central Morocco24 (14)2 (1)6 (3)32 (18)
North Morocco3 (2)12 (6)11 (8)3 (1)1 (1)30 (18)
Spain1 (1)1 (1)2 (1)4 (3)
Hungary5 (4)5 (4)
Mainland Greece38 (21)6 (4)1 (1)45 (26)
Crete14 (9)2 (2)16 (11)
Total from all regions28 (17)2 (1)18 (9)11 (8)3 (1)1 (1)1 (1)2 (1)57 (34)2 (2)6 (4)1 (1)132 (80)
Table 2.  Variant nucleotides in an alignment of all cytochrome b haplotypes found. All 13 polymorphic characters were included in the parsimony analysis (Fig. 3) and four were parsimony informative.
Haplotype codeNucleotide positionCountryRegion
 22233455566
5747959425617
0912754565806
CB_magn02CATGCTTGGTGTTSpain, MoroccoWest
CB_magn03CATGCTTAGTGTTMorocco
CB_magn04CATGCTTGGCGTTMorocco
CB_magn05CAGGCTTGGTGTTMorocco
CB_magn06CATACTTGGTGTTMorocco
CB_magn07CATGCTTGGCGTCMorocco
CB_magn08CATGCTTGGTATTSpain
CB_magn09CATGCCTGGTGTTSpain
CB_magn01CATGTTTGATATTHungary, GreeceEast
CB_magn10TATGTTTGATATTGreece (Crete)
CB_magn11CATGTTCGATATTGreece
CB_magn12CGTGTTTGATAGTGreece

The western and eastern Mediterranean lineages differed by at least two nucleotide changes in 715 positions (Fig. 3), equivalent to a genetic distance of 0.28%. The greatest genetic distance was between haplotypes CB_magn12 (Greece) and CB_magn07 (Morocco), which showed seven nucleotide changes, equivalent to a 0.98% difference.

image

Figure 3. Unrooted neighbour-joining tree generated by using untransformed genetic distances between the 12 cytochrome b haplotypes of Wohlfahrtia magnifica (CB_magn01–CB_magn12). The numbers of nucleotide differences between each haplotype are indicated by the branch lengths (see scale). Western haplotypes were found in Spain and Morocco; eastern haplotypes were found in Greece and Hungary.

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Spanish specimens belonged to three haplotypes, one shared with Morocco (Table 1). Hungarian specimens were of one haplotype (CB_magn01), which predominated in Crete and Greece (Table 1). The majority of specimens from mainland Greece belonged to this common haplotype of the eastern lineage of W. magnifica, but two rare haplotypes were also collected (CB_magn11 and CB_magn12).

Regional differences in genetic diversity in Morocco

There was a clear and statistically significant difference between the genetic composition of the central and northern faunas of Morocco (χ2 = 13.29, d.f. = 5, P = 0.02) (Fig. 4). In the central sampling area of Volubilis and Maaziz, the dominant cyt b haplotype was CB_magn02 (14/18 female equivalents, 77.7%). However, in the northern sampling area of Al Hoceima there were two co-dominant haplotypes, CB_magn04 (6/18, 33.3%) and CB_magn05 (8/18, 44.4%). Two haplotypes were shared between the regions, CB_magn02 and CB_magn04, but CB_magn02 was rare in the north and CB_magn04 was rare in the central region. Both sampling areas demonstrated unique or rare haplotypes: CB_magn03 in the central region, and CB_magn06 and CB_magn07 in the north.

image

Figure 4. Frequency (%) of each of the six cytochrome b haplotypes (CB_magn02–CB_magn07) of Wohlfahrtia magnifica found in northern Morocco (black bars; n= 18 adult equivalents) and central Morocco (cross-hatched bars; n= 18 adult equivalents).

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In the central region of Morocco, most wounds (10/11, 90.9%) were infested with larvae of the dominant haplotype CB_magn02. Two of those wounds were co-infested with either haplotypes CB_magn03 or CB_magn04, and a further wound was found infested by larvae just of CB_magn04. In the northern region, half the wounds (4/8) were infested with haplotype CB_magn04 and the other half (4/8) with CB_magn05. No wounds contained both these haplotypes. Haplotypes CB_magn02 and CB_magn06 were found together in a single wound with haplotype CB_magn05. Haplotype CB_magn07 was detected from a single adult.

