Molecular and Biochemical Parasitology, 36 (1989) 201-208 201 Elsevier

MOLBIO (11195

Restriction enzyme mapping of ribosomal DNA can distinguish between fasciolid (liver fluke) species

D a v i d B l a i r a n d D o n a l d P. M c M a n u s "

Department of Pure and Applied Biology, Imperial College of Science and Technology, London, U.K.

(Received 15 December 1988: acccptcd 4 April !989)

Recognition sites for nine different restriction cndonucleascs were mapped on rDNA genes of fasciolid species. Southern blots of digested DNA from individual worms were probed sequentially with three different probes derived from rDNA of Schistosoma mansoni and known to span between them the entire rDNA repeat unit m that species. Eighteen recognition sites were mapped for Fasciola hepatica, and seventeen for Fasciola gigantica and Fascioloides magna. Each fasciolid species had no more than two unique recognition sites, the remainder being common to one or both of the other two species. No intraspecific variation in re- striction sites was noted in F. hepatica (individuals from 11 samples studied: hosts were sheep, cattle and laboratory animals: geo- graphical origins, Australia, New Zealand. Mexico, U.K., Hungary and Spain), or in F. gigantica (two samples: Indonesia and Malaysia). Only one sample of F. magna was available. One specimen ot Fasciola sp. from Japan (specific identity regarded in the literature as uncertain) yielded a restriction map identical to that of I:. gigantica.

Almost all recognition sites occurred in or near the putative rRNA coding regions. The non-transcribed spacer region had few or no cut sites despite the fact that this region is up to about one half of the entire repeat unit in length. Length heterogeneity was noted in the ram-transcribed spacer, even within individual worms.

Key words: Fa.wiola hepatica; Fasciola gigantica: kascioloides magna; Restriction map; rDNA; Schistosoma mansoni

Introduction

In recent years, advances made in DNA tech- nology have permitted exploration of the struc- ture of genes in a way never previously possible. Parasitologists have been quick to apply such techniques to distinguish between species and

Correspondence address: David Blair, Department of Zoo- logy, James Cook University, Townsville, Queensland 4811, Australi~.

"Present address: Queensland Institute of Medical Research, Bramston Terrace, Hcrston, Brisbane, Queensland 4[~)6, Australia.

Abbreviations: ETS, external transcribed spacer: ITS, inter- nal transcribed spacer; NTS, non-transcribed spacer; rDNA. ribosomal DNA (here referring to entire repeat unit of gene): rRNA coding region, length of rDNA coding for rRNA (and therefore excluding spacers); SDS, sodium dodecyl sulphate; SSC, solution of sodium chloride and tri-sodium citrate; TBE. Tris-borate-EDTA buffer.

strains of organisms (see, e.g., ref. 1). One approach has been to prepare restriction

endonuclease maps of specific genes. Ribosomal RNA genes (rDNA) have often been used be- cause: (a) they occur in multiple copies in tan- dem arrays on one or more chromosomes; (b) they consist of two or more highly conserved cod- ing regions separated by poorly conserved spacers; and (c) ribosomal DNA has proved relatively easy to clone (such clones can be studied directly, or used as probes as in the present study).

Previous studies on the schistosomes (see ref. 2 and references therein) demonstrated that the rDNA repeat unit in Schistosoma mansoni is about 10 kb in length. Digestion with BamHI yielded three fragments which spanned the entire repeat and were cloned in pBR322. These were used as probes to investigate othcr Schistosoma species [1,2]. Species-specific restriction sites were found and inter-specific variations noted in the lengths of the spacer regions, in one case (Schis-

0166-6851/89;$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

202

tosoma margrebowiei), the non-transcribed spacer (NTS) in DNA extracted from pooled worms ex- hibited intraspecific length variations [2].

Our aim was to investigate inter- and intra- specific variation in the rDNA repeat unit of cer- tain fasciolids, using as probes the three cloned fragments of S. mansoni rDNA mentioned above.

Members of the genus Fasciola have an almost cosmopolitan distribution. F. hepatica occurs as adults in many species of mammal. Although its presumed centre of origin is Eurasia [3], it has been introduced to the Americas and Australa- sia. F. gigantica is more tropical in distribution, occurring in sub-Saharan Africa and tropical and subtropical Asia. Other species have been de- scribed within Fasciola. While a few are undoubt- edly valid (e.g., Fasciola nyanzae Leiper, 1910 from the African hippopotamus), many are prob- ably synonyms of F. hepatica and F. gigantica [4,51.

