A comparative study of the egg morphology in four species of Eubothrium (Cestoda: Pseudophyllidea) with comments on their early development

Abstract. Freshly released eggs from four species of the cestode Eubothrium ( Eubothrium crassum, Eubothrium fragile, Eubothrium rugosum , and Eubothrium salvelini ) were subjected to morphological and morphometric analysis. The eggs of the two freshwater species, E. rugosum and E. salvelini , were ovoid with a lobed embryophore whereas the eggs of the two marine species, E. crassum and E. fragile , were more circular with a smooth embryophore. However, the morphological differences between species were not readily evident to permit their clear distinction from one another. To discriminate species, a forward stepwise linear discriminant analysis, using six of the seven measured metric characters made on the eggs, was used, which gave 100% correct classification of two species, E. rugosum and E. salvelini , and a high proportion of correct classification for E. crassum (98%) and E. fragile (83%). Of the latter two species, one specimen of E . crassum and five specimens of E. fragile were misclassified between the respective groups. The principal characters used in the classification of the species were the width of the egg, the length of the mediolateral hooks, and the width of the oncosphere. To provide more information on the life cycle of each species, the eggs were used in a series of infection trials to identify appropriate intermediate hosts. Experimental infections with freshwater copepods were successful when exposed to the eggs of E . salvelini , partially successful when exposed to the eggs of marine E. crassum with 10% of the copepods becoming infected, but no infections were obtained when the eggs of E. fragile were used.     

Host specificity and geographical distribution of Eubothrium in European salmonid fish

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. trutta fario), and lake trout (S. trutta lacustris), 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.     

Morphometric analysis of four species of Eubothrium (Cestoda: Pseudophyllidea) parasites of salmonid fish: an interspecific and intraspecific comparison

Four species of the genus Eubothrium (E. crassum, E. fragile, E. rugosum and E. salvelini) were subjected to morphometric comparison. Discriminant analysis was conducted utilising 17 characters measured on the scolex and strobila of 101 specimens. Univariate statistics were first used to detect features that were useful for separating individual Eubothrium species and two different host populations of E. salvelini. Subsequent multivariate discriminant analysis, combining all the measured variables, made it possible to separate all four species. A comparison of the four taxa revealed that (1) E. fragile is the most distinct species, possessing a much smaller scolex than the other congeners, and its similarity with the other marine species E. crassum is not proven; (2) the two freshwater taxa, E. rugosum and E. salvelini are the most similar; (3) the characters most suitable for species differentiation are the length of the scolex, the width of the apical disc, the width of the neck and its area, the width of eggs and the number of testes; (4) the width of the apical disc was confirmed to be the most stable character at the intraspecific level (within E. salvelini host populations) and is therefore considered to be a trait of the highest discriminative power in the subset of four Eubothrium species.     

Studies on the biology of Eubothrium salvelini and E. crassum in resident and migratory Salvelinus alpinus and Salmo trutta and in S. salar in North Norway and the islands of Spitsbergen and Jan Mayen

Samples of Eubothrium salvelini and E. crassum were obtained from resident and migratory Salvelinus alpinus and Salmo trutta and from S. salar at sea from North Norway and the islands of Spitsbergen and Jan Mayen. Scolex dimensions proved an unsuitable character for identifying these two parasite species, and use of alternative characters confirmed that E. salvelini is specific to Salvelinus and E. crassum to Salmo. Distributions of both species are co-extensive with their hosts, but levels of infection in any locality relate to the abundance of zooplankton. E. salvelini exhibits seasonal maturation and incidence cycles in char. It is recognised as being a freshwater parasite, although some individuals can survive the period of marine migration of sea char. Returning migrant char may contain such individuals together with small plerocerciform parasites acquired almost immediately upon their re-entry to freshwater. No evidence for marine infections with E. salvelini was found. The presence of plerocerciform and gravid adult E. crassum in salmon caught at sea confirms the existence of a marine life cycle and a marine race in this species. In salmon moving from one medium to the other the marine and freshwater races replace each other, but in sea trout, which spend shorter periods at sea, complete replacement may not occur and at any one time fish may harbour individuals of both races. Recognition of the existence of two races clarifies much of the confusion surrounding the biology of this species.     

Eubothrium crassum in migratory trout, Salmo trutta L., in the sea

The incidence and intensity of infection of various ages and conditions of migratory trout by Eubothrium crassum are described. A marine source of the worm seems likely and explanations for the characteristics of the parasite burden are given on this assumption. Within weeks of first going to sea the hosts accumulate large numbers of the parasite. One year suffices for the life cycle of Eubothrium but growth continues for at least two years and possibly for longer. The parasite burden decreases in older fish because of the stress imposed on the parasite by successive migrations of the host to and from fresh water and because older fish tend to feed earlier in the year.     

