Synonymy of Proteocephalus pollanicola Gresson, 1952 (Cestoda: Proteocephalidae), a parasite of pollan, Coregonus autumnalis pollan, with. P. exiguus La ...
Systematic Parasitology 40: 35–41, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
35
Synonymy of Proteocephalus pollanicola Gresson, 1952 (Cestoda: Proteocephalidae), a parasite of pollan, Coregonus autumnalis pollan, with P. exiguus La Rue, 1911 Tom´aš Scholz1 , Vladim´ıra Hanzelov´a2 , Ivica Kr´alov´a2 & David Griffiths3 ˇ e Budˇejovice, of Parasitology, Academy of Sciences of the Czech Republic, Branišovsk´a 31, 370 05 Cesk´ Czech Republic 2 Parasitological Institute, Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice, Slovak Republic 3 University of Ulster, Freshwater Laboratory, Traad Point, Ballyronan, Co. Londonderry BT45 6LR, Northern Ireland, UK 1 Institute
Accepted for publication 29th September, 1997
Abstract The systematic status of the tapeworm Proteocephalus pollanicola Gresson, 1952, a parasite of pollan Coregonus autumnalis pollan Thompson, was evaluated on the basis of freshly collected material from the type-locality (Lough Neagh, Northern Ireland). Comparison of morphological, biometrical and DNA data (RAPD method) from P. pollanicola and from P. exiguus La Rue, 1911, a very common parasite of coregonid fish, did not find any substantial differences between them to confirm the validity of P. pollanicola. Accordingly, P. pollanicola is considered to be a synonym of P. exiguus, a widely distributed parasite in the Holarctic.
Introduction Morphological and genetic studies on fish tapeworms of the genus Proteocephalus Weinland, 1858 (Hanzelová & Špakulová, 1992; Šnábel et al., 1994, 1996; Hanzelová et al., 1995; Králová, 1996; Králová & Špakulová, 1996) demonstrated that P. exiguus La Rue, 1911 is a polymorphic species parasitising a wide variety of coregonid and salmonid fish in the Holarctic. Some taxa, namely P. fallax La Rue, 1911, P. neglectus La Rue, 1911, P. tumidocollis Wagner, 1953, and P. albulae Freze & Kazakov, 1969, have previously been synonymised with P. exiguus (see Hanzelová & Scholz, 1993; Scholz & Hanzelová, 1994; Hanzelová et al., 1995). P. pollanicola Gresson, 1952 was very briefly described on the basis of specimens found in the intestine of pollan Coregonus autumnalis pollan Thompson, 1838, from Lough Neagh, Northern Ireland (Gresson, 1952). The tapeworm was described in more detail by Gresson & Corbett (1954). The species has not been recorded from elsewhere subsequently (Chubb et al., 1987). Anikieva (1991) also studied the morphology
of P. pollanicola from Lough Neagh, in order to explain the taxonomic position of its host, C. autumnalis pollan. This author, however, did not question the validity of P. pollanicola. Because of the unusually restricted geographical distribution of P. pollanicola and its morphological similarity to P. exiguus, the validity of P. pollanicola was assessed on the basis of new material from the type-locality evaluated by morphological, biometrical and genetic (Random Amplified Polymorphic DNA – RAPD) methods.
