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Mitochondrial DNA polymorphism within and among species of Capillaria sensu lato from Australian marsupials and rodents
International Journal for Parasitology 30 (2000) 933±938
www.elsevier.nl/locate/ijpara
Research note
Mitochondrial DNA polymorphism within and among species of Capillaria sensu lato from Australian marsupials and rodents q Xingquan Zhu a, David M. Spratt b, Ian Beveridge a, Peter Haycock b, Robin B. Gasser a,* a
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia b CSIRO Wildlife & Ecology, GPO Box 284, Canberra 2601, Australia Received 12 April 2000; received in revised form 9 June 2000; accepted 9 June 2000
Abstract The nucleotide variation in a mitochondrial DNA (mtDNA) fragment within and among species of Capillaria sensu lato from Australian marsupials and rodents was analyzed using a mutation scanning/sequencing approach. The fragment of the cytochrome c oxidase subunit I (COI) was ampli®ed by PCR from parasite DNA, and analysed by single-strand conformation polymorphism (SSCP) and sequencing. There was no signi®cant variation in SSCP pro®les within a morphospecies from a particular host species, but signi®cant variation existed among morphospecies originating from different host species. The same morphospecies was found to occur in 1±3 tissue habitats within one host individual or within different individuals of a particular species of host from the same or different geographical areas, and morphospecies appeared to be relatively host speci®c at the generic level. The results indicated that the species of Capillaria sensu lato examined, although highly variable in their host and tissue speci®city, may exhibit the greatest degree of speci®city at the level of host genus. q 2000 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. Keywords: Capillaria spp; Cytochrome c oxidase subunit I; Genetic variation; Mitochondrial DNA; Polymerase chain reaction-based single-strand conformation polymorphism
Parasitic nematodes of the genus Capillaria sensu lato (Enoplida) occur in a wide range of ®sh, reptiles, birds and mammals on all major continents [1]. Many of those occurring in domestic animals are of considerable economic importance [2,3], while others cause severe disease in humans [4,5]. The accurate identi®cation of these parasites has major implications for studying the life cycles, transmission patterns, host±parasite relationships and for the development of control strategies. Despite more than 300 described species of Capillaria from all classes of vertebrates throughout the world, their taxonomy remains controversial, with some authors recognizing a single genus and other authors recognizing from ®ve to 20 or more genera [1,2,6±8]. There are major limitations in identifying specimens of Capillaria to species level because of their small size and the limited range of characteristic morphological features available. This problem is compounded by pronounced variation in morphometrics, host speci®city and habitat q The nucleotide sequence data reported in this paper are available in the EMBL, GenBank e and DDJB databases under the accession numbers AJ288160±AJ288170. * Corresponding author. Tel.: 161-3-973-12000; fax: 161-3-973-12366. E-mail address: [email protected] (R.B. Gasser).
speci®city (site in host tissues) within one host individual and one host species, and between different host individuals and different host species. Particular problems arise when apparently the same species occurs in different, sometimes distantly related hosts or in different tissues within the same host species. Therefore, alternative approaches to morphological identi®cation are needed. The aim of this study was to investigate the genetic variation within and between morphologically identi®ed species of Capillaria sensu lato from different host species and from different tissue sites within a host species. Polymerase chain reaction-based single-strand conformation polymorphism (SSCP) [9], followed by sequencing, was used to characterize sequence variation in a portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Although many are not formally named, the diverse fauna of species of Capillaria sensu lato present in Australian mammals [10,11] presented an opportunity to investigate genetic variation within the genus under the restraints described above. Adult specimens representing Capillaria gastrica from rodents and four, yet unnamed, species of Capillaria from marsupials [10] were obtained from different hosts and geographical locations (Table 1). Samples representing a
0020-7519/00/$20.00 q 2000 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. PII: S 0020-751 9(00)00076-X
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Table 1 DNA samples representing species of Capillaria sensu lato used in this study Sample
Morphospecies
OUT
Host
Site
Locality in Australia a
AhRf b CgRf1 b CgRf2 CgRf3 CgRf4 CgRf5 CgRf6 CgRl1 b CgRl2 CgRl3 C1Dv1 b C1Dv2 C1Dv3 C1Dv4 C1Dh b C2Im1 b C2Im2 C2Im3 C2Im4 C2Im5 C2Im6 C2Im7 C2Dh1 C2Dh2 C2Dh3 b C3Io1 b C3Io2 C3Io3 C3Io4 C4Im1 C4Im2 b C4Io b
Anatrichosoma haycocki Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 3 Capillaria sp. 3 Capillaria sp. 3 Capillaria sp. 3 Capillaria sp. 4 Capillaria sp. 4 Capillaria sp. 4
1 1 1 1 1 1 2 2 2 3 3 3 3 4 5 5 5 5 5 5 5 6 6 6 7 7 7 7 8 8 9
Rattus fuscipes R. fuscipes R. fuscipes R. fuscipes R. fuscipes R. fuscipes R. fuscipes R. lutreolus Rattus lutreolus R. lutreolus Dasyurus viverrinus D. viverrinus D. viverrinus D. viverrinus D. hallucatus Isoodon macrourus I. macrourus I. macrourus I. macrourus I. macrourus I. macrourus I. macrourus Dasyurus hallucatus D. hallucatus D. hallucatus Isoodon obesulus I. obesulus I. obesulus I. obesulus I. macrourus I. macrourus I. obesulus
Paracloacal gland Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Tongue Tongue Tongue Tongue Tongue Lips Lips Lips Tongue Tongue Tongue Oesophagus Oesophagus Oesophagus Oesophagus Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine
NSW sNSW sNSW sNSW sNSW sNSW sNSW cNSW cNSW TAS TAS TAS TAS TAS NT cNSW cNSW cNSW cNSW cNSW cNSW cNSW NT NT NT TAS TAS TAS TAS cNSW cNSW sNSW
a b
TAS, Tasmania; VIC, Victoria; sNSW and cNSW, southern and central New South Wales, respectively; NT, Northern Territory. Samples sequenced.
