Host specificity and geographical distribution of Eubothrium in European salmonid fish

Abstract The host specificity and distribution of Eubothrium crassum (Bloch, 1779) and Eubothrium salvelini (Schrank, 1790), morphologically fairly similar pseudophyllidean tapeworms parasitizing salmonid fish, were critically assessed on the basis of morphological and genetic evaluation of extensive material collected from different definitive hosts and geographical regions in Europe. Eubothrium crassum occurs in fish of the genera Salmo, i.e. salmon (S. salar – both freshwater and marine), sea trout (S. trutta trutta), brown trout (S. truttafario), and lake trout (S. truttalacustris), and also in Danubian salmon (Hucho hucho) and vendace (Coregonus albula). Eubothrium salvelini parasitizes Arctic char (Salvelinus alpinus) and brook trout (Salvelinus fontinalis) in Europe, and also whitefish (Coregonus wartmanni). Rainbow trout (Oncorhynchus mykiss), which is not a native European fish species, was found to be a suitable definitive host for both Eubothrium species, which may occur simultaneously in the same fish. Previous records of E. crassum in Arctic char and brook trout, and those of E. salvelini in fish of the genus Salmo were most probably misidentifications. Most studies of Eubothrium have involved salmonids from the northern part of Europe, with few records from southern and south-eastern Europe. This study also confirmed the reliability of the morphology of the apical disc for the discrimination of E. crassum and E. salvelini.


Introduction
Cestodes of the genus Eubothrium Nybelin, 1922 (Pseudophyllidea) represent a unique group of fish helminths because some species occur in the sea, whilst others are exclusively freshwater and one species, E. crassum (Bloch, 1779), lives in both environments (Kennedy, 1978a,b;Andersen & Kennedy, 1983). Despite these differences in biology and ecology, Eubothrium tapeworms may be difficult to identify due to the uniform morphology and close similarity of most species (Andersen & Kennedy, 1983). Problems in the identification of two of the most common species, E. salvelini (Schrank, 1790) and E. crassum, parasitizing salmonid fish in the Holarctic Region (Protasova, 1977;Kennedy, 1978a,b), have contributed to an unsatisfactory knowledge of the spectrum of definitive hosts, host specificity, and geographical distribution of these parasites.
Eubothrium crassum is a typical cestode of salmon (Salmo salar L.), sea and brown trout (Salmo trutta trutta L. and S. trutta fario L.) (Kennedy, 1978a,b;Andersen & Kennedy, 1983), but it has also been reported from a wide spectrum of other salmonid fish of the genera Brachymystax, Coregonus, Hucho, Oncorhynchus, Salmo, Salvelinus and Thymallus (see table 1 for references). In Europe, E. salvelini occurs most frequently in Arctic char (Salvelinus alpinus (L.)) (Kennedy, 1978a), but it has been found in other salmonids, including those serving as the definitive hosts of E. crassum (table 1).
In the present study, the host spectrum of E. crassum and E. salvelini in Europe was critically examined on the basis of an evaluation of extensive material collected from different hosts (both freshwater and marine, wild and cultured) and regions of Europe. Besides numerous recently collected specimens of Eubothrium, voucher specimens from museum collections were also examined, with an emphasis on cestodes from 'atypical' or less common fish hosts.

Materials and methods
Freshly collected cestodes from the following hosts and localities were studied: Specimens for morphological evaluation were fixed in hot 4% formaldehyde solution Hanzelová et al., 2002).
Species identification was based on the following characters: 1. The morphology of the apical disc (see Andersen, 1979;Andersen & Kennedy, 1983;Chubb et al., 1987;Hanzelová et al., 2002): only two dorsoventral grooves (incisions) in E. salvelini versus at least four grooves, including two on the lateral sides of the disc, in E. crassum ( fig. 1). Each scolex was observed unmounted and the number of gooves was counted under the light microscope in the en face view. Some scoleces were then prepared for scanning electron microscopy (SEM) using standard procedures (see  and observed with a Jeol JSM 6300. 2. The position of the vitelline follicles (Andersen & Kennedy, 1983): cortical in E. crassum and medullary in E. salvelini (see fig. 3 in Hanzelová et al., 2002). The position of the follicles was observed in 10 mm paraffin sections stained with Heidenhain's haematoxylin-eosin.
3. The presence of species-specific alleles found at Acp, Pgm and Gpi loci that code for the above-mentioned enzymes (Král'ová & Šnábel, 2000).
The identification of museum specimens was based on the morphology of the apical disc (see above) and strobilar characters described by Hanzelová et al. (2002). The possibility of simultaneous infections in individual fish hosts with both cestode species was tested in a sample of 91 Eubothrium tapeworms from ten rainbow trout collected in Loch Awe in June 2001. Each specimen was treated separately, the scolex and posterior part of the strobila were fixed for morphological evaluation (morphology of 77 scolices and topology of vitelline follicles in cross sections of 28 specimens were examined), and the middle part of the body was frozen for isoenzyme analysis (88 tapeworms).
Eubothrium in rainbow trout (Oncorhynchus mykiss) Both cestode species were found in rainbow trout from Scotland. In rainbow trout from Loch Awe, E. crassum and E. salvelini ( fig. 1I) occurred simultaneously, but E. crassum predominated. Mixed infections occurred in five fish, four hosts harboured exclusively E. crassum, and one rainbow trout was infected with E. salvelini. Rainbow trout from Loch Earn were infected with E. salvelini only ( fig. 1F), whereas rainbow trout from Loch Leven ( fig. 1E; both localities in Scotland) and Norway were infected with E. crassum. Thus, rainbow trout seems to be a suitable (but not specific) definitive host of E. crassum and E. salvelini.

