Multi-gene phylogeny of Tetrahymena refreshed with three new histophagous species invading freshwater planarians.

Planarians represent an insufficiently explored group of aquatic invertebrates that might serve as hosts of histophagous ciliates belonging to the hymenostome genus Tetrahymena. During our extensive research on freshwater planarians, parasitic tetrahymenas were detected in two of the eight planarian species investigated, namely, in Dugesia gonocephala and Girardia tigrina. Using the 16S and 18S rRNA genes as well as the barcoding cytochrome oxidase subunit I, one ciliate species was identified as T. scolopax and three species were recognized as new forms: T. acanthophora, T. dugesiae, and T. nigricans. Thus, 25% of the examined planarian taxa are positive for Tetrahymena species and three of them represent new taxa, indicating a large undescribed ciliate diversity in freshwater planarians. According to phylogenetic analyses, histophagous tetrahymenas show a low phylogenetic host specificity. Although T. acanthophora, T. dugesiae, and T. scolopax clustered together within the "borealis" clade, the former species has been detected exclusively in G. tigrina, while the two latter species only in D. gonocephala. Tetrahymena nigricans, which has been isolated only from G. tigrina, was classified within the "paravorax" clade along with T. glochidiophila which feeds on glochidia. The present phylogenetic reconstruction of ancestral life strategies suggested that the last common ancestor of the family Tetrahymenidae was free-living, unlike the progenitor of the subclass Hymenostomatia which was very likely parasitic. Consequently, there were at least seven independent shifts back to parasitism/histophagy within Tetrahymena: one each in the "paravorax" and "australis" clades and at least five transfers back to parasitism in the "borealis" clade.

Barcodes Reveal 48 New Species of Tetrahymena, Dexiostoma, and Glaucoma: Phylogeny, Ecology, and Biogeography of New and Established Species.

Tetrahymena mitochondrial cox1 barcodes and nuclear SSUrRNA sequences are particularly effective at distinguishing among its many cryptic species. In a project to learn more about Tetrahymena natural history, the majority of >1,000 Tetrahymena-like fresh water isolates were assigned to established Tetrahymena species with the remaining assigned to 37 new species of Tetrahymena, nine new species of Dexiostoma and 12 new species of Glaucoma. Phylogenetically, all but three Tetrahymena species belong to the well-established "australis" or "borealis" clades; the minority forms a divergent "paravorax" clade. Most Tetrahymena species are micronucleate, but others are exclusively amicronucleate. The self-splicing intron of the LSUrRNA precursor is absent in Dexiostoma and Glaucoma and was likely acquired subsequent to the "australis/borealis" split; in some instances, its sequence is diagnostic of species. Tetrahymena americanis, T. elliotti, T. gruchyi n. sp., and T. borealis, together accounted for >50% of isolates, consistent with previous findings for established species. The biogeographic range of species found previously in Austria, China, and Pakistan was extended to the Nearctic; some species show evidence of population structure consistent with endemism. Most species were most frequently collected from ponds or lakes, while others, particularly Dexiostoma species, were collected most often from streams or rivers. The results suggest that perhaps hundreds of species remain to be discovered, particularly if collecting is global and includes hosts of parasitic forms.

The development of Cryptocaryon irritans in a less susceptible host rabbitfish, Siganus oramin.

Cryptocaryon irritans is a ciliated protozoan parasite infecting the gills and skin of almost all marine teleosts, resulting in severe disease. Notably, C. irritans is unable to cause significant pathogenesis in rabbitfish, Siganus oramin, a well-known less susceptible host. However, little is known about the development of C. irritans in rabbitfish. Thus, in the present study, rabbitfish were artificially infected with C. irritans at a 1/2 maximal tolerance dose (MTD), using the susceptible host, large yellow croaker, Larimichthys crocea, as a control. The daily food consumption (DFC), survival, and relative infection intensity (RII) of the fish were observed, and the relative number of tomonts (RNT), reproductive rate, and characteristics of C. irritans tomonts were measured. The lethal concentration 50 (LC50) of C. irritans on rabbitfish and large yellow croaker was 2236, and 264 theronts/g fish, respectively. The DFC of rabbitfish decreased by 14.6% at day 1 post-C. irritans infection (PI), but immediately returned to normal DFC levels by day 2 PI, and was not affected by secondary infection. However, large yellow croaker stopped feeding at day 3 PI, and all fish died following secondary infection with C. irritans. The RII of rabbitfish exhibited a significant downward trend at 6 h PI, and the reproductive rate of C. irritans was 8%; in contrast, the RII of large yellow croaker was not significantly different from 0 to 72 h PI, and the reproductive rate of C. irritans reached to 59.05-fold. Over the same time frame, the diameter of tomonts from rabbitfish was smaller compared with those from large yellow croaker. These results indicated that the rabbitfish were somehow able to limit the development of C. irritans, such that most trophonts left the host during early developmental stages, whereas those that survive could only develop into small tomonts.

