Fig 1.
Schematic representation of the respiratory chain in animal mitochondria.
The mitochondrial respiratory chain (MRC) in the inner mitochondrial membrane is formed by four main multi-subunit complexes numbered from I to IV. The presence of mitochondrial alternative oxidase (AOX) is postulated for invertebrates with exception of insects and lancelets. Electrons released due to oxidation of metabolites are transported by MRC to oxygen and this generates proton gradient due to proton pumping by the MRC complexes I, III and IV from the matrix to the intermembrane space (IMS). The proton gradient is then used among others to feed ATP synthesis by the ATP synthase. AOX introduces a branch into MRC localized at the ubiquinone/ubiquinol (Q) pool. This 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 latter forming together the MRC cytochrome pathway. BHAM, antimycin A and KCN are known inhibitors of AOX, the MRC complex III and IV, respectively.
Fig 2.
Toxicity in vivo test indicates AOX contribution to functioning of H. exemplaris specimens in the absence and in the MRC cytochrome pathway inhibition.
Adult active specimens of a comparable body length and cleaned of debris were treated with KCN (1 mM) and BHAM (3 mM) in different configurations. Animals were observed after 30 min, 45 min and 2 h, and the medium was not replaced till the end of the test. The upper and lower parts of the figure provide graphic representations and images of the treated animals’ appearance indicating their body shape changes co-occurring with mobility changes, respectively. The data represent two independent repeats of the test lasting for 2 h and each tested group consisted of 20 specimens (see also Additional file 1 for the performed controls for the MRC cytochrome pathway inhibition by KCN and applied solvents as well as images of animals during and at the end of the test; see also Additional file 2 for recorded films presenting animals after 45 min of the treatment).
Fig 3.
Measurements of the rate of oxygen consumption by intact H. exemplaris specimens confirm the tardigrade AOX activity.
(A) The oxygen consumption rate traces when BHAM was added before or after KCN, determined using Hansatech Oxygraph Plus system. (B) Differences in the oxygen consumption rates calculated for data shown in (A). Basal denotes the oxygen consumption rate after addition of animals. The applied concentrations of inhibitors were as follows 3 mM BHAM and 1 mM KCN. Individual traces were recorded for 10 000 specimens. The statistical significance of results was tested using unpaired t-test for n ≥ 3. ** p < 0.01; *** p < 0.001; n/s, not statistically significant (see also Additional file 3 for data on the performed traces including AA addition as control for the MRC cytochrome pathway inhibition by KCN).
Fig 4.
The measurements of the mitochondrial inner membrane potential (Δψ) in intact H. exemplaris specimens confirm the tardigrade AOX activity.
(A) Representative fluorescence microscopic images of animals treated with KCN and BHAM applied separately or together in the absence of FCCP. (B) Quantification of TMRM signals obtained for animals treated with KCN and BHAM applied separately or together and subsequently treated with FCCP. The FITMRM represents the value of the net transmembrane potential and defined as the value of the TMRM fluorescence level under a given condition eliminated in the presence of FCCP. The applied concentrations of compounds were as follows: 2 μM TMRM, 3 mM BHAM, 1 mM KCN and 10 μM FCCP. The statistical significance of results was tested using unpaired t-test for n = 24. *** p < 0.001; n/s, not statistically significant.