Fatty acid makeup of the cestodes, Eubothrium crassum and Diphyllobothrium dendriticum]

The fat-acidic composition of E. crassum and D. dendriticum was investigated. Lipids of E. crassum differ in greater unsaturation as compared to these of D. dendriticum as well as in greater amount of acids of type omega 3, whereas acids of type omega 6 and stearic acid were found in extracts of E. crassum in less quantity than in D. dendriticum. This phenomenon is characteristic of organisms living under conditions of low temperatures.     

Metazoan parasite community structure of sea trout on the west coast of Ireland

A total of 129 sea trout smolts from 10 locations on the west coast of Ireland were examined for metazoan parasites. Of 11 species identified only four had a prevalence >20%. Two species Hysterothylacium aduncum and Hemiuris communis have a confirmed marine origin; Eubothrium crassum has been shown to exist in both the sea and fresh water. The remaining species were fresh water. Overall the parasite community was dominated by autogenic species with only Diphyllobothrium dendriticum having allogenic transmission. Infracommunity values for mean species richness, and Brillouins index of diversity were low, indicating the presence of an impoverished helminth community with little potential for interspecific interaction. The relationship between marine helminths and sea lice was also examined.     

Helminths in an intensively stocked population of lake trout, Salvelinus namaycush, from Lake Huron

Eighty stocked lake trout Salvelinus namaycush (Salmonidae), collected from 2 locations in Lake Huron in May 1995, were examined for parasites. The parasite fauna of this top predator in Lake Huron was characterized by only 6 helminth species. Echinorhynchus salmonis infected all lake trout with a mean intensity of 163.9. The intensity of this acanthocephalan species significantly increased with host length and weight. Eubothrium salvelini infected 78 lake trout with a maximum number of 81 scoleces counted. Diplostomum sp., Cyathocephalus truncatus, Capillaria salvelini, and Neoechinorhynchus sp. infrequently infected lake trout. The low parasite species richness in these lake trout is believed to be due to their large size at stocking and to the loss of historical enzootic host-parasite relationships that followed the absence of this fish species in Lake Huron for 26 yr.     

Fractionation of δ15N and δ13C for Atlantic salmon and its intestinal cestode Eubothrium crassum

Stable isotopes of nitrogen (δ¹⁵N) and carbon (δ¹³C) were measured for Atlantic salmon Salmo salar and their intestinal cestode, Eubothrium crassum , sharing the same diet. Atlantic salmon muscle tissues were enriched in ¹⁵N and depleted in ¹³C compared to their prey (sprat Sprattus sprattus sprattus ) and their intestinal cestode. There was no significant difference in δ¹⁵N or δ¹³C between E. crassum and the sprat. Differences in nutrient uptake and intestine physiology between Atlantic salmon and E. crassum are discussed, as well as how these may give rise to different fractionations of stable isotopes between a host and its parasites. Furthermore, Atlantic salmon contained a significantly higher lipid content than their prey, which may partly explain differences in δ¹³C values between the host and its cestode. In addition, cestodes inhabiting lipid-rich hosts were also lipid rich. Larger Atlantic salmon were enriched in ¹⁵N compared to smaller fish. Cestodes inhabiting large hosts were also enriched in ¹⁵N compared to parasites living in smaller hosts. The last two results were explained by larger fish possibly feeding from a higher trophic level, or from larger and older prey, that resulted in both a higher lipid content and an enrichment in ¹⁵N.     

The occurrence of Eubothrium crassum (Cestoda: Pseudophyllidea) in salmon Salmo salar and trout S. trutta of the River Exe

The occurrence, size and maturity changes of Eubothrium crassum (Bloch) have been studied in smolts and adult Salmo salar L. and S. trutta L. of the River Exe and other Devon rivers. Adult parasites are found only in salmon and sea trout migrating up river to spawn, and range in size from small and immature to large and mature, though rarely gravid, worms. It is concluded that they are acquired and only become fully mature in the sea and gravid worms are lost on or before arrival in the rivers. During the upstream migration of salmon there is a selective loss of larger and more mature parasites and the survivors do not increase in length or mature, due to the intestine becoming a progressively more unfavourable habitat as the period of fasting continues. As few salmon return to the sea, most parasites in fresh-water die before breeding. In sea trout, which feed in fresh-water, parasites continue to grow and probably mature on return to the sea. It is suggested that parasites found in adult Atlantic and Pacific salmon in rivers are rarely, if ever, the same worms that are carried to sea by smolts but are of marine origin and are residues of a marine life cycle, and that two biological races of E. crassum exist, a marine and a fresh-water one.     

