Journal of Helminthology (1992) 66, 261-266

Comparison of restriction fragment length polymorphisms of ribosomal DNA between Diphyllobothrium nihonkaiense and D. latum T. MATSUURA, G. BYLUND* and K. SUGANE Department of Parasilology, Shinshu University School of Medicine, Asahi 3-1-1, Malsumoto City, Nagano Prefecture 390, Japan; *lnstitute of Parasitology, Abo Academy, Porthansgatan 3, SF-206000 Abo, Finland

ABSTRACT

Restriction fragment length polymorphisms (RFLPs) of ribosomal DNA (rDNA) were compared between Diphyllobothrium latum and D. nihonkaiense using seven kinds of restriction endonucleases. No intra-specific variation in restriction fragment profiles was shown within both species of Diphyllobothrium. Digestion of the genomic DNA with three endonucleases, Smal, Hinfl and Hhal, provided one or two different bands between two species, although the hybridization patterns generated with the others, Hindlll, Xbal, Styl and Haelll, were the same in both. RFLPs in the digested profiles with Smal, Hinfl and Hhal could be used as species-specific markers even if only fragments of strobilae with morphological similarity were available. Other cestodes, Spirometra erinacei and Taenia saginata, used as controls showed quite different restriction fragment patterns with all the enzymes used. KEY WORDS: Diphyllobothrium, RFLPs, rDNA, nucleotide diversity, Cestoda

INTRODUCTION

Cestodes of the genus Diphyllobothrium (Cobbold, 1958) are known to be widely distributed in circumpolar countries as causative agents of diphyllobothriasis. Three species of Diphyllobothrium; D. latum, D. dendriticum and D. ditremum have been well defined in northwestern Europe (ANDERSEN, 1977). There were some difficulties in clarifying the relationship between adults and plerocercoids of Diphyllobothrium. Plerocercoids derived from fishes have been identified only at the genus level in Canada (MARGOLIS & ARTHUR, 1979). The problems in identification of the species may be due to handling technique and morphological plasticity. Some researchers considered that the morphological characters of plerocercoids were sufficient to identify species in Europe and North America (ANDERSEN & GIBSON, 1989). The identification of the adult, however, is still difficult, and the same developmental stages of worms should be compared from the same experimental hosts due to morphological plasticity (DICK & POOLE, 1985). ANDERSEN et al. (1987) reported that the shape of scolex and the length of neck are useful characters for identification. If only fragments of a strobila are available, the identification is much more difficult. In Japan, where raw fishes are habitually eaten, diphyllobothriases are caused mainly by ingestion of plerocercoids in the cherry salmon, Oncorynchus masou. The infection is widely distributed in Japan, as fishes are delivered faster and are therefore fresher. The causative tapeworm had been identified as D. latum (Linne, 1785). YAMANE et al. (1986) proposed a new species for the Japanese broad tapeworm, Diphyllobothrium nihonkaiense, and KAMO (1988) reported the morphological differences in egg pits and plerocercoids between D. latum and D. nihonkaiense. Additionally, isozyme profiles (FUKUMOTO et al., 1987) and antigenicity (FUKUMOTO et al., 1988) have distinguished D. nihonkaiense from D. latum.

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Techniques in molecular biology are also useful in clarifying inter- and intraspecific relationships (PAINDAVOINE et al., 1986; BEVERLY et al., 1987; BARKER, 1989). Genomic DNA analysis has an advantage over biochemical analysis of gene products in identifying species according to genotypic variations. DEVOS & DICK (1989) demonstrated specific differences between the species of D. latum and D. dendriticum according to isozyme profile and rDNA fragment patterns. In cestodes such as Echinococcus granulosus, RFLP of rDNA has been extensively analysed in isolates from different geographical and host origins, and some distinct strains have been identified (CUESTA-BANDERA, et al., 1988; MCMANUS & RISHI, 1989). Southern blot analysis was carried out using an rDNA probe to compare RFLPs between and within D. latum and D. nihonkaiense, which have different geographical origins. The morphological differences between the fragments of strobilae in the two species are difficult to identify. MATERIALS AND METHODS

