The Changes in Mitochondrial Morphology and Physiology Accompanying Apoptosis in Galleria mellonella (Lepidoptera) Immunocompetent Cells during Conidiobolus coronatus (Entomophthorales) Infection

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, their role in insects is not fully understood; thus, more indepth studies of insect cell apoptosis are necessary. The present study investigates mitochondrial involvement during Conidiobolus coronatus-induced apoptosis in Galleria mellonella hemocytes. Previous research has shown that fungal infection could induce apoptosis in insect hemocytes. Our findings indicate that mitochondria undergo several morphological and physiological changes during fungal infection, e.g., loss of mitochondrial membrane potential, megachannel formation, disturbances in intracellular respiration, increased nonrespiratory oxygen consumption in mitochondria, decreased ATP-coupled oxygen consumption and increased non-ATP–coupled oxygen consumption, decreased extracellular and intracellular oxygen consumption, and increased extracellular pH. Our findings confirm that G. mellonella immunocompetent cells demonstrate Ca2+ overload in mitochondria, translocation of cytochrome c-like protein from mitochondrial to cytosol fraction, and higher activation of caspase-9-like protein after C. coronatus infection. Most importantly, several of the changes observed in insect mitochondria are similar to those accompanying apoptosis in mammalian cells, suggesting that the process is evolutionarily conserved.


Introduction
Despite causing the loss of cells, host cell death may be an important defence mechanism occurring in response to microbial infection. Indeed, apoptotic and regulated necrotic processes such as necroptosis, pyroptosis, and extracellular trap-related cell death-ETosis, can have a significant influence on the outcome of a microbial insult [1]. Although most studies of programmed cell death have examined the result of viral and bacterial infections, evidence suggests that apoptosis and regulated necrosis processes also play a key role in the interplay between pathogenic fungi and host mammalian cells [2].
Apoptosis is a highly complex form of programmed cell death involving an energydependent cascade of molecular and cellular events. It represents a vital part of the immune response to pathogens, which leads to the destruction of the intracellular niche of microbial replication. Furthermore, the elimination of pathogen-containing apoptotic bodies by secondary phagocytes and the presentation of antigens derived from the apoptotic material by dendritic cells represent important antimicrobial effector mechanisms in mammals [3]. Pathogenic fungi have, therefore, evolved multiple distinct mechanisms for modulating host-cell apoptosis.

The Changes in Mitochondrial Activity in G. mellonella Hemocytes after Fungal Infection
The activity of mitochondria was measured by fluorescence microscopy using the MITO-ID Red Detection Kit (GFP-CERTIFIED ® ). The distribution of mitochondria in hemocytes of G. mellonella is presented in Figure 1A. In the controls, high red fluorescence (mitochondrial activity) was observed in both cell types; they are visible as single redstained points in the cytoplasm in plasamtocytes and as bright red fluorescent circles around the cell nuclei in granulocytes. After fungal infection, considerable disintegration of hemocytes was observed, together with a lower intensity of red fluorescence. This lower red fluorescence was accompanied by lower green fluorescence (actin staining) in the FITC channels.

The Translocation of Cytochrome c-like Protein from Mitochondrial to Cytosol Fraction in G. mellonella Hemocytes during Fungal Infection
The changes of concentration of mitochondrial and cytosol fraction containing the cytochrome c-like protein in the G. mellonella hemocyte after fungal infection are presented in Table 1. According to the presented research, the C. coronatus infection caused the decrease in mitochondrial and an increase in cytosol fraction of protein concentration in the insect hemocyte. During fungal infection, cytochrome c-like protein was present in both the mitochondrial and cytosol fractions in G. mellonella hemocytes, as detected by Western blot ( Figure 1B). In the control cells, cytochrome c-like protein was only observed in the mitochondrial fraction. Our findings indicate that translocation of cytochrome c-like protein occurs during infection.

The Flux of Ca 2+ Level in G. mellonella Hemocytes after Fungal Infection
The changes in Ca 2+ levels in cells were measured by spectrofluorometric analysis using the FLUOFORTE Calcium Assay Kit. The time-dependent changes in Ca 2+ level in G. mellonella hemocytes after fungal infection are shown in Figure 1C. An increase in calcium level was observed in samples F24 and F48 just after the fluorescence readings were started; at the last time point (50 min), the relative fluorescence units (RFU) were 2.5 times higher than in control cells for F24 and 3.01 times higher for F48.