Identification of E. crassum and E. salvelini
This study confirmed the suitability of the morphology of the apical disc observed en face (in unmounted specimens) for the differentiation of mature E. crassum and E. salvelini (see Andersen, 1979;Chubb et al., 1987). Species discrimination was confirmed independently by isoenzyme analyses and examination of sections of respective specimens. It was found, however, that immature worms of E. crassum with poorly developed incisions (grooves) on the apical disc may sometimes be difficult to identify.
Electrophoretical comparison of E. crassum and E. salvelini Using GPI, PGM and ACP enzyme systems, the two species were readily discernible at the genetic level. With GPI and PGM, alleles attributed to E. salvelini migrated more cathodically compared to E. crassum alleles. GPI gave either single-or triple-banded phenotypes in E. salvelini, while three isoenzymes of different mobility were detected in E. crassum (fig. 2). The PGM generated pattern was characterized by two isoenzymes in E. salvelini and a more complex pattern displaying three electromorphs in E. crassum. ACP produced three patterns (represented by heterozygous and alternative homozygous forms) in E. salvelini, unlike the single monomorphic band expressed by E. crassum isolates. The mobility values of major bands scored for each enzyme are listed in table 2.

Discussion
The results of the present study correspond with the observations of some previous authors (Vik, 1963;Kennedy, 1978b;Hanzelová et al., 1999Hanzelová et al., , 2002 in that E. crassum was found in fish of the genus Salmo (salmon, sea, brown, and lake trout), whereas E. salvelini occurred predominantly in Arctic char (Salvelinus alpinus), even in those localities where these fish lived sympatrically. Although extensive Eubothrium material from different regions of Europe was evaluated (table 1), no E. crassum were found in species of Salvelinus and, vice versa, E. salvelini never infected any species of Salmo. It can be concluded, therefore, that E. crassum and E. salvelini show a strict specificity to their salmonid fish hosts. In Europe, these fish are infected with only one species of Eubothrium, either E. crassum or E. salvelini.
To date, all but one sample of Eubothrium from rainbow trout in Europe were identified as E. crassum (see table 1). The present study has shown, however, that rainbow trout, which is not native to Europe (see Froese & Pauly, 2001), may harbour both Eubothrium species and become infected simultaneously. In mixed infections (Loch Awe), E. crassum was more abundant than E. salvelini. In Loch Earn, E. salvelini was the only Eubothrium species occurring in rainbow trout and its identification was confirmed by DNA-based methods (Král'ová-Hromadová et al., 2003).
The common occurrence of E. salvelini in rainbow trout in Scotland raises the question as to whether this species has been overlooked or misidentified during previous surveys of fish parasites in the UK (see Kennedy, 1974;Holland & Kennedy, 1997), or whether it has appeared only recently in British populations of rainbow trout. The latter have been stocked in Loch Earn and Loch Awe for the past 15 -20 years and it is possible that E. salvelini, previously occurring in resident populations of char, colonized rainbow trout for the first time. In Loch Leven, famous for its unique strain of brown trout, rainbow trout were first stocked as recently as 1994, which may explain the absence of E. salvelini from rainbow trout in this locality.
The susceptibility of rainbow trout to simultaneous infection with E. crassum and E. salvelini may reflect the phylogenetic relationships of this fish with other salmonids, given that members of the genus Oncorhynchus form an intermediate clade between those including species of Salmo and Salvelinus (Phillips & Oakley, 1997). Rainbow trout occurred originally in the Russian Far East and the north-western part of North America, where salmonids of the genus Oncorhynchus harbour both Eubothrium species (Andersen & Kennedy, 1983).
Results of examining museum deposited specimens have provided evidence that E. crassum and E. salvelini may occur in other salmonid genera, such as Hucho (E. crassum) and Coregonus (E. crassum and E. salvelini). A critical evaluation of these specimens has also shown numerous misidentifications of Eubothrium and confirmed the suggestions of Kennedy (1978a) and Andersen & Kennedy (1983) about the unreliability of previous records from 'atypical' hosts (see table 1).
Available data demonstrate that Eubothrium occur in the northern part of Europe, apparently reflecting the geographical distribution of salmonid fish. The absence of recent records of E. crassum and E. salvelini in some countries where native salmonids occur (e.g. Balkan Peninsula) may reflect the lack of parasitological surveys rather than the absence of these cestodes. Eubothrium crassum seems to be more widely distributed in Europe, which may be related to the wider spectrum of its definitive hosts. In some countries, such as the Czech Republic (see Moravec, 2001), E. crassum disappeared at the beginning of the 20th century after the Atlantic salmon stopped upstream migration as a consequence of the construction of barrages and weirs on rivers.
The usefulness of isoenzyme patterns as speciesspecific markers (see Král'ová & Šnábel, 2000) was demonstrated in a study of Eubothrium from a mixed infection of rainbow trout from Loch Awe ( fig. 2; table 2). The enzyme systems used appeared to be suitable for species discrimination and results obtained were congruent with those based on morphological characters, i.e. the morphology of the apical disc and the position of vitelline follicles in cross sections.