Collection of worms Mature proglottids of D. nihonkaiense and Taenia saginata were obtained from infected patients after treatment with bithionol (4,6-dichlorophenol). D. nihonkaiense was also provided by Dr. M. Yamada and Dr. Y. Matsumoto, Kyoto Prefectural University of Medicine, Kyoto, and Dr. K. Yoshimura, School of Medicine, Kanazawa University, Kanazawa. Six strobilae of D. nihonkaiense were used in this study. Strobilae of D. latum in Finland were recovered from golden hamsters after infection with plerocercoids from pike, Esox lucius, and burbot, Lota lota. A total of six frozen D. latum were transported from Finland to Japan. However, five tapeworms were frozen together. Therefore, they were used en bloc, as one frozen sample. Plerocercoids of Spirometra erinacei were collected from Elaphe quadrivirgata. All the tapeworms were washed several times in 0-9% saline, frozen immediately in liquid nitrogen and stored at -85°C until used for DNA or RNA extraction. Extraction of genomic DNA Each 0-5 g portion of frozen worm in 5 ml NET solution ((MM NaCl, 0-1M EDTA, 005 M Tris-HCl, pH 80) was disrupted in a Freezer/Mill pulverizer (Spex industries Inc., USA) for 3 min. The disrupted materials were thawed and homogenized. Sodium dodecyl sulphate and proteinase K were added to the homogenate at a final concentration of 0 1 % and 01 mg/ml, respectively. The suspension was incubated at 65°C overnight until it became transparent. CsCl was then dissolved in the suspension (0-9 mg/ml) with ethidium bromide at a concentration of 0-6 mg/ml. The mixture was centrifuged at 30 000 rpm for 30 min at 20°C in a Hitachi SRP 70AT rotor to remove insoluble materials. The supernatant was recentrifuged at 40 000 rpm for 16 h at 20°C in a Hitachi RPV 65T rotor. The DNA band visualized under UV light was recovered. Further procedures were as described by SAMBROOK et al. (1989). More than 400 ug of genomic DNA could be extracted from each tapeworm. 25S rDNA probe Total RNA was extracted from worms by the acid-guanidinium-thiocyanatephenol-chloroform method (CHOMCZYNSKI & SACCHI, 1987). 25S rRNA was recovered from total RNA by electroelution from agarose gel as described by

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MANIATIS et al. (1982). For synthesizing the radioactive 25S rDNA probe, 50 mM Tris-HCl, pH 8-3, 100 mM KC1, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM cold dATP, dGTP, and dTTP, 50 uCi of ct-32P-dCTP (3000 Ci mmol ', Amersham International pic, UK), 2 ng random oligonucleotide primer, and 5 units of reverse transcriptase (Takara Inc, Kyoto) were mixed in a volume of 25 ui and incubated at 42°C for 60 min. More than 60% of the 32P-dCTP was incorporated into cDNA with specific activity of over 109 dpm/ug.

Southern blot analysis Genomic DNA was digested with seven kinds of restriction endonucleases, Hindlll, Xbal, Smal, Styl, Haelll, Hinfl and Hhal. Digested DNA was electrophoresed on 0-8% or 1-5% agarose gel according to SOUTHERN (1975) except for the following modifications. Separated DNA fragments in agarose gel were blotted to a Hybond-N+ membrane (Amersham International pic, UK) under alkaline conditions. The membrane was hybridized with 25S rDNA probe and washed according to the Amersham's manual. The autoradiographs were prepared with RX films (Fuji Photo Film Co., Ltd.) with intensifying screens. The films were exposed at -85°C for several hours. Calculation of nucleotide diversity The value of nucleotide diversity (JI) was calculated by the method of NEI & Li (1979) and NEI & TAJIMA (1981) to estimate the proportion of DNA fragments shared among species in each number of recognized nucleotide pairs. RESULTS

The analysis of RFLPs was applied to distinguish Japanese from Finnish Diphyllobothrium. Four of seven restriction endonucleases, Hindlll, Xbal, Styl and Haelll, yielded the same patterns in two species (Figs 1 and 2, A and B). Two 1 2 3 4 5 6 7 8 9 10

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FIG. 1. RFLPs of Diphyllobothrium (lanes 1-8). Spirometra (lane 9) and Taenia (lane 10). Genomic DNA was digested with Hindlll (A). Xbal (B) and Smal(C), respectively and analysed as described in the Materials and Methods section. Lanes 1-3, Shinshu isolates (Japan); lanes 4, 5, Kyoto isolates (Japan); lane 6, Kanazawa isolate (Japan); lane 7, Finnish isolate; lane 8. mixture of five Finnish isolates; lane 9, Gifu isolate (Japan); lane 10, Shinshu isolate (Japan). Arrows indicate positions of markers (kilobases) (lambda DNA digested with Styl).

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FIG. 2. RFLPs of Diphyllobothrium (lanes 1—8), Spiromelra (lane 9) and Taenia (lane 10). Genomic DNA was digested with Styl(A), Haelll(B), Hinfl(C) and Hhal(D), respectively and analysed as described in the Materials and Methods section. Lanes 1-3, Shinshu isolates (Japan); lanes 4, 5, Kyoto isolates (Japan); lane 6, Kanazawa isolate (Japan); lane 7, Finnish isolate; lane 8, mixture of five Finnish isolates; lane 9, Gifu isolate (Japan); lane 10, Shinshu isolate (Japan). Arrows indicate positions of markers (base pairs) (pBR322 digested with Hinfl and Alul).

