The Impact of the Entomopathogenic Fungus Conidiobolus coronatus on the Free Fatty Acid Profile of the Flesh Fly Sarcophaga argyrostoma

Simple Summary The interaction between insect and fungus is characterised on the one hand by the parasite developing more effective strategies of host exploitation, and on the other, by the host mounting increasingly robust defences though Red Queen dynamics or coevolutionary arms races. Furthermore, depending on gene flow and differences in selection pressure between sites, both host and parasite may demonstrate local adaptation to their counterpart or develop more general resistance or offensive traits. As the cuticle is considered the first line of defence of the insect, changes in the FFA profile may well influence susceptibility or resistance to fungal invasion. Our findings indicate that Sarcophaga argyrostoma demonstrates stage-specific resistance to Conidiobolus coronatus infection and suggests that FFAs play a role in resistance to fungal infection in flesh flies. These findings not only increase our knowledge of the entomopatogenic potential of fungi, but also of the growing level of infection by C. coronatus in humans and other mammals. Also, the presented research suggests that FFAs demonstrate antifungal activity which may be helpful in designing new antifungal treatments. Abstract The chemical composition of the insect cuticle varies remarkably between species and their life stages. It can affect host resistance and substrate utilization by invading entomopathogen fungi, such as the soil fungus Conidiobolus coronatus. In this study, Sarcophaga argyrostoma flies were exposed to sporulating C. coronatus colonies for 24 h; the pupae were resistant, but the adults demonstrated 60% mortality. Although the pupae demonstrated no sign of infection nor any abnormal development, our findings indicate that after 24 h of contact with the fungus, the pupae demonstrated a 25.2-fold increase in total cuticular free fatty acids (FFAs) and a 1.9-fold decrease in total internal FFAs. Also, the cuticular FFA increased from 26 to 30, while the internal FFA class increased from 13 to 23. In exposed adults, the total mass of cuticular FFAs increased 1.7-fold, while the number of FFAs stayed the same (32 FFAs). Also, the internal FFA class increased from 26 to 35 and the total FFA mass increased 1.1-fold. These considerable differences between adults and pupae associated with C. coronatus exposure indicate developmental changes in the mechanisms governing lipid metabolism and spatial distribution in the organism, and suggest that cuticular lipids play a vital role in the defence against pathogenic fungi.


Infection of Insects
S. argyrostoma flies (pupae and adults) were exposed for 24 h at 20 • C to fully grown and sporulating C. coronatus colonies, around 10 per Petri dish. The controls were exposed for 24 h to sterile SAB-GM medium. After exposure, the insects were divided into the following two groups: One was transferred to new, clean Petri dishes (imagines with appropriate food), and observed for seven days. The other was treated with water and left to dry, to remove fungal conidia from cuticle surface and then frozen after 24 h exposure to C. coronatus and kept at −20 • C until FFA composition was tested. The numbers of individuals used for experiments are presented in Table 1. Each test was performed separately. The virulence of C. coronatus colonies was confirmed by testing on G. mellonella larvae treated in the same way as the S. argyrostoma pupae and adults.

Extraction of Free Fatty Acids (FFAs)
The cuticle and internal lipid components were extracted from the pupae and adults of S. argyrostoma. Firstly, the insects were extracted in 20 mL of petroleum ether for five minutes (extract I) and then a second time in 20 mL of dichloromethane for another five minutes (extract II). These two extracts (I and II) contained the cuticular lipids. The use of petroleum ether minimizes the possible extraction of internal lipids, which are mostly FFAs and glycerides [56]. The third extract was obtained by sonification of insects in 20 mL of dichloromethane for one minute. This extract contained the internal lipids. Each extraction was performed only once due to the small number of available insects. The extracts were placed in glass flasks and then evaporated under nitrogen. The masses of insects and the extracts are presented in Table 1. These methods expand upon those detailed within our previous work [37,38,57].