We were able to complete restriction maps, us- ing nine enzymes, of rDNA from F. hepatica Lin- naeus, 1758 (11 samples), F. gigantica Cobbold, 1856 (2 samples), Fasciola sp. from Japan (1 sam- ple), F. magna (Bassi, 1875) (1 sample) and sup- plement existing maps of S. mansoni Sambon, 1907.

Materials and Methods

Fluke samples used in the study were as fol- lows: (a) F. hepatica, fresh frozen, cattle, Mex- ico, courtesy of Dr. A. Flisser. (b) F. hepatica, fresh frozen, strain maintained in laboratory rat, Belfast, U.K. courtesy of Dr. A. Trudgett. (c) F. hepatica, fresh frozen, cattle, Belfast, U.K., courtesy of Dr. A. Trudgett. (d) F. hepatica, pre- served in 70% ethanol, sheep, South Island, New Zealand, courtesy of Mr. A. Lunt. (e) F. hepa- tica, preserved in 70% ethanol, sheep: 'Hampton I strain', Central Tablelands, New South Wales, Australia. Strain maintained by Dr. J.C. Boray and known to be resistant to salicylanilides. (f) F. hepatica, preserved in 70% ethanol, sheep: "Compton strain' originating in Newbury, U.K.; selected once with salicylanilides and passaged through Lyrnnaea tomentosa in Australia by Dr. J.C. Boray. (g) F. hepatica, preserved in 70% ethanol, sheep: 'Compton strain', not selected

(susceptible to salicylanilides), passaged through Lymnaea tomentosa in Australia by Dr. J.C. Boray. (h) F. hepatica, preserved in 70% ethanol: 'North Coast Strain' (susceptible to salicylani- lides), Lismore, New South Wales, Australia, passaged through Lyrnnaea tomentosa and L. vir- idis by Dr. J.C. Boray. (i) F. hepatica, preserved in 70% ethanol, cattle: 'Grafton Strain" (suscep- tible to salicylanilides), New South Wales, Aus- tralia, passaged through L. tomentosa and L. vir- idis by Dr. J.C. Boray. (j) F. hepatica, preserved in 70% ethanol, cattle, Hungary, courtesy of Dr. O. Sey. (k) F. hepatica, preserved in 70% ethanol, cattle, Barcelona, Spain, courtesy of Dr. J. Ba- gufia. (1) Fasciola species, preserved in 70% ethanol, host unrecorded, Nagoya, Japan, cour- tesy of Dr. C. Suto and Dr. F. Kawamoto. (m) F. gigantica, preserved in 70% ethanol, cattle, East Malaysia, courtesy of Dr. A.R. Sheikh-Omar and Dr. A.S. Rehana. (n) F. gigantica, preserved in 70% ethanol, bovine host, Java, Indonesia, cour- tesy of Dr. A.J. Wilson and Mr. R. Payne. (o) Fascioloides magna, preserved in 70% ethanol, strain maintained in guinea pigs, Minnesota, U.S.A., courtesy of Dr. B. Stromberg. (p) Schis- tosoma mansoni, fresh frozen, strain originating in Puerto Rico and maintained routinely in the Department of Pure and Applied Biology, Im- perial College, courtesy of Mr. R. Webber.

Each fluke was processed individually to ob- tain its DNA. Frozen specimens were dropped into liquid nitrogen, then ground into a powder using a chilled mortar and pestle. Ethanol-pre- served specimens were shaken in about 50 ml of distilled water for a few minutes before being dropped into liquid nitrogen and ground. Follow- ing cell disruption and digestion with SDS and proteinase K, DNA was extracted using a stand- ard phenol/chloroform method and precipitated with ethanol.

