Amino Acids in Entomopathogenic Fungi Cultured In Vitro

: The content of bounded amino acids in six entomopathogenic fungi was identified and determined. Analyzing the elements characterizing the pathogenicity of individual species of fungi based on infectivity criteria, ranges of infected hosts, and the ability to induce epizootics, these can be ranked in the following order: Isaria farinosa, Isaria tenuipes, Isaria fumosorose, Lecanicillium lecanii, Conidiobolus coronatus, Isaria coleopterorum. These fungi represent two types of Hyphomycetales-Paecilomyces Bainier and Verticillium Nees ex Fr. and one type of Entomophtorales-Conidiobolus Brefeld. Our study indicates that there are signiﬁcant quantitative and qualitative di ﬀ erences of bounded amino acids in the entomopathogenic fungal strains contained in the mycelium between high and low pathogenicity strains. The richest composition of bounded amino acids has been shown in the mycelium of the Isaria farinosa strain, which is one of the most commonly presented pathogenic fungi in this group with a very wide range of infected hosts and is the most frequently recorded in nature as an important factor limiting the population of insects.


Introduction
Plant pathogens created by insects significantly reduce crop yields. These pathogens strongly impact the epidemiology of a plant disease. If we want to decrease use of pesticides, then we should offer alternative ways to resolve the problem of pest damage. The choice of an effective method depends on our knowledge of the pests biology and their behavior. The aim is to manipulate pests' physiology to decline the damage they cause. Therefore, the knowledge of insect ecology should help us identify the processes and mechanisms of insect response, which help us to propose new methods and tools that effectively decrease that response.
The use of entomopathogenic fungi as biocontrol agents instead of pesticides is an attractive and an alternative method, which can efficiently control the pest population. In addition, entomopatheogenic fungi contrary to pesticides do not have any negative hazard effect on human health and environment. Therefore, fungal pathogens of arthropods have been generally considered as beneficial organisms for agriculture and forestry because of their ability to significantly decrease the populations of many economically important pest insects and mites. Their numerous strains have been included into the integrated pest management programs [1], which offer the opportunity to eliminate or drastically reduce the use of pesticides and to minimize the toxicity of an exposure to any chemicals which are used.
World-wide, the integrated pest management programs have become the suggested strategy for plant protection. Most programs for this strategy suggest that the efficient assessment of the importance of diversity for significantly decreasing pest population fluctuation has been used for the establishment of various crops, with consequent comparison of the occurrence and multiplicity of pest 1. Isaria farinosa (synonyms: Ramaria farinosa, Clavaria farinosa, Corynoides farinosa, Spicaria farinosa, Penicillium farinosum, Paecilomyces farinosus, Isaria psychidae, Penicillium alboaurantium) (MycoBank) was isolated from an unspecified insect species collected in the forest litter of the Wielkopolski National Park (Poland). This strain is characterized by growth and sporulation on commonly used mineral-organic media in saprophyte breeding, showing high pathogenicity and intensive development during the entire vegetative season, beginning from early spring to late autumn in meadows and forests [21]. This fungus belongs to the polyphagous species and causes severe epizootics in butterflies of larvae and pupae. Isaria farinosa is one of the most common species of entomopathogenic fungi with a worldwide distribution and a comparatively wide host range [22].
2. Isaria tenuipes (synonyms: Paecilomyces tenuipes, Spicaria heliothis) (MycoBank) was collected from an undetermined insect species and isolated near Vladivostok, Primorsky Krai (Russian Federation) and obtained from Dr. V.A. Borisova. In terms of morphology, it does not correspond to any of . This strain is characterized by growth and sporulation on commonly used mineral-organic media in saprophyte breeding, showing high pathogenicity and intensive development during the entire vegetative season, beginning from early spring to late autumn in meadows and forests [21]. This fungus belongs to the polyphagous species and causes severe epizootics in butterflies of larvae and pupae. Isaria farinosa is one of the most common species of entomopathogenic fungi with a worldwide distribution and a comparatively wide host range [22].
