Philasterides dicentrarchi causes a severe disease in turbot, and at present there are no drugs available to treat infected fish. We have previously demonstrated that, in addition to the classical respiratory pathway, P. dicentrarchi possesses an alternative mitochondrial respiratory pathway that is cyanide-insensitive and salicylhydroxamic acid (SHAM)-sensitive. In this study, we found that during the initial phase of growth in normoxia, ciliate respiration is sensitive to the natural polyphenol resveratrol (RESV) and to Antimycin A (AMA). However, under hypoxic conditions, the parasite utilizes AMA-insensitive respiration, which is completely inhibited by RESV and by the antioxidant propyl gallate (PG), an alternative oxidase (AOX) inhibitor. PG caused significantly dose-dependent inhibition of the in vitro growth of the parasite under normoxia and hypoxia and an over-expression of heat shock proteins of the Hsp70 subfamily. RESV and PG may affect the protective role of the AOX against mitochondrial oxidative stress, leading to an impaired mitochondrial membrane potential and mitochondrial dysfunction, which the parasite attempts to neutralize by increasing the expression of Hsp70. In view of the antiparasitic effects induced by AOX inhibitors and the absence of AOX in their host, this enzyme constitutes a potential target for the development of new drugs against scuticociliatosis.

Alternative oxidase (AOX), a ubiquinol oxidase, introduces a branch pathway to the respiratory electron transport chain (ETC), bypassing complexes III and IV and catalysing the cyanide-resistant reduction of oxygen to water without translocation of protons across the inner mitochondrial membrane. Thus, it functions as a non-energy-conserving member of respiratory ETC. Previous studies of AOX focused on plants and some fungi, whereas data on animals are limited. In this study, full-length AOX cDNA was cloned from the Echiuran worm, Urechis unicinctus, a marine benthic invertebrate. In addition, mRNA expression pattern of combined activity of cytochrome c oxidase (CCO) in the body wall and hindgut of the worm exposed in sulphide (50 µM and 150 µM) was measured. The results revealed that AOX mRNA expression increased in a time- and concentration-dependent manner in both tissues, was significantly increased at 48 h, and continuously increased with time. In contrast, the activity of CCO decreased significantly at 24 h and was inhibited at 48 h during exposure to 150 µM sulphide. The present data indicate the expression of AOX mRNA depended on the sulphide concentration present as well as being influenced by the physical condition of the worm, especially the CCO activity.

The functional mitochondrial protein alternative oxidase (AOX) gene was used as a marker to analyse the phylogenetic relationship between Cryptosporidium isolates. This gene was characterized, and the phylogentic tree was established from Cryptosporidium isolates and compared to those generated from 18S rRNA and heat-shock protein 70 (HSP70) gene sequences. The present trial aimed at finding out whether the AOX gene is suitable for phylogenetic analysis of the Cryptosporidium genus. The results revealed that the genus Cryptosporidium contained the phylogenetically distinct species C. parvum, C. hominis, C. suis and C. baileyi, which were consistent with the biological characterization and host specificity reported earlier. Cryptosporidium species formed two clades: one included C. hominis, C. suis, C. parvum cattle genotypes and C. parvum mouse genotype; and the other comprised C. meleagridis and C. baileyi isolates. Within C. parvum, both the mouse genotype and the pig genotype I (also known as C. suis) isolates differed from cattle and human (also known as C. hominis) genotypes, based on the aligned nucleotide sequences. The sequence identity of the AOX gene was higher between C. meleagridis and C. baileyi than between C. meleagridis and C. parvum. The phylogenetic trees showed that C. meleagridis was closer to C. baileyi than to C. parvum. This result was inconsistent with the phylogenetic analysis deduced from 18S rRNA and HSP70 gene sequences, respectively. The present results suggest that the AOX gene is not only equally suitable for the phylogenetic analysis of Cryptosporidium, but also provides an outstanding and new approach in determining Cryptosporidium heredity.