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International Journal for Parasitology 30 (2000) 933±938 www.elsevier.nl/locate/ijpara Research note Mitochondrial DNA polymorphism within and among of 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 . 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 of the genus Capillaria sensu lato speci®city (site in host tissues) within one host individual () 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 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 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 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 samples between Capillaria sp. 2 samples from the oesophageal without DNA and with rat DNA were included in each mucosa of D. hallucatus (samples C2Dh1±C2Dh3; Fig. 1) PCR run. Five microlitres of each PCR product were exam- from Coomalie Creek, Northern Territory and those from ined by agarose gel electrophoresis [14]. the lips, tongue and oesophagus of Isoodon macrourus The SSCP method was carried out as described previously (samples C2Im1±C2Im7; Fig. 1) from Glenreagh and [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 accord- ing 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 ˆ 1 2 M=L† [17], where M is the number of align- ment 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 arith- metic averages (UPGMA) [18]. In a ®rst step, the COI fragment (,450 bp) was ampli®ed from individual worms and subjected to agarose gel electro- phoresis. 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 morphos- pecies 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 Fig. 1. SSCP analysis of COI PCR products representing adult individuals Glenreagh in the central coastal ranges of New South of Capillaria spp. from different host species and geographical locations Wales (CgRl1 and CgRl2; Fig. 1), and to one representative using Anatrichosoma haycocki from Rattus fuscipes (sample AhRf) for comparative purposes. Lanes represent Capillaria gastrica from Rattus from Tasmania (sample CgRl3; Fig. 1). Also, substantial fuscipes (samples CgRf1±CgRf6) or Rattus lutreolus (samples CgRl1± variation in pro®les was displayed between Capillaria sp. CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (samples C1Dv1± 1 from the epithelium of the tongue of Dasyurus viverrinus C1Dv4) or Dasyurus hallucatus (sample C1Dh), Capillaria sp. 2 from from Huonvale, Tasmania (samples C1Dv1±C1Dv4; Fig. 1) Isoodon macrourus (samples C2Im1±C2Im7) or D. hallucatus (samples and a representative from the tongue of Dasyurus hallucatus C2Dh1±C2Dh3), Capillaria sp. 3 from Isoodon obesulus (samples C3Io1±C3Io4), and Capillaria sp. 4 from I. macrourus (samples C4Im1 from Coomalie Creek, Northern Territory (sample C1Dh; and C4Im2) or I. obesulus (sample C4Io). N and R represent No-DNA and Fig. 1). A lower level of sequence variation was displayed rat DNA controls, respectively. 中国科技论文在线______http://www.paper.edu.cn

936 X. Zhu et al. / International Journal for Parasitology 30 (2000) 933±938 Dorrigo, New South Wales, as well as between samples of gastrica samples from R. lutreolus from New South Capillaria sp. 4 from the intestinal mucosa of I. macrourus Wales (sample CgRl1) and Tasmania (sample CgRl3). from Dorrigo, New South Wales (samples C4Im1 and A phenogram depicting the genetic relationships among C4Im2; Fig. 1) and one representative from the intestinal the samples is shown in Fig. 2. With the exception of Capil- mucosa of Isoodon obesulus from Sidling Swamp, south- laria sp. 4 from I. obesulus, all of the morphospecies of eastern New South Wales (C4Io; Fig. 1). However, Capil- Capillaria clustered according to host genus. There was a laria sp. 2 examined from the epithelium of the lips of I. 4±5.6% sequence difference between C. gastrica samples macrourus from Glenreagh, New South Wales, the epithe- (CgRf1 versus CgRl1 and CgRl3) from different rat hosts, lium of the tongue of an animal from Dorrigo, New South 8.6% between samples (C1Dv1 and C1Dh) of Capillaria sp. Wales and the oesophageal mucosa of a second individual I. 1, and for both Capillaria sp. 2 and Capillaria sp. 4, there macrourus from that locality were genetically the same was a difference of ,30% between samples (C2Im1 versus (samples C2Im1±C2Im7; Fig. 1), indicating