Identification of differentially expressed genes in parasitic phase Miamiensis avidus (Ciliophora: Scuticociliatia) using suppression subtractive hybridization

Abstract

Miamiensis avidus, a causative agent of scuticociliatosis in cultured marine fish, can live not only in seawater as a free-living organism but also in fish as a parasite. In this study, a cDNA library of representative mRNAs more specific to parasitic phase M. avidus was generated using suppression subtractive hybridization (SSH), and 520 clones selected from the SSH library were singlerun sequenced. The differential gene expression patterns were confirmed by semi-quantitative reverse-transcription PCR. Of the 510 SSH clones, 21 clones of 6 putative genes did not match sequences in the public database. The expectation values (E-values) of 117 clones encoding 9 putative genes were greater than 1 × 10 -5. The other 372 clones that met the criterion of E value <1 × 10 -5 were matched to 26 known sequences in the database. Genes associated with signal transduction, cell proliferation, membrane transportation, protein translocation, and transcription regulation were preferentially expressed in parasitic phase M. avidus. The differential gene expression may be needed for the ciliates to survive in the host fish, and the corresponding proteins might be used as antigen candidates for development of scuticociliatosis vaccines.

Molecular cloning and expression analysis of peptidase genes in the fish-pathogenic scuticociliate Miamiensis avidus

“…To obtain cell-fed ciliates, ciliates were inoculated in sufficiently grown CHSE-214 cells in routine MEM supplemented with 10% heat-inactivated FBS or in sufficiently grown CHSE-214 cells in filtered seawater supplemented with 10% heat-inactivated FBS or were intraperitoneally injected into olive flounder. The ciliates from different culture conditions were harvested using a method described previously [13]. Briefly, the ciliates were harvested by centrifugation at 200 × g for 5 min, and washed more than 3 times by centrifugation at 150 × g for 5 min in Hanks’ balanced salt solution (Sigma) or filtered seawater.…”