Hana Pecková

Neoparamoeba spp. and their eukaryotic endosymbionts similar to Perkinsela amoebae (Hollande, 1980): coevolution demonstrated by SSU rRNA gene phylogenies.

The molecular phylogeny of Neoparamoeba spp. based on SSU rDNA was updated by including new sequences of strains isolated from an invertebrate and an alga. In total, 59 sequences of strains representating N. pemaquidensis, N. branchiphila, N. aestuarina and N. perurans were analysed. Sequences of SSU rDNA of eukaryotic endosymbionts (Perkinsela amoebae-like organisms) were prepared from 34 samples of genomic DNA of strain-representatives of N. pemaquidensis, N. branchiphila and N. aestuarina. Comparison of phylograms reconstructed from corresponding SSU rDNA sequences of host amoebae and their symbionts revealed a high level of congruence, which argues very strongly for coevolution of these two eukaryotic organisms.

Hidden diversity and evolutionary trends in malacosporean parasites (Cnidaria: Myxozoa) identified using molecular phylogenetics.

Malacosporeans represent a small fraction of myxozoan biodiversity with only two genera and three species described. They cycle between bryozoans and freshwater fish. In this study, we (i) microscopically examine and screen different freshwater/marine fish species from various geographic locations and habitats for the presence of malacosporeans using PCR; (ii) study the morphology, prevalence, host species/habitat preference and distribution of malacosporeans; (iii) perform small subunit/large subunit rDNA and Elongation factor 2 based phylogenetic analyses of newly gathered data, together with all available malacosporean data in GenBank; and (iv) investigate the evolutionary trends of malacosporeans by mapping the morphology of bryozoan-related stages, host species, habitat and geographic data on the small subunit rDNA-based phylogenetic tree. We reveal a high prevalence and diversity of malacosporeans in several fish hosts in European freshwater habitats by adding five new species of Buddenbrockia and Tetracapsuloides from cyprinid and perciform fishes. Comprehensive phylogenetic analyses revealed that, apart from Buddenbrockia and Tetracapsuloides clades, a novel malacosporean lineage (likely a new genus) exists. The fish host species spectrum was extended for Buddenbrockia plumatellae and Buddenbrockia sp. 2. Co-infections of up to three malacosporean species were found in individual fish. The significant increase in malacosporean species richness revealed in the present study points to a hidden biodiversity in this parasite group. This is most probably due to the cryptic nature of malacosporean sporogonic and presporogonic stages and mostly asymptomatic infections in the fish hosts. The potential existence of malacosporean life cycles in the marine environment as well as the evolution of worm- and sac-like morphology is discussed. This study improves the understanding of the biodiversity, prevalence, distribution, habitat and host preference of malacosporeans and unveils their evolutionary trends.

Phylogenetic analysis of freshwater fish trypanosomes from Europe using ssu rRNA gene sequences and random amplification of polymorphic DNA

The taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. A collection of 22 cloned trypanosome isolates from 14 species of European freshwater fish and 1 species of African freshwater fish were examined by molecular phylogenetic analysis. The small subunit ribosomal RNA (ssu rRNA) genes of 8 clones were sequenced and compared with ssu rRNA gene sequences from a wider selection of vertebrate trypanosome isolates by phylogenetic analysis. All trypanosomes from freshwater fish fell in a single clade, subdivided into 3 groups. This clade sits within a larger, robust clade containing trypanosomes from marine fish and various amphibious vertebrates. All 22 trypanosome clones were analysed by random amplification of polymorphic DNA. The resulting dendrogram shows 3 groups, which are congruent with the groups identified in the ssu rRNA gene phylogeny. Two of the groups contain the majority of trypanosome isolates and within-group variation is slight. These groups do not separate purported trypanosome species distinguished by morphology or host origin, and thus these criteria do not appear to be reliable guides to genetic relationships among fish trypanosomes. However, we suggest that the 2 groups themselves may represent different species of fish trypanosomes. The polymorphic DNA markers we have identified will facilitate future comparisons of the biology of these 2 groups of fish trypanosomes.

'Who's who' in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish--molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto.

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

Living together: The marine amoeba Thecamoeba hilla Schaeffer, 1926 and its endosymbiont Labyrinthula sp.

Two protists isolated simultaneously from the same sample of gill tissue of Psetta maxima (L.) were identified as Thecamoeba hilla Schaeffer, 1926 and Labyrinthula sp. A Labyrinthula strain (LTH) derived from a mixed culture of both organisms was well established in a short time, while subcultures of T. hilla continued to be associated with Labyrinthula cells despite all efforts to eliminate them. Ultrastructural examination, repeated several times in the course of long-lasting subculturing of amoebae, revealed that trophozoites of T. hilla host in their cytoplasm multiplying labyrinthulid cells. Comparison of SSU rDNA sequences of the Labyrinthula strain LTH and those from labyrinthulid endosymbionts from T. hilla verified the assumption that the extra- and intra-cellularly multiplying Labyrinthula cells are identical organisms. The association of the marine amoeba T. hilla and Labyrinthula sp. displayed signs of mutualistic symbiosis.

