Metrics details. 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.
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Metrics details. 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.
We report the sequencing, assembly and annotation of the Ich macronuclear genome. Compared with its free-living relative T. 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.
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. The ciliates are an ancient and diverse phylogenetic group related to the largely parasitic apicomplexans, but consisting mostly of free-living heterotrophs. Some ciliates, however, have adopted a parasitic lifestyle. By far the most important of these is Ichthyophthirius multifiliis which we will refer to by its common name of Ich , an endoparasite that causes white spot disease in freshwater fish [ 1 , 2 ].
With an extremely broad host-range, Ich is responsible for large-scale die-offs in natural populations and poses a significant threat to the growing worldwide aquaculture industry. Ich has a simple life cycle with no intermediate hosts Figure 1.
The free-swimming theront form invades the epidermis of susceptible fish, feeding on host tissue and growing up to 0. Host-associated trophonts become visible as individual white spots for which this disease is named.
A severe infection, particularly of the gills, results in asphyxiation and death. Although fish that survive infection are resistant to future challenge, prophylactic and therapeutic options remain extremely limited. Life cycle of Ich. Infective theronts bore through the surface mucus and take up residence within the epithelium of susceptible fish.
Theronts differentiate into feeding trophonts that grow and exit the host as tomonts within 4 to 7 days. Tomonts swim for a brief period and then adhere to an inert support where they secrete a gelatinous capsule. Tomonts divide within the capsule to form hundreds of tomites that differentiate into infective theronts within 18 to 24 hours at room temperature.
Theronts that fail to infect fish die within 1 to 2 days. Experimental studies of Ich are limited by its obligate parasitic lifestyle and lack of genetics, and therefore genomic approaches have been pursued to identify targets for therapy and vaccines. EST projects [ 3 , 4 ] have provided partial sequences of many protein-coding genes, but to gain a complete understanding of Ich's metabolism and virulence, it is necessary to obtain and analyze its full genome sequence.
Fortunately, Ich is fairly closely related to the model organisms Tetrahymena thermophila and Paramecium tetraurelia , whose macronuclear genomes have also been sequenced and annotated [ 9 — 11 ]. As shown here, comparative genomic analysis between these free-living species and the parasitic Ich reveals extensive genome reduction and modifications associated with the adoption of a parasitic lifestyle.
There are relatively few cases of which we are aware in which the genome sequences of a parasite and a closely related free-living species are both available for such comparative analysis for example, [ 12 ].
The ciliates may represent an excellent model system in which to explore the genomic consequences of this lifestyle switch, as it appears to have occurred in multiple independent cases within the genus Tetrahymena alone [ 13 ]. In addition, the genome of zebrafish, a model organism and representative host species, has been sequenced and thoroughly annotated [ 14 ]. Metabolic reconstruction of Ich and comparison with its host's metabolic pathways reveal potential targets for combating white spot disease.
We selected for sequencing an Ich strain of the D serotype, the most prevalent in known infections. To minimize locus heterozygosity, the culture was initiated from a single parasite. Like most ciliate species, Ich is binucleate, having a presumably diploid germline micronucleus MIC and a polyploid somatic macronucleus MAC. Because the MAC is the transcriptionally active nucleus, it was the focus of our sequencing efforts. By several independent methods in particular, comparison of Southern blot hybridization intensities to known amounts of cloned and genomic DNA with a unique sequence probe , we estimated the Ich MAC genome size to be about 50 Mb TG Clark, unpublished data , consistent with the 72 Mb and Mb genome sizes of P.
In all ciliates studied to date, the MAC is derived from a copy of the MIC during sexual conjugation in a process that involves genome-wide DNA rearrangements, including chromosome fragmentation and the elimination of most or all repetitive, transposon-related sequences [ 15 ]. Therefore, we anticipated the MAC genome to consist of multiple chromosomes T. In the Tetrahymena genome project, MACs were physically separated from MICs, resulting in an assembly largely free of MIC-specific sequence contamination, but similar nuclear separation techniques have not been developed for Ich.