2. Isaria tenuipes (synonyms: Paecilomyces tenuipes, Spicaria heliothis) (MycoBank) was collected from an undetermined insect species and isolated near Vladivostok, Primorsky Krai (Russian Federation) and Agronomy 2020, 10, 1899 4 of 26 obtained from Dr. V.A. Borisova. In terms of morphology, it does not correspond to any of the species described so far, but Paecilomyces amoenoroseus (P. Henn.) Samson is very close. The range of the hosts is unrecognized. In laboratory conditions, Isaria tenuipes infects 100% of bark beetles, saprophagic and hymenoptera (Hymenoptera) larvae from Parasitica group. Development and sporulation in vitro is very fast and intensive on various media. 3. Isaria fumosorosea (synonyms: Spicaria fumosorosea, Paecilomyces fumosoroseus, Spicaria aphodii, Monilia aquatilis, Paecilomyces hibernicus, Paecilomyces hibernicum, Paecilomyces isarioides) (MycoBank) was collected from soil under the spruce stand in the floodplain forest, Strass near Laufen (Bawaria, Germany). This species is considered polyphaga but with a much narrower host range than Isaria farinosa. This species is commonly found in the soil [23], but cases of insect infections are encountered less frequently and usually locally. Moreover this fungus has been reported on plants, in water and less commonly in the air on every continent except Antarctica [4]. Some of the more commonly known susceptible organisms include weevils, ground beetles, plant beetles, aphids, whiteflies, psyllids, wasps, termites, thrips and a wide variety of butterflies and moths [24,25]. The development takes place in vitro.

4.
Lecanicillium lecanii (synonyms: Cephalosporium lecanii, Verticillium lecanii, Actostalagmus albus-minimus, Actostalagmus coccidicola, Cephalosporium coccorum) (MycoBank) was isolated with imago Aleurodidae in Wielkopolski National Park (Poland). Intensive development and sporulation was observed on media. This species is highly pathogenic to beetle larvae, dipterans and hymenoptera in laboratory conditions. Endophytic colonization has been reported by Ownley [26] for Lecanicillium lecanii and it has been suggested that induced systemic resistance may be active against powdery mildew. Mycoparasitism is the primary mechanism employed by this fungus against plant pathogens. These strains are considered to be polyphagous with broad ranges of potential hosts, but they require high humidity for the initiation of epizootics.
5. Conidiobolus coronatus (Constantin) Batko (synonyms: Boudierella coronata, Delacroixia coronata, Entomophthora coronata, Conidiobolus villosus) (MycoBank) was isolated from the soil of the shelterbelt near Sains in Normandia (France). The sporulation is intensive on all culture media but prefers the substrates enriched with peptone or yolk. Conidiobolus coronatus is a saprophytic species of entomopathogenic fungus, with a tendency to parasitism. This species is characterized by low host selectivity and strong post-infection aggressiveness caused by highly toxic metabolites of invertebrates [27]. This fungus is the opportunistic pathogen of a broad range of infected hosts eg. Galleria mellonella, Dendrolimus pini [28]. This species was also noted on mushrooms, herbaceous plants, vertebrates and individual cases on aphids [29]. Under experimental conditions, the test strain showed pathogenic properties on larvae of Tribolium destructor Uytenb. (Tenebrionidae) but only 30% of positive infections were in conditions of 80% moisture. Recent study showed that this fungus is the common pathogen of soil microflora and lives mainly on the edges of forests, meadows, gardens, and parks [27,30]. 6. Isaria coleopterorum (synonyms: Paecilomyces coleopterorum) (MycoBank) was isolated from beetle larvae of the family Cantharidae (Lampyris sp.) collected on forest in Moosen near Laufen (Bavaria, Germany). Cultural characteristics and morphology of the mycelium and spores do not correspond to any of the previously known species of the genus Paecilomyces. This fungus grows well on the mineral-organic media, but better grows abundantly and sporulates efficiently on media enriched protein substances (peptone, broth, egg yolk). The host range is unknown. Laboratory sample infection of several species larvae of beetles, flies and Hymenoptera proved unsuccessful.
