REVISION OF SANGUINICOLA PLEHN, 1905 WITH REDESCRIPTION OF SANGUINICOLA VOLGENSIS (RASÍN, 1929) MCINTOSH, 1934, DESCRIPTION OF A NEW SPECIES, PROPOSAL OF A NEW GENUS, AND PHYLOGENETIC ANALYSIS.

Sanguinicola Plehn, 1905 comprises 26 species that collectively infect fishes from 8 orders (Cypriniformes, Characiformes, Siluriformes, Esociformes, Salmoniformes, Labriformes, Centrarchiformes, and Perciformes). Its revision is warranted because several species assigned to the genus could represent new genera, nucleotide sequences are wanting, many species have incomplete descriptions, and types for most species are missing or of poor quality. Herein, we emend Sanguinicola based on morphology and the first nucleotide-based phylogenetic analysis that includes multiple sequences from morphologically identified adult specimens. We describe Sanguinicola plehnae Warren and Bullard n. sp. from the heart of northern pike, Esox lucius Linnaeus, 1758 from Russia; provide supplemental observations of Sanguinicola volgensis (Rasín, 1929) McIntosh, 1934 from the heart of sabrefish (type species), Pelecus cultratus (Linnaeus, 1758) Berg, 1949 from Russia; describe Sanguinicola cf. volgensis from the heart of ide, Leuciscus idus (Linnaeus, 1758) Berg, 1949 from Russia; and describe Pseudosanguinicola occidentalis (Van Cleave and Mueller, 1932) Warren and Bullard n. gen., n. comb. from the heart of walleye, Sander vitreus (Mitchill, 1818) Bailey, Latta, and Smith, 2004 from eastern North America. Sanguinicola plehnae differs from its congeners by having lateral tegumental spines that total 118-122, are small (3% of body width), and protrude 2-3 µm from the tegument (lacking associated conical protrusion) as well as by having a large testis (>40% of body length). Sanguinicola volgensis differs from its congeners by having posteriorly directed lateral tegumental spines encased in a tegumental conical protrusion as well as by having an ovoid egg. Specimens of S. cf. volgensis differ from those of S. volgensis by having a body that is 5-6× longer than wide (vs. 2-3× in S. volgensis) and <90 lateral tegumental spines (vs. >95). Pseudosanguinicola Warren and Bullard n. gen. differs from Sanguinicola by having densely transverse rows of lateral tegumental spines (vs. a single column of large spines). The phylogenetic analysis utilizing the large subunit ribosomal DNA (28S) failed to reject monophyly of Sanguinicola.

Morphological features of the testis of freshwater blood flukes of the genus Sanguinicola Plehn, 1905, with consideration of the testicular patterns in the Aporocotylidae.

This is the first detailed study assessing the morphological features of the testis and testicular sperm of members of the freshwater blood flukes Sanguinicola sp. from Leuciscus idus (Cyprinidae) and for S. volgensis from Pelecus cultratus (Cyprinidae), and the marine blood fluke Aporocotyle simplex from Hippoglossoides platessoides (Pleuronectidae). The present study reports a unique feature in the distribution of germinal cellular components in freshwater Sanguinicola sp., showing the presence of the individual spermatocytes or their clusters in the testicular lobes, and the gathering of spermatid rosettes and spermatozoa within middle testicular lumen, which extends along the entire length of the testis. In contrast, each testis of marine A. simplex contains the usual mixed distribution of germ cells at various stages of development. The first TEM data on spermatozoon character of studied species has shown, unusual for digenean sperm structure, the absence of cortical microtubules in sperm principal region. Moreover, a variation in axoneme patterns is revealed in the studied aporocotylids, belonging to the different aporocotylid lineages a 9 + 0 axonemal type observed for freshwater teleost-infective species, Sanguinicola sp. and S. volgensis, and a 9 + '1' axonemal type revealed in spermatozoa of marine teleost-infective species A. simplex. The results discussed with the published data on the digenean sperm structure and the testicular patterns in the Aporocotylidae likely represent additional characteristics supporting the divergent evolutionary lineages of freshwater and marine aporocotylids. We anticipate future morphological studies of the sperm structure in aporocotylids of three lineages for an understanding of their phylogenetic relationships.