particular morphospecies of Capillaria from a particular host species were treated as a separate operational taxonomic unit (OTU; Table 1). Anatrichosoma haycocki from rodents was used for comparative purposes. Trapped animals were anaesthetised with Zoletil (Virbac Australia Pty Ltd; 50 mg/kg injected i.m.) and killed by cervical dislocation. Post mortem examinations of the tissues and organs of all animals were conducted under a stereomicroscope. Specimens of Capillaria sensu lato were dissected from host epithelial tissues, washed several times in physiological saline, and separated into aliquots for morphological and molecular studies. Material for morphological study was ®xed in hot (10%), neutral buffered formalin and stored in small McCartney bottles. Individual nematodes were cleared in lactophenol and allocated to morphospecies based on a suite of morphological characteristics from male and female worms and the ornamentation of the egg shell. Specimens for molecular study were placed in individual Eppendorf tubes and frozen immediately in liquid nitrogen. In addition, small samples of liver, kidney and small intestinal content from each host animal were
placed in individual Eppendorf tubes and frozen immediately in liquid nitrogen. On return to the laboratory, all frozen specimens were transferred to a biofreezer at 2708C. Genomic DNA was isolated from individual nematodes by sodium dodecyl-sulphate/proteinase K treatment [12] and direct puri®cation over spin columns (Wizard e DNA CleanUp, Promega). A portion of the mitochondrial COI gene (,450 bp) was ampli®ed by PCR using primers JB3 (5 0 -TTTTTTGGGCATCCTGAGGTTTAT-3 0 ; forward) and JB4.5 (5 0 -TAAAGAAAGAACATAATGAAAATG-3 0 ; reverse) [13] in 10 mM Tris±HCl (pH 8.4), 50 mM KCl, 4 mM MgCl2, 250 mM of each dNTP, 100 pmol of each primer and 2 U Taq polymerase (Promega) in an automated thermocycler (Perkin±Elmer Cetus) under the following cycling conditions: 948C for 5 min (initial denaturation), then 30 cycles of 948C for 30 s (denaturation), 558C for 30 s (annealing), 728C for 30 s (extension), followed by a ®nal extension at 728C for 5 min. One microlitre of each amplicon was subjected to secondary ampli®cation using 35 pmol of [g 33P]-endlabelled primers, JB3 and JB4.5, using the same
X. Zhu et al. / International Journal for Parasitology 30 (2000) 933±938
conditions as for primary ampli®cation. Control samples without DNA and with rat DNA were included in each PCR run. Five microlitres of each PCR product were examined by agarose gel electrophoresis [14]. The SSCP method was carried out as described previously [15]. Samples displaying variable COI SSCP pro®les were subjected to automated DNA sequencing (ABI 373 DNA Sequencer, Monash University) using the same primers as for primary ampli®cation. Manual sequencing using the fmole cycle sequencing kit (Promega) was carried out according to a modi®cation of the original protocol as described previously [16] to con®rm some nucleotide positions. The sequences were aligned manually. Pairwise comparisons were made of the level of sequence differences (D) using the formula D 1 2 M=L [17], where M is the number of alignment positions at which the two OTUs have a base in common, and L is the total number of alignment positions over which the two OTUs are compared. A phenogram was constructed from these data using the unweighted pair group method using arithmetic averages (UPGMA) [18]. In a ®rst step, the COI fragment (,450 bp) was ampli®ed from individual worms and subjected to agarose gel electrophoresis. No variation in size was detected on agarose gels (not shown) among 32 samples representing the ®ve currently-recognised morphospecies of Capillaria sensu lato. We then subjected the COI amplicons to SSCP analysis to screen for sequence variation within and among OTUs. While slight variation in single-stranded pro®les was detected between/among some specimens representing an OTU (e.g. sample CgRf6 versus samples CgRf1±CgRf5; samples CgRl1 and CgRl2 versus sample CgRl3; Fig. 1), signi®cant differences in the COI pro®les among the ®ve currently-recognized morphospecies of Capillaria sensu lato from different host species were displayed, which allowed the delineation of the nine OTUs (cf. Table 1). In some cases, variation in pro®les was detected between samples of the same morphospecies from a particular host (e.g. samples CgRl1 and CgRl2 versus sample CgRl3; Fig. 1). While there was no signi®cant variation in SSCP pro®les in most OTUs for which multiple individual nematodes were available, variation was displayed between morphospecies originating from different host species. For example, the SSCP pro®les for nematodes from the gastric mucosa and identi®ed as C. gastrica Baylis, 1926 from Rattus fuscipes from Ludwigs Swamp in southeastern New South Wales (samples CgRf1±CgRf6; Fig. 1) were distinctly different to those originating from Rattus lutreolus from Glenreagh in the central coastal ranges of New South Wales (CgRl1 and CgRl2; Fig. 1), and to one representative from Tasmania (sample CgRl3; Fig. 1). Also, substantial variation in pro®les was displayed between Capillaria sp. 1 from the epithelium of the tongue of Dasyurus viverrinus from Huonvale, Tasmania (samples C1Dv1±C1Dv4; Fig. 1) and a representative from the tongue of Dasyurus hallucatus from Coomalie Creek, Northern Territory (sample C1Dh; Fig. 1). A lower level of sequence variation was displayed
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between Capillaria sp. 2 samples from the oesophageal mucosa of D. hallucatus (samples C2Dh1±C2Dh3; Fig. 1) from Coomalie Creek, Northern Territory and those from the lips, tongue and oesophagus of Isoodon macrourus (samples C2Im1±C2Im7; Fig. 1) from Glenreagh and
Fig. 1. SSCP analysis of COI PCR products representing adult individuals of Capillaria spp. from different host species and geographical locations using Anatrichosoma haycocki from Rattus fuscipes (sample AhRf) for comparative purposes. Lanes represent Capillaria gastrica from Rattus fuscipes (samples CgRf1±CgRf6) or Rattus lutreolus (samples CgRl1± CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (samples C1Dv1± C1Dv4) or Dasyurus hallucatus (sample C1Dh), Capillaria sp. 2 from Isoodon macrourus (samples C2Im1±C2Im7) or D. hallucatus (samples C2Dh1±C2Dh3), Capillaria sp. 3 from Isoodon obesulus (samples C3Io1±C3Io4), and Capillaria sp. 4 from I. macrourus (samples C4Im1 and C4Im2) or I. obesulus (sample C4Io). N and R represent No-DNA and rat DNA controls, respectively.
