Detection of heterologously expressed M. inceptum AOX activity in mitochondria of intact Saccharomyces cerevisiae cells
For heterologous expression of M. inceptum AOX, the coding sequence of the protein was identified by bioinformatics methods (for details see electronic supplementary material, figure S1 and figure S2). The expression in was perform by CRISPR/Cas9 and S. cerevisiae BY4741 strain (MATa, his3Δ, leu2Δ, met15Δ, ura3Δ) (EUROSCARF) transformation by electroporation (for details see electronic supplementary material, figure S3). Cells of the resulting BY4741 + AOX yeast strain (MATa, his3Δ, leu2Δ, met15Δ, ura3Δ, gal1Δ::Mi-AOX) were grown in YPG medium (1% yeast extract, 2% peptone, 3% glycerol, pH 5.5) containing a non-fermentable carbon source that requires functioning mitochondria [35]. The cells were grown to OD550 = 1, then galactose at a final concentration of 2% was added to induce and maintain AOX expression for 24 h. In parallel, control yeast cells were cultured in the absence of galactose. Then, yeast cells were washed twice with double distilled water (ddH2O), resuspended in 100 µL of YPG and quantified by OD550 measurement. For the same amount of cells, rates of oxygen uptake were determined in 1 mL of YPG using a Clark electrode (Hansatech Instruments). To determine bioenergetics parameters related to AOX activity, 1mM KCN (to inhibit the MRC complex IV, i.e., cytochrome c oxidase) and 3 mM BHAM (to inhibit alternative oxidase) were applied. The applied final concentrations of KCN and BHAM were verified experimentally to achieve saturation.
Detection of M. inceptum AOX presence in isolated S. cerevisiae mitochondria
Mitochondria of S. cerevisiae cells expressing AOX were isolated according to the published procedure [36]. Protein concentration was measured by the method of Bradford and albumin bovine serum (BSA), essentially fatty acid free, was used as a standard. The mitochondrial proteins were separated by SDS-PAGE in the presence of 6 M urea and gels were stained with Coomassie Brilliant Blue G-250.
Culture of M. inceptum
The initial specimens of M. inceptum were collected in a xerothermic habitat: mosses on a concrete wall in the city centre of Poznań in Poland (52°24'15"N, 16°53'18"E; 87 m asl). The extraction was performed by the standard method [37]. To maintain the culture, M. inceptum specimens were kept at 18 ºC in the darkness and at relative humidity (RH) of 40% (POL EKO KK 115 TOP+ climatic chamber) in covered Petri dishes (5.5 cm in diameter) with bottom scratched by sandpaper to allow tardigrade movement. Animals were coated with a thin layer of the culture medium being spring water (Żywiec Zdrój; Żywiec Zdrój S.A., Poland) mixed with ddH2O in 1:3 ratio. The culture medium was exchanged every week and the animals were fed with the nematode Caenorhabditis elegans Bristol N2 strain, and the rotifer Lecane inermis 1.A2.15 strain. The former was obtained from the Caenorhabditis Genetics Center at the University of Minnesota (Duluth, USA) and its culture was maintained by standard methods [38].
M. inceptum anhydrobiosis protocol
For tun formation, fully active (displaying coordinated movements of the body and legs) M. inceptum adult specimens of medium body length (approximately 500-550 µm) were extracted from the culture. After removal of debris, the animals were transferred to 3.5 cm (in diameter) covered and vented Petri dishes with bottom scratched by sandpaper. In each dish, 10 specimens were placed in 400 µl of the culture medium and then dehydrated. Each of the experimental groups included four to six dishes. This denotes, for a total of 40-60 specimens per experimental group (table 1). The dishes were allowed to dry slowly in the Q-Cell incubator (40-50% RH, 20 °C, darkness) for 72h. Tun formation was checked once per 24h period by a short observation (about 1 min) under an Olympus SZ61 stereomicroscope connected to an Olympus UC30 microscope digital camera. The dehydrated animals (tuns) were kept under the above conditions for 3, 30 or 60 days.
After 3, 30 or 60 days of the tun stage duration, the tun rehydration was performed by addition of 3 ml of the culture medium to each dish, and the tuns were transferred to small glass cubes and kept at room conditions (18 to 20 °C, 40-50% RH and light conditions regulated by seasonal changes in day/night cycle; it should be mentioned that according to our observations, photoperiod does not affect return of M. inceptum tuns to active life). The animals’ return to full activity was observed in the cubes at chosen time points (i.e., time windows: 10, 20, 30, 40, 60, 90, 120, 180, 240, 360, 480 and 1440 min) and for approximately 1 min under the stereomicroscope. The average number of specimens that recovered to full activity after 24 h (1440 min) from the onset of rehydration was defined as the final return to full activity.