Detection of Changes in ADP/ATP Ratio in G. mellonella Hemocytes after Fungal Infection
The ADP/ATP ratio assay kit was used for measuring ADP and ATP levels in G. mellonella hemocytes during C. coronatus infection. After fungal infection, an increase in the ADP/ATP ratio was detected ( Figure 1D), reaching 5.95 in control cells, 13.80 in the F24 group, and 17.99 in the F48 group; ANOVA, Tukey's HSD Test; F(2,3) = 11.84, p = 0.04, MS = 6.31, df = 3.00).
2.5. Changes in Caspase-9-like Protein Activity in G. mellonella Hemocytes after Fungal Infection The activity of the caspase-9-like protein in wax-moth hemocytes is shown in Figure 1E. After fungal infection, caspase-9-like protein level was found to fluctuate. A slight decrease in activity was detected after 24 h of infection (F24) (1.6-times lower than in the control); however, this difference was not statistically significant. In contrast, activity increased 3.5-fold when compared with untreated larvae after 48 h (F48); ANOVA, Tukey's HSD Test: F(2,6) = 160.08, p < 0.001, MS < 0.001, df = 6.00).

Changes in Membrane Potential in G. mellonella Hemocytes after Fungal Infection
The MITO-ID ® Membrane Potential Kit measures fluctuations in mitochondrial membrane potential (MMP) based on a cationic dual-emission dye that exists as green fluorescent monomers in the cytosol and accumulates as red fluorescent J-aggregates in the mitochondria. Mitochondria having a low membrane potential will accumulate low concentrations of dye and thus will exhibit green fluorescence, while more highly polarized mitochondria will exhibit red fluorescence. Cells exhibit a shift from red to green fluorescence as the mitochondrial function becomes increasingly compromised. The changes in membrane potential are shown in Figure 2.
The changes in membrane potential identified by fluorescence microscopy are shown in Figure 2A. The untreated cells demonstrated high-intensity green and red fluorescence. In contrast, hemocytes treated with CCCP and those from infected insects displayed at the low intensity of red fluorescence; this reflects a shift from red to green fluorescence, indicating changes in membrane potential. The activity of the caspase-9-like protein in wax-moth hemocytes is shown in Figure  1E. After fungal infection, caspase-9-like protein level was found to fluctuate. A slight decrease in activity was detected after 24 h of infection (F24) (1.6-times lower than in the control); however, this difference was not statistically significant. In contrast, activity increased 3.5-fold when compared with untreated larvae after 48 h (F48); ANOVA, Tukey's HSD Test: F(2,6) = 160.08, p < 0.001, MS < 0.001, df = 6.00).

Changes in Membrane Potential in G. mellonella Hemocytes after Fungal Infection
The MITO-ID ® Membrane Potential Kit measures fluctuations in mitochondrial membrane potential (MMP) based on a cationic dual-emission dye that exists as green fluorescent monomers in the cytosol and accumulates as red fluorescent J-aggregates in the mitochondria. Mitochondria having a low membrane potential will accumulate low concentrations of dye and thus will exhibit green fluorescence, while more highly polarized mitochondria will exhibit red fluorescence. Cells exhibit a shift from red to green fluorescence as the mitochondrial function becomes increasingly compromised. The changes in membrane potential are shown in Figure 2.
The changes in membrane potential detected in flow cytometry are presented in Figure 2C. After flow cytometry analysis, two populations of G. mellonella hemocytes were observed (described as A and B). In the control samples, both populations (A + B) can be seen in the double-positive top-right quadrant, as well as each individual population (A or B), indicating that both red and green fluorescence was observed in hemocytes. After CCCP treatment, and in hemocytes from infected insects, the changes were mostly detected in the B population, where more cells are placed solely in the single positive quadrant (FITC), this change indicates shifts from red to green fluorescence. In addition, the B population had disappeared during fungal infection, reflecting the death of hemocytes from this population. What is more interesting is that similar changes were not observed in hemocytes taken from fungus-treated larvae without any sign of infection (F48*). In this case, both populations' cells have similar changes similar to this observed in the control.

Detection of Mitochondrial Permeability Transition Pore (MPTP) Opening in G. mellonella Hemocytes after Fungal Infection
The mitochondrial permeability transition pore (MPT pore or MPTP) is a nonspecific channel formed by components of the inner and outer mitochondrial membranes and appears to be involved in the release of mitochondrial components during cell death. Although MPTPs alternate between open and closed states in healthy cells, they dramatically alter the permeability of the mitochondria during cell death.
The hemocytes from fungal-treated and control insects were analyzed by flow cytometry ( Figure 3). Tube 1 (M1) included unstained samples, used for instrument setup. Samples stained with MPTP staining dye (M2) showed cumulative fluorescence from both the cytoplasm and mitochondria. The tubes treated with MPTP staining dye and CoCl 2 (M3) only demonstrated mitochondrial fluorescence. The tubes treated with all reagents (M4) showed the lowest fluorescence. The difference in fluorescence between M3 and M4 indicates the degree of MPTP activation and subsequent depolarization of the mitochondrial membrane. Our findings indicate a higher intensity of fluorescence in both the cytoplasm and mitochondria (M3) in the control cells than in F24 and F48, which might indicate a higher level of hemocyte mortality after fungal infection. Moreover, small differences were found between M3 and M4 in hemocytes from fungus-treated insects, which indicates the opening of MPTP in G. mellonella hemocytes (in both A and B populations) after C. coronatus infection.

