Tracing the genus Sphaerospora: rediscovery, redescription and phylogeny of the Sphaerospora ranae (Morelle, 1929) n. comb. (Myxosporea, Sphaerosporidae), with emendation of the genus Sphaerospora.

Using a combination of morphological, life-history and molecular data, we redescribe Sphaerospora ranae (Morelle, 1929) n. comb. (previously Leptotheca ranae) and emend its taxonomic status. Renal infection was recorded in 2 spp. of frogs (out of 5 amphibian spp. examined), Rana dalmatina (proposed type host) and Rana temporaria, suggesting restricted host specifity of S. ranae. We provide a description of sporogonic stages of S. ranae for the first time and suggest possible modes of its developmental cycle. Phylogenetic analysis inferred from the small-subunit ribosomal DNA revealed a close relationship of S. ranae with piscine Sphaerospora elegans (type species of the genus) and Sphaerospora truttae, forming together with distantly related Leptotheca fugu a 'Sphaerosporid clade', the basal branch to all myxosporean species. The close relationship of the 3 Sphaerospora spp. is further supported by the presence of 2 areas with extensive nucleotide insertions in the V4 region of the SSU rDNA (absent in L. fugu), morphology and life-history features. We conclude, that the spore morphology of Sphaerospora s.l., is very simple and probably represents a 'primitive', basal morphotype retained in most myxosporean lineages. Based on presented data, we propose emendation of the genus Sphaerospora using morpological, life-history and molecular features.

Extra-intestinal localization of Goussia sp. (Apicomplexa) oocysts in Rana dalmatina (Anura: Ranidae), and the fate of infection after metamorphosis.

Although coccidia of the genus Goussia are common parasites of fish, only 2 species have been described in amphibians: G. hyperolisi from common reed frogs Hyperolius viridiflavus from Kenya and G. neglecta from unspecified European water frogs of the genus Rana from Germany. The genus Goussia is characterized by an oocyst, with a fine oocyst wall, containing 4 dizoic sporocysts that are composed of 2 valves joined by a longitudinal suture and lacking a Stieda body (typical for the genus Eimeria). To date, infections in amphibians were generally considered to be specific to the intestine of aquatic larval stages (tadpoles) of anurans. Herein, we report on: (1) the presence of oocysts of Goussia sp. in an extra-intestinal location (liver) of tadpoles of the agile frog R. dalmatina and (2) the presence of oocysts in the liver of both juvenile and subadult R. dalmatina. These observations represent novel traits for Goussia infections in amphibians; they may explain the vertical transmission of Goussia in tadpoles.

Leptomonas costaricensis sp. n. (Kinetoplastea: Trypanosomatidae), a member of the novel phylogenetic group of insect trypanosomatids closely related to the genus Leishmania.

A flagellate isolated from the intestinal tract of a reduviid bug Ricolla simillima (Heteroptera) in Costa Rica was found to represent a new trypanosomatid species by the phylogenetic analysis of small subunit ribosomal RNA (SSU rRNA), glyceraldehyde phosphate dehydrogenase (GAPDH) and large subunit of RNA polymerase II (RPOIILS) genes. The phylogenetic position of this trypanosomatid, together with its typical promastigote morphology and the host identity, allowed its classification as a species that belongs to the polyphyletic genus Leptomonas. Interestingly, the new species was revealed as a member of the novel phylogenetic clade representing the closest known relative of Leishmania. With the new species used as an outgroup to root the Leishmania RPOIILS phylogenetic tree, the lineage of the Neotropical species L. enriettii was found to branch off early, and was followed by a deep split between the Old World and the remaining New World species. This tree topology supports the hypothesis that the initial transition to dixenous parasitism in this group pre-dated the continental split and that afterwards the Neotropical and the Old World groups evolved largely independently.

Three new species of coccidia (Apicomplexa: Eimeriorina) from the Marble-throated skink, Marmorosphax tricolor Bavay, 1869 (Reptilia: Scincidae), endemic to New Caledonia with a taxonomic revision of Eimeria spp. from scincid hosts.

