Identification of Genes Related to Resistance to Ichthyophthirius multifiliis Based on Co-expression Network Analysis in Grass Carp.

The ciliate protozoan Ichthyophthirius multifiliis is an essential parasite causing white spot disease in grass carp, leading to significant economic losses. Understanding the molecular basis of grass carp's response to I. multifiliis has important scientific and environmental values. The transcriptional network analysis offers a valuable strategy to decipher the changes in gene expression in grass carp infected with I. multifiliis. Our goal was to screen the genes and pathways involved in resistance to I. multifiliis in grass carp. The different traits exhibited by grass carp infected with I. multifiliis may be caused by the differences in gene expression among grass carp individuals. Herein, to reveal those resistance-associated genes against I. multifiliis infection, we performed RNA sequencing using weighted gene co-expression network analysis (WGCNA). The biological function analysis and hub gene annotation for highly relevant modules revealed that different pathogen recognition and clearance responses resulted in different resistance to I. multifiliis infection. Furthermore, gene enrichment analysis revealed that I. multifiliis invasion in the disease-resistant group mainly activated immune pathways, including scavenger receptor activity and kappa B kinase/NF-kappa B signaling. By the annotation of the highly correlated module of the hub gene, we revealed that the apoptosis and ribosome biogenesis-related genes were enriched in the disease-resistant grass carp. The results of the dark grey module showed that several genes were mainly enriched in the two-component system (ko02020) and steroid biosynthesis (ko00100), suggesting that they are resistance-associated and energy metabolism-associated genes. In the disease resistance group, hub genes mainly included Nlrc3, fos, AAP8, HAP2, HAX, cho2, and zgc:113,036. This study revealed the gene network associated with disease resistance after I. multifiliis infection. The disease resistance-related pathways and central genes identified in this study are candidate references for breeders breeding disease-resistant. The results of this study may also provide some references for the development of drugs to antagonize I. multifiliis infection.

Integrated morphological and transcriptome profiles reveal a highly-developed extrusome system associated to virulence in the notorious fish parasite, Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is an obligate parasitic ciliate that causes severe economic damage in aquaculture. The parasite contains numerous extrusive organelles (extrusomes) that assist in its pathogenesis and reproduction. However, the structure of these extrusomes and the molecular profiles involved in exocytosis remain unclear. In the present study, through comparative ultrastructural observations across the life cycle of I. multifiliis, we demonstrated that all three of its life stages (theront, trophont, and tomont) exhibited an abundance of extrusomes. In addition, two different types of extrusomes were identified according to their unique structures. Type I extrusomes (mucocysts) are crystalline, oval-shaped, 0.7-1.4 × 0.6-1.1 µm, and distributed as "rosettes" below the trophont membrane. Type II extrusomes, 2.0-3.0 × 0.2-0.3 µm, are rod-shaped with tubular cores and identified as toxicysts, the aggregation of which in the anterior part of the theront and cortex of the trophont revealed their potential roles in I. multifiliis invasion. This was confirmed by our transcriptome investigations of the three stages of I. multifiliis, which revealed that a set of genes involved in proteolysis and DNA/protein biogenesis was highly expressed in the theront and trophont. Furthermore, to map the molecular mechanisms of extrusome release, we characterized 25 Rab family genes in I. multifiliis and determined their expression profiles across the life cycle, reflecting the distribution patterns of the two extrusomes. Collectively, our data revealed that a highly developed extrusome system could play a potential role in the virulence of I. multifiliis, which facilitates a better understanding of the parasite's development.

Integration of transcriptomic and metabolomic profiling of encystation in Cryptocaryon irritans regulated by rapamycin.

Encystation in Cryptocaryon irritans is a fundamental process for environmental resistance and development. Autophagy participates in the encystation of ciliates, and rapamycin can induce autophagy in the cells. A set of genes and metabolites related to autophagy and encystation are highly elaborative. The existence of these genes and metabolites and their role are well characterized. However, little is known about their role in protozoans such as ciliates. The newly produced C. irritans protomonts were exposed to an optimal concentration of rapamycin (1400 nM), and the survival, encystation, microstructure/ultrastructure, transcriptomic and metabolomic profile in treated and control protomonts were investigated. The results showed that exposure of protomonts to rapamycin at 4 h significantly lowered the survival and encystation rates to 91.62 % and 98.44 % compared to the control group (100 %, p ≤ 0.05). Morphological alterations observed in light microscopy and transmission electron microscopy (TEM) demonstrated that the drug significantly changed cell symmetry by causing the formation of various autophagic vacuoles/vesicles. The transcriptome sequencing of rapamycin-treated protomont revealed that 2249 (1837 up-regulated and 977 down-regulated) differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 226 DEGs were successfully annotated in 21 pathways (p˂0.05), including most enriched pathways apoptosis and phagosome with 25 and 24 DEGs, respectively. Most unigenes were assigned to autophagy-related pathways; 24 DEGs were classified into phagosomes, and 15 DEGs were assigned to lysosome pathways. Cytoskeleton and cell progression-associated genes were down-regulated. Besides, cell death-inducing proteins were up-regulated. The metabolomic analysis revealed exposure to rapamycin treatment enhanced protomont metabolites, including L-Cysteine, which is related to autophagy. Rapamycin had influenced the gene and metabolites of protomont; activating autophagy with inhibition of mechanistic target of rapamycin, (mTOR). The process negatively influences protomont morphology, encystation, and survival. Further autophagy-related gene silencing can be investigated via genome sequencing of C. irritans to study encystation.