The Changes in Oxygen Consumption in G. mellonella Hemocytes after Fungal Infection
The changes in oxygen consumption observed in G. mellonella hemocytes after fungal infection are presented in Figure 4.
indicates the opening of MPTP in G. mellonella hemocytes (in both A and B populations) after C. coronatus infection. Our data indicates that C. coronatus infection caused a decrease in both extracellular and intracellular oxygen consumption together with an increase in glycolytic flux. This indicates that extracellular acidification increased in the G. mellonella hemocytes ( Figure 4A-C).
The fluorescence profiles reflecting hemocyte oxygen consumption in the control and infected insects are presented in Figure 4D. Fungal infection was associated with a lower RFU value in both the F24 and F48 probes compared with controls, indicating a decrease in all oxygen-consumption parameters, viz. ATP-coupled respiration (oligomycin treatment), maximal respiration (FACP treatment) and nonrespiratory oxygen consumption (actinomycin A treatment). Moreover, during infection, nonrespiratory oxygen consumption is more favoured in hemocytes compared to maximal respiration ATP-coupled respiration. A significant increase was noted in nonrespiratory ATP consumption (ANOVA, Tukey's HSD Test, F(2,6) = 161.05, p < 0.001, MS = 40,390.00, df = 6.00) together with a decrease in ATP-coupled oxygen consumption (ANOVA, Tukey's HSD Test, F(2,6) = 114.86, p < 0.001, MS = 41,179.00, df = 6.00). Detailed changes in oxygen consumption are presented in Figure 4E.