Materials and methods Morphology and biometry The type-specimens of Proteocephalus pollanicola could not be located. Therefore, freshly collected material from the type-host and type-locality was used in this study. Specimens were collected from the pyloric caeca of 5 Coregonus autumnalis pollan from Lough Neagh (Toome Bay), Ireland, on May 1, 1996. The precise number of worms was not counted, but the
36 intensity of infection ranged from 0 to 15 specimens per host. For morphological studies, tapeworms were fixed with hot 4% formaldehyde solution, stained with iron hydrochloric carmine and mounted in Canada balsam. For comparison, the data on P. pollanicola and P. exiguus, previously published by Anikieva et al. (1983), Anikieva (1991), Scholz and Hanzelová (1994) and Hanzelová et al. (1995), and new data on P. exiguus from two other coregonids, Coregonus autumnalis (Lake Baikal, Russia) and C. lavaretus (Switzerland), were used. All measurements are in micrometres unless otherwise noted. DNA extraction Genomic DNA was isolated from individual P. pollanicola specimens collected on September 1, 1996 and fixed using 70% ethanol. For comparison, DNA from specimens of P. percae and different host and geographical P. exiguus isolates (see below) was also isolated as described by Králová & Špakulová (1996). Three samples of each host and geographical isolate were analysed. RAPD Total volume of reaction mixture was 25 µl, including 0.5 U of Taq DNA polymerase (Promega), 1 × Taq DNA polymerase buffer with 2.5 mM mgCl2 (Promega), 100 µm each of deoxynucleotide triphosphate (USB), 0.2 µm primer and 10 ng of genomic DNA. The following random primers were used: pQEF118 (50 -GAATTCATTAAAGAGGAGAAA0 0 3 ), p78 (5 -CTGTTACGGAGCTTAAGTCTCTC-30), p95 (50 -CACTGAGGCAAAAGATCCTGTTACG-30), GT11R (50 -TGACACCAGACCAACTCTGGTAATGG30 ). The reaction was performed in the Techno CycloGene thermal cycler programmed for 45 cycles; each cycle involved denaturation at 94 ◦ C for 1 minute, annealing at 46 ◦ C for 1 minute and primer extension at 72 ◦ C for 2 minutes. Electrophoresis was performed in 1.5% agarose gel with TBE buffer (89 mM TRIS-HCl, 2.5 mM EDTA, 89 mM boric acid, pH 8.3) for 2 h at 75 V. Ethidium bromide-stained gels were visualised on an ultraviolet transilluminator. Reference profiles of P. exiguus and P. percae from the following hosts and localities were used: 1. P. exiguus from Coregonus lavaretus (L.), South Åland, Baltic Sea, Finland; 2. P. exiguus from C. lavaretus, Lons-Le-Saunier, Jura, France; 3. P. exiguus (syn. P. albulae – see Hanzelová et al., 1995) from Coregonus albula (L.), Bothnian Bay, Finland; 4. P. exiguus (syn. P. neglectus – see Hanzelová et al., 1995) from
Oncorhynchus mykiss (Walbaum); 5. P. exiguus from Salmo trutta m. fario; 6. P. exiguus from Salvelinus fontinalis (Mitchill), 4–6 all from the Dobšiná reservoir, Slovakia; 7. P. percae from Perca fluviatilis L., Ružín reservoir, Slovakia.
Results Morphology The strobila of Proteocephalus pollanicola is long and narrow, with typically elongate mature and almost rectangular gravid proglottides (Figure 1A,B). The shape of the proglottides, however, varies greatly (Figure 1A,C). The scolex is rather small, with relatively large lateral suckers and a vestigial, but large, apical sucker (Figure 2). Mature proglottides are characterised by large testes, a thick-walled, relatively long cirrus-sac, the distal part of the vagina with a well-developed circular vaginal sphincter, and the seminal receptacle situated antero-dorsally to the ovarian isthmus (Figure 1). No significant differences were found when comparing the morphology of P. pollanicola with that of reference specimens of P. exiguus from different hosts. Biometry Measurements of P. pollanicola are summarised in Table I. It is evident that measurements of all characters of this species entirely fall within the range of intraspecific variability of P. exiguus. RAPD The RAPD fingerprints generated by 4 primers did not show any interspecific differences among the P. pollanicola and P. exiguus isolates tested. Amplified sequences ranged from 0.2 to 1.5 kb in length. Figure 3 shows amplification products with the p78 primer. With this primer, the 600-bp fragment of a good intensity was detected in all the P. pollanicola and P. exiguus isolates. This fragment was regarded as species-specific because it was detected in all samples of each host or geographical P. exiguus isolates and was absent in P. percae (see Figure 3, lane 8). Several variable (polymorphic) fragments were also amplified. The 380- and 430-bp bands were present simultaneously (Figure 3, lanes 3, 4, 8), or one of them was absent (lanes 1, 2, 5), or they were not amplified at all (lanes 6, 7). Similar results were obtained for the 800and 950-bp fragments, which were absent in P. percae.
37
Figure 1. Proteocephalus pollanicola Gresson, 1952. A,C, mature proglottides; B,D, gravid proglottides; E, terminal genitalia. Note thick-walled cirrus-sac (cs) and well-developed, circular vaginal sphincter (vs) in E, and the position of the seminal receptacle (RC), situated near ovarian isthmus in B.
