Insect cuticular compounds affect Conidiobolus coronatus (Entomopthorales) sporulation and the activity of enzymes involved in fungal infection

Mycoses are a global problem that affects humans and animals. In the present study, the entomopathogenic soil fungus Conidiobolus coronatus (Entomophthorales), infecting in tropics also humans, sheep and horses, was cultivated with the addition of insect cuticular compounds (CCs) previously detected in the cuticle of C. coronatus—resistant fly species (C10–C30 fatty alcohols, butyl oleate, butyl stearate, glycerol oleate, squalene, tocopherol acetate). Our findings indicate that CCs have diversified and complex effects on the growth and sporulation of C. coronatus and its ability to infect the larvae of Galleria mellonella (Lepidoptera). The CCs affected protein content and cuticle-degrading enzymes (CDEs) activity in the conidia. Some CCs inhibited fungal growth (0.1% C10), decreased sporulation (C12, C16, C24, C28, C30, butyl stearate, squalene), virulence (C12, C14, butyl oleate, butyl stearate) and protein content (C18). They also reduced conidial CDE activity: elastase (C24, butyl oleate, butyl stearate, squalene, tocopherol acetate), chitobiosidase (C12, C14, C20) and lipase (C12, C18, C26, squalene, tocopherol acetate). Several CCs enhanced sporulation (C14, C18, C22, C26, C30), virulence (C18, C26, squalene), conidial protein content (C16, C24, C30, squalene) and CDE activity: elastase (C10, C16, C18), NAGase (C16, C20), chitobiosidase (C16) and lipase (C10, C14, C16, C20, butyl oleate). Our findings indicate that C. coronatus colonies grown on media supplemented with CCs employ various compensation strategies: colonies grown with C16 alcohol demonstrated reduced sporulation but greater conidial protein accumulation and increased elastase, NAGase, chitobiosidase and lipase activity, thus preserving high virulence. Also, colonies supplemented with C18 alcohol demonstrated high virulence and enhanced sporulation and elastase activity but slightly decreased conidial protein content. CCs that inhibit the activity of lipases and proteases show promise in the fight against conidiobolomycosis.

Increases in transplantation surgery procedures, numbers of hospitalized and immunocompromised patients, cases of HIV infection and the need for chemotherapy have resulted in a dramatic rise in incidence of human mycosis over the past few decades. There is a pressing need to develop anti-fungal agents. As infection most commonly occurs by inhalation of conidia, the key to understanding the infection strategies adopted by mycoses, and developing treatments, may well lay in the composition of conidia, particularly the enzymes playing crucial role in the fungal infection progress.
One such fungus is the cosmopolitan soil fungus Conidiobolus coronatus (Constantin) belonging to the order Entomophthorales: a typical saprotroph and a facultative human pathogen known to cause mycoses in a broad spectrum of mammals and insects [1][2][3] . In mammals, entomophthoromycosis manifests as a chronic, inflammatory or granulomatous fungal disease limited to the subcutaneous or submucosal nasal tissue 4 . The first human case of this kind was reported in 1965 in a patient from the West Indies 5 . Most human cases of C. coronatus entomophthoromycosis, known as conidiobolomycosis, have been recorded in Africa, South and Central America and www.nature.com/scientificreports/ had been allowed to cool to their melting points (64-87 °C). After thorough mixing, the mixtures were poured onto sterile Petri dishes in a laminar flow hood and sterilized by UV (20 min). Untreated SAB culture was used as a control. SAB-GM was used as an additional control, as it is the standard medium used in our laboratory for maintaining the high virulence of C. coronatus cultures used in testing susceptibility of various insect species. Each experimental variant was performed in three or four independent replications; the controls were performed as seven replications. Cultures were performed for 7 days at the optimum temperature for fungus growth (20 °C).

Virulence of C. coronatus colonies.