Discussion
Our previous research has highlighted the important role of apoptosis, oxidative stress, and caspases 1-9-like protein activation in G. mellonella hemocyte destruction during C. coronatus infection [24][25][26] with greater activation of caspase 9-like protein than caspase 8-like protein in G. mellonella hemocytes [25]; this might suggest that the intrinsic pathway plays the dominant role in apoptosis activation in insect immunocompetent cells during C. coronatus infection.
Our present findings also confirm that the initiator of apoptosis, caspase-9-like protein ( Figure 1E), demonstrates higher activity in wax-moth hemocytes taken from insects treated with fungus. The literature data also confirm the presence of the homologue of mammalian caspase-9 in Lepidoptera (Lep-caspase-5) [43]. In S. litura, this protein (Sl-caspase-5) can cleave Sl-procaspase-1, a homologue of mammalian caspase-3, which directly causes apoptosis [44]. Our data indicate that a similar protein, demonstrating higher activity towards apoptosis and fungal infection, is also present in G. mellonella; however, more detailed research is needed to compare the structure and physiology of detected protein with mammalian caspase-9.
Such elevated caspase-9-like protein activity suggests that the intrinsic pathway might play an important role in apoptosis activation, and thus hemocyte destruction, during C. coronatus infection. This pathway is closely connected with the activity of mitochondria [45]. Our present findings indicate that C. coronatus infection resulted in the changes in mitochondria morphology and activity in G. mellonella hemocytes. Mitochondria are double membrane-bound organelles found in most eukaryotic organisms. They act as prominent energy carriers and ATP production centres [46], and influence the immune response [47,48]. Mitochondria are an important source of ROS within most mammalian cells [49][50][51]. While ROS production contributes to mitochondrial damage in a range of pathologies, it is also a key component in redox signalling from the organelle to the rest of the cell [52,53].
Mitochondria have been found to play a crucial role in apoptosis activation in an insect cell line (Sf-9) after azadirachtin (insecticidal tetranortriterpenoid) treatment [20]. Our observations confirm that several physiological and morphological changes occur in mitochondria during fungal infection and underline their pivotal role in insect death after C. coronatus treatment. They also highlight the similarities in these processes between insects and mammals.
In mammal cells, mitochondrial functions are influenced by Ca 2+ overload [54]. Similarly, higher levels of Ca 2+ were also detected in G. mellonella hemocytes after fungal infection in the present study ( Figure 1C). In mammals, the endoplasmic reticulum (ER) transmits Ca 2+ signals to the mitochondria, which decode them into specific inputs to regulate essential functions, including metabolism, energy production, and apoptosis [55]. Under physiological conditions, the accumulation of Ca 2+ in mitochondria stimulates oxidative metabolism through the modulation of Ca 2+ -sensitive dehydrogenases and metabolite carriers [56,57]. During apoptosis activation via intrinsic pathways in mammals, Ca 2+ acts as a critical sensitizing signal; the Ca 2+ level can be seen to increase at both the early and late stages of the apoptotic pathway and two apoptogenic mechanisms have been proposed: Ca 2+ release from the ER, and capacitive Ca 2+ influx through Ca 2+ release-activated Ca 2+ channels [58][59][60][61]. Taking into account the evolutionary conservatism of programmed cell death signalling pathways, the changes in Ca 2+ levels observed in insect hemocytes might suggest the presence of a similar mechanism in insect cells during fungal infection. The increased intracellular calcium concentration present in the G. mellonella hemocytes during fungal infection might be a crucial factor in apoptosis induction and, as a result, the death of immunocompetent cells. However, there are no research data about the role of mitochondrial Ca 2+ influx in insect cell death. The increase of the cytosolic free calcium ([Ca 2+ ]i) concentration was observed in the S. litura cell line (SL-1) after infection by the Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV); however, in that case, neither the elevation of the cytosolic calcium ion nor extracellular calcium entry was the inducing factor of apoptosis, which hinted that the depletion of the ER Ca 2+ store contributed to SL-1 cell apoptosis induced by SfaMNPV [62]. In Drosophila melanogaster mitochondrial Ca 2+ stores are critical for signal amplification olfactory sensory neurons [63].
In eukaryotes, most ATP is synthesized by mitochondrial oxidative phosphorylation, during which several reactive oxygen species (ROS) are produced [64]. The overload of mitochondrial Ca 2+ leads to the increased generation of ROS and decreased ATP production in mammals. Previous research indicates that oxidative stress plays an important role in G. mellonella hemocyte destruction during C. coronatus infection; Kazek et al., report the presence of an increased level of 8-hydroxy-2 -deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage in fungus-treated hemocytes together with lowered levels of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) [26].
Our present findings indicate several disturbances in ATP production, and its use as a substrate for aerobic respiration, in insect mitochondria during fungal infection. More specifically, an increase in the ADP/ATP ratio was noted, as well as a decrease in ATPcoupled oxygen consumption and an increase in non-ATP-coupled oxygen consumption in insect hemocytes (Figures 1D and 4E). The elevated level of non-ATP-oxygen consumption indicates higher proteon leaks in mitochondria and, thus, ROS generation, which might be a principal source of oxidative stress and cell death in hemocytes. The high ADP/ATP ratio results in decreased levels of ATP and increased levels of ADP, which is characteristic of apoptotic or necrotic cells. All disturbances in ATP production and metabolism result in higher ROS production and, hence, oxidative stress, triggering programmed cell death in G. mellonella hemocytes during fungal infection.