38
Table I. Measurements of Proteocephalus pollanicola Gresson, 1952 and P. exiguus La Rue, 1911. Authority Character Width of scolex (WS) Diameter of sucker (DS) DS/WS Diameter of apical sucker (DAS) DAS/DS Width of neck Length of hermaphroditic proglottis (LHP) (mm) Width of hermaphroditic proglottis (WHP) (mm) LHP/WHP Length of gravid proglottis (LGP) (mm) Width of gravid proglottis (WGP) (mm) LGP/WGP Diameter of testis Number of testes Length of cirrus-sac (LCS) Width of cirrus-sac (WCS) LCS/WCS LCS/WHP Width of ovary (mm) Length of ovarian lobe Width of ovarian lobe Diameter of vaginal sphincter Number of uterine branches
P. pollanicola Gresson & Corbett (1954)
Anikieva (1991)
Present data
100–250
226–374 78–120
100–150
71–105
202–304 86–122 0.31–0.46 52–90 0.56–0.90 202–246 0.29–1.06 0.55–1.29 0.27–1.46 0.50–1.24 0.81–1.36 0.43–1.34 30–103 15–95 191–335 59–105 2.30–5.27 0.25–0.56 0.40–0.97 49–206 141–401 34–57 6–13
0.16–0.71 0.71–1.27 0.40
c. 1 80 60–75 (up to 90)
0.25
49–99 40–93 226–331 42–85 0.24–0.35
P. exiguus Anikieva (1991) 70–370 40–165 9–114
52–120 21–113 170–340
0.24–0.50
Hanzelov´a et al. (1995) 80–560 36–173 0.22–0.67 22–87 0.25–0.90 55–530 0.12–1.49 0.18–2.00 0.23–2.78 0.22–1.98 0.26–1.90 0.23–2.78 23–168 24–116 148–584 41–130 1.93–6.49 0.18–0.68 0.22–1.26 53–327 92–557 18–100 6–23
39
Figure 2. Proteocephalus pollanicola Gresson, 1952 – scolex.
The P. exiguus-specific fragments, common for all P. exiguus isolates and P. pollanicola, were detected in amplification products obtained with 3 other primers used as well. Depending on the primer, 1–3 speciesspecific fragments were detected in all P. pollanicola and P. exiguus isolates. On the basis of the striking morphological and biometrical similarity of the taxa, detection of P. exiguusspecific fragments in P. pollanicola, and the fact that both tapeworms parasitise identical fish hosts (Coregonus autumnalis - Rusinek, 1987), P. pollanicola was not found to be a valid species and it is considered a synonym of P. exiguus, which was described earlier (La Rue, 1911).
Discussion The original description of Proteocephalus pollanicola was incomplete, providing neither details of parasite morphology nor differential diagnosis. Gresson (1952) only stated vaguely “The writer has carefully compared the characters of those related forms recorded from Coregonus and from other freshwater fish. Such a comparison showed that the specimens present in C. pollan differ in several important respects from the known species of the genus Proteocephalus. It is concluded, therefore, that the proteocephalid parasitic in the alimentary canal of Lough Neagh pollan is a species new to science.” Since this description did not fill requirements of the International Code of Zoological Nomenclature (Article 13), the authority for P. pollanicola should really be Gresson & Corbett (1954), who provided a
more detailed account of the morphological characters of the same specimens originally mentioned by Gresson (1952). Despite comprehensive description of some internal organs, mainly those of the male and female reproductive systems, few metrical data (see Table I) and little in the way of a differential diagnosis of P. pollanicola were provided. Except for the position of the seminal receptacle, no significant differences were found between the data of Gresson & Corbett (1954) and the present characteristics of P. pollanicola. Gresson & Corbett (1954) described the seminal receptacle as distant from the ovarian isthmus. This study, however, demonstrated (Figure 1B) that the receptacle was situated very close to the ovarian isthmus; this is a typical feature of P. exiguus (unpublished data). In the key to Proteocephalus spp., Freze (1965) differentiated P. pollanicola from P. exiguus only by the relative size of the cirrus-sac, which was considered never to reach the mid-line of the proglottid in P. pollanicola (representing 1/3–1/5 of the proglottis width), whereas it can reach the mid-line of the proglottid in P. exiguus (representing 1/2–1/3 of the proglottis width). However, previous studies (Scholz & Hanzelová, 1994; Hanzelová et al., 1995) demonstrated that the ratio of the length of the cirrus-sac to the width of proglottides varies considerably, depending mainly on the shape of proglottides. The RAPD method has been demonstrated to be a reliable approach to taxonomic and population studies of proteocephalideans (Králová, 1996; Králová & Špakulová, 1996; Scholz et al., 1997). RAPD markers made it possible to characterize fragments common to various P. exiguus isolates (Králová & Špakulová, 1996), and to distinguish it from its congeners (Králová, 1996; Scholz et al., 1997). In the present study, fragments detected in all P. exiguus isolates occurred in P. pollanicola as well. These fragments were scored as P. exiguus-specific because of their presence in all host and geographical isolates of P. exiguus and absence in congeners (P. macrocephalus, P. percae, P. torulosus). The number of polymorphic fragments within P. exiguus isolates was quite high, which tallies with the high genetic variability detected among P. exiguus individuals by RAPD (Králová & Špakulová, 1996). Despite this fact, it was possible to detect P. exiguus-specific fragments with all primers used. In 1991, Anikieva provided data on the morphology of P. pollanicola specimens collected in the typelocality, Lough Neagh, in Northern Ireland. Despite
40
Figure 3. Amplification products of genomic DNA using p78 primer. Lane M – Marker – 100 bp DNA Ladder (Pharmacia Biotech); lane 1 – P. pollanicola from Coregonus autumnalis pollan, Northern Ireland; lane 2 – P. exiguus from C. lavaretus, Finland; lane 3 – P. exiguus from C. lavaretus, France; lane 4 – P. exiguus from C. albula, Finland; lane 5 – P. exiguus from Oncorhynchus mykiss, Slovakia; lane 6 – P. exiguus from Salmo trutta m. fario, Slovakia; lane 7 – P. exiguus from Salvelinus fontinalis, Slovakia; lane 8 – P. percae from Perca fluviatilis, Slovakia.