enzymes, Smal and Hinfl, detected one different band. Also two different bands were observed between two species in Hhal digestion. Extra bands in Japanese and Finnish Diphyllobothrium were 1-6 kb and 1-8 kb in Smal digestion (Fig. 1C) and 1-6 kb and 2 kb in Hinfl digestion (Fig. 2C), respectively. Also, extra bands with 1150 bp and 550 bp in Japanese species and 1800 bp and 450 bp in Finnish species were detected after digestion of genomic DNAs with Hhal (Fig. 2D). The value of nucleotide diversity (JT) between D. latum and D. nihonkaiense was 002. DISCUSSION Plerocercoids of Diphyllobothrium obtained from intermediate hosts and fixed in 4% formaldehyde in 1% NaCl solution are quite helpful in identifying the species. The widest part of D. nihonkaiense is at the middle of the body, whereas the widest part of D. latum is at the scolex and the body gradually tapers to the posterior end of the larva (YAMANE et al., 1986). However, there are some difficulties in morphological identification on the basis of adult strobilae, which show wide variations (ANDERSEN et al., 1987; KAMO, 1988). RFLP analysis of rDNA is a good tool for estimating the extent of intra- and inter-specific variation without consideration of host differences and developmental stages even if only fragments of strobilae, as materials from clinical cases, could be used. RFLP analysis was used for studying the relationship between D. nihonkaiense and D. latum. No intra-specific variation was detected and three of seven restriction enzymes revealed RFLPs of rDNA between the two species when rDNA was used as a probe. There are a few hundred copies of rDNA genes which are composed of tandemly repeated units in the metazoan genome. Each rDNA gene consists of the transcription unit corresponding to the small ribosomal RNA (rRNA), 5-8S rRNA and larger rRNA. The transcription unit is flanked by nontranscribed spacer DNA. All the repeated copies of rDNA are concertedly evolved (GERBI, 1986; LONG & DAWID, 1980). The hybridization signal could be detected more strongly in rDNA

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than any other unique gene. Less than 0-1 u,g of genomic DNA was necessary to obtain good RFLP profiles of rDNA after several hours of exposure in autoradiography and provides the possibility of analysing a single plerocercoid. It may be useful to study genetic variations within populations and relationships between plerocercoids and adults. Recently, it has been shown that DNA analysis can be carried out on lyophilized or ethanol-fixed materials (MCMANUS & RISHI, 1989), which will facilitate the identification of many isolates. The nontranscribed spacer region (NTS) is known to evolve more rapidly than the transcribed region (GERBI, 1986) and the diversity of NTS arose at intraspecific level in Schistosoma, Spalax and Aedes (WALKER et al., 1986; SUZUKI et al., 1987; BLACK et al., 1989). Restriction maps of rDNA in four species of Fasciola were almost identical except near the ends of the coding regions. The NTS in fasciolids varied in length within and between species. Species-specific restriction sites were found and inter-specific variations detected as different lengths of the NTS region (BLAIR & MCMANUS, 1989). In Diphyllobothrium, RFLPs in the digested profiles with Smal, Hinfl and Hhal were caused by the different restriction sites. NTS length polymorphisms could not be detected. Length variation might be too small to detect or the rDNA probe derived from the transcribed region used in the experiment did not hybridize the DNA fragments from NTS. The result indicates inter-specific differences between the two species. Furthermore, if the spacer DNA could be cloned and used as a probe, the probe might be a good tool for analysing polymorphisms within and between species because of its high copy number, distinct variation and rapid divergence (SUZUKI, et al., 1987). A measure called 'nucleotide diversity' has been proposed to express the degree of polymorphism in a population at the nucleotide level (NEI & Li, 1979; NEI & TAJIMA, 1981). Although the number of specimens was small, the inter-specific genetic diversity between D. latum and D. dendriticum calculated by us was JT=0-06 (DE VOS & DICK, 1989). From these results, the relationship between D. latum and D. nihonkaiense are considered to have close similarity at the species level. We have no information on nucleotide diversity in other helminths because most of the genetic distances in helminths have been calculated by polymorphisms in enzyme loci (THOMPSON & LYMBERY, 1990). Mitochondrial DNA has also been used to assess the systematics ard phylogeny of a variety of plants and animals. The recovery of mitochondrial DNA, however, is too small in quantity to analyse individual isolates because 1 ng of mitochondrial DNA was yielded by 50 to 300 g of tapeworms (YAP et al., 1987). It is necessary to accumulate further data of RFLP analysis in nuclear DNA to compare nucleotide diversity among various helminths. ACKNOWLEDGEMENTS We thank Dr. T. Nakamura, Department of Parasitology, Kitasato University of Medicine for providing Spirometra erinacei-infected Elaphe quadrivirgata. We also thank Mrs. Y. Andho and M. Nalbandian for critical readings of the manuscript. REFERENCES ANDERSEN, K. (1977) A marine Diphyllobothrium plerocercoid (Cestoda, Pseudophyllidea) from blue whiting (Micromesistius poutassou). Zeitschrift fur Parasilenkunde, 52, 289-296. ANDERSEN, K., CHING, H. L. & VIK, R. (1987) A review of freshwater species of Diphyllobothrium with redescriptions and the distribution of D. dendriticum (Nitzsch. 1984) and D. ditremum (Creplin, 1825) from North America. Canadian Journal of Zoology, 65, 2216-2228. ANDERSEN, K. I. & GIBSON, D. I. (1989) A key to three species of larval Diphyllobothrium Cobbold, 1858 (Cestoda: Pseudophyllidea) occurring in European and North American freshwater fishes. Systematic Parasitology, 13, 3-9.

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Accepted 13th May, 1992.