To compensate for the decrease in ATP production resulting from mitochondrial toxicity, oxygen consumption is decreased, and glycolytic flux is increased (extracellular acidification). Our present findings confirm the presence of disturbances in ATP production due to changing oxygen consumption in the G. mellonella hemocytes during fungal infection ( Figure 4A-C). C. coronatus treatment decreased both intracellular and extracellular oxygen consumption, and at the same time increased extracellular acidification in G. mellonella hemocytes. Elevated extracellular acidification is a marker of increased anaerobic respiration; it has been shown that mitochondrial toxins (electron transport inhibitors) prevent or restrict aerobic ATP generation by oxygen consumption. To compensate for this loss of cellular ATP, cells respond by increasing their glycolytic activity which directly results in an increase in acidification. All these changes were observed in insect hemocytes during fungal infection, which indicate a failure in mitochondria physiology, resulting in the activation of apoptosis and eventual cell death.
Mitochondrial dysfunction is caused by membrane depolarization [7]. The most important triggers for MPTP opening are Ca 2+ and ROS; in living cells, these act in conjunction with a variety of pathological challenges [55]. Changes in the membrane potential, along with decreases in ATP to ADP ratios, increases in mitochondrial matrix calcium levels and oxidative stress, and the release of cytochrome c into the cytosol are all presumed to be associated with the mitochondrial permeability transition, resulting in disruption of ions and small molecule homeostasis via the MPTP [8]. Our findings confirm MPTP opening (Figure 3), and fluorescence microscopy revealed changes in membrane potential ( Figure 2) in insect hemocytes during fungal infection. The activation of mitochondrial permeability transition pores (MPTPs) and the loss of mitochondrial membrane potential (MMP) were observed also very early during apoptosis in Sf9 cells after treatment with azadirachtin and camptothecin [20].
However, spectrofluorometric research did not reveal any similar changes or only found them in a lower percentage of cells. These differences may be due to the methods of sample preparation; the spectrofluorometric analysis was based on freshly collected full hemolymph, containing all classes of hemocytes, and fluorescent analysis on cultured hemocytes, where granulocytes and plasmatocytes predominate. These two classes of immunocompetent cells are the only hemocytes adhering to foreign bodies, which enables them to participate in phagocytosis, encapsulation, and nodulation. Our results suggest that the described changes in mitochondria morphology are characteristic only for these two classes. These play an essential role in insect immunological defence, and, hence, our findings highlight the crucial impact of mitochondrial disturbance in hemocyte death during fungal infection.
This thesis is supported partly by a flow cytometry analysis, which indicated that separate populations of G. mellonella hemocytes reacted differently to C. coronatus infection.
The analysis showed the presence of two main populations of wax-moth immunocompetent cells in control insects, and that fungal infection caused a greater amount of cell death and changes in mitochondrial membrane potential in one of them ( Figure 2C); it is possible that this population contains plasmacytes and granulocytes. However, there is no literature data describing the method of hemocyte sorting and, for that reason, there is a need to conduct further research focused on checking the apoptotic changes in the two isolated populations. Moreover, the result of analysis of fungus-treated larvae with no sign of infection is similar to the control ones, which might point to the significant role of mitochondrial membrane potential disturbance in hemocyte death during fungal infection.
The different reactions of the individual classes of hemocyte might account for the statistically insignificant differences observed between the groups in changes in respiratory rate and mitochondrial capacity during fungal infection. Although these parameters have been found to decrease during fungal infection, the changes were not significant. These results also might suggest that different kinds of hemocytes react differently to fungal infection.
However, a significant decrease in ATP-coupled oxygen consumption was noted, as well as a significant increase in non-ATP oxygen consumption; these findings suggest that disturbances in ATP production and aerobic respiration might be a more universal way of hemocyte destruction and might serve as important virulence factors of entomopathogenic fungi or as insect defence mechanisms; the death of cells infected by fungus might be an essential defence mechanism.
MPTP opening induces mitochondrial swelling. These large-scale alterations of organelle morphology and mitochondrial membrane permeabilization allow the release of proapoptotic factors, such as cytochrome c, into the cytosol. As a soluble protein, cytochrome c is localized in the intermembrane space and is loosely attached to the surface of the inner mitochondrial membrane [21]. Its release from mitochondria is a key event and plays an important role in initiating apoptosis in the mammalian cell [65]; however, the role of cytochrome c is not clear in insect-cell apoptosis. There are contradictory reports on the role of cytochrome c in apoptosis in Drosophila following its release from mitochondria [66,67]. In Lepidoptera, the literature data suggest that cytochrome c plays an essential role during apoptosis (e.g., Spodoptera frugiperda, Spodoptera exigua, S. litura, and B. mori) [18,19,21,23,62,[68][69][70][71][72]. On the other hand, the silencing of the expression of cytochrome c had a remarkable effect on procaspase-3 and procaspase-9 activation and resulted in the reduction of caspase-3 and caspase-9 activity in Sl-1 cells undergoing apoptosis [21]. Our present findings confirm the presence of a cytochrome c-like protein in the cytosol fraction in G. mellonella hemocytes after fungal infection, which might suggest the transition of this protein from mitochondria. This translocation is probably a result of the mitochondrial dysfunction described above, as indicated by literature confirming the presence of MPTP-dependent cytochrome c release mechanisms in lepidopteran cells [20]. However, more data are needed to confirm this thesis.