Genetic diversity of Ichthyophthirius multifiliis (Fouquet, 1876) infecting farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792) in Turkey.

We assessed genetic diversities among Ichthyophthirius multifiliis (Ich) field isolates collected from farmed rainbow trout (Oncorhynchus mykiss) in Turkey. The overall prevalence of Ich was 35.3% (634/1798). Five novel Ich genotypes (ImulTR1 and ImulTR3-ImulTR6) were described based on mitochondrial cox-1 and nad1_b genes. The remaining genotype ImulTR2 was identical to the previously reported NY3 (or Ark9 and TW7) genotype from the United States and South Asia. Phylogenetic analysis indicated Turkish Ich isolates separated genetically into at least four distinct groups. Our study presents the first data on the genotypes of Ich in Turkey. We also provide evidence for the wide distribution of the NY3 genotype (or Ark9 and TW7) from the United States and South Asia to Turkey. Genetic diversities within the mitochondrial genes provided adequate resolution for describing novel genotypes and identifying the known genotype within Turkish Ich isolates. Description of the Ich genotypes allows for tracking of pathogen genotypes worldwide. Thus, we can better understand the connections between Ich outbreaks in the fisheries aquaculture.

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.

Epidemiology and identification of two species of Chilodonella affecting farmed fishes in China.

The genus Chilodonella includes free-living ciliated protozoa as well as pathogenic species for freshwater fish, with Chilodonella hexasticha and Chilodonella piscicola being the most important ones. These parasites cause outbreaks with high mortalities among farmed freshwater fishes with great economic losses. There are few reports of these species in China, and their identification has been based mostly on their morphological characteristics. In the present work, the parasites causing five outbreaks occurring in China between 2014 and 2017 have been identified by morphological and genetic analysis. We provide the first records of Ctenopharingodon idella and Siniperca chuatsi as hosts of C. hexasticha, and of Procypris rabaudi and Schizothorax wangchiachii as hosts of C. piscicola. There are no differences in the gross pathological findings produced by C. hexasticha and C. piscicola, consisting in desquamation and necrosis of epithelial cells in the skin and gills and in severe fusion of gill lamellae. However, both species differ in their geographic distribution: C. piscicola was found in farms located at altitudes over 1500 m above sea level and with a water temperature ≤18 °C, while C. hexasticha was found in farms located at altitudes under 50 m above sea level and with a water temperature ≥21 °C. Present results confirm that C. hexasticha and C. piscicola are two different species that can be differenced by their morphology; however, their biological variability may lead to erroneous identifications and the diagnosis should be preferably based in genetic analysis including nuclear LSU rDNA and mitochondrial SSU rDNA sequences.

High genetic diversities between isolates of the fish parasite Cryptocaryon irritans (Ciliophora) suggest multiple cryptic species.

The ciliate protozoan Cryptocaryon irritans parasitizes marine fish and causes lethal white spot disease. Sporadic infections as well as large-scale outbreaks have been reported globally and the parasite's broad host range poses particular threat to the aquaculture and ornamental fish markets. In order to better understand C. irritans' population structure, we sequenced and compared mitochondrial cox-1, SSU rRNA, and ITS-1 sequences from 8 new isolates of C. irritans collected in China, Japan, and Taiwan. We detected two SSU rRNA haplotypes, which differ at three positions, separating the isolates into two main groups (I and II). Cox-1 sequences also support the division into two groups, and the cox-1 divergence between these two groups is unexpectedly high (9.28% for 1582 nucleotide positions). The divergence is much greater than that detected in Ichthyophthirius multifiliis, the ciliate protozoan causing freshwater white spot disease in fish, where intraspecies divergence on cox-1 sequence is only 1.95%. ITS-1 sequences derived from these eight isolates and from all other C. irritans isolates (deposited in the GenBank) not only support the two groups, but further suggest the presence of a third group with even greater sequence divergence. Finally, a small Ka/Ks ratio estimated from cox-1 sequences suggests that this gene in C. irritans remains under strong purifying selection. Taken together, the C. irritans species may consists of many subspecies and/or syngens. Further work is needed to determine if there is reproductive isolation between the groups we have defined.