Derivatization Method
One milligram of each sample and 10 µL of internal standard (19-methylarachidic acid; 1 mg/mL; Merck Millipore) were silylated with 100 µL of N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA): chlorotrimethylsilane (TMCS) (99:1) (Merck Millipore) mixture for one hour at 100 • C to obtain trimethylsilyl esters (TMS) of FFAs. The TMS values of the fatty acids were then analysed by GC-MS. The GC-MS analysis used 19-methylarachidic acid as an internal standard (IS) because it separates well from all the sample constituents and was not previously present in the insect samples [58]. These methods expand upon those detailed within our previous work [37,38,42,59].

GC-MS Analyses
The samples were separated and analysed by gas chromatography-mass spectrometry (GC-MS) using high-purity solvents (≥95%, Merck Millipore, Burlington, MA, USA). The GC-MS analyses were carried out on a GCMS-QP2010 system with a mass detector (Shimadzu, Kyoto Japan,). As the carrier gas, helium was used at a column head pressure of 65.2 kPa. A DB-5 MS (Zebron, Phenomenex, Torrance, CA, USA) column was used (thickness 0.25 µm, length 30 m, diameter 0.25 µm). The column oven temperature cycle was maintained at 80 • C for 3 min, then ramped up from 80 to 310 • C at 4 • C/min; the final temperature was then held for 10 min. The ion source temperature was 200 • C and the interface temperature was 310 • C. Split mode was used with a split ratio of 10. All compounds were identified based on the fragmentation patterns and mass-to-charge ions of the TMS derivatives given in the NIST 11 library. The mass spectra of the fatty acid trimethylsilyl esters comprised M+ (molecular ion), [M-15]+, and fragment ions at m/z 117, 129, 132, and 145. The content of the compounds in the analysed samples was calculated from the chromatogram peak areas. Each sample was analysed in triplicate Insects 2021, 12, 970 5 of 25 and the results were expressed as means and standard deviation. Response factors of one were assumed for all constituents. These methods expand upon those detailed within our previous work [36][37][38]42,58,59].

Statistics
Principal component analysis (an unsupervised learning method) was used to visualize differences in the data between control and fungus treated in both cuticular and internal FFA fractions from the pupae and imagines. The test was performed using Past 4.05 software [60]. The normality of the data was checked using the Kolmogorov-Smirnov (K-S) test. As all the variables had normal distributions, they were analysed using Student's t-test (to compare susceptibility) and ANOVA analysis (to compare FFA profile). The significance level was 95% (p < 0.05). STATISTICA software (StatSoft Polska, Cracow, Poland) was used for statistical testing.

Susceptibility of S. argyrostoma to Fungal Infection
The S. argyrostoma imagines and pupae demonstrated different susceptibilities to sporulating C. coronatus colonies, with the pupae being resistant and the adults being susceptible to C. coronatus infection. The results are shown in more detail in Table 2 and  Supplementary Table S1 (raw data). The pupae did not demonstrate any signs of infection nor any signs of fungal penetration through the cuticle. In addition, the metamorphosis continued normally. In contrast, 60% mortality was observed in adults after one day following 24 h exposure to C. coronatus, resulting from the ingestion of conidia and/or excretions covering the surface of the fungal colonies; S. argyrostoma flies are eager to lick all potential food sources. The SAB-GM medium on which the fungus was grown contains the following ingredients, which attract flies and encourage licking: tryptone, yeast extract, agar, glucose, and homogenized G. mellonella larvae.