Soil samples were collected in triplicate from the field (see fungi characterization) and stored in plastic bags at the temperature of 3-4 • C. Immediately before starting the experiment in the laboratory, the soil was sieved (1 mm sieve) to separate the larger particles of impurities and dried up to a moisture content of approximately 25-30% (which is optimal for fungal growth and limits the growth of entomopathogenic nematodes) [31]. The cultures of the fungi used for the bioassays and bounded amino acids study were deposited in the collection of the Institute for Agricultural and Forest Environment, Polish Academy of Sciences in Poznań, Poland. In isolated fungi, the disease agents, their frequency, duration and succession were identified by common insect pathology techniques [32]. Each fungal suspension contained 1.0 × 10 9 spores of fungi in 1 mL. This volume was taken and the sporulation fungi from cadavers grown on sterilized Czapek s agar: saccharose, sodium nitrate, dipotassium phosphate, magnesium sulfate, potassium chloride, ferrous sulfate, agar and demineralized water at pH = 7.3.
In order to obtain cultures, small portions of external mycelium from host cadavers were inoculated on the surface of the agar. They were allowed to grow on the Petri dishes (9 cm diameter) for 5 days on a rotary shaker (90 rpm) at 20 ± 1 • C. The cultures were washed in sterile redistilled water three times (wash bottle, gentle), filtered (paper Whatman GT/C), and dried at 80 • C for 4 h. For taxonomical differentiation of fungal strains, the macro-and micro-morphological features of the mycelium and sporulation on the host mites and in cultures were compared according to the method of Bałazy et al. [33]. The resin should be converted into the H + form. The process is carried out by 15% HCl (1:5 ratios) on the water bath at 80 • C for 1 h. The suspension has to be stirred and next filtered by filter paper Whatman GT/C and washed by deionized water until pH neutral.
Resin is converted from H + form to Li + form using 2 N LiOH. After washing, resin was mixed by the first elution buffer (pH = 2.9), (ratio 1:5) and allowed to stay overnight. Then the buffer is sucked off and the dilution 1:1 is left unchanged and the column is ready for filling.
Amino acids were separated with five lithium citrate buffers ( Table 2). The buffers were prepared with water generated with Mili-Q water purification system (Millipore, Molsheim, France).  Before the next sample injection, the column was equilibrated with buffer 1 for 20 min. Chemicals were accurately weighed. Each buffer was filtered through a 0.22 µ m filter (Millipore, Molsheim, France) before use, and kept under argon in the apparatus. Throughout the elution program, the flow rate for buffer solutions and ninhydrin was 12 mL/h. During the running of the samples, the separate pump system automatically mixes buffer and ninhydrin reagent, which are kept under argon in the instrument.
All chemical analyzes were run in triplicate and the results were averaged. The confidence intervals for the mean were calculated using the following formula: ̅ tα(n−1) SE, where: ̅ -mean, tα(n−1)-value of the Student test for α = 0.05, and n−1 degree of freedom, Wpisz tutaj równanie. SEstandard error.

Results and Discussion
Infection begins with a connection of conidia or blastospores to the insect cuticle. A variety of hydrolytic enzymes, e.g., proteases, chitinases, and lipases and other factors, contribute to the germination and growth of the fungus across the surface of the host and penetration of cuticular layers. During this process the fungus produces any number of specialized infection structures that can include penetration pegs or appressoria, which enable the growing hyphae to penetrate into the host integument. It is essentially in the later stages of this process that the pathogen encounters the host immune system. The insect cuticle is a highly heterogeneous structure and can vary greatly in the composition even during the various life-stages of a particular insect. The epicuticle or outermost layer provides a hydrophobic barrier rich in amino acids in peptides and lipids and is fol-

Calculations
The determination of amino acids is based on the ninhydrin reaction: (d). Ninhydrin solution: It must be prepared in black bottle. Mix 1500 mL of methyl cellosolve C3H8O2 (ethylenglycol monomethyl ether) and 500 mL of 4 N acetate buffer. Add to this solution 40 g ninhydrin (analytical grade) and stir the solution under argon for dissolving ninhydrin. After dissolving ninhydrin, 0.8 g SnCl2 2H2O is added and the stirring is done. Keep the solution of ninhydrin in refrigerator.
