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Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)
Published on: October 7, 2012
Transcriptional profiling of stage specific gene expression in the parasitic ciliate Ichthyophthirius multifiliis
Donna M Cassidy-Hanley1, Marie-Michèle Cordonnier-Pratt, Lee H Pratt
1Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, United States. dmc4@cornell.edu
Abstract:
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.
Insights
This study generated gene expression data for the fish parasite Ichthyophthirius multifiliis (Ich). This resource aids in understanding Ich virulence and developing treatments for this devastating fish pathogen.
Area of Science:
- Ichthyophthirius multifiliis (Ich) research
- Fish parasitology
- Genomics and transcriptomics
Background:
- Ichthyophthirius multifiliis (Ich) is a major protozoan pathogen impacting freshwater fish globally.
- Limited understanding of Ich genes hinders development of effective treatments and vaccines.
- The parasite's inability to be cultured in vitro complicates research.
Purpose of the Study:
- To generate a genomic resource for Ichthyophthirius multifiliis by creating EST libraries from key life cycle stages.
- To identify genes involved in parasite virulence, differentiation, and growth.
- To provide a foundation for developing novel control strategies against Ich infections.
Main Methods:
- Expressed Sequence Tag (EST) libraries were created from infective theronts and host-associated trophonts.
- Non-normalized ESTs (25,084 total) were generated and analyzed using cluster analysis to identify unique transcripts (UniScripts).
- BLASTX comparisons and Gene Ontology (GO) analysis were performed to annotate identified transcripts.
Main Results:
- 5311 unique transcripts (UniScripts) were identified, with over half of the expected protein-coding genes represented.
- 3694 significant BLASTX hits were found, and 1178 transcripts were annotated using GO analysis.
- Stage-specific gene expression patterns were observed, correlating with the metabolic activities of theront and trophont stages.
Conclusions:
- The generated genomics data provides a valuable resource for understanding Ichthyophthirius biology.
- This study lays the groundwork for identifying potential drug targets and vaccine candidates for controlling Ich.
- Understanding stage-specific gene expression is crucial for developing targeted interventions against this fish pathogen.