Restriction endonucleascs used were pur- chased from Amersham International or Boehr- inger Corporation (London) and the manufactur- er's directions for reaction conditions followed. Nine enzymes (see legend to Fig. 1 for list) were selected partly to match those used by Walker et al. [2]. For each of the following samples of flukes, a total of 45 DNA aliquots was used in diges- tions: F. hepatica samples a - d; Fasciola sp. sam-

pie 1; F. gigantica samples m and n; F. magna sample o. This permitted aliquots to be digested singly with each enzyme, or with cvery possible combination of pairs of enzymes. In most cases, sufficient DNA was obtained from a single fluke for all 45 digests. In other cases, 2-3 flukes from the same host individual were uscd sequentially to provide sufficient DNA. In the case of F. magna, pooled DNA from eight immature worms was used.

For the remaining seven samples of F. hepatica (e-k), a reduced set of double digests was carried out (nine per sample) using the same nine en- zymes, to confirm whether or not the same re- striction map must occur as in the samples ana- lysed more completely. Samples of DNA from S. mansoni were also electrophoresed alongside these. A number of additional double digests of S. mansoni DNA were carried out to supplement the map published in [1]. Identical digests of DNA from F. hepatica sample k were clcctrophoresed alongside each schistosome digest to allow direct comparisons of fragment sizes.

Separation of fragments following digestion was done by electrophoresis in 0.8% agarosc gels in TBE buffer. Phage h DNA cut with HindlII was used to provide fragment size standards. After electrophoresis, fragments were transferred to Hybond nylon membrane (Amersham Interna- tional) by Southern blotting.

Each membrane was probed sequentially with the three rDNA probes, pSM889, pSM890 and pSM389, provided by Dr. A. Simpson. For the first few hybridisations, probes were labelled with 32p by nick translation, using a kit purchased from Bethesda Research laboratories. Subsequently, probes were labelled using Multiprime (Amer- sham International) and following the manufac- turer's instructions. DNA from phage h cut with Hindlll was added to each labelling reaction to an amount equivalent to 1-2% of the probe. This allowed the fragments of standard size to be de- tected by autoradiography. Conditions of prehy- bridisation, hybridisation and washing were as outlined in the Multiprime (Amersham Interna- tional) instructions.

Fragment sizes were determined from their gel migration distance using the computer program of Schaffcr and Sederoff [6].

203

Restriction maps were drawn up after visual in- spection of the data. Enzyme recognition sites were regarded as conserved between species or samples when (a) they mapped to the same po- sition in the gene and (b) the fragments they de- limited showed identical mobility on electropho- resis (preferably in the same gel).

Results

Restriction maps are presented (Fig. 1) for the three species of fasciolid studied and for S. man- soni. In the case of S. mansoni, we used the pre- viously published map [1,2], supplemented by our own data. Of the 22 enzyme recognition sites in S. mansoni and the 15 common to all fasciolids, 8 are conserved between schistosomes and the fasciolids. Of the conserved sites, the BamHl sites at each end of the schistosome sequence in pSM889 are the most useful for mapping (Fig. 2). The region of homology between pSM889 and the fasciolid gene can thus be indicated directly (Fig. 1). This also allows us to indicate the probable positions of the rRNA coding regions in fasciol- ids; their actual positions have not been deter- mined. Regions of presumed homology with pSM890 and pSM389 are also indicated. This ho- mology does not extend far into the NTS (see legend to Fig. 3).

Restriction maps for all samples of F. hepatica were virtually identical. Similarly, those for the two samples of F. gigantica were identical. The restriction map for the Japanese Fasciola species was identical with that for F. gigantica. Differ- ences between the species occur near the ends of the presumed coding regions. F. gigantica and the Japanese Fasciola sp. lack the HindlII and XhoI- cut sites near the 5' end of the small rRNA cod- ing region but have a PstI site near the 3' end of the large rRNA coding region. F. magna lacks the single Xhoi and Kpnl cut sites found in F. he- patica, but has an additional BamHl site and a HindllI site as indicated. Few cut sites occur within the greater part of the NTS of any species.

Even within a single worm, the NTS may not be constant in length. The schistosome-derived probes often hybridised to two or more fasciolid fragments of slightly differing size when these fragments comprised mostly NTS (e.g., frag-

204

A) Schistosoma mansoni ~.~::~ ~!~ l '"t2~9 oSM890

5' B 14 K [ I I

PsB' E ' H ' K Ps X Pv 'E H X B g * B ' P v H ' E ' X H S B H 1 I - - q l l F I-- IF- I I F I I I I - ' I lF" I I

pSM3B9

3' K Ps B ° I I I

B) Cut s i tes common to fasciolids examined , . , .