Fish Trypanosomes: Their Position in Kinetoplastid Phylogeny and Variability as Determined from 12S rRNA Kinetoplast Sequences

Fish trypanosomes have traditionally been classified according to the host species from which they were isolated, each isolate being regarded as a distinct species. To test the soundness of this practice, the genetic variabilities of the kinetoplast 12S rRNA‐encoding genes of different fish trypanosomes isolates were compared. The DNAs were extracted from trypanosomes cloned from blood samples of 15 donors representing ten different fish species in four orders from waters of three major river systems of Central and Northern Europe. Comparison with other trypanosomatid sequences revealed that the fish trypanosomes form a monophyletic group with Trypanosoma brucei as a sister group. Pairwise comparisons of genetic distances yielded a wide range of continuous variation with no indication of any discontinuities attributable to barriers to gene flow. The genetic distances did not correlate with either the identity of the host species or geography. The host specificity of fish trypanosomes appears to be limited.

Phylogeny and morphology of Glugea hertwigi from rainbow smelt Osmerus mordax found in Prince Edward Island, Canada

Infection of rainbow smelt Osmerus mordax with the microsporidian Glugea hertwigi was diagnosed for the first time in Prince Edward Island, Canada. The prevalence of infection was 24%, 45 infected out of 187 examined fish captured in February and March 2009. Both large and small xenomas of G. hertwigi observed within the submucosa of the gastrointestinal tract and along the mesentery of the host contained only mature spores. Advanced and degraded xenomas associated with host reaction were described using light and transmission electron microscopy. The first rDNA sequence of G. hertwigi prepared in the present study completed the set of sequences of Glugea spp. available for comparison. The high level of rDNA sequence identity between Glugea spp. suggests that these may be variants of a single species.

Grellamoeba robusta gen. n., sp. n., a possible member of the family Acramoebidae Smirnov, Nassonova et Cavalier-Smith, 2008.

A strain of naked amoeba isolated from pikeperch (Sander lucioperca (L.)) kidney tissue has been characterized using light- and transmission electron microscopy. Sequencing of SSU rDNA and phylogenetic analysis based on a broad dataset of sequences completed our study. All data obtained suggest that this strain belongs to a species that has not been described before. As none of the existing genera of amoebae is applicable to this organism, the new genus Grellamoeba is established and the type species Grellamoeba robusta is described. Although the phylogenetic position of the SSU rDNA sequence of the type strain of G. robusta is sensitive to the method of analysis applied, a tendency to group with Acramoeba dendroida Smirnov, Nassonova et Cavalier-Smith, 2008 is evident.

Species complexes and phylogenetic lineages of Hoferellus (Myxozoa, Cnidaria) including revision of the genus: A problematic case for taxonomy

BackgroundMyxozoans are metazoan parasites whose traditional spore morphology-based taxonomy conflicts DNA based phylogenies. Freshwater species of the genus Hoferellus are parasites of the excretory system, with several members infecting food and ornamental fish species, as well as amphibians. This study aims to increase our understanding of their molecular diversity and development, aspects about which little is known, and to generate a molecular diagnostic tool to discriminate between different pathogenic and non-pathogenic Hoferellus spp.MethodsSSU and ITS rDNA phylogeny, along with morphological descriptions using light and electron microscopy were used to identify and characterize Hoferellus species collected from the urinary system of fishes and frogs. A PCR-based diagnostic assay was designed to differentiate between cryptic Hoferellus spp in cyprinid fishes commonly cultured in Central Europe.ResultsOur phylogenetic results separate the species of Hoferellus into two phylogenetic sublineages which are indistinguishable on the basis of generic morphological traits: 1) The Hoferellus sensu stricto sublineage, which is composed of the type species Hoferellus cyprini, Hoferellus carassii and a cryptic species, Hoferellus sp. detected only molecularly in common carp. 2) The Hoferellus sensu lato sublineage into which the new species we described in this study, Hoferellus gnathonemi sp. n. from the kidney of the elephantnose fish and Hoferellus anurae from reed frogs, are placed together with Hoferellus gilsoni previously sequenced from European eel. Apart from phylogenetic analyses, we also provide novel ultrastructural data on the phagocytotic nature of some Hoferellus plasmodia and on the elusive intracellular stages ascribed to the presporogonic development of this genus.ConclusionsWe provide molecular evidence of the polyphyly of the genus Hoferellus and provide novel morphological details of its members. Based on the presented data, we revise and propose emendation of the genus Hoferellus.

Neoparamoeba branchiphila infections in moribund sea urchins Diadema aff. antillarum in Tenerife, Canary Islands, Spain

A total of 109 sea urchins from 3 species collected in 2 localities off the coast of Tenerife Island, Spain, were examined for the presence of free-living amoebae in their coelomic fluid. Amoeba trophozoites were isolated exclusively from moribund individuals of long-spined sea urchins Diadema aff. antillarum (Philippi) (Echinoidea, Echinodermata) that manifested lesions related to sea urchin bald disease on their tests (16 out of 56 examined). No amoebae were detected in Arbacia lixula (L.) and Paracentrotus lividus (Lamarck). From the former sea urchin species, 8 strains, established from 10 primary isolates, were identified as Neoparamoeba branchiphila Dyková et al., 2005 using morphological and molecular methods. Results of this study (limited to the screening for free-living amoebae) together with data on agents of sea urchin mortalities reported to date justify the hypothesis that free-living amoebae play an opportunistic role in D. aff. antillarum mortality. The enlargement of the dataset of SSU rDNA sequences brought new insight into the phylogeny of Neoparamoeba species.