Evidence of multiple species of Chilodonella (Protozoa, Ciliophora) infecting Australian farmed freshwater fishes.

Parasitic Chilodonella species, Chilodonella piscicola and Chilodonella hexasticha, cause considerable economic losses globally to freshwater farmed fish production. Some genetic studies of Chilodonella spp. have indicated that many species within the genus may form cryptic species complexes. To understand the diversity of Chilodonella spp. infecting Australian freshwater farmed fish, specimens were isolated from infected barramundi (Lates calcarifer) and Murray cod (Maccullochella peelii) from fish farms in tropical north Queensland (QLD), temperate Victoria (Vic) and New South Wales (NSW) for genetic and morphological analysis. Parasites were stained and measured for morphological description and comparative phylogenetic analyses were performed using the mitochondrial small subunit (mtSSU) rDNA marker. Morphological analyses revealed four distinct morphotypes of Chilodonella infecting farmed barramundi and Murray Cod. Three putative species were isolated from barramundi (Chilodonella hexasticha, C. acuta and C. uncinata) and one from Murray cod (C. piscicola). However, phylogenetic analyses detected only three distinct genotypes, with the putative C. hexasticha and C. piscicola sharing 100% sequence identity. This suggests that Australian isolates of C. hexasticha and C. piscicola could represent the same species and may exhibit phenotypic plasticity. Further molecular analysis, including isolates from the type localities, should be performed to support or refute the synonymy of these species.

Molecular Phylogeny and Taxonomy of a New Freshwater Hymenostomatid from Northeastern China, with the Establishment of a New Genus Anteglaucoma gen. n. (Protista, Ciliophora, Oligohymenophorea).

The morphology, infraciliature and SSU rDNA sequence of a new freshwater hymenostomatid ciliate, Anteglaucoma harbinensis gen. nov., spec. nov., collected from a farmland pond in Harbin, China, were investigated. The new genus Anteglaucoma is characterized as follows: small to medium-sized Glaucomidae with oral apparatus in anterior one-third of cell; paroral membrane composed of almost longitudinally arranged dikinetids; three adoral membranelles nearly equal in length and arranged almost longitudinally in parallel; silverline pattern tetrahymenid. The improved diagnosis of family Glaucomidae Corliss 1971 is provided based on the previous and present work. The type species Anteglaucoma harbinensis spec. nov. is defined by having 32-35 somatic kineties; four or five postoral kineties; membranelle 1 and membranelle 2 having five or six kinetosomal rows, membranelle 3 having three kinetosomal rows; single macronuclear nodule; contractile vacuole on average 15% from posterior body end; locomotion characterized by crawling with a rather hectic jerking motion; freshwater habitat. Phylogenetic analyses show that Anteglaucoma clusters in the family Glaucomidae and groups with the genera Glaucoma. The molecular and morphological data indicate that Glaucomidae is related to the family Bromeliophryidae in the phylogenetic trees.

Molecular genetic diversity and characterization of conjugation genes in the fish parasite Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is the etiologic agent of "white spot", a commercially important disease of freshwater fish. As a parasitic ciliate, I. multifiliis infects numerous host species across a broad geographic range. Although Ichthyophthirius outbreaks are difficult to control, recent sequencing of the I. multifiliis genome has revealed a number of potential metabolic pathways for therapeutic intervention, along with likely vaccine targets for disease prevention. Nonetheless, major gaps exist in our understanding of both the life cycle and population structure of I. multifiliis in the wild. For example, conjugation has never been described in this species, and it is unclear whether I. multifiliis undergoes sexual reproduction, despite the presence of a germline micronucleus. In addition, no good methods exist to distinguish strains, leaving phylogenetic relationships between geographic isolates completely unresolved. Here, we compared nucleotide sequences of SSUrDNA, mitochondrial NADH dehydrogenase subunit I and cox-1 genes, and 14 somatic SNP sites from nine I. multifiliis isolates obtained from four different states in the US since 1995. The mitochondrial sequences effectively distinguished the isolates from one another and divided them into at least two genetically distinct groups. Furthermore, none of the nine isolates shared the same composition of the 14 somatic SNP sites, suggesting that I. multifiliis undergoes sexual reproduction at some point in its life cycle. Finally, compared to the well-studied free-living ciliates Tetrahymena thermophila and Paramecium tetraurelia, I. multifiliis has lost 38% and 29%, respectively, of 16 experimentally confirmed conjugation-related genes, indicating that mechanistic differences in sexual reproduction are likely to exist between I. multifiliis and other ciliate species.

Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control.

BACKGROUND: Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism. RESULTS: We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. thermophila, the Ich genome is reduced approximately two-fold in length and gene density and three-fold in gene content. We analyzed in detail several gene classes with diverse functions in behavior, cellular function and host immunogenicity, including protein kinases, membrane transporters, proteases, surface antigens and cytoskeletal components and regulators. We also mapped by orthology Ich's metabolic pathways in comparison with other ciliates and a potential host organism, the zebrafish Danio rerio. CONCLUSIONS: Knowledge of the complete protein-coding and metabolic potential of Ich opens avenues for rational testing of therapeutic drugs that target functions essential to this parasite but not to its fish hosts. Also, a catalog of surface protein-encoding genes will facilitate development of more effective vaccines. The potential to use T. thermophila as a surrogate model offers promise toward controlling 'white spot' disease and understanding the adaptation to a parasitic lifestyle.

Redescription and neotypification of a poorly known freshwater ectocommensal ciliate, Larvulina variabilis (Penard, 122), and consideration of its systematic position.

The morphology and infraciliature of Larvulina variabilis collected from freshwater Gammarus near Boise, Idaho (USA) were investigated using live observation and silver impregnation. Larvulina variabilis was collected from three sites in Idaho and one site in Michigan, indicating a wide distribution. Larvulina variabilis is a small (21-25 x 14-19microm in vivo) hymenostome ectocommensal on freshwater amphipods. Distinguishing morphologic features include an inflexible cortex with a prominent notch in the left posterior margin, a single globular macronucleus, a single posterior contractile vacuole, a somatic ciliature with 10 kineties all consisting of ciliated dikinetids, a "6"-shaped paroral membrane of the stichodyad type, long (approximately 20microm), immobile cilia arising from three adoral membranelles, a discrete dorsal thigmotactic ciliary field consisting of five kineties, distinctive extrusomes and a coarsely polygonal silverline pattern. The forward-projecting anterior cilium emphasized in the original description is an inconstant finding not essential for identification. An improved diagnosis is provided for the genus and the species. Because no type specimen exists and prior descriptions were conflicting, a specimen from the Idaho population is designated as the neotype. The systematic position of this unusual ciliate remains obscure. Possibly, it belongs to the Pleuronematida or the Thigmotrichida.

Cross-immunity and antibody responses to different immobilisation serotypes of Ichthyophthirius multifiliis.

Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.

Differences in virulence between two serotypes of Ichthyophthirius multifiliis.

Naive channel catfish Ictalurus punctatus were infected by 2 isolates of the parasitic ciliate Ichthyophthirius multifiliis that differed in virulence. The isolates, NY1 and G5, Serotypes A and D, respectively, express different surface immobilization-antigens. The virulence of the 2 isolates was compared using tail-fin infections to quantitate parasite numbers and by analysis of the survival of infected fish. Although NY1 infected fish at a lower level than G5, all NY1-infected fish died, but 51% of G5-infected fish survived. The greater virulence of NY1 is apparently a consequence of its shorter life cycle, which results in overwhelming reinfection of fish before they can develop a protective immune response. This report represents the first experimental evidence for differences in virulence between serotypes of I. multifiliis.

Induced cross-protection in channel catfish, Ictalurus punctatus (Rafinesque), against different immobilization serotypes of Ichthyophthirius multifiliis.