Three new species of coccidia are described from Marble-throated skink Marmorosphax tricolor from New Caledonia, namely, Isospora bocagei sp. n., Acroeimeria rouxi sp. n., and Choleoeimeria sadlieri sp.n. All species differ markedly from other eimerian coccidia described from scincid hosts. Isospora marmorosphaxi develops extra-nuclearly in small intestine. A. rouxi develops epicitoplasmatically in small intestine. C. sadlieri affects the gall bladder mucosa. Generic affiliation of Eimeria-like coccidia from reptiles is discussed and all taxa (with adequate information on endogenous development available) from scincid hosts are revised and placed into genera Acroeimeria and Choleoeimeria.

Three new species of Eimeria (Apicomplexa: Eimeriidae) from the silvery mole rat Heliophobius argenteocinereus Peters, 1846 (Rodentia: Bathyergidae) from Malawi.

Three species of Eimeria Schneider are described from feces of the African bathyergid rodent, Heliophobius argenteocinereus, from Malawi. Oocysts of Eimeria heliophobii n. sp. are broadly ellipsoidal; 27.9 (22-31) x 22.3 (18-24.5) microm with a brownish, heavily pitted oocyst wall, and vacuolar oocyst residuum. Sporocysts are oval, 12.8 (12-14) x 8.4 (8-9) microm with Stieda and substieda bodies. Eimeria nafuko n. sp. has subspherical oocysts; 15.5 (15-16) x 12.8 (12-13) microm with a smooth, colorless oocyst wall. Sporocysts are oval, 9.2 (9-10) x 5.3 (5-6) microm, with a small Stieda body; the substieda body is not visible. Oocysts of Eimeria yamikamiae n. sp. are broadly ellipsoidal to subspherical; 20.8 (19-22) x 17.5 (15.5-19) microm, with slightly yellowish, very faintly pitted oocyst wall. The majority of oocysts contained a single spherical vesicular oocyst residuum and numerous very small granules. Sporocysts are oval, 10.7 (10-11) x 6.8. (6-7) microm, with a dome -like Stieda body and a subspherical to lentil-like substieda body. Typically, infected rodents shed oocysts of more than 1 species of Eimeria.

Phylogeny of mitosporic entomopathogenic fungi: is the genus Paecilomyces polyphyletic?

We analyzed sequences of the divergent domain at the 5' end of the large subunit rRNA gene from the mitosporic entomopathogenic fungi Paecilomyces sp., Paecilomyces fumosoroseus, Paecilomyces farinosus, Paecilomyces lilacinus, Verticillium lecanii, Verticillium psalliotae, Beauveria bassiana, Aschersonia sp., Aschersonia placenta, ascomycetous Cordyceps sp., and Cordyceps militaris. Phylogenetic analysis showed P. fumosorseus as the best characterized out of the analyzed species with the B. bassiana clade as its sister group. Two of the P. farinosus isolates were invariably placed within the Verticillium cluster, which also contained C. militaris. The only analyzed P. lilacinus isolate appeared on the root of the hyphomycetous fungi and was characterized as the most distinct from all the hyphomycetous fungi tested. Polyphyly of the genus Paecilomyces was well supported by the Kishino-Hasegawa test. In all trees based on the small subunit rRNA gene sequences obtained from the GenBank, V. lecanii, V. psalliotae, P. fumosoroseus, P. tenuipes and B. bassiana form, together with that of C. militaris, the best supported cluster in the tree. The rest of Cordyceps spp. constitute a distinct clade. Phylogenetic relationships derived from both tested DNA regions show polyphyly of the genus Paecilomyces and close relationships among entomopathogenic species of the genera Verticillium, Paecilomyces, and Beauveria.

Phylogenetic position of a renal coccidium of the European green frogs, 'Isospora' lieberkuehni Labbé, 1894 (Apicomplexa: Sarcocystidae) and its taxonomic implications.

