Differential expression of liver fluke β-tubulin isotypes at selected life cycle stages

We have shown that Fasciola hepatica expresses at least six beta-tubulins in the adult stage of its life cycle, designated F.hep-beta-tub1-6 (Ryan et al., 2008). Here we show that different complements of tubulin isotypes are expressed in different tissues and at different life cycle stages; this information may inform the search for novel anthelmintics. The predominant (as judged by quantitative PCR) isotype transcribed at the adult stage was F.hep-beta-tub1 and immunolocalisation studies revealed that this isotype occurred mainly in mature spermatozoa and vitelline follicles. Quantitative PCR indicated that changes occurred in the transcription levels of beta-tubulin isotypes at certain life cycle stages and may be of importance in the efficacy of benzimidazole-based anthelmintic drugs, but there were no significant differences between the triclabendazole-susceptible Leon isolate and the triclabendazole-resistant Oberon isolate in the transcription levels of each of the isotypes. When three well-characterised isolates with differing susceptibilities to triclabendazole were compared, only one amino acid change resulting from a homozygous coding sequence difference (Gly269Ser) in isotype 4 was observed. However, this change was not predicted to alter the overall structure of the protein. In conclusion, these findings indicate that there is tissue-specific expression of tubulin isotypes in the liver fluke but the development of resistance to triclabendazole is not associated with changes in its presumed target molecule. (C) 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.