Multiple infestations

The majority of wounds examined in all geographical regions (40/52, 76.9%) were infested by larvae of W. magnifica characterized by the same cyt b haplotype. Such cases could have been infested by larvae of two or more females sharing the same cyt b haplotype or, as only a small subsample of larvae in the wounds were analysed, by several females with more than one haplotype. However, despite the minimal sampling, there was clear evidence of multiple infestations of hosts, based on the detection of multiple haplotypes of larvae within a single wound, or of a single haplotype in larvae of such differing sizes or instars that they had clearly been deposited by different females. Thus, almost a quarter of wounds (12/52, 23.1%) contained larvae of at least two females and 7.7% (4/52) contained larvae of at least three females. The greatest number of female larvipositions detected in a single wound found in Morocco was four (1/52, 1.9%). The greatest number of cyt b haplotypes detected in a single wound was three (CB_magn02, CB_magn05 and CB_magn06), found in a dog in Al Hoceima. Two or more cyt b haplotypes were detected in 15.4% of wounds (8/52).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

The analysis of the cytochrome b mtDNA haplotypes confirmed the presence of two lineages of W. magnifica reported by a preliminary analysis of a shorter region of the same gene (Hall et al., 2009). The most likely hypothesis to explain the geographical distributions of the two lineages is that they were isolated in western and eastern Pleistocene glacial refugia and did not disperse far afterwards, maintaining allopatry. There is good evidence for post-glacial expansion of a number of Mediterranean species similar to that hypothesized here for W. magnifica (Schmitt, 2007). The Balkans and the Iberian and Italian peninsulas are recognized as important glacial refugia (Schmitt, 2007). In our study we did not have access to Italian specimens, but an earlier study using shorter sequences did link specimens from Italy with those from the eastern lineage (Hall et al., 2009). A detailed survey for W. magnifica in central Western Europe, including the Alps and Pyrenees, which may act as barriers to spread, is necessary to determine the extent of expansion and possible areas of sympatry of the two lineages.

In addition to post-glacial expansion of Mediterranean species, Schmitt (2007) considered the expansion of continental and Arctic/Alpine species. Although it occurs in the Mediterranean region, W. magnifica is also considered to be a continental species, adapted to the open steppe regions of Europe and Asia, tolerant of hot and dry summers and able to diapause through cold winters (Portchinsky, 1916; Ternovoy, 1978; Valentin et al., 1997). It is possible, therefore, that W. magnifica also occupied glacial refugia in the eastern Palaearctic region. The suggestion that extra-Mediterranean ice-age refugia were more common in Europe than previously considered deserves more investigation (Schmitt, 2007). Wohlfahrtia magnifica would be an ideal species with which to study this in view of its present Mediterranean and continental distribution, using a more complete phylogeographic analysis based on nuclear loci as well as mtDNA.

The haplotype profile in Crete was quite different to that in Morocco. Only two haplotypes were detected, of which one was predominant and the other was found in only 20% of AEs (Table 1). The predominant haplotype was identical to that found in greatest abundance in Hungary and mainland Greece. This result emphasizes the relative lack of genetic diversity in Eastern Europe, contrasting with that in Morocco. That the rare haplotype CB_magn10 was found only in Crete is not proof of an old and diverging population, but more probably reflects our under-sampling elsewhere, especially in the south of Greece, which should be a focus of further surveys. The prevalence of wohlfahrtiosis in sheep in Crete was up to 15% (Sotiraki et al., 2003, 2005a, 2005b), which is much higher than the equivalent figure for Morocco and lends support to the hypothesis that this infestation represents a recent introduction. Farmers and veterinarians had no previous experience of wohlfahrtiosis on Crete and no specimens of W. magnifica were found in the collections of the Natural History Museum in Heraklion. The data show that the W. magnifica introduced to Crete came from somewhere in the central or eastern part of the species’ range, but certainly not from the Iberian peninsula or Morocco. Early after its introduction it was speculated that the fly could have been introduced from Spain because Crete imported approximately 2000 live sheep from Spain each year in 1998 and 1999 for the tourist trade. Approximately 80% were slaughtered within 2 days of importation, but the remainder were slaughtered up to 1 month later (Z. Somaras, National Veterinary Services, Heraklion, Crete, personal communication, 2007). This time period would have enabled any undetected and untreated larvae on the imported sheep to mature, leave the host and, potentially, establish a breeding population of flies on Crete. However, our results show that the W. magnifica found in Crete had clearly not been imported from Spain and thus confirm the value of molecular genetic studies in evaluating the origin of invading species.

