____________________________________________________________________________http://www.paper.edu.cn 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 rodentsq Xingquan Zhua, David M. Sprattb, Ian Beveridgea, Peter Haycockb, Robin B. Gassera,* aDepartment of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia bCSIRO 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 conforma- tion polymorphism Parasitic nematodes of the genus Capillaria sensu lato speci®city (site in host tissues) within one host individual (Enoplida) occur in a wide range of ®sh, reptiles, birds and one host species, and between different host individuals and mammals on all major continents [1]. Many of those and different host species. Particular problems arise when occurring in domestic animals are of considerable economic apparently the same species occurs in different, sometimes importance [2,3], while others cause severe disease in distantly related hosts or in different tissues within the same humans [4,5]. The accurate identi®cation of these parasites host species. Therefore, alternative approaches to morpho- has major implications for studying the life cycles, transmis- logical identi®cation are needed. sion patterns, host±parasite relationships and for the devel- The aim of this study was to investigate the genetic varia- opment of control strategies. Despite more than 300 tion within and between morphologically identi®ed species described species of Capillaria from all classes of verte- of Capillaria sensu lato from different host species and from brates throughout the world, their taxonomy remains contro- different tissue sites within a host species. Polymerase chain versial, with some authors recognizing a single genus and reaction-based single-strand conformation polymorphism other authors recognizing from ®ve to 20 or more genera (SSCP) [9], followed by sequencing, was used to character- [1,2,6±8]. ize sequence variation in a portion of the mitochondrial There are major limitations in identifying specimens of cytochrome c oxidase subunit I (COI) gene. Although Capillaria to species level because of their small size and many are not formally named, the diverse fauna of species the limited range of characteristic morphological features of Capillaria sensu lato present in Australian mammals available. This problem is compounded by pronounced [10,11] presented an opportunity to investigate genetic variation in morphometrics, host speci®city and habitat variation within the genus under the restraints described above. q The nucleotide sequence data reported in this paper are available in the Adult specimens representing Capillaria gastrica from EMBL, GenBanke and DDJB databases under the accession numbers rodents and four, yet unnamed, species of Capillaria from AJ288160±AJ288170. marsupials [10] were obtained from different hosts and * Corresponding author. Tel.: 161-3-973-12000; fax: 161-3-973-12366. geographical locations (Table 1). Samples representing a E-mail address: [email protected] (R.B. Gasser). 0020-7519/00/$20.00 q 2000 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. PII: S0020-7519(00)00076-X 中国科技论文在线___________________________________________________________________________http://www.paper.edu.cn 934 X. Zhu et al. / International Journal for Parasitology 30 (2000) 933±938 Table 1 DNA samples representing species of Capillaria sensu lato used in this study Sample Morphospecies OUT Host Site Locality in Australiaa AhRfb Anatrichosoma haycocki Rattus fuscipes Paracloacal gland NSW CgRf1b Capillaria gastrica 1 R. fuscipes Stomach sNSW CgRf2 Capillaria gastrica 1 R. fuscipes Stomach sNSW CgRf3 Capillaria gastrica 1 R. fuscipes Stomach sNSW CgRf4 Capillaria gastrica 1 R. fuscipes Stomach sNSW CgRf5 Capillaria gastrica 1 R. fuscipes Stomach sNSW CgRf6 Capillaria gastrica 1 R. fuscipes Stomach sNSW CgRl1b Capillaria gastrica 2 R. lutreolus Stomach cNSW CgRl2 Capillaria gastrica 