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Dorrigo, New South Wales, as well as between samples of Capillaria sp. 4 from the intestinal mucosa of I. macrourus from Dorrigo, New South Wales (samples C4Im1 and C4Im2; Fig. 1) and one representative from the intestinal mucosa of Isoodon obesulus from Sidling Swamp, southeastern New South Wales (C4Io; Fig. 1). However, Capillaria sp. 2 examined from the epithelium of the lips of I. macrourus from Glenreagh, New South Wales, the epithelium of the tongue of an animal from Dorrigo, New South Wales and the oesophageal mucosa of a second individual I. macrourus from that locality were genetically the same (samples C2Im1±C2Im7; Fig. 1), indicating that a single morphospecies of Capillaria sensu lato may occur in various epithelial tissues of different individuals within a single host species from different geographical locations. On the other hand, Capillaria sp. 2 occurring in the oesophageal epithelium of I. macrourus from Dorrigo, New South Wales, had similar SSCP pro®les to Capillaria sp. 4 occurring in the intestinal epithelium of that same individual (sample C2Im7 versus samples C4Im1 and C4Im2; Fig. 1). Having demonstrated that SSCP could detect genetic variation within and among species of Capillaria, we wanted to quantitate the levels of sequence difference in the COI. For this purpose, samples (CgRf1, CgRl1, CgRl3, C1Dv1, C1Dh, C2Im1, C2Dh3, C3Io1, C4Im2, C4Io and AhRf; Table 1) representing different SSCP pro®les were subjected to cycle sequencing. A 374 bp sequence of COI was obtained for all samples and aligned (available from the authors upon request). No polymorphism (more than one base at any one position) was detected in the COI sequence within any OTU. The G 1 C content of the COI sequences for all OTUs were similar, ranging from 33.2±34.8%. Pairwise comparison of the sequences between the OTUs revealed differences ranging from 0.5±32.6% (Table 2). For instance, sequence differences of 4.0±5.6% existed among C. gastrica samples from R. fuscipes and R. lutreolus (samples CgRf1, CgRl1 and CgRl3), whereas a nucleotide difference of 4.8% was detected between C.
gastrica samples from R. lutreolus from New South Wales (sample CgRl1) and Tasmania (sample CgRl3). A phenogram depicting the genetic relationships among the samples is shown in Fig. 2. With the exception of Capillaria sp. 4 from I. obesulus, all of the morphospecies of Capillaria clustered according to host genus. There was a 4±5.6% sequence difference between C. gastrica samples (CgRf1 versus CgRl1 and CgRl3) from different rat hosts, 8.6% between samples (C1Dv1 and C1Dh) of Capillaria sp. 1, and for both Capillaria sp. 2 and Capillaria sp. 4, there was a difference of ,30% between samples (C2Im1 versus C2Dh3, and C4Im2 versus C4Io, respectively) from different hosts (Table 2; Fig. 2). The variation within a morphospecies from different host species was frequently greater than differences between different morphospecies from the same host species (Table 2; Fig. 2). In addition to the morphological and speci®city constraints referred to previously, the controversy surrounding the taxonomy of the nematode genus Capillaria sensu lato and relationships among its many species stems in part from the fact that no major revision has involved a thorough study of type specimens or specimens from type hosts [19]. Many descriptions of species are inadequate. Consequently, a review of the literature has been an inappropriate basis for revision. We sought an alternative approach by examining genetic variation within and between morphologically identi®ed species of Capillaria sensu lato from different rodent and marsupial host species and from different tissue sites within a host species. A constraint in this study, which was not encountered in previous reports (e.g. [17,20]), was that it was not possible to identify the nematodes frozen for molecular work to morphospecies under a dissecting microscope, given their small size. Consequently, in each instance, an aliquot of nematodes was frozen and an aliquot from the same tissue site and host individual was ®xed. Determination of the morphospecies was based on the morphological features of nematodes in the ®xed aliquot. In two instances, we suspect that an error has
Table 2 Pairwise comparison of nucleotide differences (%) in the COI sequence among representatives of Capillaria gastrica from Rattus fuscipes (sample CgRf1) or Rattus lutreolus (samples CgRl1 and CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (sample C1Dv1) or Dasyurus hallucatus (sample C1Dh), Capillaria sp. 2 from Isoodon macrourus (sample C2Im1) or D. hallucatus (sample C2Dh3), Capillaria sp. 3 from Isoodon obesulus (sample C3Io1), and Capillaria sp. 4 from I. macrourus (sample C4Im2) or I. obesulus (sample C4Io), using Anatrichosoma haycocki from Rattus fuscipes (sample AhRf) for comparative purposes Sample
AhRf
CgRf1
CgRl1
CgRl3
C1Dv1
C1Dh
C2Im1
C2Dh3
C3Io1
C4Im2
C4Io
AhRf CgRf1 CgRl1 CgRl3 C1Dv1 C1Dh C2Im1 C2Dh3 C3Io1 C4Im2 C4Io
± 28.9 29.4 28.6 27.5 28.6 34.2 28.6 32.6 34.2 31.0
± 5.6 4.0 15.5 14.4 31.6 14.2 30.5 30.7 20.9
± 4.8 16.8 14.2 32.6 13.9 32.6 32.6 21.1
± 15.0 14.4 32.6 14.7 32.4 32.6 20.9
± 8.6 30.5 8.8 29.4 30.2 19.5
± 29.9 1.3 29.7 29.9 19.5
± 29.4 7.0 0.5 29.9
± 28.5 29.4 19.0
± 7.0 29.1
± 30.2
±
X. Zhu et al. / International Journal for Parasitology 30 (2000) 933±938