The resin should be converted into the H + form. The process is carried out by 15% HCl (1:5 ratios) on the water bath at 80 °C for 1 h. The suspension has to be stirred and next filtered by filter paper Whatman GT/C and washed by deionized water until pH neutral.
Resin is converted from H + form to Li + form using 2 N LiOH. After washing, resin was mixed by the first elution buffer (pH = 2.9), (ratio 1:5) and allowed to stay overnight. Then the buffer is sucked off and the dilution 1:1 is left unchanged and the column is ready for filling.
Amino acids were separated with five lithium citrate buffers ( Table 2). The buffers were prepared with water generated with Mili-Q water purification system (Millipore, Molsheim, France).

Calculations
The determination of amino acids is based on the ninhydrin reaction: The ninhidrin-CO2 method is highly specific and used in detection and assay of amino acids. It requires a free NH2 group adjacent to a -COOH group. The control of pH has high restriction for this determination. Ninhydrin creates NH3 blue colored compound in higher pH value shown below.
The ninhidrin-CO 2 method is highly specific and used in detection and assay of amino acids. It requires a free NH 2 group adjacent to a -COOH group. The control of pH has high restriction for this determination. Ninhydrin creates NH 3 blue colored compound in higher pH value shown below. This analysis is recommended for the material, where amino acids are in low amounts (body fluids, mineral and organic soils, feed etc.).
The retention time of each amino acid seems to be a characteristic value for its identification. It is possible to identify these amino acids and/or ninhydrin positive substances present in the calibration solution.
The method is characterized by high specificity. The differences between the retention times of amino acids in hydrolysates mycelium and in standard mixtures were below 2%, for wide range of concentrations the linear correlation coefficients ranged from 0.992 to 0.997, high accuracy (recovery 92.9-108.5%), the precision of the results below 4.2% in case of repeatability.
The column (resin Ostion LGFA) is designed for rapid, high-resolution amino acids separations. Column has high capacity and reproducibility, making them ideal for the intermediate and polishing steps of the laboratory-scale amino acids separation workflow. The connection of the column to the sequence of lithium-citrate buffers is responsible for the large number of separated amino acids.
Precautions All buffers have to be adjusted precisely, however, ninhydrin solution should be aged at least 14 h. Flow rates of the buffers and ninhydrin as well as temperatures used for separation of amino acids have to be set. The standard solution must be prepared quantitatively, using those amino acids, which are not represented in commercial standards. The control of instrument has to be checked every day.

Method for the Separation and Determination of Amino Acids in Mycelium Hydrolysates
The concentrations of amino acids were determined in hydrolysates mycelium. To obtain hydrolysates mycelium, 20 mL 6 M HCl was added to 0.2 g of sample in the test tube. An amount of 6 N HCl was used to perform the hydrolysis of proteins and peptides. This chemical agent was prepared by diluting the concentrated HCl 1:1 distilled water. The tube was allowed to stay overnight. The next day, the test tube was put on the microwave water bath to remove gasses. Then argon was babbled for 10 min to remove oxygen. Tubes were closed tightly and heated for 24 h at 15 °C. Solutions from the test tubes were transferred to 50 mL flasks. The solution was evaporated to dry mass. Dry mass was dissolved in 0.6 mL ethyl alcohol (96%) and added to 0.4 mL 40% trichloroacetic acid and 1 mL deionized water. The solution was centrifuged and the supernatant was used for analysis.
Separation and determination of 30 bounded amino acids was carried out on T 339 Amino Acids Analyzer (Mikrotechna-Prague) (ion-exchange chromatography, the strong cation exchange resin), [34]. The separation of the bound amino acids was achieved by injecting 100 mL samples into an Ostion LGFA (0.37 × 20 cm) column. Lithium-citric buffers of the following pHs were used: 2.90, 3.10, 3.35, 4.05 and 4.90, and the absorbance of the eluent-ninhydrin complex was monitored at λmax = 520 nm (Szajdak 1996). The mobile phase was pumped at 12 cm 3 h −1 and developed a pressure of 2.5 MPa. A full-range recorder span of 100 mV was used to provide on-scale peaks. With this system, adequate resolution of a large number of amino acids up to and including arginine can be achieved in a chromatographic run of 4 h (total run time) (Figure 2).
