Transcriptome analysis of the Cryptocaryon irritans tomont stage identifies potential genes for the detection and control of cryptocaryonosis

Abstract

Background: Cryptocaryon irritans is a parasitic ciliate that causes cryptocaryonosis (white spot disease) in marine fish. Diagnosis of cryptocaryonosis often depends on the appearance of white spots on the surface of the fish, which are usually visible only during later stages of the disease. Identifying suitable biomarkers of this parasite would aid the development of diagnostic tools and control strategies for C. irritans. The C. irritans genome is virtually unexplored; therefore, we generated and analyzed expressed sequence tags (ESTs) of the parasite to identify genes that encode for surface proteins, excretory/secretory proteins and repeat-containing proteins.

Results: ESTs were generated from a cDNA library of C. irritans tomonts isolated from infected Asian sea bass, Lates calcarifer. Clustering of the 5356 ESTs produced 2659 unique transcripts (UTs) containing 1989 singletons and 670 consensi. BLAST analysis showed that 74% of the UTs had significant similarity (E-value < 10-5) to sequences that are currently available in the GenBank database, with more than 15% of the significant hits showing unknown function. Forty percent of the UTs had significant similarity to ciliates from the genera Tetrahymena and Paramecium. Comparative gene family analysis with related taxa showed that many protein families are conserved among the protozoans. Based on gene ontology annotation, functional groups were successfully assigned to 790 UTs. Genes encoding excretory/secretory proteins and membrane and membrane-associated proteins were identified because these proteins often function as antigens and are good antibody targets. A total of 481 UTs were classified as encoding membrane proteins, 54 were classified as encoding for membrane-bound proteins, and 155 were found to contain excretory/secretory protein-coding sequences. Amino acid repeat-containing proteins and GPI-anchored proteins were also identified as potential candidates for the development of diagnostic and control strategies for C. irritans.

Conclusions: We successfully discovered and examined a large portion of the previously unexplored C. irritans transcriptome and identified potential genes for the development and validation of diagnostic and control strategies for cryptocaryonosis.

Figure 1

Length of unique transcripts versus BLAST hit. The relationship between the length of the unique transcripts (UTs) and the number of UTs with BLAST hits.

Figure 2

Distribution of best BLASTX matches according to organisms. A diagrammatic representation of organism distribution according to UTs with the best match to the NCBI non-redundant protein database.

Figure 3

Phylogenetic analysis of C. irritans. A maximum parsimony tree inferred from the complete β-tubulin amino acid sequence of C. irritans and other alveolates, with Mus musculus as an outgroup. The I. multifiliis β-tubulin amino acid sequence was inferred from the EST dataset. The numbers below branches are the bootstrap values of 1000 iterations of the data file.

Figure 4

Proteins shared among C. irritans, T. thermophila and P. falciparum. Venn diagram summary of C. irritans translated UTs comparison with T. thermophila and P. falciparum peptide sequences. The numbers at the overlapping area represent matching peptides (BLASTX of E < 10^-5) in the relevant organism with the query 2659 translated UTs of C. irritans.

Figure 5

GO annotation. GO annotation was done with the Blast2GO tool. The level 2 GO terms which are (a) biological process, (b) molecular function, and (c) cellular component are shown.