Lipomyces yamadae (UOFS Y-2824), preserved in the UNESCO MIRCEN culture collection at the University of the Free State, South Africa, was used in this study. Directly after isolation and again after one year of preservation through continuous sub-culturing (every three months) at 25 °C on yeast malt (YM) agar slants [7], L. yamadae was streaked out on YM agar plates and incubated at 25 °C to obtain growth for bio-assay experiments.

Lipomyces yamadae was suspended in sterile distilled water (dH₂O), whereafter 200 μL of the suspension was spread out on YM agar plates. Central wells were made in the plates with sterile Pasteur pipettes. To these wells 46 μL of either 4% or 8% (m/v) acetylsalicylic acid (ASA; Sigma-Aldrich, Steinheim, Germany) dissolved in 96% ethanol (EtOH; Merck, Darmstadt, Germany) were added. An EtOH control was included [2]. All plates were incubated for 11 days at 25 °C. The same technique was used to prepare the L. yamadae bio-assay after one year of preservation.

To evaluate whether the sensors of the yeast L. yamadae are associated with increased mitochondrial transmembrane potential (Δψ_m), cells of this organism (grown for 11 days at 25 °C on YM agar plates), were washed with Phosphate Buffered Saline (PBS; Oxoid, Hampshire, England) and treated with 31 μL of the mitochondrial stain Rhodamine 123 (Rh123; Molecular Probes, Invitrogen Detection Technologies, Eugene, OR, USA). These were incubated for 1 h in the dark at 21 °C, washed with PBS and subsequently fixed in Dabco (Sigma-Aldrich) on microscope slides. The cells were then viewed with CLSM (Nikon TE 2000, Tokyo, Japan) to visualize Δψ_m [8]. Green fluorescence indicates areas of increased Δψ_m and therefore mitochondrial activity.

The protocol included the preparation of bio-assay plates treated with EtOH alone (Control) and chloroquine dissolved in EtOH respectively, all prepared according to Section 2.2. Here ASA was replaced with 8% (m/v) Chloroquine diphosphate (Sigma-Aldrich) in EtOH. After incubation, samples were drawn from two areas of each plate. Each area was sampled at five different points spaced equally across the specific area tested on the plates. Each experiment was performed on separate bio-assay plates in triplicate. A total of 10 fields (40× magnification) was analyzed per sample point and the percentage sensors (i.e., asci with ascospores) relative to the total number of asexual cells plus sensors of L. yamadae was calculated using a Light Microscope (Axioplan, Zeiss, Göttingen, Germany) coupled to a Colourview Soft Digital Imaging System (Münster, Germany). Therefore, a total of 1 (area) × 5 (sample points in each area) × 10 (fields per sample point) × 3 (triplicate) = 150 fields were analysed per area to obtain a representative relative sensor count. This experiment was repeated after one year of preservation to determine the effects of storage and sub-culturing on sensor development.

Scanning Electron Microscopy was performed according to the method of Swart and co-workers [9]. Cells from different zones of the plate were chemically fixed with 3% (v/v) glutardialdehyde (Merck) in a sodium phosphate buffer for 3 h. The suspension was rinsed with buffer followed by fixation with 1% (m/v) osmium tetroxide (Merck), also in sodium phosphate buffer for 90 min. The suspension was again washed with buffer and dehydrated in a graded series of EtOH: 30%, 50%, 70% and 90% for 30 min each, with centrifugation between each step, and 2 × 100% EtOH for 90 min each, with centrifugation between each step. The dehydrated yeast cells were submitted to critical point drying, mounted on stubs and coated with a layer of gold—making it electrically conductive. These preparations were viewed with SEM (Shimadzu SSX-550 Superscan, Tokyo, Japan).

This was performed according to the method of Swart and co-workers [9]. Cells from the dark brown zone of the Lipomyces bio-assay were first prepared for SEM observation according to the protocol in Section 2.5 and then subjected to NanoSAM Ar⁺ etching. Here, a PHI 700 Nanoprobe (Chigasaki, Japan) with SAM and SEM facilities was used. The field emission electron gun employed for SEM and SAM analyses was set at 2.45 A filament current, 4 kV extractor voltage and 238.1 μA extractor current, thereby obtaining a 20 kV, 10 nA electron beam for the Auger analyses and SEM imaging. The diameter of the electron beam was 12 nm. The electron gun unit's upper pressure was 8.8E-10 Torr and that in the main chamber was 2.29E-10 Torr. All measurements were performed using aperture A. An 8 μm Field of View (FOV) was used for SEM and the number of frames used was 4. The Auger point analyses were acquired by using 10 cycles per survey, 1 eV per step and 20 ms per step. The Nanoprobe was equipped with an Ar⁺ ion sputtering gun that operated at a beam voltage of 2 kV, an ion beam current of 2 μA and a raster area of 1 × 1 mm. This gave a sputter rate of about 27 nm/min. The ion emission current operated at 15 mA. Finally, an alternating sputter mode with 1 min sputter intervals and 2 min sputter time was used with no rotation.