Insects
A culture of the wax moth, G. mellonella, was maintained and reared in temperature and humidity-controlled chambers (30 • C, 70% r.h.) in constant darkness on an artificial diet [73]. Fully-grown larvae were collected before pupation, surface-sterilized, and homogenized, and then used as a supplement in the fungal cultures. Five-day-old last instar larvae were used to analyse the influence of fungal infection on the morphology and functioning of mitochondria in insect hemocytes.

Fungus
C. coronatus (isolate number 3491), originally isolated from Dendrolaelaps spp., was obtained from the collection of Prof. Bałazy (Polish Academy of Sciences, Research Center for Agricultural and Forest Environment, Poznań, Poland). It was routinely maintained in 90 mm Petri dishes at 20 • C with cyclic changes of light (L:D 12:12) on Sabouraud agar medium (SAM) with the addition of homogenized G. mellonella larvae to a final concentration of 10% wet weight. The sporulation and virulence of the SAM C. coronatus cultures were enhanced with the addition of homogenized G. mellonella larvae.

Infection of Insects with C. coronatus
G. mellonella larvae (five-day-old last instar) were exposed for 24 h at a temperature of 20 • C to fully-grown and sporulating C. coronatus colonies. Fifteen individuals were maintained in each Petri dish. A control group was formed of larvae exposed for 24 h to sterile Sabouraud agar medium (Merck Millipore, Darmstadt, Germany). After exposure, the insects were transferred to new, clean Petri dishes on an artificial diet [73], and kept at 20 • C for one day. Following this 24-h exposure to the fungus, one group of insects was collected immediately for examination (F24 group) while the rest were left for another 24 h before collection (F48 group).

Larval Hemolymph Collection
G. mellonella hemolymph was collected from both the control and infected (F24 and F48) larvae. Before bleeding, the insects were disinfected in 70% ethanol, and immersed in distilled water to reduce the contamination of hemolymph samples. Hemolymph was taken from the larvae through an incision made in the last proleg. The hemolymph was prepared in different ways depending on the planned method.
For spectrofluorometric analysis, 20 drops of fresh hemolymph collected from 20 larvae were suspended in 100 µL of phosphate-buffered saline (PBS) with PTU on ice. This suspension was used to determine the ADP/ATP ratio. For other analyses, the hemolymph was centrifuged (300× g, 4 • C, 5 min), and the hemocyte pellet was taken for further testing.
For flow cytometric analysis, 100 µL of fresh hemolymph collected from ten larvae were suspended in 100 µL of supplemented GIM with 10 mM EDTA (ethylenediaminetetraacetic acid) and 30 mM sodium citrate. The hemolymph was centrifuged at 300× g at room temperature (5 min) and the pellet was collected for future analysis.

Staining of Mitochondria in Fluorescent Microscopy
The hemocyte cell cultures were prepared according to Section 2.4. After 24 h of incubation, the cells were fixed in 4% paraformaldehyde (Sigma Aldrich, München, Germany; PFA) in phosphate-buffered saline (PBS) and permeabilized in 0.1% Triton X-100 (Sigma Aldrich, München, Germany) in PBS. Mitochondria were detected with a MITO-ID Red Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA). The cells were incubated for 30 min with Dual Detection Reagent and ActinGreen 488 ReadyProbes Reagent (Invitrogen, Thermofisher, Carlsbad, CA, USA) was used to label the actin fibres. The cell nuclei were stained with Hoechst (Enzo Life Sciences). Fluorescence signals were analysed by fluorescent microscopy using an Axio Vert.A1 fluorescence microscope (Carl Zeiss, Jena, Germany) with Axio Cam ICc 5 (Carl Zeiss, Jena, Germany).

The Calculation of Ca 2+ Concentration in Hemocytes
The changes in Ca 2+ concentration in hemocytes were detected by spectrofluorometric analysis using the FLUOFORTE Calcium Assay Kit (Enzo Life Sciences).
The hemocytes, prepared as above, were resuspended in FLUOFORTE Dye-Loading Solution and then plated to a black 96-well plate (nest) in a plating volume of 100 µL. The cell suspensions were then incubated for one hour at room temperature. The calcium flux was monitored as the fluorescence insensitivity at Ex = 490 nm and Em = 525 nm, measured using a Synergy HT Microplate Reader (BioTek, Instruments, Inc., Winooski, VT, USA) every 10 min for 50 min. The test was conducted as three independent replicates.

The Detection of Changes in ADP/ATP Ratio
To detect changes in the ADP/ATP ratio, the ADP/ATP Ratio Assay Kit (Sigma Aldrich, München, Germany) was used. Briefly, 10 µL amounts of freshly prepared hemolymph were plated in a white 96-well plate (CytoGen, Zgierz, Poland) and ATP reagent was then added to each well. The luminescence was read after one-minute incubation at room temperature (RLU A ). The plate was incubated for another 10 min, and the luminescence was read again (RLU B ). Immediately afterwards, the ADP reagent was added to each well and mixed by tapping the plate or pipetting. After another minute, the luminescence (RLU C ) was read. The ADP/ATP ratio was calculated as:

The Measurement of Protein Concentration-The Bicinchoninic Acid Assay (BCA Method)
Protein concentration was measured using the commercial Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the samples were prediluted 100Xin PBS (this dilution was included in the final calculations) and 25 µL of each dilution were transferred to a 96-well plate (NEST, Wuxi, China); following this, 200 µL of "working reagent", prepared according to the manufacturer's instructions, was added to each well. The samples were incubated at 37 • C for 30 min. After this time, the plate was cooled to room temperature and the absorbance was read at 562 nm in a BioTek HT spectrofluorometer. The protein concentration in the tested samples was measured based on a calibration curve. Each sample was prepared in three replications.