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.

Approaches towards DNA vaccination against a skin ciliate parasite in fish.

Rainbow trout (Oncorhynchus mykiss) were immunized with plasmid DNA vaccine constructs encoding selected antigens from the parasite Ichthyophthirius multifiliis. Two immobilization antigens (I-ags) and one cysteine protease were tested as genetic vaccine antigen candidates. Antigenicity was evaluated by immunostaining of transfected fish cells using I-ag specific mono- and polyclonal antibodies. I. multifiliis specific antibody production, regulation of immune-relevant genes and/or protection in terms of parasite burden or mortality was measured to evaluate the induced immune response in vaccinated fish. Apart from intramuscular injection, needle free injection and gene gun delivery were tested as alternative administration techniques. For the I-ags the complement protein fragment C3d and the termini of the viral haemorrhagic septicaemia virus glyco(G)protein (VHSV G) were tested as opsonisation and cellular localisation mediators, respectively, while the full length viral G protein was tested as molecular adjuvant. Expression of I-ags in transfected fish cells was demonstrated for several constructs and by immunohistochemistry it was possible to detect expression of a secreted form of the Iag52B in the muscle cells of injected fish. Up-regulations of mRNA coding for IgM, MHC I, MHC II and TCR ß, respectively, were observed in muscle tissue at the injection site in selected trials. In the spleen up-regulations were found for IFN-γ and IL-10. The highest up-regulations were seen following co-administration of I-ag and cysteine protease plasmid constructs. This correlated with a slight elevation of an I. multifiliis specific antibody response. However, in spite of detectable antigen expression and immune reactions, none of the tested vaccination strategies provided significant protection. This might suggest an insufficiency of DNA vaccination alone to trigger protective mechanisms against I. multifiliis or that other or additional parasite antigens are required for such a vaccine to be successful.

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.

Gene expression profiling of a fish parasite Ichthyophthirius multifiliis: Insights into development and senescence-associated avirulence.

The ciliate parasite Ichthyophthirius multifiliis (Ich) infects many freshwater fish, causing white spot disease that leads to heavy economic losses to aquaculture and ornamental industries. Despite its economic importance, molecular studies examining fundamental processes such as life stage regulation and infectivity have been scarce. In this study, we developed an oligo microarray platform using all available I. multifiliis expressed sequence tag (EST) information as well as probes designed through comparative genomics to other protozoa. Gene expression profiling for developmental and virulence factors was conducted using this platform. For the developmental study, the microarray was used to examine gene expression profiles between the three major life stages of Ich: infective theront, parasitic trophont, and reproductive tomont. A total of 135 putative I. multifiliis genes were found to be differentially expressed among all three life-stages. Examples of differentially expressed transcripts among life stages include immobilization antigens and epiplasmin, as well as various other transcripts involved in developmental regulation and host-parasite interactions. I. multifiliis has been shown to lose infectivity at later cell divisions potentially due to cellular senescence. Therefore, the microarray was also used to explore expression of senescence-associated genes as related to the passage number of the parasite. In this regard, comparison between tomont early and late passages yielded 493 differently expressed genes; 1478 differentially expressed genes were identified between trophont early and late passages. The EST-derived oligo microarray represents a first generation array of this ciliate and provided reproducible expression data as validated by quantitative RT-PCR.

Transcriptional profiling of stage specific gene expression in the parasitic ciliate Ichthyophthirius multifiliis.

The parasitic ciliate, Ichthyophthirius multifiliis (Ich), is among the most important protozoan pathogens of freshwater fish. Ichthyophthirius cannot be grown in cell culture, and the development of effective prophylactic and therapeutic treatments has been hampered by a lack of information regarding genes involved in virulence, differentiation and growth. To help address this issue, we have generated EST libraries from the two major stages of the parasite life cycle that infect and develop within host tissues. A total of 25,084 ESTs were generated from non-normalized libraries prepared from polyA+ RNA of infective theronts and host-associated trophonts, respectively. Cluster analysis identified 5311 unique transcripts (UniScripts), of which 2091 were contigs and 3220 singletons. Extrapolation of the data based on rates of EST discovery suggests that more than half the expected protein-coding genes of I. multifiliis are represented in this data. BLASTX comparisons against GenBank nr, UniProtKB (SwissProt and TrEMBL), as well as Tetrahymena thermophila, Plasmodium falciparum, and Paramecium tetraurelia protein databases produced 3694 significant (E-value ≤1e(-10)) hits, of which 1178 were annotated using gene ontology (GO) analysis. A high proportion of UniScripts (63%) showed similarity to other ciliate proteins. When combined with expression profiling data, GO ontology analysis of Biological Process, Cellular Component, and Molecular Function revealed interesting differences in gene families expressed in the two stages. Indeed, the most abundant transcripts were highly stage-specific and coincided with the metabolic activities associated with each stage. This work provides an effective genomics resource to further our understanding of Ichthyophthirius biology, and lays the groundwork for the identification of potential drug targets and vaccines candidates for the control of this devastating fish pathogen.