that a single C2Dh3, and C4Im2 versus C4Io, respectively) from differ- morphospecies of Capillaria sensu lato may occur in ent hosts (Table 2; Fig. 2). The variation within a morphos- various epithelial tissues of different individuals within a pecies from different host species was frequently greater single host species from different geographical locations. than differences between different morphospecies from the On the other hand, Capillaria sp. 2 occurring in the oeso- same host species (Table 2; Fig. 2). phageal epithelium of I. macrourus from Dorrigo, New In addition to the morphological and speci®city South Wales, had similar SSCP pro®les to Capillaria sp. 4 constraints referred to previously, the controversy surround- occurring in the intestinal epithelium of that same individual ing the taxonomy of the genus Capillaria sensu (sample C2Im7 versus samples C4Im1 and C4Im2; Fig. 1). lato and relationships among its many species stems in part Having demonstrated that SSCP could detect genetic from the fact that no major revision has involved a thorough variation within and among species of Capillaria,we study of type specimens or specimens from type hosts [19]. wanted to quantitate the levels of sequence difference in Many descriptions of species are inadequate. Consequently, the COI. For this purpose, samples (CgRf1, CgRl1, a review of the literature has been an inappropriate basis for CgRl3, C1Dv1, C1Dh, C2Im1, C2Dh3, C3Io1, C4Im2, revision. We sought an alternative approach by examining C4Io and AhRf; Table 1) representing different SSCP genetic variation within and between morphologically iden- pro®les were subjected to cycle sequencing. A 374 bp ti®ed species of Capillaria sensu lato from different rodent sequence of COI was obtained for all samples and aligned and marsupial host species and from different tissue sites (available from the authors upon request). No polymorph- within a host species. ism (more than one base at any one position) was detected in A constraint in this study, which was not encountered in the COI sequence within any OTU. The G 1 C content of previous reports (e.g. [17,20]), was that it was not possible to the COI sequences for all OTUs were similar, ranging from identify the nematodes frozen for molecular work to morphos- 33.2±34.8%. Pairwise comparison of the sequences between pecies under a dissecting microscope, given their small size. the OTUs revealed differences ranging from 0.5±32.6% Consequently, in each instance, an aliquot of nematodes was (Table 2). For instance, sequence differences of 4.0±5.6% frozen and an aliquot from the same tissue site and host indi- existed among C. gastrica samples from R. fuscipes and R. vidual was ®xed. Determination of the morphospecies was lutreolus (samples CgRf1, CgRl1 and CgRl3), whereas a based on the morphological features of nematodes in the nucleotide difference of 4.8% was detected between C. ®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 28.9 ± CgRl1 29.4 5.6 ± CgRl3 28.6 4.0 4.8 ± C1Dv1 27.5 15.5 16.8 15.0 ± C1Dh 28.6 14.4 14.2 14.4 8.6 ± C2Im1 34.2 31.6 32.6 32.6 30.5 29.9 ± C2Dh3 28.6 14.2 13.9 14.7 8.8 1.3 29.4 ± C3Io1 32.6 30.5 32.6 32.4 29.4 29.7 7.0 28.5 ± C4Im2 34.2 30.7 32.6 32.6 30.2 29.9 0.5 29.4 7.0 ± C4Io 31.0 20.9 21.1 20.9 19.5 19.5 29.9 19.0 29.1 30.2 ± 中国科技论文在线______http://www.paper.edu.cn

X. Zhu et al. / International Journal for Parasitology 30 (2000) 933±938 937 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

Financial support was provided by the Australian Biolo- gical Resources Study (DMS), the Department of Industry, Science and Technology (IS&T) and the Australian Fig. 2. A phenogram depicting the genetic relationships among representa- tives of Capillaria gastrica from Rattus fuscipes (sample CgRf1) or Rattus Research Council (RBG). lutreolus (samples CgRl1 and CgRl3), Capillaria sp. 1 from Dasyurus viverrinus (sample C1Dv1) or Dasyurus hallucatus (sample C1Dh), Capil- laria sp. 2 from Isoodon macrourus (sample C2Im1) or D. hallucatus References (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 [1] Moravec F. Proposal of a new systematic arrangement of nematodes haycocki from R. fuscipes (sample AhRf) for comparative purposes. of the family . Folia Parasitol (Praha) 1982;29:119±32. [2] Skrjabin