Surviving in the fish gut: Comparative inhibitory capacities against the host proteinases in cestodes of the genus Proteocephalus.

Currently, little is known about inhibitory substances enabling tapeworms to settle in fish intestines thereby avoiding proteolysis. Contrary to previous studies with certain host-parasite pairs, this research compares the inhibitory capacities in three tapeworm species of the same genus Proteocephalus from four different fishes (P. torulosus from dace and zope, P. sagittus from stone loach and P. cernuae from ruffe). The tapeworm extracts studied significantly reduced the activity of commercial trypsin (although to a lesser degree than the synthetic inhibitor of serine proteinases PMSF), displaying clear inter-specific variation in worms' inhibitory ability. We also measured the proteolytic activity of the host intestinal mucosa exposed to tapeworm extracts which served as inhibitors. Based on per cent inhibition values, all tapeworm extracts significantly suppressed the mucosal proteolytic activity, with marked differences between certain host-parasite pairs. SDS-PAGE electrophoresis of the incubation media and extracts detected in each tapeworm species 20-36 protein bands with apparent molecular weights from 10-12 to 312.5 kDa, mostly below 50 kDa. The incubation medium and extract of each parasite shared one to six bands ranging from 12 to 35 kDa, depending on its species, with only four bands common for two or more species. The band profiles suggest that in various Proteocephalus species inhibitory capacities against host proteinases can be ensured by different proteins.

Phyllodistomum kupermani n. sp. from the European perch, Perca fluviatilis L. (Perciformes: Percidae), and redescription of Phyllodistomum macrocotyle (Lühe, 1909) with notes on the species diversity and host specificity in the European Phyllodistomum spp. (Trematoda: Gorgoderidae).

BACKGROUND: European species of the large genus Phyllodistomum Braun, 1899 had historically been erected based solely on morphological characters. Unfortunately, many of them are still poorly known and inadequately described. Molecular approaches are critical to delineate species which were impossible to differentiate based on morphology alone. METHODS: New samples of adult Phyllodistomum spp. were collected from the urinary bladder and/or ureters of European freshwater fishes and fixed to conduct a light and scanning electron microscopy study, and to obtain sequences of nuclear (ITS2 spacer and 28S rRNA gene), to be analysed in the context of a molecular phylogeny. RESULTS: Based on new findings, a new species of Phyllodistomum from the urinary bladder of the European perch, Perca fluviatilis, in Volga River basin, Russia, is described. Additionally, new data on the morphology and tegumental surface topography of P. macrocotyle (Lühe, 1909) Odhner, 1911 from ureters of the common rudd, Scardinius erythrophthalmus, is presented. The host range of P. folium, confirmed by DNA analysis, is extended to other cyprinid fish species. CONCLUSIONS: The present study has again shown that species of the genus Phyllodistomum are in dire need of revision based on both molecular analysis and detailed morphological redescriptions of the forms attributed to the genus. Morphologically, P. kupermani n. sp. most closely resembles P. pseudofolium, a highly host-specific parasite of Gymnocephalus cernuus (L.), but molecular phylogenetic analyses based on ITS2 and 28S rDNA sequences showed that these species are distantly related. Phyllodistomum kupermani n. sp. was found to be phylogenetically most closely related to the type-species of Phyllodistomum, P. folium. Phylogenetic analyses revealed that Phyllodistomum kupermani n. sp. and P. folium formed a clade with other freshwater species for which cystocercous cercariae develop in bivalves of the family Sphaeriidae. The micromorphology and tegumental surface topography of P. macrocotyle revealed in the present study provide a valuable taxonomic criterion for congeneric species differentiation.

Diversity and phylogenetic relationships of European species of Crepidostomum Braun, 1900 (Trematoda: Allocreadiidae) based on rDNA, with special reference to Crepidostomum oschmarini Zhokhov & Pugacheva, 1998.