BHAM, a known AOX inhibitor (prepared in methanol), and MitoTEMPO, a known mitochondria-specific superoxide scavenger (prepared in ddH2O), were added to the culture medium at the beginning of the dehydration or rehydration stage. The applied final concentrations of BHAM (0.1 and 0.2 mM) and MitoTEMPO (0.01 mM) were verified experimentally to avoid lethal effect. For that, we analysed animal mobility after addition of the compounds to the culture medium containing active animals. We assumed the absence of a lethal effect when approximately 90% of the treated animals moved their legs and body in a coordinated way 24 h after addition of the compounds. Because two different solvents were applied for BHAM and MitoTEMPO, two different controls were used in experiments: control 1 (C1) included animals dehydrated or rehydrated in the culture medium in the presence of suitably diluted methanol solution (0.3% v/v), and control 2 (C2) included animals dehydrated in the culture medium. To test for differences in the animals’ return to full activity, we compared the animals’ recovery at the applied time windows (10, 20, 30, 40, 60, 90, 120, 180, 240, 360, 480 and 1440 min) between experimental groups (table 1) and for different time spent in the tun stage. For animals treated with BHAM, during both dehydration and rehydration, we distinguished three experimental groups (C1, 0.1 mM BHAM and 0.2 mM BHAM) and for animals treated with MitoTEMPO during dehydration, we distinguished two experimental groups (C2 and 0.01 mM MitoTEMPO).
Table 1. Experimental groups applied in the studies. They included two different concentrations of BHAM during M. inceptum dehydration and rehydration, and one concentration of MitoTEMPO during M. inceptum dehydration (including proper controls) as well as different durations of the tun stage. The tested conditions are marked with the X.
TUN DURATION
(days)
|
EXPERIMENTAL GROUPS
|
DEHYDRATION
|
REHYDRATION
|
control 1 (0.3% v/v methanol)
|
0.1 mM BHAM
|
0.2 mM BHAM
|
control 2 (culture medium)
|
0.01 mM MitoTEMPO
|
control 1 (0.3% v/v methanol)
|
0.1 mM BHAM
|
0.2 mM BHAM
|
3
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
30
|
x
|
x
|
x
|
|
|
|
|
|
60
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
Chemicals
Manufacturers of the chemicals used are listed in electronic supplementary material, table S1.
Statistical analysis
Effects of BHAM and KCN on the rate of oxygen uptake displayed by S. cerevisiae cells were analysed by t-test.
For testing differences in number of tuns formed under different conditions (in the presence of MitoTEMPO or BHAM as well as for proper controls) one-way ANOVA [39] was applied.
To analyse the effect of BHAM presence during tun formation or rehydration and of MitoTEMPO presence during tun formation on anhydrobiotic animals’ recovery to full activity after different duration of the tun stage, Factorial ANOVA [39] was applied. For this analysis we used two grouping independent variables: (I) time windows of animal observations following rehydration and (II) the applied compound concentrations. The latter denotes two different concentrations of BHAM versus control conditions and one concentration of MitoTEMPO versus control conditions, where control conditions denote the absence of BHAM or MitoTEMPO. In this way we distinguished three experimental groups for testing of BHAM presence (C1, 0.1 mM BHAM and 0.2 mM BHAM) or two experimental groups for testing of the MitoTEMPO (C2 and 0.01 mM MitoTEMPO) (table 1). Thus, differences in animals’ return to full activity were tested between the mentioned two grouping independent variables: (I) 12 time windows (10, 20, 30, 40, 60, 90, 120, 180, 240, 360, 480 and 1440 min) and (II) three experimental groups distinguished for BHAM (C1, 0.1 mM BHAM and 0.2 mM BHAM) or (III) two experimental groups distinguished for MitoTEMPO (C2 and 0.01 mM MitoTEMPO). Because of the application of the two grouping variables, it was impossible to simply test the differences between each group, as the effect of multiple comparisons would decrease the biological value of the results [40]. We used the standard presentation of results for Factorial ANOVA analysis [39, 40]. For each model we calculated F-statistics and R2 as a measure of variance explained [40]. For ANOVA models, when F was statistically significant (i.e., α-probabilities (p) < 0.05), in the next step we deepened that analysis by determination of statistically significant factors in the model (i.e., time window or experimental groups). For this purpose we performed t-test for each independent variable (electronic supplementary material, table S2 and table S3). If this test for the factor 'experimental groups' was statistically significant, it provided (from a methodical point of view) an authorization to perform Tukey post-hoc test [39]. Then the post-hoc test results were visualized in figures.
To compare effects of BHAM and MitoTEMPO on animals’ return to full activity, we analysed the ratios of average numbers of fully active animals obtained for BHAM- or MitoTEMPO-treated tardigrades to average numbers of fully active appropriate control animals for different duration of the tun stage. The analysis was performed, regardless of the time windows, using developed Linear Mixed Models for the duration of the tun stage (3 and 60 days) and the applied concentrations of BHAM (0.1 mM and 0.2 mM) and MitoTEMPO (0.01 mM). This procedure was justified by our observation that revival time was influenced by the time window (see Results). To control for that factor we developed models with time window as a random factor and concentration as a fixed effect. All calculations were performed using R [41].