To determine the ability of C. coronatus to infect G. mellonella, 40 G. mellonella larvae were exposed to each fungus SAB culture supplemented with the CCs at final concentrations of 0.1-0.0001%. All larvae were 5 days old, in their last (VII-th) instar (i.e. at the beginning of the wandering stage, 5 days after last larval molt and 2 to 3 days before pupation), and were maintained and reared in temperature and humidity-controlled chambers (30 °C, 70% r.h.) in constant darkness on an artificial diet 36 . After exposure (20 h), the insects were transferred to clean Petri dishes with food and kept under their growing conditions for 7 days. The larvae at this stage physiologically cease feeding and the food serves them as a safe environment for pupation. The condition of the exposed larvae was monitored daily. The control insects were exposed for 20 h to sterile SAB plates. The exposure of tested insects to a C. coronatus colony has been found to be the most efficient method resembling the natural infection process 37 . Virulence was determined based on the percentage of infected larvae. Insect infection was recognized by immobilization of larvae and melanization of the cuticle as black spots. The percentage of dead insects was not taken into account as this work concerns the effect of CCs added to the SAB medium on the levels of cuticle-degrading enzymes (essential for the first phase of infection) in the spores produced by the mycelia grown on such media; in G. mellonella, the death of larvae following C. coronatus infection is mainly caused by the toxic metabolites released by the fungus after invasion [8][9][10][11][12][13][14][15]38 .
The conidia of C. coronatus were stained with Calcofluor White, which binds to chitin in the fungal cell wall. Conidia washed off the fungus cultures were placed on a µ-Slide 8-well cell culture plate (Ibidi). Two drops of Calcofluor White and two drops of 10% potassium pydroxide (both from Merck) were added to each well. The slide was incubated for one minute at room temperature. To visualize the fungal spores and hyphae on the surface of infected G. mellonella, the larvae were placed in the well of a 6-well, flat bottom, sterile plate (Corning). Six drops of Calcofluor White and six drops of 10% potassium pydroxide were added to each well and incubated for one minute at room temperature. The microscope observations and photo documentation were performed using an Axio Vert.A1 fluorescence microscope (Zeiss) with Axio Cam ICc 5 (Zeiss) and ZEN 3.2 lite software with Modul Image Analysis (Zeiss).

Sporulation efficiency.
To estimate the efficiency of C. coronatus sporulation, 7-day-old solid cultures were briefly washed with 2 ml sterile water, and the number of harvested conidia was determined under the microscope in a Bürker chamber. Briefly, 50 µl of conidia suspension was thoroughly mixed and taken for counting. For all culture variants, the conidia were counted in three or four independent samples; the controls (SAB and SAB-GM) were counted in seven independent samples. The result was converted into 2 ml and then 1 cm 2 colony area.
Protein concentration. Samples with the known number of conidia were sonicated until all conidia were homogenized (inspection under microscope), then the protein contents were measured with the Protein Assay (Bio-Rad). Bovine serum albumin (BSA; Sigma-Aldrich) was used as a standard. The results were based on the total protein content in the entire sample and then calculated as a value per single conidium.
Enzymatic assays in C. coronatus conidia. The sonicated samples of conidia were centrifuged (4 °C, 20 min, 1500 × g). Activities of the cuticle degrading enzymes (CDEs) were measured in supernatants as described earlier 31 . The activities of elastase, N-acetylglucosaminidase (NAGase), chitobiosidase and lipase, all considered as determinants of fungal virulence, were measured spectrophotometrically and spectrofluorimetrically (BioTek Synergy HT) in 96-well polystyrene plates (Corning) using suitable synthetic substrates (all from Sigma-Aldrich) made up to a final volume of 200 μl with reaction buffer (Tris-HCl pH 7.0, 8.0 or 10.0-depending on the substrate used). All supernatant samples were thoroughly mixed for one minute before adding to the appropriate substrate solutions.