BACKGROUND: Within the genus Crepidostomum Braun, 1900, identification of species and taxonomic decisions made only on the basis of adult morphology have resulted in great problems associated with evaluating actual diversity and validity of species. Life-cycle data, while equal in importance to adult characters, are scarce, controversial or incomplete for most Crepidostomum spp. In this study, rDNA sequences generated from adult and larval Crepidostomum spp. and some other allocreadiid species were analysed to reveal the diversity and phylogenetic relationships of the species and their host range. Detailed morphological description based on light microscopy, SEM tegumental surface topography and genetic data are provided for the poorly known trematode C. oschmarini Zhokhov & Pugacheva, 1998 found in the intestine of two teleost fish species, Barbatula barbatula (L.) and Cottus gobio L. RESULTS: We characterized 27 isolates of adult and larval parasites. Based on newly obtained 28S and ITS1-5.8S-ITS2 rDNA sequences, new intermediate and final hosts were ascertained, and life-cycles clarified for some allocreadiids. New knowledge on the diversity and phylogenetic relationships of European Crepidostomum spp. was gained. The validity of C. oschmarini was verified based on comparative sequence analysis. Ophthalmoxiphidiocercariae of C. oschmarini were recorded in sphaeriid bivalves Pisidium (Euglesa) casertanum (Poli). Additionally, morphological differences between gravid specimens of C. oschmarini and other related species were observed. CONCLUSIONS: Species of the Allocreadiidae parasitizing fishes in Europe are distributed among two monophyletic genera, Allocreadium and Bunodera, and two paraphyletic Crepidostomum clades. A complex of Crepidostomum metoecus (syn. C. nemachilus), C. oschmarini and Crepidostomum sp. 2 clustered in one clade, and a complex of C. farionis, Crepidostomum sp. 1 and, probably, C. wikgreni in the other. Molecular data indicated that C. oschmarini and Crepidostomum sp. 2 presumably have a wide geographical distribution in Europe. The new data provided evidence that Crepidostomum is a more diverse genus than can be judged from morphological data and host switching in this genus may occur independently of fish-host phylogeny.

Host specificity, molecular phylogeny and morphological differences of Phyllodistomum pseudofolium Nybelin, 1926 and Phyllodistomum angulatum Linstow, 1907 (Trematoda: Gorgoderidae) with notes on Eurasian ruffe as final host for Phyllodistomum spp.

BACKGROUND: Host-specificity patterns are not well-defined for trematodes of the genus Phyllodistomum Braun, 1899. The Eurasian ruffe, Gymnocephalus cernuus L., has been recorded as a definitive host for Phyllodistomum folium (Olfers, 1816), P. angulatum Linstow, 1907 and P. megalorchis Nybelin, 1926 and as the type-host for P. pseudofolium Nybelin (1926). A wide range of other host fishes have been recorded for these species as well. All present host records have been based on light microscopy and the life-cycles of P. pseudofolium, P. angulatum and P. megalorchis are unknown. The validity of P. pseudofolium and P. megalorchis require verification. In this study, rDNA sequences generated from adult Phyllodistomum spp., as well as from larval stages developing in Pisidium amnicum Müller, were analysed to establish the real number of Phyllodistomum species utilizing G. cernuus, and to associate larvae with the corresponding adult forms. RESULTS: Phylogenetic analyses of adult and larval stages of Phyllodistomum spp. based on ITS2 and partial 28S rDNA data allowed the confirmation of the validity of P. pseudofolium. A macrocercous cercaria, known as Phyllodistomum sp. from P. amnicum is genetically identical to adult P. pseudofolium. Phyllodistomum megalorchis obtained from its type-host, Lota lota L., showed no genetic differences from P. angulatum parasitizing Sander lucioperca L. In our analysis, P. pseudofolium, P. angulatum and P. macrocotyle formed a highly supported clade despite the fact that these species appear to be associated with distinct patterns of first intermediate host identity and cercarial morphology. Some morphological differences between gravid specimens of P. pseudofolium and P. angulatum were observed and their SEM tegumental surface topography is described. CONCLUSIONS: The results lead us to the perception that macroevolutionary host switching in the genus Phyllodistomum is independent of host phylogeny. This study suggests strict host-specificity (oioxeny) for P. pseudofolium using one first intermediate host species (P. amnicum) and one definitive host species (G. cernuus). Phyllodistomum megalorchis is to be regarded as a synonym of P. angulatum. The close phylogenetic relatives, P. pseudofolium and P. angulatum, can be differentiated by morphological traits, the micromorphology and tegumental surface topography of these two species is intended to provide useful data for their identification and support the use of such features as a valuable taxonomic criterion. Molecular data showed that G. cernuus is a definitive host for two species: the oioxenous P. pseudofolium and the euryxenous P. folium.