Effectiveness of Extraction Process
Three types of extraction were performed for the pupal and adult material. The cuticular lipids were obtained in the petroleum ether and dichloromethane extracts (extract I and II, respectively), and the internal lipids in the dichloromethane extracts after sonification (extract III). The total masses of the extracts are shown in Table 1.
Exposure to C. coronatus caused changes in the masses of the cuticular and internal extracts in both pupae and adults. A 1.2-fold increase in cuticular lipids and a 16.1-fold decrease in internal lipids per insect was observed in the extracts from the fungus-treated pupae, compared to the untreated pupae. Exposure to the fungus resulted in the proportion of cuticular to internal extracts changing from 1:3.08 in the control pupae to 1:0.16 in the treated pupae.
The fractions from the untreated adults also demonstrated a higher mass of internal lipids (0.44 mg per insect) than cuticular lipids (0.25 mg per insect). Similarly to the pupae, the exposure of adults to C. coronatus caused a 1.8-fold increase in the mass of the cuticle extracts and a 3.9-fold decrease in the internal extracts per insect. The ratio of the cuticular to internal fractions was 1:1.76 in the control insects, and 1:0.25 after exposure to fungus.
These extracts were further analysed by GC-MS. A comparison of the FFA profiles of the cuticle surface (sum of extracts I and II), and the internal structures of the pupae and imagines, is given in Tables 3-6; the raw data are appended in Supplementary Table S2.

GC-MS Analyses
The principal component analysis (PCA, Figure 1) showed a clear distinction between the concentrations of FFAs in the control group and the group treated with the fungus. The concentrations of FFAs following fungal exposure significantly overlapped in the pupae and imagines; this confirms that the FFA profiles of the cuticular and internal fractions converged after fungal exposure in both developmental stages. The first component explained 99.04% of the variation, and C18:1 and C16:0 represented the largest contribution. The second component explained 0.84% of the variation, with the largest contributions from C16:1. FFAs were extracted from control and exposed to C. coronatus pupae and adults. The first two principal components cover 99.04% and 0.84% of the variance, respectively, for a total of 99.88% variance.

GC-MS Analyses of Compounds Extracted from Pupae
The concentrations of the individual FFAs extracted from the pupae are presented in Table 3. Table 3. Fatty acid content of the cuticular and internal lipids extracted from the pupae of Sarcophaga argyrostoma (μg/g FFAs were extracted from control and exposed to C. coronatus pupae and adults. The first two principal components cover 99.04% and 0.84% of the variance, respectively, for a total of 99.88% variance.

GC-MS Analyses of Compounds Extracted from Pupae
The concentrations of the individual FFAs extracted from the pupae are presented in Table 3. Table 3. Fatty acid content of the cuticular and internal lipids extracted from the pupae of Sarcophaga argyrostoma (µg/g of insect body ± SD). The highest total FFA content was observed in the cuticular fraction of the pupae after fungal treatment (1189.69 ± 57.97 µg/g of the insect body); the value was 28.1 times higher than in the controls (47.27 ± 0.26 µg/g of the insect body, F(3,8) = 952.35, p < 0.001). However, in the internal fraction, the total FFA content was 1.8 times lower in the extract Insects 2021, 12, 970 8 of 25 from the exposed insects (8.53 ± 0.07 µg/g of the insect body) than in the untreated ones (15.78 ± 0.45 µg/g of the insect body).

FFA
The FFAs C16:0, C18:1, and C18:0 were found to be dominant in the cuticular and internal extracts from both the untreated and exposed pupae.
The concentrations of glycerol, cholesterol, β-sitosterol, and stigmastanol in the extracts from the pupae are presented in Table 4. Glycerol and cholesterol were observed in all the extracts. Similarly to the FFA content, their levels were elevated 2.7-fold and 2.2-fold in the cuticular fraction following exposure to C. coronatus, but decreased 10.  SD-standard deviation; ND-not detected; statistically significant differences (ANOVA, post hoc Tukey's HSD) between concentration of compounds are marked with the same letters ( A,B ); see Supplementary Table S2 for raw data.
The total ion current (TIC) chromatograms of the fatty acids (TMS esters) of the ether extract (extract I), dichloromethane extract (extract II), and dichloromethane extract after sonification (extract III), from both the control and exposed pupae, are given in

GC-MS Analyses of Compounds Extracted from Adults
The untreated adults contained, respectively, 3.0-and 3.4-times higher total masses of FFAs in the cuticular and internal extracts compared to the untreated pupae. In contrast to the pupae, the exposure of adult flies to C. coronatus resulted in elevations in the total FFA masses in both the cuticular (1.7-fold) and internal fractions (1.1-fold).

