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Molecular & Biochemical Parasitology 159 (2008) 59–63
Short communication
Microsatellite genotyping supports the hypothesis that Teladorsagia davtiani and Teladorsagia trifurcata are morphotypes of Teladorsagia circumcincta Victoria Grillo a,∗ , Barbara H. Craig b,2 , Barbara Wimmer b,1 , John Stuart Gilleard a a
Division of Infection and Immunity, Institute of Comparative Medicine, Faculty of Veterinary Medicine, Bearsden Road, University of Glasgow, Glasgow G61 1QH, UK b Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK Received 9 October 2007; received in revised form 8 January 2008; accepted 8 January 2008 Available online 17 January 2008
Abstract Traditionally nematode species designations have been based on morphological criteria. However, it is has long been recognised that valid species designations are critical for basic biological and epidemiological studies. The ever increasing use of molecular and genetic techniques has allowed traditional classifications to be more closely examined. The sub-family Ostertagiinae is of particular interest as many of the species within this group are of economic importance worldwide, with unresolved classification complicating epidemiology, management, control and genetic studies. This study examines genetic differences between three morphological variants (morphotypes) within the genus Teladorsagia (sub-family: Ostertagiinae) using a multi-locus population genetic analysis approach. Five microsatellites were used to genotype a total of 31 T. davtiani (ScKiTD), 30 T. trifurcata (ScKiTT), and 31 T. circumcincta (ScKiTC). Population genetic analysis detected no genetic differentiation between T. davtiani, T. trifurcata, and T. circumcincta supporting the hypothesis that these are morphotypes of the same species. © 2008 Elsevier B.V. All rights reserved. Keywords: Nematode; Teladorsagia; Ostertagia; Microsatellites; Species identification
Valid species designations are of fundamental importance to basic biological and epidemiological studies of parasites as well as for their diagnosis, treatment and control [9,24]. Defining a species is a notoriously difficult and controversial matter. There are many different species concepts in use, for example species may be defined on the basis of diagnostic traits [19], cladistic classification or biological concepts [3]. For parasitic nematodes, species classification has traditionally employed cladistic taxonomy with morphological features and morphometric measurements used to distinguish between nema-
∗
Corresponding author at: School of Animal and Veterinary Sciences, E H Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW 2678, Australia. Tel.: +61 2 6933 4128; fax: +61 2 6933 2991. E-mail address:
[email protected] (V. Grillo). 1 Present address: Eurofins Medigenomix, Fraunhoferstr. 22, 82152 Martinsried, Germany. 2 Present address: Wildlife, Ecology and Management Group, Central Science Laboratory, York, YO41 1LZ, UK. 0166-6851/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2008.01.001
tode species [11,15]. However, it is often unclear as to whether particular morphological features reflect genuine species differences or are simply morphological variations within the same species. In addition, these criteria tend to be limited to the identification of adult stages. Whereas identification of eggs and larvae, found in the more readily available source of material e.g. faeces, can be time consuming, requires highly trained specialists, and in some cases is unreliable and limited to family level [25]. This has not only been widely debated but has also hindered the progress of researchers studying nematodes within the superfamily Trichostrongyloidea, and in particular the sub-family Ostertagiinae, an economically important group of pathogens of ruminant livestock throughout the world [10,14,20,21,25,34]. It is widely recognised that the increasing use of molecular and genetic techniques, sometimes in combination with complex morphological datasets, has allowed traditional classifications to be more closely examined. In particular, molecular and genetic techniques will allow improved identification and classification of species applicable to all nematode life stages.