Elastase activity was measured in 2 μl of supernatant samples mixed with 0.5 mM N-Succinyl-Ala-Ala-Pro-Leu-p-Nitroanilide in a 100 mM Tris-HCl buffer containing 20 mM CaCl 2 (pH 8.0). Absorbance was read at 410 nm. NAGase activity was measured using 5 μl of supernatant samples incubated with 0.3 mM 4-Nitrophenyl-N-acetyl-β-d-glucosaminide in a 10 mM Tris-HCl buffer (pH 7.0). Absorbance was read at 405 nm. Chitobiosidase activity was measured in 6 μl of supernatant samples added to 0.003 mM 4-Methylumbelliferyl β-d-N-N′diacetylchitobioside in a 50 mM Tris-HCl buffer (pH 7.0). Fluorescence was read at Ex = 340 nm, Em = 450 nm. Lipase activity was measured using 10 μl of supernatant samples incubated with 0.01 mM 4-Methylumbelliferyl oleate in a 50 mM Tris-HCl buffer (pH 10.0). Fluorescence was read at Ex = 360 nm and Em = 450 nm. All reaction mixtures were incubated at 30 °C. The assays were prepared in three or four independent replicates.
CDEs activities were presented as: (a) total activity of the whole sample calculated per ng of conidial protein present in the sample, (b) activity calculated per ng of proteins present in single conidium, (c) activity calculated per single conidium.
Statistics. Statistical

Sporulation of C. coronatus.
Cultivation on solid media, as described in Methods, is a standard procedure used to determine the ability of a filamentous fungus to sporulate. Figure 1 presents C. coronatus cultures on Sabouraud agar (SAB) (Fig. 1A) and SAB medium enriched with a homogenate of G. mellonella (SAB-GM) (Fig. 1B). The mycelia growing on SAB-GM had a more undulating surface than those on the SAB alone. In contrast, supplementation of SAB medium with all tested cuticular compounds (CCs) had no influence on the mycelium appearance. No differences were found in the appearance and size of spores produced by mycelia grown on control SAB and SAB-GM media and on media supplemented with the CCs. Sporulation appeared to be influenced by the type of CC used to supplement the SAB medium and their applied concentrations ( Fig. 2; see also Table 1); however, the differences in sporulation between SAB and SAB-GM were statistically insignificant. The highest conidia production (24,809 ± 2155 conidia/cm 2 ) was observed after SAB supplementation with 0.0001% C30 alcohol; this value was about 2.3-fold higher than in the case of fungus cultured on SAB alone (10,900 ± 6282 conidia/cm 2 ; p < 0.001) and 2.0-fold higher than in SAB-GM cultures (11,880 ± 4345 conidia/cm 2 ). In contrast, the addition of 0.1% C10 alcohol completely inhibited fungal growth and sporulation, but further dilutions had no effect on sporulation. Similarly, SAB supplemented with 0.1% C24 alcohol significantly inhibited sporulation (1609 ± 434 conidia/cm 2 ) by 6.8-fold compared with SAB (p = 0.008). A low concentration (0.0001%) of C16 also significantly reduced sporulation (2293 ± 1038 conidia/ cm 2 ; p = 0.020).

Virulence of C. coronatus.
Larval infection was found to occur during 2 days after contact with C. coronatus ( Supplementary Fig. 1). In the first stage of infection, black melanized spots were observed on the cuticle followed by immobilization of larvae and the cessation of silk spinning and construction of cocoons. Infection results in the death of the insect and its mummification.
Protein content in C. coronatus conidia. The effect of SAB supplementation with tested CCs on protein content in C. coronatus conidia is shown in Fig. 4 and Table 1. The conidia collected from SAB enriched with G. mellonella larval homogenate (SAB-GM) displayed 1.4-fold higher protein content compared with SAB alone, however the differences in protein content between conidia from SAB and SAB-GM were statistically insignificant. More precise protein content values in C. coronatus conidia are given in Supplementary Table 1.
No significant decrease in protein content was observed after SAB supplementation with any CC.