GC-MS Analyses of Compounds Extracted from Adults
The untreated adults contained, respectively, 3.0-and 3.4-times higher total masses of FFAs in the cuticular and internal extracts compared to the untreated pupae. In contrast to the pupae, the exposure of adult flies to C. coronatus resulted in elevations in the total FFA masses in both the cuticular (1.7-fold) and internal fractions (1.1-fold).
Glycerol and cholesterol were observed in all the extracts (Table 6). β-sitosterol and stigmastanol were found to be absent from the adults, but present in the pupal extracts. The highest concentrations of glycerol and cholesterol were observed in the cuticular fraction after exposure to C. coronatus, with these being 1.9-times higher (0.19 µg/g insect body mass) and 1.3 times higher (8.50 µg/g insect body mass) than in the cuticle fraction from the control insects. A higher concentration of cholesterol was also observed in the internal fraction from the imagines exposed to C. coronatus; however, no statistically significant differences in glycerol concentration were observed between the internal fractions of the control and exposed adults (0.08-0.07 µg/g insect body mass, respectively). The total ion current (TIC) chromatograms of fatty acids (TMS esters) of the ether extract (extract I), dichloromethane extract (extract II), and dichloromethane extract after sonification (extract III), from control and exposed adults, are given in Figures 5-7.

Discussion
The structure of the host exoskeleton, particularly the composition of lipids present on the cuticle, seems to be a major factor determining the susceptibility or resistance of insect species to C. coronatus infection [31,[34][35][36]46,61]. Research on C. vicina has shown the larvae to be resistant to fungal infection after exposure to sporulating fungal colonies; however, injection of C. coronatus conidia resulted in 100% mortality in 24 h. The flies exposed to the fungus did not demonstrate any signs of fungal penetration through the fly cuticle, nor any changes to the internal organs, nor any mobilization of haemocytes to eliminate the fungal pathogen; in contrast, those injected with C. coronatus conidia suffered profound damage to the internal organs. Hence, in these Dipteran flies, it appears that a pivotal role in the resistance to fungal infection was played by the cuticle [62]; this is supported by further observations that C. vicina larvae have thicker cuticles than those of G. mellonella or D. pini, which are also more susceptible to fungal infection [62]. The role of cuticular FFAs in the resistance to fungal infection by C. vicina, D. pini, and G. mellonella larvae is also described by Gołębiowski et al. [31].
Our findings indicate that the effects of C. coronatus exposure on S. argyrostoma vary considerably between developmental stages, with pupae being resistant to infection and adults being susceptible. This result is consistent with previous studies of other flies from