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different morphospecies may occur in the same host species from different geographical areas. Low-level variation in morphospecies may occur in closely-related host species. Morphospecies may be reasonably host speci®c at the generic level. These results imply that species of Capillaria sensu lato, although highly variable in their host and tissue speci®city, may nevertheless exhibit the greatest degree of speci®city at the level of host genus. Acknowledgements
Fig. 2. A phenogram depicting the genetic relationships among representatives of Capillaria gastrica from Rattus fuscipes (sample CgRf1) or Rattus lutreolus (samples CgRl1 and CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (sample C1Dv1) or Dasyurus hallucatus (sample C1Dh), Capillaria sp. 2 from Isoodon macrourus (sample C2Im1) or D. hallucatus (sample C2Dh3), Capillaria sp. 3 from Isoodon obesulus (sample C3Io1), and Capillaria sp. 4 from I. macrourus (sample C4Im2) or I. obesulus (sample C4Io) based on COI sequence data, using Anatrichosoma haycocki from R. fuscipes (sample AhRf) for comparative purposes.
occurred and these can be recognized in both Fig. 1 and Table 2. Individuals C2Im1 and C4Im2 could both represent Capillaria sp. 2 (OTU5) because they have almost identical SSCP pro®les and have a 0.5% nucleotide difference in the COI sequence. This is supported by the 7% nucleotide difference in the COI sequence for both C2Im1 versus C3Io1 and C3Io1 versus C4Im2, indicating minor variation between morphospecies from these two closely-related species of bandicoot hosts. A morphological study (D.M. Spratt, unpublished) has demonstrated that Capillaria sp. 2 and Capillaria sp. 4 frequently co-occur in I. macrourus. Similarly, C1Dh and C2Dh3, which both represented Capillaria sp. 2 (OTU 6), appeared very similar electrophoretically and exhibited only 1.3% nucleotide difference in the COI sequence. This is supported by the 8.6 and 8.8% nucleotide difference in the COI sequence for C1Dh versus C1Dv1 and C2Dh3 versus C1Dv1, respectively, again suggesting a minor variation between morphospecies from these two closely-related quoll hosts. Morphological study has shown that Capillaria sp. 1 and Capillaria sp. 2 frequently co-occur in D. hallucatus. Consequently, there is actually a delineation of seven rather than nine OTUs as stated previously, and the following conclusions stem from this. There is no signi®cant variation in SSCP pro®les within a morphospecies within a particular host species, but signi®cant variation occurs between morphospecies originating from different host species. The same morphospecies may occur in at least three or four tissue habitats within one host individual or within different individuals of one host species from the same or different geographical areas. Different morphospecies may co-occur in the same or different tissue sites or habitats in individual hosts. Genetic variants representing
Financial support was provided by the Australian Biological Resources Study (DMS), the Department of Industry, Science and Technology (IS&T) and the Australian Research Council (RBG). References [1] Moravec F. Proposal of a new systematic arrangement of nematodes of the family Capillariidae. Folia Parasitol (Praha) 1982;29:119±32. [2] Skrjabin KI, Shikhobalova NP, Orlov IV. Trichocephalids and capillariids of animals and man and the diseases caused by them (English edition, Birron A, Greenberg D, editors. Jerusalem: Israel program for scienti®c translations, 1970). In: Skrjabin KI, editor. Essentials of nematology, Moscow, The Academy of Sciences of the USSR, vol. 6, 1957. p. 599. [3] Beck JW, Beverley-Burton M. The pathology of Trichuris, Capillaria and Trichinella infections. Helminthol Abstr 1968;37:1±26. [4] Chitwood MB, Velasquez C, Salazar NG. Capillaria philippinensis sp. n. (Nematoda: Trichinellida), from the intestine of man in the Philippines. J Parasitol 1968;54:368±71. [5] Cross JH, Banzon T, Murrell KD, Watten RH, Dizon JJ. A new epidemic diarrheal disease caused by the nematode Capillaria philippinensis. Ind Trop Health 1970;7:124±31. [6] Lopez-Neyra R. Los Capillariinae. Memorias de la Rea. Academia de Ciencias exactas, ®sicas y naturales de Madrid 1947;12:1±248. [7] Freitas TJF. EsboÃcËo de novo arranjo sistematico para os nematoÂdeos capilariõÂneos (Trichuroidea). Atas da Sociedade de Biologia do Rio de Janeiro 1959;3:4±6. [8] Anderson RC, Bain O. Keys to genera of the superfamilies Rhabditoidea, Dioctophymatoidea, Trichinelloidea and Muspiceoidea. In: Anderson RC, Chabaud AG, Willmott S, editors. CIH keys to the nematode parasites of vertebrates, No. 9. Farnham Royal: Commonwealth Agricultural Bureaux, 1982. p. 26. [9] Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 1989;5:874±9. [10] Spratt DM, Beveridge I, Walter EL. A catalogue of Australasian monotremes and marsupials and their recorded helminth parasites. Records of the South Australian Museum 1991;1:1±105 Monograph series. [11] Smales LR. A review of the helminth parasites of Australian rodents. Aust J Zool 1997;45:505±21. [12] Gasser RB, Monti JR, Qian B-Z, Polderman AM, Nansen P, Chilton NB. A mutation scanning approach for the identi®cation of hookworm species and analysis of population variation. Mol Biochem Parasitol 1998;92:303±12. [13] Bowles J, Blair D, McManus DP. Genetic variants within the genus Echinococcus identi®ed by mitochondrial sequencing. Mol Biochem Parasitol 1992;54:165±74. [14] Zhu XQ, Jacobs DE, Chilton NB, Sani RA, Cheng NABY, Gasser RB.