Measurement of Caspase Activity
Caspase-9-like protein activity was measured using the commercial Caspase-9 colourimetric assay kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer's protocol. Briefly, the samples were collected as described above (Section 2.4). The haemocyte pellets were dissolved in cell lysis buffer, incubated for 10 min on ice and then centrifuged (10,000× g, 4 • C, 1 min). The protein content was measured in the supernatant using the BCA method. From each sample, 200 µg of protein (experimentally selected amount) were added to separate wells in a 96-well plate (Nest, Wuxi, China), then mixed with 50 µL of 2X reaction buffer (containing 10 mM DTT, dithiothreitol) and 5 µL of 200 µM substrate (4 mM LEHD-pNA). The samples were incubated at 37 • C for two hours. After this time, the absorbance was read at 400 nm. Fold increase in caspase activity was determined by comparing the level of infected larvae and the noninfected control.

Detection of the Changes in Membrane Potential in G. mellonella Hemocyte Mitochondria
The changes in the membrane potential were determined with the MITO-ID ® membrane potential detection kit and MITO-ID ® membrane potential cytotoxicity kit (both Enzo Life Sciences, Farmingdale, NY, USA). As a positive control, the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used.
Changes in membrane potential were determined by fluorescent microscopy and flow cytometry using hemocytes from the control and infected insects, prepared as described above. Hemolymph from fungus-treated insects with no sign of infection was used additionally for flow cytometry. To the positive control, 1 µL of 200 µM CCCP was added 15 min before the experiment and incubated at 27 • C. The controls and infected cells were washed in a washing buffer and then incubated with the dual detection reagent for 15 min at room temperature; the samples were protected from light. Fluorescence signals were analysed by fluorescent microscopy using an Axio Vert.A1 fluorescence microscope (Zeiss) with Axio Cam ICc 5 (Zeiss) and by flow cytometry using a CyFlow Cube 8 (Sysmex, Norderstedt, Germany). The readings were analysed with FCS Express 7 (DeNovo Software).
The changes in membrane potential were detected by spectrofluorometry. The hemocytes were resuspended in MITO-ID ® MP Dye Loading Solution, and then plated in a 96-well dark plate (Sigma Aldrich, München, Germany), at a volume of 100 µL per well (in three replicates). The positive control was treated with 1 µL of 200 µM CCCP 15 min before the experiment and incubated at 27 • C. The membrane potential was monitored as the fluorescence insensitivity at Ex = 490 nm and Em = 590 nm using a Synergy HT Microplate Reader (BioTek) every 10 min for 90 min. For each sample, 1 mL aliquots of hemocyte suspension were added to four separate tubes using MPTP Wash Buffer: one tube did not receive any treatment (tube 1), one tube with MPTP Staining Dye only (1:500 in wash buffer; tube 2), one tube with MPTP staining dye and cobalt (II) chloride (5 µL of CoCl 2 ; tube 3) and one tube with MPTP staining dye, CoCl 2 and Ionomycin (5 µL of Ionomycin; tube 4).
The samples were then incubated at 37 • C for one hour, protected from light. After incubation, the cells were pelleted by centrifugation at 1000× g for 5 min in RT and then resuspended in 1 mL of wash buffer to remove excess staining and quenching reagents. After staining, the cells were kept on ice and analyzed within one hour by flow cytometry on a CyFlow Cube 8 (Sysmex, Norderstedt, Germany) and analysed with FCS Express 7 (DeNovo Software).