This analysis is recommended for the material, where amino acids are in low amounts (body fluids, mineral and organic soils, feed etc.).
The retention time of each amino acid seems to be a characteristic value for its identification. It is possible to identify these amino acids and/or ninhydrin positive substances present in the calibration solution.
The method is characterized by high specificity. The differences between the retention times of amino acids in hydrolysates mycelium and in standard mixtures were below 2%, for wide range of concentrations the linear correlation coefficients ranged from 0.992 to 0.997, high accuracy (recovery 92.9-108.5%), the precision of the results below 4.2% in case of repeatability.
The column (resin Ostion LGFA) is designed for rapid, high-resolution amino acids separations. Column has high capacity and reproducibility, making them ideal for the intermediate and polishing steps of the laboratory-scale amino acids separation workflow. The connection of the column to the sequence of lithium-citrate buffers is responsible for the large number of separated amino acids.
Precautions All buffers have to be adjusted precisely, however, ninhydrin solution should be aged at least 14 h. Flow rates of the buffers and ninhydrin as well as temperatures used for separation of amino acids have to be set. The standard solution must be prepared quantitatively, using those amino acids, which are not represented in commercial standards. The control of instrument has to be checked every day.

Method for the Separation and Determination of Amino Acids in Mycelium Hydrolysates
The concentrations of amino acids were determined in hydrolysates mycelium. To obtain hydrolysates mycelium, 20 mL 6 M HCl was added to 0.2 g of sample in the test tube. An amount of 6 N HCl was used to perform the hydrolysis of proteins and peptides. This chemical agent was prepared by diluting the concentrated HCl 1:1 distilled water. The tube was allowed to stay overnight. The next day, the test tube was put on the microwave water bath to remove gasses. Then argon was babbled for 10 min to remove oxygen. Tubes were closed tightly and heated for 24 h at 15 • C. Solutions from the test tubes were transferred to 50 mL flasks. The solution was evaporated to dry mass. Dry mass was dissolved in 0.6 mL ethyl alcohol (96%) and added to 0.4 mL 40% trichloroacetic acid and 1 mL deionized water. The solution was centrifuged and the supernatant was used for analysis.
Separation and determination of 30 bounded amino acids was carried out on T 339 Amino Acids Analyzer (Mikrotechna-Prague) (ion-exchange chromatography, the strong cation exchange resin) [34]. The separation of the bound amino acids was achieved by injecting 100 mL samples into an Ostion LGFA (0.37 × 20 cm) column. Lithium-citric buffers of the following pHs were used: 2.90, 3.10, 3.35, 4.05 and 4.90, and the absorbance of the eluent-ninhydrin complex was monitored at λ max = 520 nm (Szajdak 1996). The mobile phase was pumped at 12 cm 3 h −1 and developed a pressure of 2.5 MPa. A full-range recorder span of 100 mV was used to provide on-scale peaks. With this system, adequate resolution of a large number of amino acids up to and including arginine can be achieved in a chromatographic run of 4 h (total run time) (Figure 2).
Before the next sample injection, the column was equilibrated with buffer 1 for 20 min. Chemicals were accurately weighed. Each buffer was filtered through a 0.22 µm filter (Millipore, Molsheim, France) before use, and kept under argon in the apparatus. Throughout the elution program, the flow rate for buffer solutions and ninhydrin was 12 mL/h. During the running of the samples, the separate pump system automatically mixes buffer and ninhydrin reagent, which are kept under argon in the instrument. All chemical analyzes were run in triplicate and the results were averaged. The confidence intervals for the mean were calculated using the following formula: Xt α(n−1) SE, where: X-mean, t α(n−1) -value of the Student test for α = 0.05, and n − 1 degree of freedom, Wpisz tutaj równanie. SE-standard error.