Elicited cross-protection and specific antibodies in Mozambique tilapia (Oreochromis mossambicus) against two different immobilization serotypes of Cryptocaryon irritans isolated in Hawaii.

The objective of this study was to determine whether immunization of Mozambique tilapia with different Cryptocaryon irritans i-antigen serotypes elicited cross-protection against challenge infection by both serotypes. Fish were directly exposed to live theronts of isolate W1 or isolate K1, that express different surface i-antigens. There was no significant difference in the number of trophonts infecting the fish between the two isolates, W1 and K1, following primary exposure. Serum from immunized fish exposed to live theronts showed higher immobilization titres and ELISA values against homologous isolates than to heterologous isolates after the primary exposure. However, mucus antibody did not immobilize theronts although the ELISA results clearly indicated that mucus antibodies recognizing C. irritans were generated. In a study with Western blot analyses, serum antibodies recognized only an antigen of the corresponding serotype and no proteins common to both serotypes were identified. Sequence analyses of 754 bases of rDNA nucleotide sequence including complete nuclear ribosomal ITS-1-5.8S rDNA-ITS-2 region were conducted and found to be identical for W1- and K1-isolates. These findings confirmed that both isolates were members of the species, C. irritans, and that rDNA analysis would not distinguish the two isolates. In conclusion, despite the fact that the immobilization assays and ELISA detected two serotypes in vitro, challenge assays provided evidence for only one type of C. irritans.

Generation and analysis of expressed sequence tags from the ciliate protozoan parasite Ichthyophthirius multifiliis.

BACKGROUND: The ciliate protozoan Ichthyophthirius multifiliis (Ich) is an important parasite of freshwater fish that causes 'white spot disease' leading to significant losses. A genomic resource for large-scale studies of this parasite has been lacking. To study gene expression involved in Ich pathogenesis and virulence, our goal was to generate expressed sequence tags (ESTs) for the development of a powerful microarray platform for the analysis of global gene expression in this species. Here, we initiated a project to sequence and analyze over 10,000 ESTs. RESULTS: We sequenced 10,368 EST clones using a normalized cDNA library made from pooled samples of the trophont, tomont, and theront life-cycle stages, and generated 9,769 sequences (94.2% success rate). Post-sequencing processing led to 8,432 high quality sequences. Clustering analysis of these ESTs allowed identification of 4,706 unique sequences containing 976 contigs and 3,730 singletons. These unique sequences represent over two million base pairs (~10% of Plasmodium falciparum genome, a phylogenetically related protozoan). BLASTX searches produced 2,518 significant (E-value < 10-5) hits and further Gene Ontology (GO) analysis annotated 1,008 of these genes. The ESTs were analyzed comparatively against the genomes of the related protozoa Tetrahymena thermophila and P. falciparum, allowing putative identification of additional genes. All the EST sequences were deposited by dbEST in GenBank (GenBank: EG957858-EG966289). Gene discovery and annotations are presented and discussed. CONCLUSION: This set of ESTs represents a significant proportion of the Ich transcriptome, and provides a material basis for the development of microarrays useful for gene expression studies concerning Ich development, pathogenesis, and virulence.

Non-invasive detection and quantification of the parasitic ciliate Ichthyophthirius multifiliis by real-time PCR.

The main parasitic threat to freshwater fish is the ciliate Ichthyophthirius multifiliis. We developed a real-time PCR assay using SYBR Green intercalating fluorescent dye for rapid detection and quantification of I. multifiliis. This non-invasive assay was based on the quantification of I. multifiliis free-swimming stages from filtered water samples, and thus made it possible to preserve host individuals. An alignment of 18S rDNA sequences of I. multifiliis and related species of the ciliate order Hymenostomatida was used to design amplification primers specifically targeting the I. multifiliis 18S rDNA gene. Different standard curves consisting of 2-fold serial dilutions of DNA extracted from 20, 60, 100 and 1000 I. multifiliis cells were constructed. The assay was able to detect less than 0.5 cell equivalent and showed a strong linearity (R2 = 0.984). Water samples were collected from 2 tanks containing heavily infected and apparently uninfected Carassius auratus specimens and were used to test this technique. Positive signals were obtained from water samples collected from both tanks, with a deduced concentration ranging from 3 to 58 I. multifiliis cells l(-1). The assay can detect low concentrations of the parasite in water, presumably corresponding to an early phase of the disease. It may, thus, be a valuable tool in assisting in the monitoring and control of ichthyophthiriasis in aquaculture.

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.