The Detection of Changes in Oxygen Consumption
Any changes in oxygen consumption were detected using MITO-ID Extracellular To detect changes in extracellular oxygen consumption and glycolytic flux (extracellular pH changes) in cells, hemocytes (preparation described above-Section 2.4) were resuspended in PBS to measure extracellular oxygen consumption; to detect pH changes, they were placed in respiration buffer (1 mM K-phosphate, 20 mM glucose, 0.07 M NaCl, 0.05 M KCl, 0.8 mM MgSO 4 , 2.4 mM CaCl 2 , and pH = 7.4), mixed with oxygen or pH extracellular sensor probe. The suspensions were then plated in a black 96-well plate (Sigma Aldrich, München, Germany) to a volume of 100 µL. Following this, one drop of mineral oil (Enzo Life Science) was added to each well and the result was immediately read on a fluorescence plate reader.
To detect changes in intracellular oxygen consumption, hemocytes were cultured in a black 96-well sterile plate for 24 h, as described in Section 2.4. Following this, 10 µL of intracellular sensor probe were added and the cells were incubated for another 24 h and then read on a fluorescence plate reader.
Real-time analysis of cellular respiration and mitochondrial function was performed using the Mitochondrial Stress Test Complete Assay Kit (Abcam, Cambridge, UK). Briefly, the hemocytes were resuspended in a 96-well plate (Nest) to reach a concentration of 4 × 10 5 cells in a 90 µL medium (GIM). Following this, 10 µL of the extracellular O 2 probe were added to each well, apart from the wells used as blank controls. Next, to wells was added: 10 µL of oligomycin working stock (1.5 µM final concentration/well to measure ATP-coupled respiration), 10 µL of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone working stock (FCCP, 2.5 µM final concentration/well to measure maximal respiration), 10 µL of antimycin A working stock (1 µM final concentration/well, to measure nonrespiratory oxygen consumption), or 10 µL reconstituted glucose oxidase (1.5 mg/mL) to the signal control wells, respectively. Each well was overlaid with two drops of HS mineral oil and immediately read on a fluorescence plate reader.

Isolation of Cytochrome c-like Protein from G. mellonella Larvae' Hemolymph
Cytochrome c-like protein was isolated from the hemolymph of infected and noninfected G. mellonella larvae using a commercial isolation kit (Cytochrome c releasing apoptosis assay kit, Enzo Life Sciences, Farmingdale, NY, USA). The samples were collected from 100 larvae for each variant, as described in Section 2.4. Then, the hemolymph was centrifuged (600× g for 5 min. at 4 • C); the pellets were resuspended with 1 mL of 1Xcytosol extraction buffer mix containing DTT and Protease Inhibitors and then incubated on ice for 10 min. The samples were then homogenized in an ice-cold Kimble dounce tissue grinder (Merck Millipore, Darmstadt, Germany) (30 passes) and the homogenates were transferred to a fresh 1.5 mL tube and centrifuged at 700× g for 10 min. at 4 • C. The supernatants were transferred again to a fresh 1.5 mL tube and centrifuged at 10,000× g for 30 min at 4 • C. The supernatants were collected as a cytosolic fraction and pellets were resuspended in 0.1 mL of the mitochondrial extraction buffer mix containing DTT and protease inhibitors, vortexed for 10 s and saved as the mitochondrial fraction. After isolation, the protein content of both fractions was checked using the BCA method (as described above) and frozen at −80 • C.

Detection of Cytochrome c-like Protein in the Mitochondrial and Cytosolic Fractions (Western Blot Method)
An equal number of proteins (30 µg) from both treated and untreated cells were loaded and electrophoresed on 4-20% SDS-polyacrylamide gels (Mini-Protean Gels, 4-20%, Bio-Rad Laboratories Inc., Hercules, CA, USA). At the end of the electrophoresis, the proteins were blotted onto the nitrocellulose membrane (0.45 µm, Thermo Fisher, Carlsbad, CA, USA). The efficiency of the process was checked using a commercial membrane stain kit (MemCode Reversible Protein Stain Kit, Thermo Fisher, Carlsbad, CA, USA). Then the membrane was dried and preblocked with 5% skimmed milk prior to incubation with the primary antibodies: 1 µg/mL, murine monoclonal antibody, attached to the Cytochrome c releasing apoptosis kit assay kit (Enzo Life Sciences). The membrane was then probed with secondary antimouse antibodies conjugated to horseradish peroxidase (final antibody concentration-0.13 µg/mL, Enzo Life Sciences, Farmingdale, NY, USA). The immunoreactions between the antibodies were detected by the Covalight Detection System-enhanced chemiluminescence apparatus (Enzo Life Sciences) and recorded using the ChemiDoc MP gel visualization system (Bio-Rad).

Statistical Analysis
All data were expressed as mean ± standard deviation. The normality of their distribution was checked using the Kolmogorov-Smirnov (K-S) test. The ANOVA test and Tukey's HSD Test were used to compare them. The significance level was assumed to be 95% (p < 0.05). STATISTICA 6.1 software (StatSoft Polska, Kraków, Poland) was used for all statistical testing.

Conclusions
The research presented in this paper indicates that during fungal infection, several changes in mitochondrial physiology and morphology take place in G. mellonella hemocytes and that these disturbances accompany apoptosis in insect immunocompetent cells. Most of them are similar to changes occurring in mammalian cells, which would reflect the considerable evolutionary conservatism regarding the role of mitochondria in apoptosis activation in insects and mammals. However, further research is needed to determine