Discussion
The structure of the host exoskeleton, particularly the composition of lipids present on the cuticle, seems to be a major factor determining the susceptibility or resistance of insect species to C. coronatus infection [31,[34][35][36]46,61]. Research on C. vicina has shown the larvae to be resistant to fungal infection after exposure to sporulating fungal colonies; however, injection of C. coronatus conidia resulted in 100% mortality in 24 h. The flies exposed to the fungus did not demonstrate any signs of fungal penetration through the fly cuticle, nor any changes to the internal organs, nor any mobilization of haemocytes to eliminate the fungal pathogen; in contrast, those injected with C. coronatus conidia suffered profound damage to the internal organs. Hence, in these Dipteran flies, it appears that a pivotal role in the resistance to fungal infection was played by the cuticle [62]; this is supported by further observations that C. vicina larvae have thicker cuticles than those of G. mellonella or D. pini, which are also more susceptible to fungal infection [62]. The role of cuticular FFAs in the resistance to fungal infection by C. vicina, D. pini, and G. mellonella larvae is also described by Gołębiowski et al. [31].
Our findings indicate that the effects of C. coronatus exposure on S. argyrostoma vary considerably between developmental stages, with pupae being resistant to infection and adults being susceptible. This result is consistent with previous studies of other flies from the order Diptera, such as C. vomitoria, C. vicina, L. sericata, and M. domestica [32,34,41,61,62]. While there was no apparent sign of infection on the cuticle of the S. argyrostoma imagines, studies have shown a significant mortality rate of adult flies. Assuming that the adult flies were licking the sporulating fungus during the present study, it is likely that the route of infection was by ingestion of fungal spores, which would quickly germinate inside the gut. The growing hyphae release toxic metabolites [42,53,[63][64][65], which could promptly kill the flies. In contrast, fungus can only invade the pupae via the cuticle.
Changes in cuticle lipid profiles, occurring during normal development and after contact with the fungus, are the basis for selecting antifungal substances. The fatty acid contents of insects can vary according to their developmental stage, temperature, and dietary regime [38,42,57,59]. A previous study describing the metamorphosis-related changes in the FFA profiles of S. argyrostoma indicated the presence of C23:0 and C25:0 only in larvae, C28:0 in the pupal cuticle, and C12:1 and C18:3 in internal extracts from adults. The present research confirms the presence of C28:0 in both the pupae and adults. The occurrence of this FFA is quite unique for insects. It is an aliphatic primary acid, which has been shown to be an antibiofilm and anti-adherence agent against Streptococcus mutans (Lactobacillales: Streptococcaceae) [66]; it has, so far, only been detected in the cuticular wax of the honey bee Apis mellifera (Hymenoptera: Apidae) [67] and in the cuticular fraction from the larvae and pupae of Dendrolimus pini (Lepidoptera: Lasiocampidae) [33]. It is important to note that this FFA is absent in extracts from species considered as significant tools in forensics, such as C. vicina [68], C. vomitoria [34], and S. carnaria [46].
In turn, the FFA C13:0 was observed in the extract from G. mellonella imagines [42] and the internal extract from B. orientalis oothecae [37], in the larvae and puparia of C. vicina [31], and in three development stages (larvae, pupae, and imagines) of C. vomitoria [34]. Although this FFA has been found to demonstrate antifungal activity, and to have a negative impact on the growth and sporulation of C. coronatus [43,46], Wrońska et al. reported a high positive correlation between the concentration of C13:0 in the cuticle of G. mellonella imagines and the effectiveness of the enzyme cocktail produced by C. coronatus when breaching the cuticle; this may suggest that it has a positive impact on the fungus [42]. A strong positive corelation was also observed between the concentration of C13:0 in the cuticle of C. vicina and the effectiveness of proteases, chitinases, and lipases produced by C. coronatus in degrading the main cuticular constituents, viz. the proteins, chitin, and lipids [41].