Abstract Channel catfish, Ictalurus punctatus (Rafinesque), were immunized with Ichthyophthirius multifiliis (Ich) theronts and trophonts, and the immune response and host protection against both homologous and heterologous serotypes of Ich were evaluated. Immunizations were done with two immobilization serotypes (ARS4 and ARS6) of live theronts by bath immersion (trial I) and with sonicated trophonts by intraperitoneal (i.p.) injection (trial II). Cutaneous and serum antibody titres against Ich following immunization were measured and survival of catfish was determined after theront challenge. Theronts were immobilized by the antiserum from fish immunized with homologous theronts or trophonts, but not by the serum of fish immunized with the heterologous serotype. Serum from fish immunized by immersion with live theronts showed higher enzyme-linked immunosorbent assay titres against both homologous and heterologous serotypes than fish immunized by i.p. injection of trophonts. Channel catfish immunized by immersion with live theronts or by i.p. injection with sonicated trophonts developed an immune response against Ich and provided cross-protection against challenge from both serotypes (ARS4 and ARS6) of the parasite. Sonicated trophont antigens in aqueous solution by i.p. injection could stimulate an immune response in fish, but the immunity was of short duration.

Ophryoglena hemophaga n. sp. (Ciliophora: Ophryoglenidae): a parasite of the digestive gland of zebra mussels Dreissena polymorpha.

Ophryoglena hemophaga n. sp. is described from a freshwater Dreissena polymorpha population in the Rhine delta of the Netherlands. This is the first ophryoglenine species (order Hymenostomatida, suborder Ophryoglenina) recorded as a molluscan parasite. As is typical of ciliates in the suborder Ophryoglenina, O. hemophaga exhibits a polymorphic life history with cystment and reproduction by palintomy. Trophonts were observed within digestive gland lumina, and zebra mussel hemocytes were present in some of their digestive vacuoles. The presence of a single, longitudinal tract of multiple contractile vacuoles represents its most unique feature and distinguishes it from all other described Ophryoglena spp. The number of somatic kineties of O. hemophaga (range 100 to 124) is also distinguishing as it is one of the lowest for [corrected] an Ophryoglena sp. Other characteristics of this species include: ovoid to elongate trophonts 96 to 288 microm in length, with an elongate macronucleus 41 to 65 microm in length; tomonts 50 to 150 microm in diameter producing a clear mucous cyst envelope, whose thickness is approximately half of the tomont diameter; elongated theronts 96 to 131 microm in length which emerge after 1 to 3 cell divisions taking 36 to 48 h at 20 +/- 3 degrees C. Protomonts and theronts are, respectively, negatively and positively phototactic--characteristics that likely aid in maintenance of infection in zebra mussel populations.

Immunisation of channel catfish, Ictalurus punctatus, with Ichthyophthirius multifiliis immobilisation antigens elicits serotype-specific protection.

Surface immobilisation antigens (i-antigens) were purified from two strains of Ichthyophthirius multifiliis (NY1 and G5) that represent different i-antigen serotypes, namely A and D, respectively. The efficacy of the purified antigens as subunit vaccines was then tested in challenge studies using parasites of the homologous or heterologous serotype. Three groups of juvenile channel catfish (70 animals per group) were immunised with i-antigens from either the G5 or NY1 isolates, or with bovine serum albumin (BSA) as a control. Proteins were injected intraperitoneally (i.p.) at a dose of 10 microg/fish with complete Freund's adjuvant on day 1, followed by a second injection in incomplete Freund's adjuvant on day 15. Fish immunised with the purified i-antigens developed high titres of serum immobilising antibodies whereas sera from BSA-injected control fish did not. Fish antisera immobilised parasites of the homologous, but not the heterologous strain, and recognised the corresponding i-antigens on Western blots run under non-reducing conditions. On day 36, each group was divided into two subgroups (n=30). One subgroup was challenged with G5 parasites, and the other was challenged with NY1 parasites. When challenged with G5 parasites, 70% of fish immunised with the G5 i-antigens survived. When challenged with NY1 parasites, 33.3% of fish immunised with the NY1 i-antigens survived. All BSA-injected control fish died, as did all fish injected with the purified antigens and challenged with the non-homologous parasite strain. Statistical analyses indicated significant differences among test and control groups with regard to the mean days to death (MDD). While the results of these studies clearly support a role for i-antigens in protection, active immunity in response to natural infection is not serotype-specific. The utility of i-antigens, as well as the existence of other potential vaccine candidates for the prevention of 'white-spot' disease, are discussed.

Variation in primary sequence and tandem repeat copy number among i-antigens of Ichthyophthirius multifiliis.