Therefore, we relied on natural enrichment of the MAC genome; during the host-associated trophont stage of parasite development Figure 1 , endoduplication of the MAC genome occurs, leading to an estimated ploidy of up to 12, C, in the absence of MIC genome duplication [ 16 ]. Whole cell DNA was prepared from trophonts, taking care to minimize contamination from fish tissue or other associated microbes. Plasmid libraries were prepared with 2 to 4 kb and 4 to 6 kb insert size ranges for paired end sequencing.
However, initial quality control of these libraries revealed a high proportion of reads with higher than expected GC content Figure 2a and sequence similarity to bacteria. Further analysis [ 17 ] made it clear that this Ich strain harbors multiple species of intracytoplasmic bacteria which we will refer to as endosymbionts, although the nature of their relationship to their Ich host is unclear.
Efforts to purify or selectively clone Ich DNA were unsuccessful, and therefore we decided to shotgun sequence and assemble the mixture and separate the genomes bioinformatically. Presumably because of a bias against stable maintenance of AT-rich DNA in Escherichia coli , the plasmid libraries, especially the larger insert library, were heavily contaminated with bacterial sequence. We therefore focused most sequencing effort on pyrosequencing FLX Titanium supplemented by 2 to 4 kb paired end Sanger reads.
GC content of reads and scaffolds. All good quality Sanger and reads were assembled using Celera Assembler Version 5. As shown by Figure 2b , these scaffolds can be almost completely partitioned on the basis of average GC content into two separate bins, one representing the very AT-rich ciliate genome and the other representing the genomes of endosymbiotic bacteria. Assembly and analysis of the endosymbiont reads will be described in a separate paper.
We also searched for MIC contamination by BLAST-searching all contigs against known ciliate transposase sequences, but could detect no clear contamination. We cannot rule out the possibility of some MIC contamination, but available evidence suggests any such contamination would likely be less than that found in the initial T. We can also not entirely rule out the presence of contamination from other sources, such as bacterial symbionts or fish host, in the current assembly; further efforts in genome closure would likely be the most effective means of eliminating any such contamination.
The span of the final set of scaffolds was Because these sequences were represented among the reads in much higher numbers than the average locus, the Celera Assembler excluded them as repetitive DNA, but they were assembled 'manually' as described in the Materials and methods section.
During T. Palindrome formation and gene amplification are characteristic of a number of developmental and disease-associated genomic events [ 21 ]. The Ich rDNA is also a palindrome, but lacks a non-palindromic center. The non-telomeric portion of the molecule is 47, bp in length. Its structure and coding potential are described below. Linear mtDNAs found in ciliates and other species are capped by telomeres of varying lengths that consist of tandemly repeated units ranging up to bp in length [ 23 , 24 ].
It is thought that these telomeres are maintained by unequal crossing over, which keeps their repeat sequences homogeneous but allows the rapid accumulation of interspecies differences. The mitochondrial telomeres of several Tetrahymena species have been sequenced [ 25 , 26 ].
Each species' characteristic repeat unit is between 31 and 53 bp and shares no identifiable sequence similarity with the others, except for the most closely related species. In some species, each end of the mtDNA is capped by a repeat unit unrelated to that found at the other end. The Ich mtDNA is terminated by identical repeat units at each end, in an inverted orientation.
The repeat unit is bp in length, substantially longer than those of known Tetrahymena species. Following the initial assembly and partitioning, standard autoclosure efforts resulted in closing of the Ich intra-scaffold gaps. Additional assembly statistics are presented in Additional file 1. We plotted the mean depth of coverage for all Ich scaffolds against their sizes and found that they do not vary greatly, except for the expected stochastic variation found among the smallest scaffolds Additional file 2.
Thus, it appears that Ich chromosomes are also present in roughly equal copies, indicating that they are amplified to the same extent during trophont growth.