Bio-assays were prepared as described in Section 2.2. The disappearance of pigmentation on bio-assay plates was regarded as a positive hit, i.e., selective inhibition of sensor development. This was verified by Light Microscope observation. Various compounds were screened for anti-mitochondrial activity (Table 1). These included Non-Steroidal Anti-Inflammatory drugs (NSAIDs) [46 μL of an 8% (m/v) solution dissolved in 96% EtOH] (Sigma-Aldrich), anti-malarial drugs [46 μL of an 8% (m/v) solution dissolved in 96% EtOH] (Sigma-Aldrich), antifungal drugs (added as E-Test strips) (Davies Diagnostics, Randburg, South Africa), anticancer drugs [46 μL of an 8% (m/v) solution dissolved in 96% EtOH] (Sigma-Aldrich) and three classic respiratory inhibitors (Sigma-Aldrich). The effect of oxygen limitation was also investigated according to the method of Ncango and co-workers [10].

Eremothecium ashbyi (UOFS Y-630), preserved in the UNESCO MIRCEN culture collection at the University of the Free State, South Africa, was cultivated as described in Section 2.1.

The same protocol described in Section 2.2 was used to construct the Eremothecium bio-assay [11]. Only an 8% (m/v) in EtOH solution [not 4 % (m/v) EtOH solution as in Lipomyces] of the tested compounds was added while the plates were incubated under continuous inspection for pigmentation for 48 h at 25 °C. The screening of compounds was according to Table 1. Results for oxygen limitation and other compounds indicated by an asterisk in Table 1 were obtained from [2,5].

CLSM: Cells from the yellow zone were mounted on a microscope slide whereafter one drop of Calcofluor White Stain (Fluka, Buchs, Switzerland) and one drop of 10% potassium hydroxide (Merck) were added. The specimen was covered with a cover slip and after 1 min the slide was examined with CLSM (Nikon TE 2000) using ultraviolet (UV) light at 407 nm.

LM: The same cells as above were studied using a Light Microscope (Axioplan, Zeiss, Göttingen, Germany) coupled to a Colourview Soft Digital Imaging System (Münster, Germany).

SEM: This was performed as described in Section 2.5 on yeast cells from different zones of the plate.

NanoSAM: Cells, including ascospores from the yellow zone of the Eremothecium bio-assay were first prepared for SEM observation according to the protocol in Section 2.5 and then subjected to NanoSAM Ar⁺ etching as described in Section 2.6.

Dipodascopsis uninucleata var. uninucleata (UOFS Y-0128), preserved in the UNESCO MIRCEN culture collection at the University of the Free State, South Africa, was cultivated as described in Section 2.1.

The same protocol used to construct the bio-assay of L. yamadae (Section 2.2) was used except that only an 8% (m/v) solution of chloroquine in EtOH was added and compared to an EtOH control. The bio-assay plates were incubated for 72 h at 25 °C. After incubation, samples were drawn from three areas (1-3) of each plate as depicted in Figure 4(a,b). Each area was sampled at three points spaced equally across the specific area tested on the plate. Experiments were performed in triplicate on EtOH (Control = Contr.) and chloroquine in EtOH (CQ) bio-assays. A total of 25 fields in each area point were analyzed by using a haemacytometer (40 × magnification) and Light Microscope (Axioplan, Zeiss) coupled to a Colourview Soft Digital Imaging System. This was performed by counting the number of empty sensors (ascospores discharged) as well as sensors filled with ascospores. Therefore, a total of 1 (area) × 3 (sample points in each area) × 25 (fields per sample point) × 3 (triplicate) = 225 fields were analysed per area to obtain a representative relative empty sensor count that depicts percentage discharged sensors.

One drop of a 1% (m/v) Orange G (Molecular Probes) was added to cells with ascospore filled sensors mounted on a microscope slide and incubated for 5 min at 21 °C to allow the stain to penetrate the cells. The stained cells were then examined using a Nikon TE 2000 CLSM.

Cells, including ascospores from zone 3 of the Dipodascopsis bio-assay were first prepared for SEM observation according to the protocol in Section 2.5 and then subjected to NanoSAM Ar⁺ etching as described in Section 2.6.

Nadsonia fulvescens (UOFS Y-0705), preserved in the UNESCO MIRCEN culture collection at the University of the Free State, South Africa, was cultivated as mentioned in Section 2.1 except that the incubation temperature was 21 °C.