Differentiation among three species of bovine Thelazia (Nematoda: Thelaziidae) by polymerase chain reaction-restriction fragment length polymorphism of the first internal transcribed spacer ITS-1 (rDNA).

Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini are nematodes transmitted by some species of Musca (Diptera: Muscidae) which cause ocular infestations in bovines. Differences in the rDNA of these species were determined by a PCR using different sets of relatively conserved oligonucleotide primers. PCR on the first internal transcribed spacer (ITS-1) revealed differences in size in Thelazia species (437 bp for T. gulosa, 370 bp for T. rhodesi and 506 bp for T. skrjabini) while the DNA control of Musca spp. was not amplified. The ITS-1 amplicons of the three species were sequenced and then analysed. The GC contents ranged from 26 to 36% and the level of differences in the nucleotide sequences of ITS-1 was lower between T. skrjabini and T. gulosa (39%) than the latter and T. rhodesi (49–56%). Restriction fragment length polymorphism (RFLP) of ITS-1 amplicons was also carried out and the restriction profiles compared. Clear genetic differences among the three Thelazia examined were demonstrated by using the enzymes HpaII, CpoI and SspI. This PCR–RFLP for the delineation of T. gulosa, T. rhodesi and T. skrjabini offers prospects as a molecular epidemiological tool to study parasite transmission patterns and prevalence.     

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.     

The metazoan parasites of freshwater fishes in the Corrib catchment area, Ireland

A survey of the parasites of 243 fish belonging to 13 species, from the Corrib catchment area, western Ireland, was undertaken. At least 32 species of metazoan parasites were shown to be present. Diplostomum gasterostei, Tylodelphys clavata, Eubothrium salvelini, Cucullanus truttae, Raphidascaris acus, Ergasilus gibbus and Salmincola gordoni appear to be first records for Ireland and Phyllodistomum conostomum has not previously been reported from the British Isles. New host records involving Lamptera planeri, Salvelinus alpinus, Abramis brama, Noemacheilus barbatulus, Anguilla anguilla and Pungitius pungitius for the British Isles are given.     

Molecular characterization of hard tick <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> from China by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of Haemaphysalis longicornis from China were amplified by polymerase chain reaction. The 45 representative amplicons were sequenced, and sequence variation in the ITS was examined. The ITS sequences of H. longicornis were 3644 bp in size, including the part of 18S rDNA, 28S rDNA sequences and the complete ITS-1, 5.8S rDNA and ITS-2 sequences. Sequence analysis revealed that the ITS-1, 5.8S rDNA and ITS-2 of this hard tick were 1582, 152, and 1610 bp in size, respectively. The intra-specific sequence variations of ITS-1 and ITS-2 within H. longicornis were 0–2 and 0–2.2%; however, the inter-specific sequence differences among members of the genus Haemaphysalis were significantly higher, being 35.1–55.2 and 37–52% for ITS-1 and ITS-2, respectively. The molecular approach employed in this study provides the foundation for further studies of the genetic variation of H. longicornis from different hosts and geographical origins in China.     

Systematics of the Genus Eubothrium Nybelin (Cestoda, Pseudophyllidea), with Partial Re-description of the Species

Material from as many species of the cestode genus Eubothrium as could be obtained (six out of nine existing species), from a range of hosts and from both marine and freshwater habitats, were examined both by light and scanning electron microscopy. The scolex shape as seen with SEM turned out to be a very valuable specific character. Particular attention was also paid to the possibility that intra-specific races, distinguishable on biological criteria, might in addition exhibit characteristic morphological differences.     

The first internal transcribed spacer (ITS-1) of Melipona species (Hymenoptera, Apidae, Meliponini): characterization and phylogenetic analysis

Summary. Internal transcribed spacer 1 (ITS-1) sequences of the nuclear rDNA of eight bee species of the genus Melipona were studied. Complete ITS-1 sequence and flanking regions from three Melipona species were PCR-amplified, cloned, sequenced, and their variability compared. These sequences show length variation (1391 to 1417 bp), several repeated elements of one, two, three, and four nucleotides, and a repeated tandem sequence of approximately 80 bp. The low variation level between M. quadrifasciata and M. mandacaia sequences supports the hypothesis that they diverged recently. PCR-amplification, cloning, and sequencing of a partial ITS-1 sequence (394 to 496 bp) of eight Melipona species and two outgroups were performed and the obtained sequences used for phylogenetic analysis. The single tree estimated from parsimony analysis recovered four well-defined clades and monophyly of the genus Melipona. The phylogenetic relationships derived from sequences of ITS-1 fragments corroborate the taxonomic classification of Melipona based on morphological characters.Received 17 July 2003; revised 10 May 2004; accepted 1 June 2004.     