Results and Discussion
Infection begins with a connection of conidia or blastospores to the insect cuticle. A variety of hydrolytic enzymes, e.g., proteases, chitinases, and lipases and other factors, contribute to the germination and growth of the fungus across the surface of the host and penetration of cuticular layers. During this process the fungus produces any number of specialized infection structures that can include penetration pegs or appressoria, which enable the growing hyphae to penetrate into the host integument. It is essentially in the later stages of this process that the pathogen encounters the host immune system. The insect cuticle is a highly heterogeneous structure and can vary greatly in the composition even during the various life-stages of a particular insect. The epicuticle or outermost layer provides a hydrophobic barrier rich in amino acids in peptides and lipids and is followed by the procuticle that contains chitin and sclerotised protein [9,[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50]. Therefore, hydrophobic mycotoxins will be easily transported throughout this barrier and the knowledge about the content of bounded amino acids is needed.
The total amount of bounded amino acids in the mycelium of the individual fungal strains tested was clearly diverse. The highest total content of bounded amino acids (14,963.0 µg g −1 ) was related to Isaria tenuipes (Paecilomyces sp.II), while the lowest (390.0 µg g −1 ) was measured in mycelium of Conidiobolus coronatus (Table 3).
In the mycelium samples, the following groups of bounded amino acids were tested: sulfuric, acidic, neutral and basic (Table 4). Neutral amino acids dominated. Their contents in the individual strain of entomathogenic fungi ranged from 38 to 57% of all amino acids determined. However, the content of acidic amino acids ranged from 17 to 30% of the total amino acids identified. The lowest percentage content was found for basic amino acids which amounted to from 8 to 32%. The concentrations of sulfuric amino acids in all analyzed strains of fungi were the lowest among these groups and their contents ranged from 4 to 11%.
In Isaria farinosa, Isaria tenuipes, Isaria fumosorosea, Leanicillium lecanii a group of hydrophobic amino acids dominated and their content ranged from 53 to 66% (Table 5). On the other hand, hydrophilic amino acids were found to be from 34 to 47% in the tested entomathogenic fungi. With reference to Conidiobolus coronatus and Isaria coleopterorum, the dominant groups in the hydrolysates from their mycelium were generally hydrophilic amino acids, whose percentages were close to approximately 60% and hydrophobic amino acids at 40%, respectively. From 21 to 24 amino acids were identified in various strains of the mycelium samples. This number is significantly longer than in published studies (Table 6).  In all analyzed fungi samples, aspartic acid, hydroksyproline, glycine, alanine, isoleucine and arginine dominated (Table 3). In the studied entomopathogenic fungi, except Isaria farinosa, the highest concentration measured for arginine was from 11 to 19%. Its highest content among all identified amino acids was found in Isaria coleopterorum, while the lowest was in Isaria fumosorosea. Arginine is one of a number of isoenzymes presenting in the fungal cell walls [8].
In the mycelium of Isaria farinosa hydroxyproline dominated. Its content was about 20% of all amino acids determined. This amino acid is formed from proline as a result of metabolic pathways in the mycelium [8].
Among neutral amino acids, the highest content for isoleucine and glycine was determined. The concentration of isoleucine in mycelium ranged from 6 to 11% and its highest concentration was demonstrated in Isaria coleopterorum. The percentage of glycine in the tested fungi ranged from 5 to 8% and its highest concentrations were found in Isaria tenuipes, while the lowest contents, as in the case of isoleucine, were reported for Isaria coleopterorum.
Aspartic acid dominated among the acidic amino acids except for Isaria farinosa. The highest percentage of its content was found in the mycelium of Isaria coleopterorum (6.7% of total amino acids).