Elastolytic activity in conidia of C. coronatus. Elastase plays a key role in the infection process. Only few of the tested CCs affected elastase activity in C. coronatus conidia ( Fig. 5; see also Table 1). More precise values are given in Supplementary Table 2. Differences in total elastase activity observed between conidia from SAB and SAB-GM cultures (601 ± 472 and 80 ± 74 pM/min/ng of protein, respectively) were statistically irrelevant. In this context, total elastase activity is understood as the elastase activity of all sonicated conidia present in the sample calculated per protein content in the sample. Interestingly although fungal growth was observed in the presence of 0.1% C24 alcohol, no elastolytic activity was detected. Furthermore, while the addition of 0.1% C10 alcohol inhibited fungal growth, lower concentrations, viz. 0.001 and 0.0001%, caused a significant increase of elastase activity (in both cases, p < 0.001). For alcohol C10, the greatest elastase activity was measured at a concentration of 0.001%, i.e. a 4.9-fold increase compared with untreated SAB, while for C18, the highest elastase activity was measured at 0.01%: a 5.5-fold increase compared with SAB (p < 0.001).
It should be pointed out that these results were acquired by measuring the activity of elastase in homogenates made from all fungal conidia collected from mycelia (Fig. 5). However, the fungus produced different numbers of conidia under different culture conditions (Fig. 2) and the conidia had different protein contents (Fig. 4). The general elastolytic activity measured in the conidia was influenced by three variables: the number of conidia produced by the mycelium, the protein content of each conidium, and the proportion of proteins with elastolytic activity in each conidium. To correctly determine the contribution of each of these factors to the overall enzymatic activity, the elastolytic activity is presented in three ways: (a) total activity of the whole sample calculated   Tables 3-5).
In general, the total elastolytic activity was found to be higher than the equivalent values expressed as activity calculated per single spore and per protein content per single spore. In most cases, activity calculated per protein content per spore equaled those calculated per single spore, suggesting that proteins with elastolytic activity and other proteins were evenly distributed between the spores. However, in C14 (0.1%), C16 (0.0001%), C18 (0.1%), BS (0.0001%) and GO (0.0001%) appeared to vary between spores ( Fig. 5; see also Supplementary Table 2).
NAGase activity in C. coronatus conidia. NAGase activity did not differ significantly between SAB and SAB-GM conidia ( Fig. 6; see also Table 1 and Supplementary Table 3). Only C16 had a significant effect on total NAGase activity in C. coronatus conidia, resulting in elevated NAGase activity at all applied concentrations (0.1-0.0001%; in all cases, p < 0.001) The highest increase, sixfold compared to SAB controls, was observed at 0.01% C16. A slight (statistically irrelevant) elevation of total NAGase activity was observed after SAB supplementation with C20 (0.1 and 0.001%).
Similarly to elastase, total NAGase activity tended to be higher than both the activity calculated per protein content per conidium and the NAGase calculated per conidium; both of the latter being equal. Only a couple of exceptions were observed: C16 (0.001% and 0.0001%) and C24 (0.1%) ( Fig. 6; see also Supplementary Table 3).
Chitobiosidase activity of C. coronatus. No significant differences were observed between SAB and SAB-GM with regard to total chitobiosidase activity ( Fig. 7; see also Table 1 and Supplementary Table 4). Supplementation of SAB with C16 at all concentrations elevated total chitobiosidase activity compared with SAB alone (in all cases, p < 0.001). The greatest (7.2-fold) increase was noticed at the concentration of 0.001% C16. Only a few alcohols slightly (i.e. statistically insignificantly) decreased total chitobiosidase activity: C12 (0.01, 0.001 and 0.0001%), C14 (all concentrations), C20 (0.001 and 0.0001%).
The activity of chitobiosidase resembled that of elastase and NAGase: total chitobiosidase activity was higher than activity calculated per protein content per conidium and higher than enzyme activity calculated per conidium: both values calculated per conidium were equal. However, exceptions were observed in the case of: C16 (0.001 and 0.0001%), C18 (0.1%) and C24 (0.1%) suggesting an uneven distribution of chitobiosidase and other proteins in these conidia ( Fig. 7; see also Supplementary Table 4).