The same protocols for bio-assay construction and screening described in Sections 2.2 and 2.7 respectively, were followed except that only an 8% m/v in EtOH solution of the tested compounds was added. The compounds screened are listed in Table 1. The plates were incubated for 7 days at 21 °C to observe sensor response. Results for compounds indicated by a double asterisk in Table 1 were obtained from [8].

Results of the anti-mitochondrial drug ASA, obtained with the Lipomyces, Eremothecium and Nadsonia bio-assays were compared (Figure 1). In all cases, the bio-assay sensors are pigmented which facilitates screening of compounds that stimulate (increase in colour intensity) or inhibit (decrease in colour intensity) sensor development. As was expected from the hypothesis, increased mitochondrial activity was observed in sensors of Lipomyces as well as Eremothecium [2] and Nadsonia [8] (Figure 2(d)). This increase was mainly associated with the ascospores inside the sensors. In all bio-assays studied, the EtOH controls developed homogeneously pigmented lawns, Lipomyces–brown (Figure 1(b)); Eremothecium–yellow (Figure 1(f)) and Nadsonia–brown (Figure 1(j)). These lawns contained matured pigmented sensors (Figure 1(a,e,i)) as observed by SEM (Figure 1(a,e)) and Transmission Electron Microscopy (TEM) (Figure 1(i)). When the anti-mitochondrial ASA was added, all bio-assays developed light zones (L) where pigmentation and hence sensor development were inhibited (Figure 1(c,g,k)). Ultra-structural imaging showed that sensors were absent or malformed in these zones (Figure 1(d,h,l)) and that ASA selectively inhibited these sensors at sub-lethal concentrations (Figure 1(g,k)).

As expected, we found that the Lipomyces bio-assay was more than twice as sensitive to ASA compared to the other two bio-assays. A larger sensor inhibition zone (Figure 1(c)) in the Lipomyces assay was observed, when 4% (m/v) ASA solution was added compared to when an 8% (m/v) ASA solution was added to the other two fermenting yeasts. This was to be expected since Lipomyces can only obtain energy from an aerobic respiratory metabolism and not from fermentation as well [5]. It is interesting to note that when Lipomyces was grown on 8% (m/v) ASA, still no growth inhibition was observed (results not shown). This means that the asexual cells of L. yamadae are more resistant towards this anti-mitochondrial drug compared to E. ashbyi and N. fulvescens. This cannot be explained at present.

Various compounds with known and unknown anti-mitochondrial activities were screened with the Lipomyces, Eremothecium and Nadsonia bio-assays and compared with reports from the Food and Drug Administration (FDA) on drugs posing mitochondrial liabilities [4,12] (Table 1). No discrepancies between screening results of the three yeast bio-assays could be found.

Compounds posing mitochondrial liabilities according to FDA Black Box Warnings as well as those that gave positive hits in all three bio-assays include diclofenac, ibuprofen and indomethacin. Compounds that tested positive for Black Box Warnings as well as Lipomyces and Eremothecium bio-assays include naproxen, peroxicam and sulindac. However, diflunisal and fenoprofen tested negative with Black Box Warnings but positive with the bio-assays. Interestingly, these NSAIDs were also reported to be anti-mitochondrial in literature [2,12,13]. The NSAIDs, ASA and salicylic acid tested positive for all three the bio-assays. These results also corroborate previous mammalian and plant studies suggesting that the NSAIDs, ASA, ibuprofen, indomethacin and salicylic acid also have anti-mitochondrial activity [14–17]. The anti-malarial drug quinine tested negative for both Lipomyces and Eremothecium bio-assays, while chloroquine tested negative for all bio-assays. Artemisinin gave a positive hit when tested with the Eremothecium bio-assay. This was to be expected since the anti-mitochondrial activity of this important anti-malarial drug is well reported in yeasts [18]. Thapsigargin, capable of collapsing membrane potential of mitochondria in the malaria parasite [19] also gave a positive hit with the Eremothecium bio-assay. In addition, all antifungal agents submitted to the bio-assays tested positive with all three yeast bio-assays (Table 1).

The anti-mitochondrial anticancer drug lonidamine [20] tested positive with the Lipomyces and Eremothecium bio-assays. In addition, CD 437 and betulinic acid also tested positive with the Lipomyces bio-assay. Both of these anticancer drugs are known to have anti-mitochondrial activity [21,22].

The effect of three classic respiratory inhibitors was also investigated in order to serve as benchmark for the efficacy of these bio-assays. All three bio-assays were sensitive to antimycin A and oxygen limitation, while rotenone also tested positive with the Lipomyces bio-assay.