'Isospora' lieberkuehni, an unusual isosporoid renal coccidium that parasitizes the European water frog was isolated from the edible frog, Rana kl. esculenta, in the Czech Republic. Sequencing of the small-subunit (SSU) rRNA gene showed that it belongs to the family Sarcocystidae, being closely related to a clade comprising members of the subfamily Toxoplasmatinae. The position within Sarcocystidae correlates with the mode of excystation via collapsible plates as postulated by previous authors. Phylogenetic, morphological and biological differences between 'Isospora' lieberkuehni and the other Stiedabody-lacking members of the genus Isospora justify separation of this coccidium on a generic level. Hyaloklossia Labbé, 1896 is the oldest available synonym and is herein re-erected. The original definition of the genus Hyaloklossia is emended based on recent observations.

Multiple origin of the dihomoxenous life cycle in sarcosporidia.

Although their ssrRNA gene sequences are closely related, the lizard sarcosporidia (Apicomplexa, Sarcocystidae) Sarcocystis lacertae and Sarcocystis gallotiae posses heteroxenous and dihomoxenous life cycles, respectively. When aligned with available sarcosporidian ssrRNA genes, both species constitute a monophyletic clade that is only distantly related with sarcosporidia that have a viperid snake as their definitive host (Sarcocystis sp., Sarcocystis atheridis). To test the phyletic status of the dihomoxenous life style, Sarcocystis rodentifelis and Sarcocystis muris, two dihomoxenous parasites of mammals were included into this study. All studied species group together with former Frenkelia spp., Sarcocystis neurona and related marsupial and bird sarcosporidia in a monophyletic clade. However, the available dataset supports independent appearance of the dihomoxenous life cycle at least twice during the evolution of the Sarcocystidae.

Eimeria telekii n.sp. (Apicomplexa: Coccidia) from Lemniscomys striatus (Rodentia: Muridae): morphology, pathology and phylogeny.

Using a combination of morphological, life-cycle and molecular data, we describe a new apicomplexan parasite Eimeria telekii n.sp. from a striped grass mouse Lemniscomys striatus captured in Kenya. Oocysts are oval to spherical or ellipsoidal, 20.4 x 15.7 (15.5-25.0 x 12.0-20.0) microm with a colourless, smooth and bilayered wall. Sporocysts are ellipsoidal, 11.2 x 7.8 (10.0-12.0 x 7.0-9.0) microm with a small Stieda body and granular sporocyst residuum and contain 2 elongated, banana-shaped sporozoites with a single refractile body. Life-cycle, pathogenicity and host specificity of this parasite were studied in laboratory-bred Lemniscomys barbarus and BALB/c mice. Two asexual stages and the sexual phase took place within the enterocytes of the caecum and colon of L. barbarus but not in inoculated BALB/c mice. An infectious dose of 5000 oocysts caused severe clinical illness and mortality in 2/2 (100%) L. barbarus. Phylogenetic analysis of the small subunit rRNA gene of E. telekii and members of the genera Eimeria, Cyclospora and Isospora placed E. telekii within the eimerian rodent clade.

Phylogeny of the bodonid flagellates (Kinetoplastida) based on small-subunit rRNA gene sequences.

The phylogeny of kinetoplastid flagellates was investigated by determining the sequences of the small-subunit (18S) rRNA from Bodo designis, Bodo saltans K, Bodo saltans P, Bodo sorokini, Bodo sp. (cf. uncinatus), Cruzella marina, Cryptobia helicis, Dimastigella mimosa and Parabodo nitrophilus and analysing these data together with several previously obtained sequences. The root of the kinetoplastid tree was tentatively determined to be attached to the branch of B. designis and/or Cruzella marina. Within this topology, the suborder Trypanosomatina appears as a late-emerging monophyletic group, while the suborder Bodonina is paraphyletic. Within the bodonid subtree, the branches of parasitic organisms were intermingled with free-living ones, implying multiple transitions to parasitism. The tree indicates that the genera Cryptobia and Bodo are artificial taxa. In addition, the separation of the fish cryptobias and Trypanoplasma borreli as different genera was not supported.

