Project description:The protozoan Ichthyophthirius multifiliis (Ich) is a eukaryotic ciliate parasite of freshwater fish. Ich causes ichthyophthiriosis or ‘white spot disease’ characterized by white cysts covering the host skin and gills. The parasite is responsible for high mortalities and severe economic losses to farmed species as well as to ornamental species of fish. Despite the global importance of Ich, little is known about the genetic processes underlying its development and infectivity. Ich has three main life-stages, an infective theront, a parasitic trophont, and a reproductive tomont. To compare gene expression among Ich life-stages, oligonucleotide microarrays were constructed and utilized. All publicly-available Ich ESTs (~35K) were clustered to generate 9,129 unique consensus sequences represented as probes on custom microarrays produced in coordination with Roche NimbleGen. To facilitate comparative genomic analysis and to potentially increase gene content through cross-hybridization, gene coding sequences of related protozoans Tetrahymena thermophila and Plasmodium falciparum were also added to the microarrays. Gene expression was analyzed in samples taken from each of the three Ich life-stages. The results of this study will add in the understanding of protozoan global gene regulation and biology and should aid in the development of strategies aimed at the control of this important fish parasite. Submitted is a nine chip oligo array design using 385 K Nimblegen arrays. A total of nine microarrays were used for the experiment: three replicates from each of the three Ich life-stages (tomont, trophont, and theront life-stages). Probes were designed using 9,129 unique Ich ESTs (clustered contigs and singletons) as well as 26,273 Tetrahymena thermophila and 5,184 Plasmodium falciparum coding sequences. The probe design strategy was to create 12 60-mer oligonucleotide probes per I. multifiliis sequence, and 10 60-mer oligonucleotide probes for both T. thermophila and P. falciparum sequences. Total RNA was isolated in triplicate from the three life-stages of I. multifiliis and submitted to Nimblegen for labeling, hybridization, and scaning.

Project description:The protozoan Ichthyophthirius multifiliis (Ich) is a eukaryotic ciliate parasite of freshwater fish. Ich causes ichthyophthiriosis or ‘white spot disease’ characterized by white cysts covering the host skin and gills. The parasite is responsible for high mortalities and severe economic losses to farmed species as well as to ornamental species of fish. Despite the global importance of Ich, little is known about the genetic processes underlying its infectivity. Ich has three main life-stages, an infective theront, a parasitic trophont, and a reproductive tomont. Further, Ich has been demonstrated previously to display a loss of infectivity as the number of lab-passages on a fish increase, presumably relating to senescence of the organism. To compare gene expression among two of the three Ich life-stages (the tomont and trophont life-stages) at different passages, oligonucleotide microarrays were utilized. Gene expression was analyzed in samples taken from two of the three Ich life-stages (the tomont and trophont life-stages) at the first serial passage on channel catfish in the lab (P1), and at serial passage 100 (P100). The results of this study will add in the understanding of protozoan global gene regulation and biology and should aid in the development of strategies aimed at the control of this important fish parasite. Submitted is a 12 chip oligo array design using 385 K Nimblegen arrays. A total of 12 microarrays were used for the experiment: three replicates from two of the three Ich life-stages (tomont and trophont life-stages) at serial passage #1 (P1) and serial passage #100 (P100). Probes were designed using 9,129 unique Ich ESTs (clustered contigs and singletons) as well as 26,273 Tetrahymena thermophila and 5,184 Plasmodium falciparum coding sequences. The probe design strategy was to create 12 60-mer oligonucleotide probes per I. multifiliis sequence, and 10 60-mer oligonucleotide probes for both T. thermophila and P. falciparum sequences. Total RNA was isolated in triplicate from the three life-stages of I. multifiliis and submitted to Nimblegen for labeling, hybridization, and scaning. This microarray study is based on the GPL9449 platform built for the developmental stages of the parasite.

Project description:Purpose: a transcriptomic analysis was performed to extend our understanding on the immune response picture of rainbow trout exposed to I. multifiliis. Methods: Gill samples were collected from fish in each tank (control and infected group) at day 8 after infection. Total RNA was extracted using RTN350 (Sigma-Aldrich), according to the manufacturer’s instruction and subsequently, DNase treated with DNase I (Thermo Scientific, USA). Quality and integrity of the total RNA were checked on an Agilent Bioanalyzer 2100 total RNA Nano series II chip (Agilent, Amstelveen, Netherlands). Illumina RNAseq libraries were prepared from 500 ng total RNA using the Illumina TruSeqTM Stranded mRNA LT Sample Prep Kit according to the manufacturer’s instructions (Illumina Inc. San Diego, CA, USA). All RNAseq libraries (150-750 bp inserts) were sequenced on an Illumina HiSeq2500 sequencer as 1 x 50 nucleotides single-end reads according to the manufacturer’s protocol. Image analysis and base calling were done using the Illumina pipeline. Reads were aligned to the Rainbow trout reference genome (http://www.genoscope.cns.fr/trout/data/) using TopHat (version 2.0.5) and on average 53.4% of the RNAseq reads could be mapped. The resulting files were filtered using SAMtools (version 0.1.18) to exclude secondary alignment of reads. For statistical comparison of gene expression levels between groups, aligned fragments per predicted gene were counted from SAM alignment files using the Python package HTSeq (version 0.5.3p9). To make comparisons across samples possible, these fragment counts were corrected for the total amount of sequencing performed for each sample. As a correction scalling factor, we employed library size estimates determined using the R/Bioconductor (release 2.11) package DESeq. Read counts were normalized by dividing the raw counts obtained from HTSeq by its scale factor. Correction for false positives is included in the statistical analysis of gene expression through DESeq. The cut-off for significance was set to adjusted p<0.05 and at least 2-fold change. Results: a transcriptomic analysis was performed on infected rainbow trout gills and it showed that a total of 3,352 (7.2%) out of 46,585 identified genes were revealed significantly expressed after parasite infection. Of differentially expressed genes, 1.796 genes were up-regulated and 1.556 genes down-regulated. These were classified into 61 Gene Ontology (GO) terms and mapped to 282 reference canonical pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Infection of I. multifiliis induced a clear differential expression of immune genes, related to both innate and adaptive immunity. A total of 268 (6.86%) regulated genes was known to take part in 16 immune-related pathways. These involved pathways related to the innate immune system such as Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration. Conclusion: a transcriptomic profile of rainbow trout gills exposed to the parasitic I. multifiliis was reported for the first time. A total of 3,355 differentially expressed unigenes were identified. Of these were 1,184 unigenes (mapped to 952 genes) annotated 282 KEGG pathways and 268 unigenes were associated with 16 immune pathways. Most unigenes were related to innate immune system pathways (Chemokine signaling pathway, Platelet activation, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, and Leukocyte transendothelial migration) although a number of unigenes was related to adaptive responses (antigen processing and presentation, T and B cell receptor signaling pathway). The present study gave a far better resolution of the immune response of rainbow trout exposed to a parasitic protozoan than has ever been presented previously. The identification of a series of immune genes involved in several but important was useful for understanding of immune mechanism of the rainbow trout responding to the parasite I. multifiliis. Our results provide tools to link innate and adaptive immune elements in the process and present basic information which will be useful in the future studies related to immunoprophylaxis.

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Ichthyophthirius multifiliis strain:G5

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