Lipase activity in conidia of C. coronatus. No significant differences in total lipase activity were observed between SAB and SAB-GM. The CCs demonstrated a wider range of effects on lipase activity than on elastase or the two chitinases ( Fig. 8; see also Table 1 and Supplementary Table 5). Increased total lipolytic activity was observed after SAB supplementation with C10 (0.01 and 0.001%; p < 0.001 and p = 0.005, respectively), C14 (0.1%; p = 0.002), C20 (0.01%; p < 0.001) and BO (0.001 and 0.0001%; p < 0.001 and p = 0.016, respectively). Increased lipolytic activity in the spores collected from SAB supplemented with C16 (0.001 and 0.0001%) insignificant in total activity was evident after conversion to one spore or the protein content in one spore.
The trends described in the case of elastase and two chitinolytic enzymes (NAGase, chitobisidase) were also observed for lipolytic activity: total enzyme activity was higher than activity calculated per protein content per conidium and higher than enzyme activity calculated per conidium, both values being equal. However, exceptions were observed: C16 (at all concentrations) and C24 (0.1%) ( Fig. 8; see also Supplementary Table 5). Fig. 9) showed a clear distinction between the effects of CCs on the sporulation of C. coronatus and the ability to infect G. mellonella larvae. After analysis of all CCs, some parameters overlapped with those of the controls: GO, TA and C28 overlap with SAB, while BO and C20 overlap with SAB-GM. Interestingly, the SAB and SAB-GM parameters do not overlap. However, others were found to significantly differ from controls. This confirms that in most cases, the addition of a CC affected the sporulation of C. coronatus and the ability of spores to infect G. mellonella larvae. The first component (PC1) explained about 72% of the variation (the exact values depend on the used method of enzymatic activity calculation; Supplementary Table 6), with the values for sporulation (after adding all concentrations of CCs) representing the largest contribution. The second component (PC2) explained about 20% of the variation, with the largest contributions also being from sporulation.

Principal component analysis (PCA). The principal component analysis (PCA,
The PCA of the correlations between the effects of CCs on the growth and sporulation of C. coronatus and its ability to infect the larvae of G. mellonella are given in Fig. 10. The sum of its components is dependent on the method used for enzymatic activity calculation. When total enzymatic activity is considered, the first component (PC1) explained 49.26% of the variation, with the largest contribution for elastase, NAGase and chitobiosidase activity, as well as protein content (positive correlation) and sporulation (negative correlation). The second component (PC2) explained 14.92% of the variation, with high contributions from lipase activity (the high positive correlation) and virulency (negative correlation).
The first component (PC1) explained 58.50% of the variation, when the enzymatic activity was calculated per protein content per conidium. The largest contribution was observed for elastase, NAGase and chitobiosidase activity (positive correlations) and sporulation (negative correlation). The second component (PC2) explained 12.81% of the variation, with high contributions made by virulence and sporulation (positive correlation) and lipase activity (negative correlation). www.nature.com/scientificreports/ When enzymatic activity is calculated as activity per conidium, PC1 explained the 59.38% of the variation, with the largest contribution observed for elastase, NAGase, chitobiosidase and lipase activity (positive correlations) and sporulation (negative correlation). The second component (PC2) explained 10.91% of the variation, with the high contribution made by virulency and sporulation (positive correlation).

Discussion
The insect-fungus model is not only beneficial for studies evaluating the use of entomopathogenic fungi to control populations of insect pests, but it may also bring new ideas in the control of mycosis in humans, livestock and domestic animals. The object of our research is the cosmopolitan soil fungus C. coronatus, selectively acting entomopathogen capable of infecting also humans, dogs, horses and sheep. C. coronatus infects susceptible insect hosts via direct cuticle penetration by invasive hyphae formed after the germination of the spores on the cuticle. Penetration of the host integument is achieved by the mechanical pressure imposed by growing hyphae and the enzymatic degradation of major cuticle components (proteins, chitin and lipids) by proteases, chitinases and lipases produced by the fungus 31,32,40 . Upon invasion of the host hemocoel, hyphae expand but do not infest internal organs due to rapid host death caused by toxic metabolites of the fungus which disorganize functioning of Malpighian tubules, incapacitate immune system and affect serotonin-regulating enzymes 8,[10][11][12][14][15][16]38 .