KI, Shikhobalova NP, Orlov IV. Trichocephalids and capil- lariids of animals and man and the diseases caused by them (English occurred and these can be recognized in both Fig. 1 and Table edition, Birron A, Greenberg D, editors. Jerusalem: Israel program for scienti®c translations, 1970). In: Skrjabin KI, editor. Essentials of 2. Individuals C2Im1 and C4Im2 could both represent Capil- nematology, Moscow, The Academy of Sciences of the USSR, vol. laria sp. 2 (OTU5) because they have almost identical SSCP 6, 1957. p. 599. pro®les and have a 0.5% nucleotide difference in the COI [3] Beck JW, Beverley-Burton M. The pathology of Trichuris, Capillaria sequence. This is supported by the 7% nucleotide difference and Trichinella infections. Helminthol Abstr 1968;37:1±26. in the COI sequence for both C2Im1 versus C3Io1 and C3Io1 [4] Chitwood MB, Velasquez C, Salazar NG. Capillaria philippinensis sp. n. (Nematoda: Trichinellida), from the intestine of man in the versus C4Im2, indicating minor variation between morphos- Philippines. J Parasitol 1968;54:368±71. pecies from these two closely-related species of bandicoot [5] Cross JH, Banzon T, Murrell KD, Watten RH, Dizon JJ. A new hosts. A morphological study (D.M. Spratt, unpublished) has epidemic diarrheal disease caused by the nematode Capillaria philip- demonstrated that Capillaria sp. 2 and Capillaria sp. 4 pinensis. Ind Trop Health 1970;7:124±31. frequently co-occur in I. macrourus. Similarly, C1Dh and [6] Lopez-Neyra R. Los Capillariinae. Memorias de la Rea. Academia de Ciencias exactas, ®sicas y naturales de Madrid 1947;12:1±248. C2Dh3, which both represented Capillaria sp. 2 (OTU 6), [7] Freitas TJF. EsboÃcËo de novo arranjo sistematico para os nematoÂdeos appeared very similar electrophoretically and exhibited only capilariõÂneos (Trichuroidea). Atas da Sociedade de Biologia do Rio de 1.3% nucleotide difference in the COI sequence. This is Janeiro 1959;3:4±6. supported by the 8.6 and 8.8% nucleotide difference in the [8] Anderson RC, Bain O. Keys to genera of the superfamilies Rhabdi- COI sequence for C1Dh versus C1Dv1 and C2Dh3 versus toidea, Dioctophymatoidea, Trichinelloidea and Muspiceoidea. In: Anderson RC, Chabaud AG, Willmott S, editors. CIH keys to the C1Dv1, respectively, again suggesting a minor variation nematode parasites of vertebrates, No. 9. Farnham Royal: Common- between morphospecies from these two closely-related quoll wealth Agricultural Bureaux, 1982. p. 26. hosts. Morphological study has shown that Capillaria sp. 1 [9] Orita M, Suzuki Y, Sekiya T, Hayashi K. Rapid and sensitive detec- and Capillaria sp. 2 frequently co-occur in D. hallucatus. tion of point mutations and DNA polymorphisms using the polymer- Consequently, there is actually a delineation of seven ase chain reaction. Genomics 1989;5:874±9. [10] Spratt DM, Beveridge I, Walter EL. A catalogue of Australasian rather than nine OTUs as stated previously, and the follow- monotremes and marsupials and their recorded helminth parasites. ing conclusions stem from this. There is no signi®cant varia- Records of the South Australian Museum 1991;1:1±105 Monograph tion in SSCP pro®les within a morphospecies within a series. particular host species, but signi®cant variation occurs [11] Smales LR. A review of the helminth parasites of Australian rodents. between morphospecies originating from different host Aust J Zool 1997;45:505±21. [12] Gasser RB, Monti JR, Qian B-Z, Polderman AM, Nansen P, Chilton species. The same morphospecies may occur in at least NB. A mutation scanning approach for the identi®cation of hook- three or four tissue habitats within one host individual or worm species and analysis of population variation. Mol Biochem within different individuals of one host species from the Parasitol 1998;92:303±12. same or different geographical areas. Different morphospe- [13] Bowles J, Blair D, McManus DP. Genetic variants within the genus cies may co-occur in the same or different tissue sites or Echinococcus identi®ed by mitochondrial sequencing. Mol Biochem Parasitol 1992;54:165±74. habitats in individual hosts. Genetic variants representing [14] Zhu XQ, Jacobs DE, Chilton NB, Sani RA, Cheng NABY, Gasser RB. 中国科技论文在线______http://www.paper.edu.cn

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