Despite the impression given by veterinarians in Morocco that wohlfahrtiosis was a new problem in the northern province of Al Hoceima, our molecular studies do not support this. Five different haplotypes were found in a relatively small geographical area (20 × 5 km) and three of those were unique to that region (Fig. 2). If there had been a recent introduction of W. magnifica, it would most likely have come from a single source, such as a consignment of infested animals, with one or, at most, two haplotypes, because infestations with more than two haplotypes were rare in our study (1/52, 1.9%). Hence a recent introduction would be unlikely to have shown the genetic diversity we found in Al Hoceima Province. Therefore, the situation in Al Hoceima appears most likely to be the result of resurgence from extant populations of W. magnifica, for reasons at present unknown. That we found W. magnifica in a museum collection in Morocco demonstrates that it has been present in the country for at least 30 years.

It is interesting that northern and central Morocco showed significantly different haplotype diversities. The most common haplotype in central Morocco (CB_magn02: 77.8%) was comparatively rare in northern Morocco (11.1%; Table 1). This suggests that there is limited genetic exchange between these two regions. The Rif Mountains, which reach over 1800 m above sea level, are a likely barrier to the movement of adult flies between these regions. In this regard, it is interesting to note that the greatest altitude at which we recorded cases of wohlfahrtiosis in Morocco was 360 m a.s.l. However, elsewhere in the distribution of W. magnifica, cases have been recorded at much greater altitudes, such as 1200 m a.s.l. in Mongolia (Valentin et al., 1997) and up to 2600 m a.s.l. in the French Pyrenees (Ruíz Martínez & Leclercq, 1994). We were unable to find evidence of wohlfahrtiosis in the northern foothills of the Middle Atlas, at approximately 1000–1700 m a.s.l. Reasons for this cannot include altitude, unless the populations in Morocco are less adapted to high altitudes and the associated climate than populations in Mongolia and the Pyrenees. The cold winter temperatures at these altitudes would also not normally be a contra-indicator for W. magnifica, which endures a much harsher winter in Eastern Europe. The region had many large flocks of sheep and goats and appeared ideal for W. magnifica. It is possible that some unique aspect of animal husbandry in the region limits wohlfahrtiosis, but this needs further study and the apparent lack of the species at higher altitudes in Morocco is, at present, a mystery.

The sampling of Hall et al. (2009) was more widespread than the present survey in geographical terms, but it looked at only a 273-bp sequence. The present analysis looked at 715 bp, but only in populations from Morocco, Spain, Hungary and Greece. Clearly, a much broader survey for the longer sequence is needed throughout the distribution range of W. magnifica to fully explore its genetic diversity and post-glacial expansion. Reasons for the comparatively high diversity of W. magnifica in Morocco compared with other areas studied to date in the Mediterranean basin and Europe deserve further study as they could provide indicators for how the species has spread with human interventions. Thus, the commonality of haplotypes on Crete and in mainland Greece and Hungary (Table 1) may have resulted from more extensive trade over centuries within Europe compared with that in Morocco. In this connection, W. magnifica could be a useful tool for examining the potential spread of insect pests of animals along the Eurasian ruminant road (Slingenbergh et al., 2004). It is possible that the degree of genetic diversity in any area may be linked to factors, such as animal trade, that can overcome natural barriers to pest dispersal.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References

We are grateful to Dr H. Benazzou and his staff at the Moroccan Ministry of Agriculture, government veterinarians in Meknes, Drs S. Filali and N. Bikri, and Drs Y. Lhor, A.K. Bouzagou and B.Y. Lotfi, all in Morocco, and to Dr J.F. Graf, farm owners Manolis Stefanakis and Nikos Spondidakis, and the staff of the National Agricultural Research Foundation (NAGREF) Research Station in Asomaton, especially animal technician Nikos Christodoulakis, all in Crete, for facilitating the Moroccan and Cretan studies, respectively. We are also grateful to Javier Lucientes Curdi for the provision of Spanish specimens and to Gillian Watson and Anny Bivand for contributions to the molecular studies. We acknowledge the Food and Agriculture Organization of the United Nations (FAO), the International Atomic Energy Agency (IAEA), the Ministry of Agriculture, Morocco, NAGREF and the British Council for funding different aspects of this collaborative research.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflicts of interest
  9. References
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