A gene-expression oligo microarray for the three developmental stages of Ichthyophthirius multifiliis

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.