To gain a bigger picture of Ich MAC genome organization and lay the groundwork for future genome finishing efforts, we contracted with Opgen, Inc.
This map revealed 69 complete linear chromosomes and four partial single-ended chromosomes; these four most likely represent the individual ends of two complete chromosomes that the mapping algorithm was unable to join. Thus, it appears that the Ich MAC genome consists of 71 chromosomes of between 1. The total length of the optical restriction map was We next attempted to map as many of our scaffolds as possible to the optical restriction map on the basis of their predicted restriction digest fragmentation patterns using two independent algorithms, OpGen's MapSolver and SOMA [ 28 ].
Although the two algorithms generally agreed on the placement of larger scaffolds, there was disagreement in the placement of many smaller contigs with fewer diagnostic restriction sites. To evaluate scaffold placement further, we identified scaffolds that ended in multiple copies of the telomeric repeat unit GGGGTT, close to the expected total of excepting the rDNA. Of these, 46 were found on scaffolds not placed on the optical map by either algorithm Table S2b in Additional file 3.
The remaining 75 mapped scaffolds ideally should only be found at the ends of chromosomes and in their proper orientation, but we found that almost one in four was either misplaced internal to the optical chromosome map or in improper orientation. By extension, we expect that many of the other placements, especially those with lower confidence see Materials and methods were also misplaced. We examined the scaffold and found no indication of misassembly. Because the scaffold is large, contains a number of diagnostic restriction sites and maps uniquely by both algorithms, we suspect a misassembly of the optical map in this region resulted in its misplacement at a chromosome-internal position.
This was a region of relatively lower fragment coverage in the map, which may be related to the failure to assemble a complete chromosome. This optical mapping analysis provides substantial linkage information not discernible from the draft assembly and suggests multiple targets for future directed genome closure efforts by inter-scaffold PCR.
This method also proved to be an efficient means of determining the total number and sizes of Ich MAC chromosomes. Optical map coverage appeared to be generally equal across all chromosomes, consistent with our conclusion from sequence coverage data that Ich MAC chromosome copy number does not vary widely. We annotated the Ich mitochondrial genome to identify 41 protein-coding genes, five tRNA genes, one split gene for small subunit rRNA and two inverted terminal copies of the split large subunit rRNA gene.
Table 1 presents the full ordered list of predicted genes in the Ich mitochondrial genome in comparison with that of T. While the nuclear genome of Ich has undergone significant contraction compared to its free-living relative see below , the mitochondrial genome size, content and gene order are strikingly similar to those of Tetrahymena spp.
Between 38 and 41 depending on whether three poorly conserved gene pairs are indeed homologous of T. Ich also retains five of the eight predicted Tetrahymena tRNA genes, all in nearly the same locations and orientations, as well as the same configuration of rRNA genes, although the tRNA genes found between the split portions of the large subunit rRNA genes of Tetrahymena spp. Thus, parasitic adaptation by Ich has resulted in no significant minimization of mitochondrial functions compared to its free-living relatives.
The Fish Parasite Ichthyophthirius Multifiliis - Host Immunology, Vaccines and Novel Treatments
Ichthyophthirius multifiliis, the causative agent of white spot disease ichthyophthiriasis is a major burden for fish farmers and aquarists globally. The parasite infects the skin and the gills of freshwater fish, which may acquire a protective adaptive immune response against this disease, making vaccine strategies feasible. However, there is no prophylactic treatment available and repetitive treatments with auxiliary substances are needed to control the infection. Historically, a variety of drugs and chemicals have been used to combat the disease but due to changing regulations and recognition of carcinogenic and environmentally damaging effects the most efficient compounds are prohibited.
Only one species is found in the genus which also gave name to the family. The parasite can infect most freshwater fish species and, in contrast to many other parasites, shows very low host specificity. It penetrates gill epithelia, skin and fins of the fish host and resides as a feeding stage the trophont inside the epidermis. It is visible as a white spot on the surface of the fish but, due to its internal microhabitat, it is a true endoparasite and not an ectoparasite.