The susceptibility or resistance of various insect species to C. coronatus invasion results from several factors, including the structure of the host's exoskeleton and the composition of its cuticle, as well as the efficiency of the host's immune system 13 . It seems however, that the composition of lipids present in the epicuticle is a key factor in protecting insects against fungal assault [19][20][21][22][23][24][25][26] . Previous studies demonstrating differential efficiency in hydrolyzing in vitro cuticles from larvae of three insect species representing various susceptibilities to C. coronatus infection (resistant: C. vicina, susceptible: G. mellonella and Dendrolimus pini) by enzyme cocktail secreted by the fungus into incubation medium, indicate a C. coronatus host specificity based on differential All these three FFAs when added to C. coronatus cultures retarded or totally inhibited fungus growth. Another FFAs effects: decrease of sporulation and virulence (C14:0, C20:0), and lower toxicity of metabolites released into culture medium (C16:1, C20:0) but surprisingly supplementation of culture medium with FFA C16:1 resulted in elevated virulence 30 . Further experiments proved that the effectiveness of C. coronatus enzymes (proteases, chitinases and lipases) hydrolyzing cuticle of several other insect species, both susceptible and resistant to that fungus, is correlated with concentrations of compounds detected in the cuticles of tested insects. Positive correlations indicate compounds used by the fungus as nutrients, whereas negative correlations those engaged in insect resistance 31,33,34 . Our attention was especially drawn to scavenger flies and houseflies, whose larvae and pupae are completely resistant to C. coronatus attack. The prompt death of adult flies exposed to C. coronatus colonies results from the ingestion of conidia or fungal excretions, as adult flies willingly lick all surfaces, including these covered by fungal spores 31   www.nature.com/scientificreports/ The present study examines the impact of these CCs on fungal growth, sporulation, and virulence, as well as activity of elastase, two chitinases (NAGase, chitobiosidase) and lipase in spores produced by C. coronatus colonies cultivated on media supplemented with CCs. Our findings indicate that tested CCs have diversified and complex effects on the growth and sporulation of C. coronatus and virulence toward the larvae of G. mellonella. CCs applied at various concentrations inhibited fungal growth (0.1% C10 only), decreased sporulation (C12, C16, C24, C28, C30, BS, S), virulence (C12, C14, BO, BS) and protein content in conidia (C18). They also reduced activity of cuticle degrading enzymes (CDEs) present in conidia: elastase (C24, BO, BS, S, TA), chitobiosidase (C12, C14, C20) and lipase (C12, C18, C26, S, TA). On the other hand, several CCs enhanced sporulation (C14, C18, C22, C26, C30), virulence (C18, C26, S), conidial protein content (C16, C24, C30, S) and CDE activity: elastase (C10, C16, C18), NAGase (C16, C20), chitobiosidase (C16) and lipase (C10, C14, C16, C20, BO). This variety of action on various elements determining the effectiveness of fungal infection indicates a high degree of complexity of the C. coronatus infection strategy. A good example is C10 alcohol, which totally inhibited fungus growth at the maximum concentration of 0.1%, probably by preventing conidia germination, but strongly stimulated the activity of lipase (0.01%) and elastase (0.001 and 0.0001%) at lower concentrations. None of the other tested CCs prevented C. coronatus growth, but in Saccharomyces cerevisiae C10 and C11 alcohols demonstrated antifungal effects, probably by disrupting native membrane proteins as nonionic surfactants 41 .