the close similarity of P. pollanicola to populations of P. exiguus from Coregonus albula, C. autumnalis and C. lavaretus, the author did not doubt the validity of the former taxon. Anikieva (1991) found P. pollanicola to be most similar to P. exiguus from C. lavaretus and considered this to support the notion that the pollan is only a form of C. lavaretus. However, there is still a question concerning the taxonomic status of pollan (C. autumnalis pollan). On morphological (Behnke, 1972), allozyme (Ferguson et al., 1978; Bodaly et al., 1991) and mitochondrial DNA (Bernatchez et al., 1991) evidence it is regarded as conspecific with C. autumnalis (Pallas). However, Bodaly et al. (1994) cite DNA hybridisation work by Kaukorante & Mednikov (1988) which indicates that the pollan is closer to C. lavaretus than to C. autumnalis. Whatever its precise taxonomic status it is apparent that the pollan is closely related to some Coregonus spp. which have been reported to harbour P. exiguus (see Schmidt, 1986). Freze (1965) listed as many as 13 species of coregonids, including C. lavaretus, as hosts of P. exiguus and Rusinek (1987) reported C. autumnalis as a host of P. exiguus. The present results do not provide any support for considering P. pollanicola a valid species. In addition
to its morphological and morphometric similarity to P. exiguus, the presence of analogous RAPD profiles for P. pollanicola and P. exiguus from different hosts in all primers used confirms their synonymy.
Acknowledgements The authors are very grateful to Assoc. Prof. Štefan Vilˇcek, University of Veterinary Medicine, Košice, Slovakia, for enabling us to perform DNA experiments in his laboratory; to Drs Edoardo Pozio and Giuseppe La Rosa, Istituto Superiore di Sanita, Roma, Italy, for providing primers; to Drs Alain de Chambrier, Museum d’Histoire Naturelle, Geneva, Switzerland; Hans-Peter Fagerholm, Åbo University, Abo, Finland; Daniel Gerdeaux, INRA, Thonon, France; Marc Morand, Laboratoire Vétérinaire Départemental, Lons-Le-Saunier, France; and E. Tellervo Valtonen, University of Jyväskylä, Jyväskylä, Finland, for providing P. exiguus specimens. Dr Josef Matˇena, Institute of Hydrobiology, Academy of Sciences of the ˇ Czech Republic, Ceské Budˇejovice, is acknowledged for providing information about Coregonus autum-
41 nalis pollan, and Mrs Martina Borovková, Institute of ˇ Parasitology, Ceské Budˇejovice, for technical help. Financial support was given by the Grant Agency of the Czech Republic (project No. 508/95/0294) and the Grant Agency of the Slovak Republic VEGA (No. 2/1364/97).