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Molecular characterization of a Toxocara variant from cats in Kuala Lumpur, Malaysia. Parasitology 1998;117:155±64. [15] Zhu XQ, Gasser RB. Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to ®ngerprint sequence variation in ribosomal DNA of ascaridoid nematodes. Electrophoresis 1998;19:1366±73. [16] Gasser RB, Chilton NB, Hoste H, Beveridge I. Rapid sequencing of rDNA from single worms and eggs of parasitic helminths. Nucleic Acids Res 1993;21:2525±6. [17] Chilton NB, Gasser RB, Beveridge I. Differences in a ribosomal DNA sequence of morphologically indistinguishable species within the
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Research note
Mitochondrial DNA polymorphism within and among species of Capillaria sensu lato from Australian marsupials and rodents q Xingquan Zhu a, David M. Spratt b, Ian Beveridge a, Peter Haycock b, Robin B. Gasser a,* a
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia b CSIRO Wildlife & Ecology, GPO Box 284, Canberra 2601, Australia Received 12 April 2000; received in revised form 9 June 2000; accepted 9 June 2000
Abstract The nucleotide variation in a mitochondrial DNA (mtDNA) fragment within and among species of Capillaria sensu lato from Australian marsupials and rodents was analyzed using a mutation scanning/sequencing approach. The fragment of the cytochrome c oxidase subunit I (COI) was ampli®ed by PCR from parasite DNA, and analysed by single-strand conformation polymorphism (SSCP) and sequencing. There was no signi®cant variation in SSCP pro®les within a morphospecies from a particular host species, but signi®cant variation existed among morphospecies originating from different host species. The same morphospecies was found to occur in 1±3 tissue habitats within one host individual or within different individuals of a particular species of host from the same or different geographical areas, and morphospecies appeared to be relatively host speci®c at the generic level. The results indicated that the species of Capillaria sensu lato examined, although highly variable in their host and tissue speci®city, may exhibit the greatest degree of speci®city at the level of host genus. q 2000 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. Keywords: Capillaria spp; Cytochrome c oxidase subunit I; Genetic variation; Mitochondrial DNA; Polymerase chain reaction-based single-strand conformation polymorphism
Parasitic nematodes of the genus Capillaria sensu lato (Enoplida) occur in a wide range of ®sh, reptiles, birds and mammals on all major continents [1]. Many of those occurring in domestic animals are of considerable economic importance [2,3], while others cause severe disease in humans [4,5]. The accurate identi®cation of these parasites has major implications for studying the life cycles, transmission patterns, host±parasite relationships and for the development of control strategies. Despite more than 300 described species of Capillaria from all classes of vertebrates throughout the world, their taxonomy remains controversial, with some authors recognizing a single genus and other authors recognizing from ®ve to 20 or more genera [1,2,6±8]. There are major limitations in identifying specimens of Capillaria to species level because of their small size and the limited range of characteristic morphological features available. This problem is compounded by pronounced variation in morphometrics, host speci®city and habitat q The nucleotide sequence data reported in this paper are available in the EMBL, GenBank e and DDJB databases under the accession numbers AJ288160±AJ288170. * Corresponding author. Tel.: 161-3-973-12000; fax: 161-3-973-12366. E-mail address: [email protected] (R.B. Gasser).