Ultrastructural features of the spermatozoon and its differentiation in Brandesia turgida (Brandes, 1888) (Digenea, Microphalloidea, Pleurogenidae).

Spermatological characters of the digenean Brandesia turgida (Brandes, 1888), an intestinal parasite of the frog Pelophylax ridibundus (Pallas, 1771), have been investigated by means of transmission electron microscopy for the first time. The process of the spermatozoon formation begins with the appearance of the differentiation zone bordered by cortical microtubules and containing two centrioles associated with striated rootlets and with an intercentriolar body. The intercentriolar body is made up of seven distinct electron-dense plates, two less electron-dense, and four electron-lucent zones. The orthogonal development of the two flagella is followed by a flagellar rotation and their proximodistal fusion with the median cytoplasmic process. This process is accompanied by an extension of both the mitochondrion and nucleus into the median cytoplasmic process. The mature spermatozoon of B. turgida contains two parallel axonemes of unequal lengths with the 9 + "1" trepaxonematan pattern, mitochondrion, nucleus, parallel cortical microtubules, four electron-dense attachment zones, an external ornamentation of the plasma membrane, and electron-dense glycogen granules. The anterior extremity of the male gamete contains one complete centriole, a small component of the central element of the second centriole, and peripheral cortical microtubules (up to 45). The posterior extremity of the mature spematozoon exhibits tubular elements of the disorganized axoneme. The present study provides the first data on spermiogenesis within the family Pleurogenidae. Variations of the spermatozoa ultrastructural characters within Digenea, in particular, between different families of the superfamily Microphalloidea, are discussed.

Ultrastructural features of the tegumental surface of a new metacercaria, Nematostrigea sp. (Trematoda: Strigeidae), with a search for potential taxonomically informative characters.

The tegumental surface of a new strigeid metacercaria, Nematostrigea sp., which is a parasite of the freshwater fish Channa gachua (Hamilton) in central Vietnam, is described for the first time using scanning (SEM) and transmission (TEM) electron microscopy. In addition to the general tegumental surface in various parts of the body, details of the surface of the suckers, lappets and holdfast organ are presented, as are variations in the form and distribution of the body spines. As good taxonomic criteria are few in diplostomoid metacercariae at both specific and generic levels, a number of the ultrastructural features revealed may prove to represent taxonomically informative characters. These include the presence of: two rings of dome-shaped papillae localised at different levels on the rim of the oral sucker, a single ring of ciliated papillae on the inner margin of the ventral sucker and a band of dome-shaped papillae along the lateral margins of the broad body-fold in the ventral forebody; an unarmed oral sucker and anteroventral surface of the forebody, although the latter bears protuberant secretory pores; an armed ventral sucker covered by six-pointed spines, except on its rim; multi-pointed spines along the dorsal and ventral sides of the forebody, with the number of their teeth increasing posteriorly; multi-pointed spines on the forebody which gradually transform into single-pointed, more widely distributed spines on the hindbody, disappearing completely at posterior end of the body; the surface of the lappets with a particular distribution of pores leading to three types of secretory glands and three topographical modifications (areas where the surface is smooth, bears digitiform processes or bears recurved, dagger-shaped spines); and the surface of the holdfast organ which is covered with densely packed, straight or slightly curved, simple spines on its lateral surface but is smooth medially.