. . . . . . . . . I - 1 1 / C IF-- I --1[ I - - 1 1 I I jL~H H~,) [; s,,:8 ~!) I)SM890 . . . . . . . . . . . ~SM3,8 9

B ~ I

"':; ' ~:"~' I ";':".~:: '~""~' I : ~ I ' ~ g ~ ~ : '^ I .~'s , ~'~'~ I I

Cut sites unique to:

C) Fasciola hepatica H X (B °) K (B') H X (B')

. . . . . . . . . IF- I I / . . . . . . . . IF" I

D ) Fasciola gigan tica

K (BI') Ps (B ' ) (~ ' ) I I J

E) I-'asdoloides magna

t{ B (B') (B') H B (B'I) . . . . . . . . . 1 I I I . . . . . . . . I I

[ : ~:: I Fig. 1. Restriction maps of the rDNA of S. mansoni, F. hepatica, F. gigantica and F. magna. Approximately 1.3 repeat units are shown to illustrate the tandem arrangement of the genes. Cleavage sites of 9 enzymes, all with hexameric recognition sites, are shown. B, BamHI; Bg, BgIII" E, EcoRl; H, ttindIII; K, Kpnl; Ps, Pstl; Pv, Pvu lh S, Sail; X, Xhol. (A) S. rnansoni, based on [1] with positions of additional sites marked. The extents of the three fragments of the S. mansoni gene, cloned in plasmids as pSM389, pSM889 and pSM890 and used as probes in this study, are shown above the map. The three fragments are bounded by BarnHI cleavage sites. Likewise, the extents of the large and small rRNA coding regions are shown as boxes below the map. Cleavage sites conserved between S. rnansoni and the fasciolids are indicated by asterisks in (A) and (B). (B) Cleavage sites com- mon to the fasciolids examined. The region recognised by pSMS89 is bounded by Baml l l sites conserved between schistosomes and fasciolids (see also Fig. 2) and has a high degree of homology with the probe. This has allowed us to indicate the probable locations of the large and small rR NA coding regions. The remaining probes, pSM389 and pSM890, extend into the NTS, a region in which there is relatively little homology between schistosomes and fasciolids (see legend to Fig. 3). The variable length of the NTS + ETS (an ETS is presumed to occur at the 3' end of the NTS) is indicated by the dashed line on the map. (C - E) cleavage sites unique to each of the three indicated fasciolid species. All the cleavage sites shown in (B) also occur in (C - E). To help

align the maps, the BamHI sites conserved between S. mansoni and the fasciolids are indicated in parentheses.

205

A 8 C - - 1 2 1 2 1 2

~ ? ' ' > Q 411 Q < 9 . ~ . ~ ,,1t~.4.~ 41. ,: . -

• :.3;~ > 1 ,rod ~ <1.3i: II. ;..~,:

! qlD

i d >

i <.;.3.~ 4 . - . 2 [ :

4

41. " . ) .

Fig. 2. Comparisons of restriction patterns seen following di- gests of 1'. hepatica and S. mansoni DNA with BamHl and probed with sehistosome-derivcd probes. Same Southern blot probed with (A) pSM389: (B) pSM889; (C) pSM890. Lane 1, F. hepatica (sample k); lane 2. S. mansoni. Sizes of fragments indicated by open arrows. Positions of phage k/ltindlll size markers indicated by closed arrows. In each case, as ex- pected, the pSM-probc hybridises to a fragment the same size as itself in S. mansoni. Probe pSM889 also hybridiscs to a fragment its own size in F. hepatica: the BamHl site at each end of pSM889, and in the homologous fasciolid rcgion, is conserved between the species. There are no other BamHI sites in the rDNA of F. hepatica. As a consequence, pSM389 and pSM890 both hybridise to the same large fragment (about

9.87 kb) in this individual (scc also Fig. 1).

m e n t s cut by a d o u b l e d iges t o f B a m H I / H i n d I l l

f r o m F. hepatica; Fig. 3). B e c a u s e the l eng th o f

the N T S was k n o w n f r o m o t h e r da ta , it was c l ea r

that t h e s e f r a g m e n t s w e r e no t due to a d d i t i o n a l

cut s i tes wi th in t he space r , but to l eng th v a r i a t i o n

wi th in this r e g i o n in D N A f r o m a s ingle w o r m .