Another example of the multilevel effects of the tested substances on the components of the insect destruction system owned by C. coronatus is provided by the application of C16 alcohol. Supplementation of culture medium with low concentrations (0.001 and 0.0001%) of C16 alcohol reduced sporulation, but all applied concentrations strongly increased protein content in conidia, as well as the activity of elastase and both chitinases, while lipase activity was elevated to a lesser extent and only at low C16 concentrations (0.001 and 0.0001%). As virulence remained unchanged despite the reduced number of conidia, it appears that the fungus compensated for the quantitative loss in "ammunition" with more heavily-loaded "bullets". The fact that C10 alcohol stimulated an increase in elastase and lipase activity without increasing the protein content in spores, while C16 alcohol induced elastase and chitinase activity with a simultaneous high accumulation of proteins in the spores demonstrated that the processes responsible for the synthesis and deposition of cuticle-degrading enzymes in conidia are complex and require further detailed research. From our data it appears that cuticle degrading enzymes are not always evenly deposited in the spores produced by the same mycelium. Moreover, their proportions with regard to other conidial proteins varies depending on CC type and concentration in the culture medium, indicating a high complexity of regulatory processes controlling the synthesis of functionally different fungal proteins and their storage in the spores. Considering the fact that single conidium of C. coronatus contains from several to even a hundred cell nuclei which are genetically diverse 42,43 , determination of the mechanisms controlling C. coronatus genes encoding proteins with proteolytic, chitinolytic and lipolytic activities (226, 299 and 532 genes, respectively 44,45 ) is a big challenge.
Two controls were used in the studies assessing the effect of CC on fungal colonies: SAB medium without any additives and SAB enriched with homogenized G. mellonella larvae (SAB-GM). The addition of homogenate of whole larvae to SAB is a routine procedure to preserve the virulence of the cultured fungus over subsequent generations 37 . The insect homogenate contains high concentrations of proteins, chitin and lipids, which serve as excellent nutrients for the fungus; therefore, is not surprising that the colonies grown on SAB-GM demonstrated slightly higher spore production with slightly higher virulence, as well as greater protein content, compared with SAB cultures. However, the spores produced by SAB-GM colonies demonstrated lower activity of cuticledegrading enzymes than those from SAB colonies. Although these differences are clearly visible and frequently seen in previous studies concerning C. coronatus grown on media with various nutrient content under optimal and stressful conditions 31,46-48 , they turned out to be statistically insignificant in the current study. Although growth substrates such as the lipids commonly found on insect cuticles generally increase fungal infectivity 49 , conidia derived from media containing lower carbon/nitrogen ratios frequently are found to be more virulent than those harvested from nutrient-rich media [49][50][51] . Cuticle-degrading enzymes play a key role in determining virulence as they allow the fungus to disrupt the integument of the insect and enter the body cavity. The fungal proteases, chitinases and lipases digesting the major components of insect cuticle demonstrate significantly higher activity in C. coronatus cultures propagated in liquid minimal medium (MM) than in those prepared in rich Luria Broth (LB) medium 48 . Furthermore, post-incubation MM is characterized by higher toxicity than LB 8 , partly due to the accumulation of higher amounts of β-carboline alkaloids (harman and norharman); these disorganize the development and immune system of G. mellonella larvae by affecting serotonin-regulating enzymes 10,11 . Similarly, basidiomycetes grown in media with low C/N ratios demonstrate reduced mycelial growth but increased production of laccase and other enzymes hydrolyzing lignin and cellulose 52,53 . This common phenomenon results from carbon catabolic repression (CCR) mechanism helping fungi to precisely adapt their physiology to the environment. CCR switches off the enzymes needed to utilize less-favored carbon sources when a more readily available carbon source is present in the medium 54 .
Results indicating that some compounds present in the cuticle of the C. coronatus-resistant flies completely inhibit elastase and lipase activity (alcohols: C24 and C12, respectively) or only slightly reduce their activities (squalene and tocopherol acetate) may be a good starting point for the development of new methods of combating conidiobomycosis in humans and animals. Further studies based on other concentrations, vehicles and accompanying substances than those used in the present study, intended to confirm the inhibitory effect of squalene and tocopherol acetate are merited, as these substances demonstrate particular promise. While fatty alcohols can be irritating to human skin, especially the skin of people attacked by the fungus, the therapeutic use of tocopherol acetate and squalene seems safe because these two compounds are natural products of sebocytes and are essential constituents of human sebum 55

Conclusion
Our findings can be used to identify compounds which inhibit or stimulate the development of mycosis, and thus describe the infection strategies employed by C. coronatus. The insect-fungus research model is very convenient, as it demonstrates high metabolic flexibility of the fungus and easy adaptation to different media in terms of C and/or N composition or biotic and abiotic stress 56 .