THERMOPHILA FOR NEW CRYOPRESERVATION METHODS

Ichthyophthirius multifiliis is an obligate protozoan parasite of freshwater fishes that has a complex developmental cycle. It has not been successfully cryopreserved, so management studies are restricted to parasites obtained during outbreaks or perpetuated by passage in live fishes. To overcome this serious limitation, free-swimming I. multifiliis parasites were tested in a cryopreservation protocol routinely used for a related ciliate, Tetrahymena. In this protocol, L multifiliis theronts retained infec- tivity for 3 days, although the protocol itself was ultimately lethal. Exposure of I. multifiliis and Tetrahymena thermophila to a battery of media and cryopreservative reagents showed that L multifiliis was less hardy than T. thermophila and likely had significant biological and cytoskeletal differences. No combination of reagents, media, freezing rates, or dilution media permitted cryopreservation of I. multifiliis parasites that could then undergo development or infect fish. However, a vitrification protocol was formulated using Ficoll, 1,2-propanediol, and N,N-dimethylacetamide from which intact cryopreserved theronts with some motility were recovered. Understanding the effects of these reagents may lead to both a cryopreservation method for L multifiliis and to improved understanding of the biology of ciliates. Ichthyophthirius multifiliis is an obligate protozoan parasite of freshwater fishes that undergoes developmental replication. When L multifiliis trophonts are feeding on the skin or fins of fish, the large (up to 500 pm) single-celled parasites are easily visible and lend the infection its name, white spot disease. In