speci®city (site in host tissues) within one host individual and one host species, and between different host individuals and different host species. Particular problems arise when apparently the same species occurs in different, sometimes distantly related hosts or in different tissues within the same host species. Therefore, alternative approaches to morphological identi®cation are needed. The aim of this study was to investigate the genetic variation within and between morphologically identi®ed species of Capillaria sensu lato from different host species and from different tissue sites within a host species. Polymerase chain reaction-based single-strand conformation polymorphism (SSCP) [9], followed by sequencing, was used to characterize sequence variation in a portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Although many are not formally named, the diverse fauna of species of Capillaria sensu lato present in Australian mammals [10,11] presented an opportunity to investigate genetic variation within the genus under the restraints described above. Adult specimens representing Capillaria gastrica from rodents and four, yet unnamed, species of Capillaria from marsupials [10] were obtained from different hosts and geographical locations (Table 1). Samples representing a
0020-7519/00/$20.00 q 2000 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. PII: S 0020-751 9(00)00076-X
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Table 1 DNA samples representing species of Capillaria sensu lato used in this study Sample
Morphospecies
OUT
Host
Site
Locality in Australia a
AhRf b CgRf1 b CgRf2 CgRf3 CgRf4 CgRf5 CgRf6 CgRl1 b CgRl2 CgRl3 C1Dv1 b C1Dv2 C1Dv3 C1Dv4 C1Dh b C2Im1 b C2Im2 C2Im3 C2Im4 C2Im5 C2Im6 C2Im7 C2Dh1 C2Dh2 C2Dh3 b C3Io1 b C3Io2 C3Io3 C3Io4 C4Im1 C4Im2 b C4Io b
Anatrichosoma haycocki Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria gastrica Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 1 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 2 Capillaria sp. 3 Capillaria sp. 3 Capillaria sp. 3 Capillaria sp. 3 Capillaria sp. 4 Capillaria sp. 4 Capillaria sp. 4
1 1 1 1 1 1 2 2 2 3 3 3 3 4 5 5 5 5 5 5 5 6 6 6 7 7 7 7 8 8 9
Rattus fuscipes R. fuscipes R. fuscipes R. fuscipes R. fuscipes R. fuscipes R. fuscipes R. lutreolus Rattus lutreolus R. lutreolus Dasyurus viverrinus D. viverrinus D. viverrinus D. viverrinus D. hallucatus Isoodon macrourus I. macrourus I. macrourus I. macrourus I. macrourus I. macrourus I. macrourus Dasyurus hallucatus D. hallucatus D. hallucatus Isoodon obesulus I. obesulus I. obesulus I. obesulus I. macrourus I. macrourus I. obesulus
Paracloacal gland Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Tongue Tongue Tongue Tongue Tongue Lips Lips Lips Tongue Tongue Tongue Oesophagus Oesophagus Oesophagus Oesophagus Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine
NSW sNSW sNSW sNSW sNSW sNSW sNSW cNSW cNSW TAS TAS TAS TAS TAS NT cNSW cNSW cNSW cNSW cNSW cNSW cNSW NT NT NT TAS TAS TAS TAS cNSW cNSW sNSW
a b
TAS, Tasmania; VIC, Victoria; sNSW and cNSW, southern and central New South Wales, respectively; NT, Northern Territory. Samples sequenced.
particular morphospecies of Capillaria from a particular host species were treated as a separate operational taxonomic unit (OTU; Table 1). Anatrichosoma haycocki from rodents was used for comparative purposes. Trapped animals were anaesthetised with Zoletil (Virbac Australia Pty Ltd; 50 mg/kg injected i.m.) and killed by cervical dislocation. Post mortem examinations of the tissues and organs of all animals were conducted under a stereomicroscope. Specimens of Capillaria sensu lato were dissected from host epithelial tissues, washed several times in physiological saline, and separated into aliquots for morphological and molecular studies. Material for morphological study was ®xed in hot (10%), neutral buffered formalin and stored in small McCartney bottles. Individual nematodes were cleared in lactophenol and allocated to morphospecies based on a suite of morphological characteristics from male and female worms and the ornamentation of the egg shell. Specimens for molecular study were placed in individual Eppendorf tubes and frozen immediately in liquid nitrogen. In addition, small samples of liver, kidney and small intestinal content from each host animal were
placed in individual Eppendorf tubes and frozen immediately in liquid nitrogen. On return to the laboratory, all frozen specimens were transferred to a biofreezer at 2708C. Genomic DNA was isolated from individual nematodes by sodium dodecyl-sulphate/proteinase K treatment [12] and direct puri®cation over spin columns (Wizard e DNA CleanUp, Promega). A portion of the mitochondrial COI gene (,450 bp) was ampli®ed by PCR using primers JB3 (5 0 -TTTTTTGGGCATCCTGAGGTTTAT-3 0 ; forward) and JB4.5 (5 0 -TAAAGAAAGAACATAATGAAAATG-3 0 ; reverse) [13] in 10 mM Tris±HCl (pH 8.4), 50 mM KCl, 4 mM MgCl2, 250 mM of each dNTP, 100 pmol of each primer and 2 U Taq polymerase (Promega) in an automated thermocycler (Perkin±Elmer Cetus) under the following cycling conditions: 948C for 5 min (initial denaturation), then 30 cycles of 948C for 30 s (denaturation), 558C for 30 s (annealing), 728C for 30 s (extension), followed by a ®nal extension at 728C for 5 min. One microlitre of each amplicon was subjected to secondary ampli®cation using 35 pmol of [g 33P]-endlabelled primers, JB3 and JB4.5, using the same
X. Zhu et al. / International Journal for Parasitology 30 (2000) 933±938