The FFA C24:1 is an uncommon lipid in insect tissues. In the present study, it was only detected in the fractions from imagines exposed to infection by C. coronatus. However, it has previously been observed in extracts from Sphex flavipennis (Hymenoptera: Apocrita), mostly in the heads of wasps [70], and in powders obtained from the house cricket Acheta domesticus (Orthoptera: Gryllidae) and mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) [71]. The FFA C24:1, n-9 ∆15 (cis-15-tetracosenoic acid), or nervonic acid (NA), is an important component in myelin biosynthesis in the central and peripheral nervous system. Myelin is generally localized to the sphingomyelin of animal cell membranes, where it has been proposed to enhance human brain function [72]. Several filamentous fungi and bacteria, such as Macrophomina phaseolina (Botryosphaeriales: Botryosphaeriaceae), Francisella tularensis (Thiotrichales: Francisellaceae), or Mortierella capitata (Mortierellales: Mortierellaceae), are capable of accumulating NA [73], which might suggest that the C24:1 observed in the extracts from adults is produced by C. coronatus during fungal exposure (data not presented).
Changes in the thickness and composition of the cuticle can increase the resistance of insects by slowing the penetration of a pathogen or insecticide. In addition, transporters of cuticular lipids can be overexpressed in the epidermis, which can improve insecticide resistance by inhibiting insecticide penetration [80].
In the present study, all the pupal and adult extracts demonstrated a gain in mass in the cuticular fraction, following exposure to the fungus, and a loss of mass in the internal extracts. Similarly, the total concentration of all the cuticular FFAs was higher after fungal treatment. In the pupae, which were resistant to the fungal infection, the extract mass increased by only 25 µg per insect; however, the sum of FFAs increased 28 times. This result might suggest that exposure to C. coronatus promotes the translocation of FFAs from inside the body to the cuticle of S. argyrostoma, and this may serve as a protective mechanism against fungal infection in the pupae. An important finding is that exposure of the pupae to the fungus resulted in an increase in the concentrations of C6:0, C9:0, C12:0, C14:0, C15:0, C16:1, C16:0, C18:1, and C18:0 in the cuticular fraction, accompanied by a decrease in the internal fraction, which might suggest the translocation of these FFAs from the insect body to the cuticle in response to C. coronatus. However, more research is needed to confirm this.
After exposure, C11:0, C15:1, C24:0, C23:0, and C23:0 were detected in the cuticular fraction of the pupae, and C17:1, C17:0, and C20:0-C30:0 in the internal fraction. In addition, ß-sitosterol and stigmastanol were observed in both the cuticular and internal fractions. These changes may also be associated with fungal exposure, but, again, more research is needed to confirm this. The lack of C20:5 and C20:1 in the exposed pupae might be due to these acids being used by the fungus as a nutrient; however, this also requires further study.
In the present study, the imagines demonstrated increases in FFA levels in both the cuticular and internal fractions. This change may be the response of the insect to counter fungal infection, as it could protect the insect from both cuticle penetration and the activity of fungal toxins or infection inside the body. In addition, the exposed adults demonstrated the presence of C13:0 and C24:1 in the cuticle, and C11:0, C12:1, C13:0, C19:1, C19:0, C20:3, C20:1, and C24:1 in the internal fraction; as well as this, C12:1 and C32:0 were absent from the cuticle, which suggests that the imagines have a different reaction to C. coronatus exposure than the pupae. However, more research is needed to conclusively determine the high susceptibility of adult flies to C. coronatus infection.
The observed increase in FFA concentration might be connected with the lipolytic activity of enzymes produced by C. coronatus. Previous studies have found that the enzyme cocktail produced by C. coronatus demonstrates low efficacy against cuticles extracted from the pupae, thoraces, and wings of adults from four Diptera flies, viz. M. domestica, C. vomitoria, C. vicina, and L. sericata; all of these demonstrate similar resistance to C. coronatus infection as S. argyrostoma [41]. Histological studies have also found that the conidia of C. coronatus did not germinate on the cuticle of C. vicina larvae, which are resistant to fungal infection [62]. Hence, we suppose that the detected FFAs are not released in a result of lipolytic activity of fungus from the cuticle of S. argyrostoma, a species with similar resistance to fungal infection as Dipteran flies; however, the efficiency of the conidia against internal tissues and organs remains unknown.