A Comparative Study of Eubothrium Salvelini and E. crassum (Cestoda: Pseudophyllidea) Parasites of Arctic Charr and Brown Trout in Alpine Lakes

We compared the morphology, morphometry, and some aspects of the biology and ecology of Eubothrium crassum and E. salvelini, two pseudophyllidean tapeworms in brown trout, Salmo trutta m. lacustris, and Arctic charr, Salvelinus alpinus, in lakes of the western part of the Alps. The taxonomic importance of the apical disc for the discrimination of both taxa was confirmed. The apical disc of E. crassum is rectangular, possessing two deep grooves above the bothriae and 2–16 additional indentations. In E. salvelini, the apical disc is bilobed and bilaterally symmetric. We found new and significant interspecific differences in the length and width of the scolex, the diameter of the apical disc, and the width of the neck, with all measurements being larger in E. crassum. The two species differ in the number and size of the testes (fewer and larger in E. salvelini), the length of the cirrus sac (longer in E. crassum), and the size of vitelline follicles (larger in E. salvelini). Vitelline follicles of E. crassum are always cortical and sometimes enter into the outermost muscle fibres, whereas follicles of E. salvelini are situated largely medullary with few follicles entering between the innermost bundles of muscles. The eggs and the oncospheres of E. salvelini are larger. The embryonic hooks of E. crassum measure 14–18 m whilst those of E. salvelini are 18–26 m. For both taxa, the intraspecific variability in morphometric characters was fairly low. The prevalence and intensity of infection of E. crassum and E. salvelini in their respective fish hosts were very high (prevalence 90–94% mean intensity 36.3 and 6.6, maximums of 172 and 63 tapeworms per fish, respectively). E. salvelini eggs were spontaneously released throughout the year except for winter months; E. crassum laid eggs only in summer (June–August). Natural infection of copepods with Eubothrium procercoids was very low (prevalence 0.002%). The morphogenesis of Eubothrium procercoids was studied in an intermediate host, the copepod Cyclops prealpinus, that had been simultaneously infected with both species.     

Discrimination between <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis> and <Emphasis Type="Italic">H. qinghaiensis</Emphasis> based on the partial 16S rDNA and the second internal transcribed spacer (ITS-2)

In the present study, two hard tick species, Haemaphysalis longicornis and H. qinghaiensis from North-western China were characterized genetically by the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and partial 16S rDNA. Based on a fragment within the hypervariable region of 16S rDNA with the length of approximately 453 bp, the phylogenetic trees were constructed by Neighbor-Joining and Maximum-parsimony methods. The results indicated that the phylogenetic status of H. qinghaiensis was distant from that of H. longicornis and closer to H. flava. Furthermore, the ITS-2 rDNA was amplified by PCR and sequenced from individual ticks. The length of ITS-2 is 1,606 bp for H. longicornis and 1,162 bp for H. qinghaiensis. Although sequence variation between the immature stages of H. longicornis was 0.1–0.4%, nucleotide differences between the tested species ranged 2.1–23.2%, indicating that ITS-2 rDNA sequences are genetic markers for the differentiation of the two hard ticks in China. Hence, a PCR-linked restriction fragment length polymorphism (RFLP) approach was developed for their unequivocal differentiation based on ITS-2 rDNA, which provides the foundation for further studies on ticks in China and has implications for studying the population genetic structure of the ticks and for identification and differentiation of closely related ticks.     

Parasites of arctic charr Salvelinus alpinus (Linnaeus) and their use in separating sea-run and non-migrating charr

Metazoan parasites of arctic charr, Salvelinus alpinus , from the Nettilling Lake–Koukdjuak River system, Baffin Island, were studied. Non-migrating charr hosted 9 species of parasites while sea-run charr hosted 15 species of parasites. Diphyllobothrium spp., Eubothrium salvelini , and Proteocephalus longicollis were found to be good indicators of non-migrating charr and Brachyphallus crenatus, Bothrimonus sturionis and Prosorhynchus squamatus were good indicators of sea-run charr. Using these parasites as biological tags, the proportion of charr caught by the commercial fishery at the outlet of the Koukdjuak River were determined to be approximately 80% sea-run and 20% non-migrating charr. Step-wise discriminant statistical analysis on morphic characters of charr, designated as sea-run and non-migrating by their parasites, showed these charr to differ morphologically.