The immobilization antigens (i-antigens) of Ichthyophthirius multifiliis are potential vaccine candidates for the prevention of 'white spot' disease in freshwater fish. These antigens vary with respect to antigenicity and molecular mass, and at least five i-antigen serotypes have been identified among parasite isolates thus far. In previous studies, the gene and corresponding cDNA encoding a approximately 48 kDa i-antigen from parasite isolate G1 (serotype A), had been cloned and sequenced. We now report on the isolation of two new genes, designated IAG52A[G5] and IAG52B[G5], encoding approximately 52/55 kDa i-antigens from a parasite isolate representing a different serotype, namely, D. Based on their deduced sequences, the approximately 52/55 kDa gene products have the same structural features as the 48 kDa protein including hydrophobic N- and C-termini, periodic cysteine residues with the potential for metal binding, and tandemly repetitive amino acid sequence domains that span their length. Nevertheless, the products of these genes vary in their tandem repeat copy number, and share only approximately 50% homology overall. When expressed in heterologous systems, the products of the newly described genes react strongly with monospecific polyclonal antisera against the i-antigens of serotype D and are clearly i-antigens. It would nevertheless appear that mRNA transcripts from the two genes are present at widely different levels within parasites themselves. Analysis at the protein level using 2-D SDS-PAGE would further suggest that multiple i-antigens are expressed within the same serotype at any given time.

Characterization of histone H3/H4 gene region and phylogenetic affinity of Ichthyophthirius multifiliis based on H4 DNA sequence variation.

We sequenced the amino-terminal third of the histone H3 and H4 genes and the intergenic region from Ichthyophthirius multifiliis. Fourteen recombinant clones of 646 bp were sequenced and the level of sequence variation detected among these clones was similar to that reported among closely related species of Tetrahymena and to levels of sequence variation detected within other ciliates. The intergenic region is 417 bp and approximately 92% AT rich, making it the longest and most AT-rich ciliate H3/H4 intergenic region yet identified. Similar to Tetrahymena, the intergenic region of Ichthyophthirius contains two CCAAT regions arranged in a complementary orientation. A neighbor-joining tree was constructed based on nucleotide sequence variation among H4 genes to evaluate evolutionary relationships within and among six classes of Ciliophora. The single shortest neighbor-joining tree depicted a sister-group relationship of Ichthyophthirius with taxa of Tetrahymenina, thereby supporting monophyly of Oligohymenophorea.

Phylogeny of the fish parasite Ichthyophthirius and its relatives Ophryoglena and Tetrahymena (Ciliophora, Hymenostomatia) inferred from 18S ribosomal RNA sequences.

Phylogenetic relationships within the subclass Hymenostomatia were inferred from the comparisons of three new SSrRNA gene sequences from Ichthyophthirius multifiliis (1,751 bp), Ophryoglena catenula (1,748 bp), and Tetrahymena corlissi (1,753 bp). Using maximum-parsimony and distance-matrix methods, Ichthyophthirius and Ophryoglena were consistently paired and formed a sister group to the tetrahymenines, consistent with their placement in the Ophryoglenina. Tetrahymenids formed a monophyletic group that was divided into main lineages: T. corlissi diverged from the base of the lineage that included T. thermophila.

Serotypic variation among isolates of Ichthyophthirius multifiliis based on immobilization.

Efforts have been made to determine whether surface antigens could be used as biochemical markers to define strain differences in the parasitic ciliate Ichthyophthirius multifiliis. In previous studies, a wild-type isolate designated G1 was found to have surface proteins analogous to the immobilization antigens of Paramecium and Tetrahymena; rabbit antiserum against this strain immobilizes homologous cells in vitro. It has now been shown for two additional Ichthyophthirius isolates (designated G1.1 and G2) that immobilization antigens are both present and serologically distinct. Proteins of similar size, which cross-react in Western blots with rabbit antisera against immobilization antigens of the G1 strain, are nevertheless found in the G1.1 and G2 isolates. As shown by Southern blotting analysis, the G1.1 and G2 strains also contain genomic DNA sequences which hybridize with an immobilization antigen cDNA from G1 when probed under conditions of reduced stringency. The serotypic differences in immobilization between I. multifiliis isolates appear to be stable over time and provide a means of discriminating strains. In addition to providing a basis for comparative studies, the work described here has implications for the development of vaccines against this important fish parasite.