D i f f e r e n t d e g r e e s o f l e n g t h v a r i a t i o n in the N T S

w e r e also n o t e d b e t w e e n ind iv idua l w o r m s f r o m

the s a m e s a m p l e ( T a b l e l and Fig. 3).

Discussion

lntra-specif ic variability. A l t h o u g h no in t r a spe -

cific v a r i a t i o n was n o t e d in e n z y m e r e c o g n i t i o n

si tes, the s p a c e r r e g i o n in fasc io l ids is o f va r i ab l e

l eng th w i th in and b e t w e e n spec ies and wi th in in-

d iv idua l s ( T a b l e I). Suzuk i et al. [7] w e r e ab le to

c h a r a c t e r i s e s e v e r a l c h r o m o s o m a l spec ies o f t he

m o l e rat s u p e r s p e c i e s Spalax ehrenbergi on the

basis o f N T S l e n g t h p o l y m o r p h i s m s wi th in indi-

v idua l an ima l s . S imi l a r ly , it m a y p r o v e poss ib le

to c h a r a c t e r i s e s t ra ins o f fasc io l ids . O n e ind iv id -

|D .bDi 423.72

~ 9.*16

"11-6.67

"44.26

~2.25

~I" I. 96

" 4 0 . 5 9

Fig. 3. Demonstration of length variability of the non-tran- scribed spacer region in individual F. hepatica from the same sample. Each lane contains DNA from a different individual of sample (e) double-digested with Barn}II and ttindIII and probed with pSM89t). The seven lanes show DNA from in- dividuals 1-7 in Table I, in that order. The size of one frag- ment is indicated by an open arrow. Positions of phage ,k/HindIII size markers are indicated by closed arrows.

The small (0.91 kb) fragment seen in each lane maps to a position within the 3' half of the large rRNA coding region (see Fig. 1). The intense signal given by this fragment indi- cates a high degree of homology between the schistosomc-dc- rived probe and the corresponding region in F. hepatica. The fragment between the 3' HindIII site in the large rRNA cod- ing region and the HindIII site upstream of the next small rRNA coding region (see Fig. 1) consists of spacer. The probe pSM890 recognises several fragments of slightly different lengths but of approximately the expected size (6--8 kb). This suggests length heterogeneity in this region, even within an individual worm. Moreover, the signal is relatively faint, sug- gesting poor homology between the probe and the spacer re-

gion in F. hepatica.

ual F. hepatica f r o m ca t t le f r o m N o r t h e r n I r e l and

( s amp le c) had a s p a c e r c o n s i d e r a b l y s h o r t e r t han

tha t in o t h e r s a m p l e s ( T a b l e 1). F u r t h e r w o r k o n

fasc io l id space r s is h a m p e r e d at p r e s e n t by the

lack of known c leavage sites within the reg ion and

by lack o f a m o r e h o m o l o g o u s p r o b e . Schis to-

s o m e - d e r i v e d p r o b e s s h o w re l a t ive ly l i t t le ho-

206

TABLE I

Spacer lengths (3' end of large rRNA coding region - 5' cnd of next small rRNA coding region) for species studied, indicating variability within and between species and individuals

Species Samplc Individual No. Spaccr lcngths (kb) Species Sample Individual No. Spacer lengths (kb)

F. hepatica a 1 7.13 F. hepatica f 3 7.19 6.15

b 1 7.48 4 7.34

c 1 5.38 g 1 6.51

d 1 6.42 5.25 2 7.99

7.49 e 1 6.64 6.51

5.38 6.04

2 6.71 h 1 7.19

3 6.64 i 1 8.74 5.93 6.77

4 7.19 j 1 7.34 6.114

k 1 7.16 5 7.05

6.15 F. gigantica m 1 6.77 6.19

6 7.19 5.46

7 6.27 n 1 6.30

f 1 7.05 Fasciola sp. 1 1 7.23 6.04 (Japan) 6.61

6.02 2 7.19 4.66

6.15 F. magna o (pooled) 5.48

S. rnansoni p (pooled) 2.82

mology with the fasciol id spacer (Fig. 3). More de ta i l ed work is l ikely to reveal a ' l a d d e r ' of var- iants, each di f fer ing by a length r ep resen t ing a r e p e a t e d subuni t (see also refs. 1 and 2).