2 Rattus lutreolus Stomach cNSW CgRl3 Capillaria gastrica 2 R. lutreolus Stomach TAS C1Dv1b Capillaria sp. 1 3 Dasyurus viverrinus Tongue TAS C1Dv2 Capillaria sp. 1 3 D. viverrinus Tongue TAS C1Dv3 Capillaria sp. 1 3 D. viverrinus Tongue TAS C1Dv4 Capillaria sp. 1 3 D. viverrinus Tongue TAS C1Dhb Capillaria sp. 1 4 D. hallucatus Tongue NT C2Im1b Capillaria sp. 2 5 Isoodon macrourus Lips cNSW C2Im2 Capillaria sp. 2 5 I. macrourus Lips cNSW C2Im3 Capillaria sp. 2 5 I. macrourus Lips cNSW C2Im4 Capillaria sp. 2 5 I. macrourus Tongue cNSW C2Im5 Capillaria sp. 2 5 I. macrourus Tongue cNSW C2Im6 Capillaria sp. 2 5 I. macrourus Tongue cNSW C2Im7 Capillaria sp. 2 5 I. macrourus Oesophagus cNSW C2Dh1 Capillaria sp. 2 6 Dasyurus hallucatus Oesophagus NT C2Dh2 Capillaria sp. 2 6 D. hallucatus Oesophagus NT C2Dh3b Capillaria sp. 2 6 D. hallucatus Oesophagus NT C3Io1b Capillaria sp. 3 7 Isoodon obesulus Small intestine TAS C3Io2 Capillaria sp. 3 7 I. obesulus Small intestine TAS C3Io3 Capillaria sp. 3 7 I. obesulus Small intestine TAS C3Io4 Capillaria sp. 3 7 I. obesulus Small intestine TAS C4Im1 Capillaria sp. 4 8 I. macrourus Small intestine cNSW C4Im2b Capillaria sp. 4 8 I. macrourus Small intestine cNSW C4Iob Capillaria sp. 4 9 I. obesulus Small intestine sNSW a TAS, Tasmania; VIC, Victoria; sNSW and cNSW, southern and central New South Wales, respectively; NT, Northern Territory. b Samples sequenced. particular morphospecies of Capillaria from a particular placed in individual Eppendorf tubes and frozen immedi- host species were treated as a separate operational taxo- ately in liquid nitrogen. On return to the laboratory, all nomic unit (OTU; Table 1). Anatrichosoma haycocki from frozen specimens were transferred to a biofreezer at rodents was used for comparative purposes. 2708C. Genomic DNA was isolated from individual nema- Trapped animals were anaesthetised with Zoletil (Virbac todes by sodium dodecyl-sulphate/proteinase K treatment Australia Pty Ltd; 50 mg/kg injected i.m.) and killed by [12] and direct puri®cation over spin columns (Wizarde cervical dislocation. Post mortem examinations of the DNA CleanUp, Promega). tissues and organs of all animals were conducted under a A portion of the mitochondrial COI gene (,450 bp) was stereomicroscope. Specimens of Capillaria sensu lato were ampli®ed by PCR using primers JB3 (50-TTTTTTGGG- dissected from host epithelial tissues, washed several times CATCCTGAGGTTTAT-30; forward) and JB4.5 (50-TA- in physiological saline, and separated into aliquots for AAGAAAGAACATAATGAAAATG-30; reverse) [13] in morphological and molecular studies. Material for morpho- 10 mM Tris±HCl (pH 8.4), 50 mM KCl, 4 mM MgCl2, logical study was ®xed in hot (10%), neutral buffered forma- 250 mM of each dNTP, 100 pmol of each primer and 2 U lin and stored in small McCartney bottles. Individual Taq polymerase (Promega) in an automated thermocycler nematodes were cleared in lactophenol and allocated to (Perkin±Elmer Cetus) under the following cycling condi- morphospecies based on a suite of morphological character- tions: 948C for 5 min (initial denaturation), then 30 cycles istics from male and female worms and the ornamentation of 948C for 30 s (denaturation), 558C for 30 s (annealing), of the egg shell. Specimens for molecular study were placed 728C for 30 s (extension), followed by a ®nal extension at in individual Eppendorf tubes and frozen immediately in 728C for 5 min. One microlitre of each amplicon was liquid nitrogen. In addition, small samples of liver, kidney subjected to secondary ampli®cation using 35 pmol of and small intestinal content from each host animal were [g 33P]-endlabelled primers, JB3 and JB4.5, using the same 中国科技论文在线___________________________________________________________________________http://www.paper.edu.cn X. Zhu et al. / International Journal for Parasitology 30 (2000) 933±938 935 conditions as for primary ampli®cation. Control