At this point, we cannot exclude the possibility that the observed increase in internal FFAs may not only be a result of the insect defence mechanism, but also might be connected with fungal lipolytic activity against internal structures. In the case of the latter, the higher concentrations of the released FFAs would be accompanied with higher glycerol concentrations; lipases liberate free fatty acids and glycerol as a result of hydrolysing lipids at lipid-water interfases, functioning as glycerol ester hydrolases acting on mono-, di, and tri-glycerides [81]. In the present study, the exposure of adults to C. coronatus resulted in an increase in FFA concentration, accompanied, in one case, by a decrease in glycerol content; this might suggest that even the digestion of internal lipids is not a very effective process, or that the digested glycerol is quickly transported to other parts of the body. More research is needed to confirm these theses.
Cuticular fatty acids have a profound effect on fungal spore germination, and have different effects depending on the species of invading fungus, being either toxic, fungistatic, or, occasionally, stimulatory. Previous studies have shown that the FFAs C16:0, C16:1, C18:0, and C18:1 inhibit the growth and sporulation of C. coronatus [43,47]. The FFAs C18:3, C20:0, and C20:1 have been found to inhibit not only the growth and sporulation of C. coronatus, but also the virulence and toxicity of fungal metabolites released into the medium, even at a concentration of 0.0001% [43,47]. In addition, previous studies on insects that were resistant or susceptible to C. coronatus infection have identified a correlation between the concentrations of those FFAs (C16:0, C16:1, C18:0, C18:1 C18:3, C20:0, and C20:1) in the cuticle and the effectiveness of the protease, chitinase and lipase enzyme cocktail produced by C. coronatus, degrading the main cuticular constituents (proteins, chitin, and lipids) [37,41,42]. On the other hand, C16:0, C18:1, and C18:0 are favoured by another insect fungal pathogen, B. bassiana, and research has shown that supplementing the medium with these FFAs results in increased virulence. Moreover, decreased virulence was observed in a mutant with a loss of CYP P450 function, an enzyme known to display terminal hydroxylation activity against fatty acids. B. bassiana mutants also demonstrated reduced virulency in percutaneous infections against G. mellonella larvae, but displayed no such reduction when the insect cuticle was bypassed in intra-hemocoel injection assays; this suggests that cuticular FFAs may act as susceptibility factors [81][82][83].
In the present study, C16:0, C16:1, C18:0, and C18:1 FFAs are dominant in both the cuticular and internal fractions, and their concentrations increase after exposure to C. coronatus. A considerable increase in their concentration in the cuticle was also observed in C. vicina larvae and adults after treatment with the C. coronatus metabolite dodecanol [84]. Moreover, research comparing the FFA profiles of three species with differing susceptibility to fungal infection confirmed the presence of C16:0 only in C. vicina, which is highly resistant to fungal infection [31].
C. coronatus infection also caused changes in the concentration of glycerol. Similar to the FFAs, its accumulation was observed in the cuticular fraction from S. argyrostoma pupae and adults exposed to the fungus. The presence of glycerol in invertebrates prevents them from freezing and protects them from osmotic stress [88][89][90]; the increase in glycerol concentration in non-diapausing larvae of the flesh fly S. bullata occurs as a response to several forms of short-term environmental stress, such as low temperature, anoxia, and desiccation [91]. The accumulation of glycerol in the cuticle might be used to reduce water loss through evaporation [34,91]; however, increased glycerol and lipid mobilization, at least in part, is responsible for the increased appressorial turgor pressure of entomopathogenic Metarhizium spp. [92,93].
Another important group of compounds present in insect lipids are sterols. Of these, S. argyrostoma mostly displayed accumulation of cholesterol after C. coronatus exposure. Similar increases in cholesterol concentration have also been observed in the fungussensitive pine weevil H. abietis after B. bassiana infection [79]; however, cholesterol content was found to decrease in fungus-sensitive male Tettigonia viridissima (Orthoptera: Tettigoniidae) after C. coronatus treatment [94].
Being an important component of cellular membranes [95], and the precursor for many hormones [96,97], and due to its role in regulating genes involved in developmental processes [98], cholesterol is essential for the metabolism of insects. It has also been found to accumulate in membranes at low temperatures [99]. Insects are obligate sterol auxotrophs and must obtain cholesterol or its precursors from their diet. The fact that arthropods expend considerable energy in sequestering cholesterol in their cuticle suggests that it has functional significance; however, its role, if any, remains obscure [98,100].