lnterspecific variability. By vir tue of thei r r D N A res t r ic t ion maps , we have been able to dist in- guish c lear ly b e t w e e n the th ree n a m e d species of fasciolid ut i l ised in this s tudy. W h e r e species can be ident i f ied by morpho log i ca l means , the l abour and expense of constructing restriction maps is not necessary . H o w e v e r , there are c i rcumstances where m o r p h o l o g y canno t be used or is unrel ia-

ble. For example , the cercariae of F. hepatica and F. gigantica cannot be d is t inguished on morpho - logical g rounds (Boray , pe rsona l communica t i on ) but D N A from snails infected with e i ther should be readi ly ident i f ied by res t r ic t ion f ragment com- par isons . The J apanese Fasciola species p rov ides another example. A range of morphological types of Fasciola occurs in Sou thcas t Asia ( Japan , Tai- wan, Phi l ippines) . A t ex t r emes of this morpho - logical range , ear ly au thors desc r ibed some as re- sembl ing F. hepatica on the one hand and F. gi- gantica on the o ther , with an i n t e rmed ia t e form also occurr ing [4]. K imura et al. [8] r epo r t ed dif-

ficulty in clearly distinguishing between the three forms. Huang et al. [9] considered that the three forms in Taiwan represented a single variable species, a conclusion also reached in Japan by Oshima et al. [10]. Watanabe [11,12] carried out an extensive biological and morphological study and concluded that two species occur in Japan: F. gigantica which is a rare and geographically lo- calised, and Fasciola sp. (possibly F. indica Varma, 1953) which is by far the more common.

Studies of Fasciola karyotypes have not clari- fied the situation in Japan. Moriyama et al. [13] and Sakaguchi [14] reported that the Japanese Fasciola sp. has 20 or 30 chromosomes, appar- ently corresponding to diploid and triploid sets. Some worms exhibited a mosaic of ploidy ('mixo- ploid'). Spermatogenesis was aberrant and sperm generally absent. Oogenesis was also aberrant with primary oocytes containing 20 or 30 chro- mosomes. Sakaguchi [14] concluded that the Jap- anese Fas'ciola sp. is parthenogenetic. Diploid and triploid forms differed in body size [14] and egg sizes [13]. It is not clear from the literature whether the various ploidy states correspond with the morphological types recognised by some au- thors.

The single representative of the Japanese Fas- ciola sp. included in this study belongs to F. gi- gantica according to its rDNA rcstriction map. It would be of considerable interest to prepare re- striction maps of a more extensive series of the variants discussed above.

Data from restriction maps of rDNA genes have been used to construct phylogenetic trees (see, e.g., ref. 15 for a study of the frog genus Rana). Restriction sites which are conserved among all species in a group being studied are of

21)7

no value in constructing a phylogeny: only vari- able sites are of use. The fasciolid species studied here have too few variable sites for a meaningful phylogcnetic reconstruction on the basis of the nine enzymes used. Even between the fasciolids and the distantly related S. mansoni, many of the restriction sites appear to be conserved.

Most of the restriction sites mapped here are in, or very close to, regions coding for rRNA. These regions are known to be highly conserved, and such a finding is no surprise. What is more sur- prising is the relative lack of sites within the NTS and the cxternal transcribed spacer (ETS), which together comprise about half of the total repeat unit length in many cascs. It was largely within this region in thcir frog species that Hillis and Davis [15] found the variable sites needed for their data analysis. 6-bp cutting enzymes should recog- nise a cut site approximatcly evcry 4 kb in ran- dom sequence DNA. Howcver, rDNA NTS re- gions are not random in sequence [16]. Rather, they tend to consist of numerous repeats of a smaller subunit. The likelihood of any given rec- ognition site occurring within a short subunit is not great.

Acknowledgements

We wish to thank the Wellcome Trust for their generous support during this project. Our cordial thanks also go to those persons, named earlier, who provided specimens of fasciolids. Dr. A.J.G. Simpson made available the cloned probes, pSM389, pSM889 and pSM890. Thanks are also due to the Nuffield Foundation, Miss P.J. Ingles, Miss L. Renton, Mr. G. Medley, Mr. A. Reid, Dr. D. Rollinson and Mr. S. Stammers.

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