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The Ostertagiinae are a sub-family of bursate nematodes in which species designations are often based on a number of morphological and morphometric features of the adult male tail [5,10,14,24–26]. Species designations within the genus Teladorsagia (previously Ostertagia) have been particularly confused and remain to be completely defined. Teladorsagia circumcincta was traditionally defined, on morphological criteria, as a single species that could infect sheep, goats and muskoxen. However, recent molecular studies suggest that the population structure is more complex with both morphologically discernable and cryptic species being identified, with the genus Teladorsagia actually composed of several species with differing host specificities [12,16,18,22]. Hoberg et al. [18] discovered that nematode parasites of muskoxen (Ovibos moschatus) from the central Canadian Artic, previously regarded as T. circumcincta, were a separate species which they named Teladorsagia boreoarcticus, based on a number of morphological measurements and mtDNA ND4 sequences. T. boreoarcticus is distinguishable from T. circumcincta by virtue of a 13% sequence divergence of the mtDNA gene ND4. There is also strong evidence that some T. circumcincta populations found in goats differ genetically from those found in sheep, using several independent molecular markers; the mtDNA ND4 gene, the -tubulin isotype 1 gene, the second transcribed region of the rRNA cistron (ITS-2) and microsatellite loci [12,16,22]. It has long been recognised that some genera of Ostertagiinae consist of morphological variants (morphotypes) rather than separate species [10,20,21,24,36]. For each polymorphic species, only morphologically discrete males are recognised, mainly based on the generic male characteristics, e.g. structure of the spicules, gubernacula and genital cone [10]. In spite of this, the clear morphological differences in the spicules and genital cone of the male tail originally led to T. circumcincta, Teladorsagia trifurcata, and Teladorsagia davtiani being considered as three separate species [5,14]. Indeed, this view still persists in some circles as illustrated by the fact that commercial companies often list each species morphotype separately in their claims for the spectrum of activity of anthelmintic products [29]. T. circumcincta is characterised by slender spicules in comparison to the shorter stouter spicules of T. trifurcata and T. davtiani [5]. The latter two are differentiated by morphological variation of the genital cone [5]. A number of authors have suggested that T. trifurcata and T. davtiani are in fact morphotypes of T. circumcincta [4,24,33,34]. This is based on a number of observations also observed for other genera within the sub-family Ostertagiinae. Firstly, the three male morphotypes appear always to occur together, with T. circumcincta constituting the major proportion of the population [8,20,21,34]. Secondly, no morphological differences in female worm morphology have been identified within populations containing the three male morphotypes [24]. Thirdly, characteristics of the oesophagus and surface cuticular ridges (synlopes) are uniform between the morphotypes [26]. Fourthly, cross-breeding experiments have produced males of the different morphotypes either from a cross between a female T. circumcincta (monomorphic lab strain), and male T. trifurcata or from unknown Teladorsagia females mating with males of one
of the morphotypes [21,24,34]. These genetic crossing experiments are not absolutely conclusive since the species identity of the females used in crosses must be presumed to be T. circumcincta since there is no female morphological variation known to correspond to the three different male morphotypes [24]. Comparatively few molecular approaches utilising a relatively limited number of specimens have been used for genetic comparisons of the three morphotypes. Allozyme electrophoresis studies revealed no fixed differences between the three morphotypes at twelve polymorphic markers. However this was limited to the simple presence or absence of alleles with no information on allele frequencies, because pooled material was examined [4]. These results were supported by a subsequent study using five of these allozyme markers on individual adult worms of T. circumcincta and T. trifurcata [13]. Comparisons have also been made using sequences of the second transcribed spacer of the ribosomal DNA (ITS-2). No fixed differences were detected between the ITS-2 sequences from three T. circumcincta, two T. davtiani and a single T. trifurcata individual examined [33]. It is also worth noting that T. circumcincta, T. davtiani and T. trifurcata all have indistinguishable karyotypes when metaphase spreads are microscopically examined [28]. Hence the morphological, experimental and genetic evidence to date suggest that these three morphotypes do not represent separate species. The genus Teladorsagia represents just one example of an issue contributing to the taxonomic complexity and confusion of many nematode species. There is not only a requirement to form a strong phylogenetic baseline for the subfamily Ostertagiinae [25] but also a need to complement this with population genetic analysis. An understanding of the genetic structure of parasite populations is necessary to appreciate important evolutionary processes such as adaptation to host defences, speciation and the evolution of resistance to drugs and vaccines [7,9]. The use of multi-locus markers is critical to such studies. Anderson [1] demonstrated that for species or populations which have only recently diverged, analysis using a single locus can cause misleading information regarding species or population differences. Prior to each new species become fixed for alternative alleles, all loci may be in a state of polyphyly or paraphyly and, as a result, no one marker can be used as a diagnostic