Following fungal exposure, the presence of β-sitosterol and stigmastanol was observed in S. argyrostoma pupae, but not in control pupae or in adults. Insects use β-sitosterol as a substrate for cholesterol synthesis, and it has been found to contribute to the activation of biting and dietary selection in the silkworm Bombyx mori (Lepidoptera: Bombycidae). In addition, the inhibition of β-sitosterol metabolism results in growth inhibition in the tobacco hornworm M. sexta [101][102][103]. β-sitosterol demonstrates antibacterial and antifungal activity against Salmonella typhi (Enterobacterales: Enterobacteriaceae), Corynebacterium diphtheriae, Bacillus subtilis (Bacillales: Bacillaceae), and Fusarium spp (Hypocreales: Nectriaceae), and inhibits spore germination and germ-tube elongation in Aspergillus niger (Eurotiales: Trichocomaceae) and Botryodiplodia theobromae (Botryosphaeriales: Botryosphaeriaceae) [104,105]. The addition of β-sitosterol to A. caespitosus growth medium increases its inhibitory activity against C. albicans [106].
Both β-sitosterol and stigmastanol (5,6-dihydro-β-sitosterol) are intermediate substrates in the metabolism of cholesterol in the Mexican bean beetle Epilachna varivestis (Coleoptera: Coccinellidae) [107]. This might suggest that the presence of these sterols in pupae is connected with their role as substrates for cholesterol synthesis; however, again, more research is needed to confirm this thesis. Stigmastanol was also observed in the internal extract from T. molitor, after treatment with cyfluthrin-containing insecticide [108].
Fungal infection changes not only the composition of the cuticle, but also activates the immune system and induces the production of various immune molecules, including antibacterial proteins. Research has shown the presence of antifungal protein in the hemolymph of S. peregrina larvae (AFP). This protein significantly inhibits the growth of C. albicans, but has no effect on bacterial growth; its antifungal activity is increased when it forms a complex with the bactericidal protein sarcotoxin IA [109]. Two other important components of the immune system are nitric oxide (NO) and immune-reactive lysozymes. NO is an immune response molecule against bacteria that is mainly biosynthesized in the haemocytes of S. argyrostoma, while lysozymes support early immune responses in flesh fly larvae against invading bacteria by degrading bacterial cells and debris [110].
The biosynthesis of NO and lysozymes in S. argyrostoma larvae, arising in response to infection, is mediated and coordinated by eicosanoids [110], which are mediators of insect cellular and humoral immunity, and various metabolites of C20:4 (arachidonic acid) [111,112]. They not only mediate this biosynthesis of NO and lysozyme [110], but also participate in the LPS-dependent activation of the IMD pathway in S. peregrina [113]. C20:4 has also been found to be present at 90-times higher concentrations in the internal extracts of female S. carnaria than males; it has been proposed that the compound may play an important role in vitellogenesis [46]. Clements et al. [114] propose that arachidonic acid may also play a role in the resistance of the Colorado potato beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) to neonicotinoid insecticide, and suggest that this may be associated with its regulatory role in cytochrome P450-dependent insecticide detoxification pathways; however, Stanley and Kim postulate that it is evolutionarily advantageous for insects to maintain a low level of polyunsaturated fatty acid (PUFA), to reduce oxidative stress at the intracellular level, due to their high sensitivity to oxygen damage [115].

Conclusions
In conclusion, the internal and surface FFA compositions of S. argyrostoma pupae and adults changed after exposure to C. coronatus; most strikingly, the developmental stages demonstrated considerable discrepancy in the reactions to fungus exposure, with the pupae being resistant and the adults being susceptible to infection. As the cuticle is considered the first defence mechanism of the insect, changes in the FFA profile may well influence the susceptibility or resistance of the insects to fungal invasion. In extracts from pupae exposed to fungus, the concentrations of individual FFAs (C6:0-C10:0, C12:0, C14:0, C15:0, C16:1, C16:0, C18:2, C18:1, and C18:0) increased in the cuticular fraction, but decreased in the internal fraction. This might suggest that C. coronatus treatment promotes the translocation of these FFAs, and this may be one of the defence mechanisms used against fungal infection. In imagines, the increase in FFAs observed in both the cuticular and internal fractions might reflect the response to fungal infection, not only to protect against cuticle penetration, but also to protect from spore poisoning or infection inside the body. The increase in the FFA C20:4, which has immunomodulatory activities in internal extracts, might also suggest activation of the insects immune system.