conditions as for primary ampli®cation. Control samples without DNA and with rat DNA were included in each PCR run. Five microlitres of each PCR product were examined by agarose gel electrophoresis [14]. The SSCP method was carried out as described previously [15]. Samples displaying variable COI SSCP pro®les were subjected to automated DNA sequencing (ABI 373 DNA Sequencer, Monash University) using the same primers as for primary ampli®cation. Manual sequencing using the fmole cycle sequencing kit (Promega) was carried out according to a modi®cation of the original protocol as described previously [16] to con®rm some nucleotide positions. The sequences were aligned manually. Pairwise comparisons were made of the level of sequence differences (D) using the formula D 1 2 M=L [17], where M is the number of alignment positions at which the two OTUs have a base in common, and L is the total number of alignment positions over which the two OTUs are compared. A phenogram was constructed from these data using the unweighted pair group method using arithmetic averages (UPGMA) [18]. In a ®rst step, the COI fragment (,450 bp) was ampli®ed from individual worms and subjected to agarose gel electrophoresis. No variation in size was detected on agarose gels (not shown) among 32 samples representing the ®ve currently-recognised morphospecies of Capillaria sensu lato. We then subjected the COI amplicons to SSCP analysis to screen for sequence variation within and among OTUs. While slight variation in single-stranded pro®les was detected between/among some specimens representing an OTU (e.g. sample CgRf6 versus samples CgRf1±CgRf5; samples CgRl1 and CgRl2 versus sample CgRl3; Fig. 1), signi®cant differences in the COI pro®les among the ®ve currently-recognized morphospecies of Capillaria sensu lato from different host species were displayed, which allowed the delineation of the nine OTUs (cf. Table 1). In some cases, variation in pro®les was detected between samples of the same morphospecies from a particular host (e.g. samples CgRl1 and CgRl2 versus sample CgRl3; Fig. 1). While there was no signi®cant variation in SSCP pro®les in most OTUs for which multiple individual nematodes were available, variation was displayed between morphospecies originating from different host species. For example, the SSCP pro®les for nematodes from the gastric mucosa and identi®ed as C. gastrica Baylis, 1926 from Rattus fuscipes from Ludwigs Swamp in southeastern New South Wales (samples CgRf1±CgRf6; Fig. 1) were distinctly different to those originating from Rattus lutreolus from Glenreagh in the central coastal ranges of New South Wales (CgRl1 and CgRl2; Fig. 1), and to one representative from Tasmania (sample CgRl3; Fig. 1). Also, substantial variation in pro®les was displayed between Capillaria sp. 1 from the epithelium of the tongue of Dasyurus viverrinus from Huonvale, Tasmania (samples C1Dv1±C1Dv4; Fig. 1) and a representative from the tongue of Dasyurus hallucatus from Coomalie Creek, Northern Territory (sample C1Dh; Fig. 1). A lower level of sequence variation was displayed
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between Capillaria sp. 2 samples from the oesophageal mucosa of D. hallucatus (samples C2Dh1±C2Dh3; Fig. 1) from Coomalie Creek, Northern Territory and those from the lips, tongue and oesophagus of Isoodon macrourus (samples C2Im1±C2Im7; Fig. 1) from Glenreagh and
Fig. 1. SSCP analysis of COI PCR products representing adult individuals of Capillaria spp. from different host species and geographical locations using Anatrichosoma haycocki from Rattus fuscipes (sample AhRf) for comparative purposes. Lanes represent Capillaria gastrica from Rattus fuscipes (samples CgRf1±CgRf6) or Rattus lutreolus (samples CgRl1± CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (samples C1Dv1± C1Dv4) or Dasyurus hallucatus (sample C1Dh), Capillaria sp. 2 from Isoodon macrourus (samples C2Im1±C2Im7) or D. hallucatus (samples C2Dh1±C2Dh3), Capillaria sp. 3 from Isoodon obesulus (samples C3Io1±C3Io4), and Capillaria sp. 4 from I. macrourus (samples C4Im1 and C4Im2) or I. obesulus (sample C4Io). N and R represent No-DNA and rat DNA controls, respectively.
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Dorrigo, New South Wales, as well as between samples of Capillaria sp. 4 from the intestinal mucosa of I. macrourus from Dorrigo, New South Wales (samples C4Im1 and C4Im2; Fig. 1) and one representative from the intestinal mucosa of Isoodon obesulus from Sidling Swamp, southeastern New South Wales (C4Io; Fig. 1). However, Capillaria sp. 2 examined from the epithelium of the lips of I. macrourus from Glenreagh, New South Wales, the epithelium of the tongue of an animal from Dorrigo, New South Wales and the oesophageal mucosa of a second individual I. macrourus from that locality were genetically the same (samples C2Im1±C2Im7; Fig. 1), indicating that a single morphospecies of Capillaria sensu lato may occur in various epithelial tissues of different individuals within a single host species from different geographical locations. On the other hand, Capillaria sp. 2 occurring in the oesophageal epithelium of I. macrourus from Dorrigo, New South Wales, had similar SSCP pro®les to Capillaria sp. 4 occurring in the intestinal epithelium of that same individual (sample C2Im7 versus samples C4Im1 and C4Im2; Fig. 1). Having demonstrated that SSCP could detect genetic variation within and among species of Capillaria, we wanted to quantitate the levels of sequence difference in the COI. For this purpose, samples (CgRf1, CgRl1, CgRl3, C1Dv1, C1Dh, C2Im1, C2Dh3, C3Io1, C4Im2, C4Io and AhRf; Table 1) representing different SSCP pro®les were subjected to cycle sequencing. A 374 bp sequence of COI was obtained for all samples and aligned (available from the authors upon request). No polymorphism (more than one base at any one position) was detected in the COI sequence within any OTU. The G 1 C content of the COI sequences for all OTUs were similar, ranging from 33.2±34.8%. Pairwise comparison of the sequences between the OTUs revealed differences ranging from 0.5±32.6% (Table 2). For instance, sequence differences of 4.0±5.6% existed among C. gastrica samples from R. fuscipes and R. lutreolus (samples CgRf1, CgRl1 and CgRl3), whereas a nucleotide difference of 4.8% was detected between C.