for differentiating between species or populations [2]. However, if many independent loci are used differentiation between populations will be more accurate and reliable [30]. Microsatellite markers are ideal as they occur in multiple numbers fairly evenly spread throughout the genome, are polymorphic and generally considered to be neutral markers [6,35]. They can be used for multi-locus genotyping allowing powerful analysis of population differentiation and species fingerprinting. We have undertaken a multi-locus population genetic approach to further test the hypothesis that T. circumcincta, T. davtiani and T. trifurcata are morphotypes of the same species, investigating this genus at a more detailed scale. A previously characterised panel of five microsatellite markers which have high levels of polymorphism and that have already uncovered genetic differentiation between standard T. circumcincta and a
V. Grillo et al. / Molecular & Biochemical Parasitology 159 (2008) 59–63 Table 1 Number of individuals of each Teladorsagia morphotype taken from each of the 12 Soay sheep hosts listed Host ID number
Teladorsagia davtiani
Teladorsagia trifurcata
Teladorsagia circumcincta
YR514 YO517 NP089 YY557 NW538 YB160 AY058 AY069 NG528 OP553 YL072 AY151 AY151
3 7 1 6 2 2 1 1 1 6 – 1 1
3 7 1 5 3 2 1 1 1 6 1 – –
3 7 1 5 3 2 1 1 1 6 1 – –
cryptic species in goats was utilised [16]. We have examined sympatric populations of T. circumcincta, T. davtiani and T. trifurcata since the presence of genetic differentiation in this situation would provide direct evidence of a lack of gene flow between the three morphological types consistent with a mating barrier. Individual adult males were collected at post mortem from Soay sheep and identified as being one of the three Teladorsagia morphotypes, based on male tail and spicule morphology in accordance with Becklund and Walker and the Manual of Veterinary Parasitology Laboratory Techniques [5,27]. The three populations consisted of 31 T. davtiani (ScKiTD), 30 T. trifurcata (ScKiTT), and 31 T. circumcincta (ScKiTC). Equal numbers of each type were, as far as possible, collected from a total of 12 Soay sheep to avoid any host bias (Table 1). Individual worm lysates were stored at −80 ◦ C. One microlitre of a 1:30 dilution of neat lysate was used as DNA template. Several aliquots of “blank” dilution water (no DNA) were made in parallel of individual worm dilutions, and were used in addition to standard PCR negative controls (no DNA).
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All three populations were genotyped using the following five microsatellite markers; MTG15, MTGHC3, MTG67, MTG73 and MTG74. The primers and PCR conditions for microsatellite genotyping were as previously described [17]. Accurate sizing of microsatellite PCR products by capillary electrophoresis was performed using an ABI Prism 3100 genetic analyser (Applied Biosystems). Individual chromatograms were analysed using Genotyper 3.7 NT software (Applied Biosystems) to determine the genotype of each sample. Expected heterozygosity (He ), unbiased for sample size, observed heterozygosity (Ho ) and average number of alleles per locus were calculated by GDA (Genetic Data Analysis) Version 1.1 [23]. Exact tests for Hardy–Weinburg equilibrium (HWE) and pairwise linkage disequilibrium were tested for using Fisher’s Exact Test (10,000 runs) using GDA Version 1.1. Pairwise FST values were calculated using GDA Version 1.1 [23]. Analysis of molecular variance (AMOVA) was conducted to test for population differentiation of samples at various levels, locus by locus, using Arlequin Version 2.0 [32]. Data were defined as ‘standard’ rather than ‘microsatellite’, as loci did not necessarily adhere to the stepwise mutation model. Principle coordinate analysis (PCA) was conducted and the data for individual worms was plotted for the first two principal coordinates using GenAlEx Version 5.1 [31]. All three morphotypes were equally polymorphic at all five microsatellite loci, with the average expected heterozygosity >0.5; T. davtiani (0.682), T. trifurcata (0.628) and T. circumcincta (0.653). The average number of alleles per locus was also similar; T. davtiani (8.2), T. trifurcata (7.6), and T. circumcincta (7.8), with the number of alleles per locus per morphotype ranging from 3 to 16. Alleles unique to any one morphotype were of extremely low frequency (frequency < 0.05) and have been detected in other T. circumcincta populations from other geographical origins [16]. There were no major differences between allele frequencies between morphotypes for any of the markers. This was reflected by the AMOVA analysis conducted to examine the partitioning of variation within and between the three morphotypes, in which the majority of the total genetic varia-
Table 2 Pairwise FST values between morphotypes of T. circumcincta sampled on St. Kilda, Outer Hebrides, Scotland Pairwise Fst using all Loci ScKiTT vs. ScKiTC ScKiTT vs. ScKiTD ScKiTD vs. ScKiTC
−0.0068 0.0214 0.0250
Pairwise Fst for each microsatellite ScKiTT vs. ScKiTC ScKiTT vs. ScKiTD ScKiTD vs. ScKiTC
MTG15
MTGHC3
MTG 67
MTG 73
MTG 74
−0.0031 0.0178 0.0093
−0.0107 −0.0113 −0.0001
−0.0012 0.0797 0.0837
0.0071 −0.0156 −0.0130
−0.0210 0.0133 0.0258
Pairwise Fst analysis after sequential omission of each microsatellite No MTG 15 No MTGHC3 ScKiTT vs. ScKiTC ScKiTT vs. ScKiTD ScKiTD vs. ScKiTC
−0.0082 0.0227 0.0306
0.0320 0.0332 −0.0054
No MTG 67
No MTG 73
No MTG 74
0.0043 0.0076 −0.0083
0.0243 0.0285 −0.0082
0.0232 0.0248 −0.0039
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V. Grillo et al. / Molecular & Biochemical Parasitology 159 (2008) 59–63
Fig. 1. Principle coordinate analysis plotting the data from each individual worm using the microsatellites MTG15, MTGHC28, MTG67, MTG73 and MTG74. Percentage of variation explained by the first two coordinates is shown on X- and Y-axis of the graph. Symbols indicate the morphotype of each worm.
