Currently, it is suggested that the mitochondrial respiratory chain (MRC) of most invertebrates contains the alternative oxidase (AOX), an enzyme which provides a secondary oxidative pathway to the classical cytochrome pathway (e.g. ) but till now, natively expressed AOX level and activity have not been detected simultaneously in intact animals. Since the enzyme is not present in mitochondria of vertebrates, it is hypothesized that AOX-based respiration was critical to the evolution of animals by enabling oxidative metabolism during the transition to a fully oxygenated Earth . Thus, AOX may be treated as an adaptation to oxygen shortage which, in present-day organisms, may be caused by MRC deficiency or blockage. This in turn implies AOX application in treatment of mitochondrial diseases. As summarized by Saari et al. , the diseases may range from primary mitochondriopathies to common disease entities where mitochondrial disruption is due to ischemia/reperfusion injury, oxidative or proteotoxic stress, toxic damage or other external causes. However, the relevant data has been obtained by heterologous AOX expression in cells and animals that do not have native AOX although it appears to be essential for understanding of AOX contribution to animal physiology and consecutive development of AOX-based therapeutic strategies. The AOX activity can be understand by detailed investigation of effects imposed by heterologous AOX expression on physiological responses of model organisms under stressful environmental conditions . However, the research could be greatly simplified by application (as a model) of animals natively expressing AOX.
According to the definition, AOX is the mitochondrial inner membrane enzyme introducing a branch into the canonical animal MRC formed by four main multi-subunit complexes numbered from I to IV (Fig. 1). The branching occurs before the MRC complex III, at the ubiquinone/ubiquinol pool, and results in transferring of electrons to oxygen with sustained proton pumping by the MRC complex I but without proton pumping by the MRC complexes III and IV. The transfer is antimycin A (AA)- and cyanide-insensitive because AOX is not inhibited by AA and cyanides which are frequently used as inhibitors of the MRC complexes III and IV, i.e. ubiquinol–cytochrome c reductase and cytochrome c oxidase, respectively (e.g. [1, 4–8]). Consequently, proton-pumping by MRC is confined to complex I which results in cyanide- and AA-resistant respiration. Since proton gradient, formed due to proton pumping, is required for ATP synthesis, this enables fine tuning of ATP synthesis as well as modulation of reactive oxygen species (ROS) and calcium ion levels (e.g. ). Resultantly, AOX is regarded to provide metabolic plasticity being useful for adaptation to variable biotic and abiotic stress factors (e.g. [3, 7]). Moreover, it is suggested that AOX can bypass blockade or deficiency of the MRC complexes III and IV by restoring electron flow upstream of the MRC complex III. Consequently, AOX is considered to be an important element of a therapeutic strategy against impairment of these complexes [1, 3, 9–12].
At present genomes of about 160 animal species representing 16 phyla, from Placozoa to Chordata with exception of insects, lancelets and vertebrates, are proposed to contain AOX encoding gene [1, 12–14]. However, the enzyme functionality was only tested in the case of a few species. The best known examples are AOXs of the pacific oyster Crassostrea gigas  and the tunicate Ciona intestinalis . The first one was studied in isolated C. gigas mitochondria  and after heterologous expression in the yeast Saccharomyces cerevisiae  cells . The second one was heterologously expressed in mitochondria of cultured human cells [9, 20–21] as well as in the fruit fly Drosophila (Sophophora) melanogaster  [23–27] and mouse [9, 12, 28–29]. Additionally, some experimental data concerning AOX protein are available for crustaceans, the brine shrimp Artemia franciscana , the white shrimp Litopenaeus vannamei   and the marine copepod Tigriopus californicus  . In the case of both the shrimps the data concerns AOX activity in isolated mitochondria whereas in the case of the copepod amounts of AOX mRNA and protein in various life stages of the animal and under stress temperature conditions were studied.
As it has been summarized by Rajendran et al. , C. intestinalis AOX expressed in human cells, fruit flies and mice is not active when the MRC complex III and/or IV work properly, but inhibition or overload of these complexes triggers the enzyme activity. In the case of human cell lines, C. intestinalis AOX expression was shown to confer spectacular cyanide resistance of mitochondrial respiration and compensate for both the growth defect and the pronounced oxidant-sensitivity caused by the MRC complex IV deficiency [20–21]. The possibility to provide a complete or substantial protection against a range of phenotypes induced by the MRC complex IV deficiency or inhibition was also observed after expression of C. intestinalis AOX in mitochondria of fruit flies [23–25] and mice [28–29]. The same applies to the mouse MRC complex III deficiency [9, 11]. .
Thus, the available data indicates that functional studies of animal AOX are based on models that do not contain native AOX with exception of T. californicus, C. gigas, A. franciscana and L. vannamei enzyme. The molecular weight (MW) detected for the AOX protein reported for T. californicus is not convincing and its predicted amino acid sequence does not contain the proper C-terminus motif . In the case of C. gigas, AOX contribution to the oyster adjustments to short-term hypoxia and re-oxygenation was considered in isolated mitochondria  but the presented data do not allow for clear conclusions as the observed AA-resistant respiration was not shown to be eliminated by AOX inhibitor, for example by benzohydroxamic acid (BHAM) . Moreover, to observe the enzyme activity in the shrimp isolated mitochondria the authors applied an uncoupler to increase the recorded oxygen uptake rate . Resultantly, the measurements were performed for uncoupled mitochondria, i.e. in the absence of ubiquinone reduction to ubiquinol, regarded as crucial for AOX activity [9, 12, 24, 29]. Thus, data concerning animal AOX expression and activity regulation is still scarce (e.g. [1, 3, 34]).
In accordance with recent sequencing data, putative AOX encoding genes have been also identified in tardigrades [1, 36]. This concerns three species, namely Hypsibius exemplaris  (former Hypsibius dujardini ), Ramazzottius varieornatus  and Milensium inceptum  (former Milnesium tardigradum . Tardigrades are microscopic invertebrates with a body length ranging from 50 µm to 1200 µm although very few species exceed 800 µm. They inhabit marine, freshwater and terrestrial habitats, but all are regarded to be aquatic animals because they require a water-film surrounding the body to be active. The most known feature of tardigrades is the ability to enter cryptobiosis when environmental conditions are unfavorable (for review, see, for example, [40–46]).
Here, we set out to test whether AOX activity of H. exemplaris, as a part of the mitochondrial respiratory system, can be observed at the level of intact organisms. We also estimated whether the activity requires the MRC complex III and/or IV blockage. To that end, we determined the animal viability and respiration in the presence or absence of inhibitors of the MRC complexes and AOX as well as verified the AOX protein presence. The obtained data points at an emergence of a whole-animal model suitable to study activity and expression regulation of natively expressed animal AOX. According to our best knowledge we demonstrated, for the first time, that AOX activity of small aquatic invertebrates can be monitored by measurement of whole-body respiration due to registration of the oxygen uptake rate by intact specimen mitochondria. Moreover, we demonstrated that the enzyme contributed to animal functioning also in the absence of the MRC complex III and/or IV blockage previously described as precondition to observe animal AOX activity.