gastrica samples from R. lutreolus from New South Wales (sample CgRl1) and Tasmania (sample CgRl3). A phenogram depicting the genetic relationships among the samples is shown in Fig. 2. With the exception of Capillaria sp. 4 from I. obesulus, all of the morphospecies of Capillaria clustered according to host genus. There was a 4±5.6% sequence difference between C. gastrica samples (CgRf1 versus CgRl1 and CgRl3) from different rat hosts, 8.6% between samples (C1Dv1 and C1Dh) of Capillaria sp. 1, and for both Capillaria sp. 2 and Capillaria sp. 4, there was a difference of ,30% between samples (C2Im1 versus C2Dh3, and C4Im2 versus C4Io, respectively) from different hosts (Table 2; Fig. 2). The variation within a morphospecies from different host species was frequently greater than differences between different morphospecies from the same host species (Table 2; Fig. 2). In addition to the morphological and speci®city constraints referred to previously, the controversy surrounding the taxonomy of the nematode genus Capillaria sensu lato and relationships among its many species stems in part from the fact that no major revision has involved a thorough study of type specimens or specimens from type hosts [19]. Many descriptions of species are inadequate. Consequently, a review of the literature has been an inappropriate basis for revision. We sought an alternative approach by examining genetic variation within and between morphologically identi®ed species of Capillaria sensu lato from different rodent and marsupial host species and from different tissue sites within a host species. A constraint in this study, which was not encountered in previous reports (e.g. [17,20]), was that it was not possible to identify the nematodes frozen for molecular work to morphospecies under a dissecting microscope, given their small size. Consequently, in each instance, an aliquot of nematodes was frozen and an aliquot from the same tissue site and host individual was ®xed. Determination of the morphospecies was based on the morphological features of nematodes in the ®xed aliquot. In two instances, we suspect that an error has
Table 2 Pairwise comparison of nucleotide differences (%) in the COI sequence among representatives of Capillaria gastrica from Rattus fuscipes (sample CgRf1) or Rattus lutreolus (samples CgRl1 and CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (sample C1Dv1) or Dasyurus hallucatus (sample C1Dh), Capillaria sp. 2 from Isoodon macrourus (sample C2Im1) or D. hallucatus (sample C2Dh3), Capillaria sp. 3 from Isoodon obesulus (sample C3Io1), and Capillaria sp. 4 from I. macrourus (sample C4Im2) or I. obesulus (sample C4Io), using Anatrichosoma haycocki from Rattus fuscipes (sample AhRf) for comparative purposes Sample
AhRf
CgRf1
CgRl1
CgRl3
C1Dv1
C1Dh
C2Im1
C2Dh3
C3Io1
C4Im2
C4Io
AhRf CgRf1 CgRl1 CgRl3 C1Dv1 C1Dh C2Im1 C2Dh3 C3Io1 C4Im2 C4Io
± 28.9 29.4 28.6 27.5 28.6 34.2 28.6 32.6 34.2 31.0
± 5.6 4.0 15.5 14.4 31.6 14.2 30.5 30.7 20.9
± 4.8 16.8 14.2 32.6 13.9 32.6 32.6 21.1
± 15.0 14.4 32.6 14.7 32.4 32.6 20.9
± 8.6 30.5 8.8 29.4 30.2 19.5
± 29.9 1.3 29.7 29.9 19.5
± 29.4 7.0 0.5 29.9
± 28.5 29.4 19.0
± 7.0 29.1
± 30.2
±
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different morphospecies may occur in the same host species from different geographical areas. Low-level variation in morphospecies may occur in closely-related host species. Morphospecies may be reasonably host speci®c at the generic level. These results imply that species of Capillaria sensu lato, although highly variable in their host and tissue speci®city, may nevertheless exhibit the greatest degree of speci®city at the level of host genus. Acknowledgements
Fig. 2. A phenogram depicting the genetic relationships among representatives of Capillaria gastrica from Rattus fuscipes (sample CgRf1) or Rattus lutreolus (samples CgRl1 and CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (sample C1Dv1) or Dasyurus hallucatus (sample C1Dh), Capillaria sp. 2 from Isoodon macrourus (sample C2Im1) or D. hallucatus (sample C2Dh3), Capillaria sp. 3 from Isoodon obesulus (sample C3Io1), and Capillaria sp. 4 from I. macrourus (sample C4Im2) or I. obesulus (sample C4Io) based on COI sequence data, using Anatrichosoma haycocki from R. fuscipes (sample AhRf) for comparative purposes.
occurred and these can be recognized in both Fig. 1 and Table 2. Individuals C2Im1 and C4Im2 could both represent Capillaria sp. 2 (OTU5) because they have almost identical SSCP pro®les and have a 0.5% nucleotide difference in the COI sequence. This is supported by the 7% nucleotide difference in the COI sequence for both C2Im1 versus C3Io1 and C3Io1 versus C4Im2, indicating minor variation between morphospecies from these two closely-related species of bandicoot hosts. A morphological study (D.M. Spratt, unpublished) has demonstrated that Capillaria sp. 2 and Capillaria sp. 4 frequently co-occur in I. macrourus. Similarly, C1Dh and C2Dh3, which both represented Capillaria sp. 2 (OTU 6), appeared very similar electrophoretically and exhibited only 1.3% nucleotide difference in the COI sequence. This is supported by the 8.6 and 8.8% nucleotide difference in the COI sequence for C1Dh versus C1Dv1 and C2Dh3 versus C1Dv1, respectively, again suggesting a minor variation between morphospecies from these two closely-related quoll hosts. Morphological study has shown that Capillaria sp. 1 and Capillaria sp. 2 frequently co-occur in D. hallucatus. Consequently, there is actually a delineation of seven rather than nine OTUs as stated previously, and the following conclusions stem from this. There is no signi®cant variation in SSCP pro®les within a morphospecies within a particular host species, but signi®cant variation occurs between morphospecies originating from different host species. The same morphospecies may occur in at least three or four tissue habitats within one host individual or within different individuals of one host species from the same or different geographical areas. Different morphospecies may co-occur in the same or different tissue sites or habitats in individual hosts. Genetic variants representing
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