tion was distributed within populations (98.07%) compared to between morphotypes (1.93%; variance: 0.029; P < 0.01). Quantitative analysis, using pairwise FST values calculated from allele frequencies using all five microsatellites, showed very little genetic differentiation between the three morphotypes (FST values: −0.007 (T. circumcincta–T. trifurcata), 0.0214 (T. davtiani–T. trifurcata), and 0.0250 (T. davtiani–T. circumcincta) (Table 2). Pairwise FST values calculated using each individual marker and after sequential omission of each marker (the pairwise FST calculated from the remaining four markers) were similarly low (Table 2). For four out of five markers the FST values were well below 0.05 with only marker MTG67 showing FST values >0.05 between T. davtiani and the other two populations. PCA was conducted using multi-locus genotypes based on the five markers to plot and compare the genetic distance between individual worms of each morphotype (Fig. 1). The plot suggests a lack of between morphotype differentiation since individual worms of each morphotype are randomly distributed throughout the plot with no obvious clustering of particular morphotypes. This multi-locus genotype analysis suggests there is little genetic difference between the three morphotypes of the genus Teladorsagia based on a panel of five polymorphic microsatellites. These morphotypes have similar pairwise FST values to those found between morphologically identical populations of T. circumcincta taken from a range of U.K. farms (−0.001 to 0.065) [16]. This is consistent with them being morphotypes of the same species rather than being separate species. It is of course possible that the use of a larger marker panel could uncover more subtle genetic differences. However, it is noteworthy that this same panel of five microsatellite markers showed clear genetic differentiation between sympatric populations of T. circumcincta and a putative cryptic species in French goats [16]. The cryptic French species showed obvious genetic differentiation with pairwise FST values ranging between 0.081–0.157 and clear clustering of multi-locus genotypes on PCA analysis, despite the lack of obvious morphological differences to T. circumcincta [16]. In the present study, the only marker that potentially showed a low level of genetic differentiation between the three morphotypes was MTG67 with FST values >0.05 between T. davtiani and the other two morphotypes (Table 2). Genetic differences at a single marker does not in itself imply
these morphotypes are likely to be separate species. For example, the marker could be genetically linked to a locus involved in determining male tail morphology. Furthermore, the pairwise FST values at the MTG67, although >0.05, are still low and so genotyping a much larger number of worms would be required to determine its significance. In summary, the genotyping of sympatric T. circumcincta, T. davitiani and T. trifurcata populations with a panel of five polymorphic microsatellite markers shows an absence of genetic differentiation between these three morphotypes. This provides further support that these are morphotypes of a single species rather than separate species. This approach of multi-locus genotyping of sympatric populations is a potentially powerful method to help resolve questions concerning the species status of many parasitic nematodes. The main limitation at present is the lack of markers available for many species and the labour intensive nature of marker development for organisms for which there is little genome sequence. However, genome sequencing projects are now underway for a variety of trichostrongyloid nematode species which will soon allow much larger sets of markers to be more easily developed and applied. Acknowledgements The project was also supported by a Wellcome Trust Entry Level Fellowship and Wellcome Trust Project Grant 067811/Z/02/Z. Victoria Grillo was also funded by a University of Glasgow Postgraduate Scholarship. Josephine M. Pemberton for access to samples from St. Kilda & comments on manuscript. References [1] Anderson TJ. The dangers of using single locus markers in parasite epidemiology: Ascaris as a case study. Trends Parasitol 2001;17(4): 183–8. [2] Anderson TJ, Blouin MS, Beech RN. Population biology of parasitic nematodes: applications of genetic markers. Adv Parasitol 1998;41:219– 83. [3] Anderson TJ, Jaenike J. Host specificity, evolutionary relationships and macrogeographic differentiation among Ascaris populations from humans and pigs. Parasitology 1997;115(Pt 3):325–42.
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