On the phylogenetic positions of the Caryophyllidea, Pseudophyllidea and Proteocephalidea (Eucestoda) inferred from 18S rRNA.

A phylogenetic analysis of tapeworms (Eucestoda) based on complete sequences of the 18S rRNA genes of 43 taxa (including new sequences of 12 species) was carried out, with the emphasis on the groups parasitising teleost fish and reptiles. Spathebothriidea and Trypanorhyncha (the latter group being paraphyletic) appeared as basal groups of the Eucestoda but their position was not stable. The tetrafossate orders (Litobothriidea, Lecanicephalidea, Tetraphyllidea, Proteocephalidea, Nippotaeniidea, Tetrabothriidea and Cyclophyllidea) were well separated from the remaining groups. Results supported polyphyly of the Pseudophyllidea formed by two distinct clades: one with diphyllobothriids (Diphyllobothrium, Schistocephalus, Spirometra and Duthiersia) and another including Abothrium, Probothriocephalus, Eubothrium and Bothriocephalus. The former pseudophyllidean clade formed a separate branch with the Caryophyllidea (Khawia and Hunterella) and Haplobothriidea (Haplobothrium), the latter taxon being closely related to either caryophyllideans or diphyllobothriids in different analyses. Proteocephalideans formed a monophyletic group in all analyses and constituted a clade within the Tetraphyllidea thus rendered paraphyletic. Within the Proteocephalidea, the Acanthotaeniinae (Acanthotaenia from reptiles in Africa) and Gangesiinae (Gangesia and Silurotaenia from silurid fish in the Palearctic Region) were separated from parasites of freshwater fish and mammals. The family Proteocephalidae was found to be paraphyletic due to the placement of a monticelliid species, Monticellia sp., in a clade within the former family. The genus Proteocephalus appeared as an artificial assemblage of unrelated taxa which is congruent with previous molecular analyses.

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks.

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.

RNA editing in the free-living bodonid Bodo saltans.

In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids. In contrast, B.saltans cytochrome c oxidase (cox) subunit 2 and maxicircle unidentified reading frame (MURF) 2 RNAs display limited editing in the same regions as their trypanosomatid counterparts. A putative intramolecular cox2 gRNA and the gene for gMURF2-I directing the insertion of only one U in the 5' editing domain of MURF2 RNA, are conserved in B.saltans. This lends (further) evolutionary support to the proposed role of these sequences as gRNAs. Phylogenetic analysis showed that B.saltans is more closely related to trypanosomatids than the cryptobiids Trypanoplasma borreli and Cryptobia helicis, in line with the trypanosomatid-like cox2 and MURF2 RNA editing patterns. Nevertheless, other features like the apparent absence of a catenated mtDNA network, are shared with bodonid and cryptobiid species. ND5 RNA editing may represent yet another example of editing 'on the way out' during kinetoplastid evolution, but in view of the fact that cox2 RNA is unedited in T. borreli and C.helicis, we infer that the editing of this RNA may have arisen relatively recently. Our results provide the first examples of RNA editing in a free-living kinetoplastid, indicating that there is no direct link between U-insertion/deletion editing and a parasitic lifestyle.

Pankinetoplast DNA structure in a primitive bodonid flagellate, Cryptobia helicis.

The mitochondrial DNA (mtDNA) of a primitive kinetoplastid flagellate Cryptobia helicis is composed of 4.2 kb minicircles and 43 kb maxicircles. 85% and 6% of the minicircles are in the form of supercoiled (SC) and relaxed (OC) monomers, respectively. The remaining minicircles (9%) constitute catenated oligomers composed of both the SC and OC molecules. Minicircles contain bent helix and sequences homologous to the minicircle conserved sequence blocks. Maxicircles encode typical mitochondrial genes and are not catenated. The mtDNA, which we describe with the term 'pankinetoplast DNA', is spread throughout the mitochondrial lumen, where it is associated with multiple electron-lucent loci. There are approximately 8400 minicircles per pankinetoplast-mitochondrion, with the pan-kDNA representing approximately 36% of the total cellular DNA. Based on the similarity of the C.helicis minicircles to plasmids, we present a theory on the formation of the kDNA network.