References Anikieva, L.V. (1991) The use of morphological indices of Proteocephalus pollanicola (Cestoda: Proteocephalidea) for a more precise definition of the origin of its host, Coregonus pollan Thompson. Parazitologiya, 25, 228–233. (In Russian). Anikieva, L.V., Malakhova, R.P. & Ieshko, E.P. (1983) Ecological analysis of parasites of coregonid fish. Leningrad: Nauka, 168 pp. (In Russian). Behnke, R.J. (1972) The systematics of salmonid fishes of recently glaciated lakes. Journal of the Fisheries Research Board of Canada, 29, 639–671. Bernatchez, L., Colombani, F. & Dodson, J.J. (1991) Phylogenetic relationships among the subfamily Coregoninae as revealed by mitochondrial DNA restriction analysis. Journal of Fish Biology, 39 (Supplement A), 283–290. Bodaly, R.A., Vuorinen, J., Ward, R.D., Luczynski, M. & Reist, J.D. (1991) Genetic comparisons of New and Old World coregonid fishes. Journal of Fish Biology, 38, 3752. Bodaly, R.A., Vuorinen, D.A., Reshetnikov, Y.S. & Reist, J.D. (1994) Genetic relationships of five species of coregonid fishes from Siberia. Journal of Ichthyology, 34, 117–130. Chubb, J.C., Pool, D.W. & Veltkamp C.J. (1987) A key to the species of cestodes (tapeworms) parasitic in British and Irish freshwater fishes. Journal of Fish Biology, 31, 517–543. Ferguson, A., Himberg, K.J. & Svardson, G. (1978) The systematics of the Irish pollan (Coregonus pollan Thompson): an electrophoretic comparison with other holarctic Coregoninae. Journal of Fish Biology, 12, 221–233. Freze, V.I. (1965) [Proteocephalids – tapeworm helminths of fish, amphibians and reptiles.] Essentials of Cestodology, Vol. V. Moscow: Nauka, 540 pp. (In Russian). Gresson, R.A.R. (1952) A new species of Proteocephalus from Coregonus pollan Thompson. Irish Naturalists Journal, 10, 308–309. Gresson, R.A.R. & Corbett, M.P. (1954) A morphological study of
a fish tapeworm, Proteocephalus pollanicola. Parasitology, 44, 34–49. Hanzelová, V. & Scholz, T. (1993) Systematic status of Proteocephalus tumidocollis (Cestoda: Proteocephalidae), a parasite of salmonid fishes in North America. Helminthologia, 30, 157–161. Hanzelová, V., Scholz, T. & Fagerholm, H.-P. (1995) Synonymy of Proteocephalus neglectus La Rue, 1911 with P. exiguus La Rue, 1911, two fish cestodes from the Holarctic Region. Systematic Parasitology, 30, 173–185. Hanzelová, V. & Špakulová, M. (1992) Biometric variability of Proteocephalus neglectus (Cestoda: Proteocephalidae) in two different age groups in the rainbow trout from the Dobšiná dam (East Slovakia). Folia Parasitologica, 39, 307–316. La Rue, G.R. (1911) A revision of the cestode family Proteocephalidae. Zoologischer Anzeiger, 38, 473–482. Kaukorante, M. & Mednikov, B. M. (1988) Genetic divergence of whitefishes in Fennoscandia. In: Biologia sigovych ryb [Biology of coregonid fishes]. Moscow: Nauka, pp. 31–48. (In Russian). Králová, I. (1996) A total DNA characterization in Proteocephalus exiguus and P. percae (Cestoda: Proteocephalidae): random amplified polymorphic DNA and hybridization techniques. Parasitology Research, 82, 668–671. Králová, I. & Špakulová, M. (1996) Intraspecific variability of Proteocephalus exiguus La Rue, 1911 (Cestoda: Proteocephalidae) as studied by the random amplified polymorphic DNA method. Parasitology Research, 82, 542–545. Rusinek, O.T. (1987) On the cestodes of the genus Proteocephalus, parasites of fishes of Lake Baikal. Parazitologiya, 21, 127–133. (In Russian). Schmidt, G.D. (1986) Handbook of tapeworm identification. Boca Raton, Florida: CRC Press, 675 pp. Scholz, T. & Hanzelová, V. (1994) Taxonomic study of two Proteocephalus species (Cestoda: Proteocephalidae) parasitizing coregonid fishes: the synonymy of P. fallax La Rue, 1911 with P. exiguus La Rue, 1911. Systematic Parasitology, 26, 1–12. Scholz, T., Špakulová, M., Šnábel V., Králová, I., & Hanzelová, V. (1997) A multidisciplinary approach to the systematics of Proteocephalus macrocephalus (Cestoda: Proteocephalidae). Systematic Parasitology, 37, 1–12. Šnábel, V., Hanzelová, V. & Fagerholm, H.-P. (1994) Morphological and genetic comparison of two Proteocephalus species (Cestoda: Proteocephalidae). Parasitology Research, 80, 141–146. Šnábel, V., Hanzelová, V., Mattiucci, S., D’Amelio, S. & Paggi, L. (1996) Genetic polymorphism in Proteocephalus exiguus shown by enzyme electrophoresis. Journal of Helminthology, 70, 345– 349.