Molecular phylogenetic relatedness of Frenkelia spp. (Protozoa, Apicomplexa) to Sarcocystis falcatula Stiles 1893: is the genus Sarcocystis paraphyletic?

The coccidians Frenkelia microti and F. glareoli (Apicomplexa: Sarcocystidae) form tissue cysts in the brain of small rodents (intermediate hosts) while oocysts are formed in the intestine of final hosts, buzzards of the genus Buteo. The inclusion of the small subunit ribosomal RNA gene sequences (SSU rRNA) of both Frenkelia species into the SSU rRNA trees of other, tissue cyst-forming coccidia strongly supports paraphyly of the genus Sarcocystis. Frenkelia spp. exhibit close relatedness to Sarcocystis falcatula Stiles 1893, a bird-opossum parasite, recognized under its junior synonym S. neurona Dubey et al. 1991, as the causative agent of equine protozoan myeloencephalitis on the American continent. As the definition of the genus Frenkelia is based on a plesiomorphic character (affinity to the neural tissue) of supposedly low phylogenetic value, the synonymization of the genus Frenkelia with Sarcocystis is proposed. This renders the genus Sarcocystis monophyletic.

Analysis of ribosomal RNA genes suggests that trypanosomes are monophyletic.

To further investigate the phylogeny of protozoa from the order Kinetoplastida we have sequenced the small subunit (SSU) and a portion of the large subunit (LSU) nuclear rRNA genes. The SSU and LSU sequences were determined from a lizard trypanosome, Trypanosoma scelopori and a bodonid, Rhynchobodo sp., and the LSU sequences were determined from an insect trypanosomatid, Crithidia oncopelti, and a bodonid, Dimastigella trypaniformis. Contrary to previous results, in which trypanosomes were found to be paraphyletic, with Trypanosoma brucei representing the earliest-diverging lineage, we have now found evidence for the monophyly of trypanosomes. Addition of new taxa which subdivide long branches (such as that of T. brucei) have helped to identify homoplasies responsible for the paraphyletic trees in previous studies. Although the monophyly of the trypanosome clade is supported in the bootstrap analyses for maximum likelihood at 97% and maximum parsimony at 92%, there is only a small difference in ln-likelihood value or tree length between the most optimal monophyletic tree and the best suboptimal paraphyletic tree. Within the trypanosomatid subtree, the clade of trypanosomes is a sister group to the monophyletic clade of the nontrypanosome genera. Different groups of trypanosomes group on the tree according to their mode of transmission. This suggests that the adaptation to invertebrate vectors plays a more important role in the trypanosome evolution than the adaptation to vertebrate hosts.

Phylogeny of trypanosomes as inferred from the small and large subunit rRNAs: implications for the evolution of parasitism in the trypanosomatid protozoa.

Sequences of the small rRNA genes and partial sequences of the large rRNA genes were obtained by PCR amplification from a variety of vertebrate trypanosomes. The trypanosome species and hosts included Trypanosoma avium from a bird, T. rotatorium from an amphibian, T. boissoni from an elasmobranch, T. triglae from a marine teleost and T. carassii from a freshwater teleost. Phylogenetic relationships among these species and other representatives of the family Trypanosomatidae were inferred using maximum likelihood, maximum parsimony and evolutionary parsimony. The trypanosomatid tree was rooted using rRNA sequences from two species from the suborder Bodonina. All methods showed that the mammalian parasite, Trypanosoma brucei, constitutes the earliest divergent branch. The remaining trypanosomes formed a monophyletic group. Within this group, the bird trypanosome was grouped with T. cruzi, while the elasmobranch trypanosome and the two fish trypanosome species formed a group with an affinity to T. rotatorium. Our results provide no evidence for co-evolution of trypanosomatids and their hosts, either vertebrate or invertebrate. This suggests that evolution of trypanosomatids was accompanied by secondary acquisitions of hosts and habitats.