Analyzing the elements characterizing the pathogenicity of individual species of fungi based on infectivity criteria, ranges of infected hosts and the ability to induce epizootics, these can be ranked in the order shown in Table 3. The high infectivity of the strain Isaria tenuipes (Paecilomyces sp.II) has been identified only in experimental conditions, but there are no studies on its pathogenicity in nature [33]. Therefore, the richest and the largest amounts of amino acids are formed by strains of the species demonstrating the strongest entomopathogenic properties (Table 3).  The largest number of amino acids was isolated from the mycelium strain of Isaria farinosa (Paecilomyces farinosus), wherein the four compounds: cysteine, methionine, β-alanine and β-aminobutyric acid do not occur in any of the other tested strains. This may suggest that their presence in the composition of the fungus metabolites affects the scope of its affinity for the host pathogenic because both the number and taxonomic differentiation of arthropods infected by Isaria farinosa (Paecilomyces farinosus) are the greatest of all the tested strains.
Special attention is given to the very high content of hydroxyproline in the mycelium strain of Isaria farinosa in comparison with the other species (Isaria tenuipes, Isaria fumosorose, Lecanicillium lecanii, Conidiobolus coronatus, Isaria coleopterorum). This amino acid is created from proline as a result of metabolic conversions in the mycelium.
However, α-aminobutyric acid, threonine, and 1-methylohistidine not produced by the Isaria farinosa (Paecilomyces farinosus) strain occurred in all other species. α-aminobutyric acid belongs to dominants in three strains of high pathogenicity. Another characteristic feature is the similar and proportionally high share of five amino acids (glycine, alanine, valine, isoleucine, leucine) in hydrolysates of the four most pathogenic fungal strains [31,33].
The strains of Conidiobolus coronatus, Isaria coleopterorum are slightly different from the rest of the composition and the percentage share of the produced amino acids. However, the absolute amount of these compounds in hydrolysates of mycelia was over 10 to 40 times lower than in highly virulent species. In comparison with Isaria farinosa amino acids, the composition of the mycelium Conidiobolus coronatus was poorer for six of the compounds (25%) and seven compounds (29%) relative to that of the mycelium of Isaria coleopterorum. The last two differ from all of the highly pathogenic phosphoethanolamines and lack of production of glutamic acid; in Isaria coleopterorum the presence of leucine was not observed. In addition, there was a lack of ornithine in the mycelia hydrolysates of both strains of weak pathogenicity and also in Lecanicillium lecanii, which is considered a conditional pathogen.
Growth in the hemocoel includes various hydrolytic enzymes and may lead to the creation of smaller compounds with harmful effects on the host [35]. They are simple organic poisons, e.g., oxalic acid, but these are mainly larger compounds, e.g., depsipeptides. These substances such as metabolites of entomopathogenic fungi are investigated as potential chemical insecticides and as pharmacological agents.
On the basis of the obtained results, it cannot be concluded whether the low content of amino acids in the mycelium of Conidiobolus coronatus and Isaria coleopterorum is the result of their poor pathogenicity or the use of too poor a composition of the culture medium. The latter hypothesis seems more probable. Czapek s mineral medium is widely used primarily as a standard culture medium for the saprophytic Hyphomycetes and Zygomycetes, especially Mucorales [40]. However, it is not considered optimal for pathogenic fungi.
For fungus, some authors postulated a varied composition of medium which modifies the quantity and quality of metabolites and mycotoxins. Therefore, the composition of the medium may be an important factor in regulating the pathogenicity of the fungus.
For example, Koval [41] recommends enriching it with insect decoction. This medium, however, is often used in various modifications due to the strictly defined chemical composition, allowing to observe a number of metabolites. For this reason, it was also used in these studies.
In addition, the development of Conidiobolus coronatus and Isaria coleopterorum on media with various composition shows that their nutritional requirements are greater than the four strains showing high pathogenicity. The study results on the physiology of highly specialized entomopathogenic fungi, especially Entomophthorales and Ascomycetes of Cordyceps genus and related ones including theirs anamorphs belonging to the group of Hyphomycetales, indicate that in most cases, these fungi require for development an in vitro of the substrates enriched with various organic compounds for their development, such as, e.g., fatty acids, vitamins, meat extracts and egg yolks. With these substances, numerous amino acids are also introduced into the media among other chemical compounds. Their impact on developing cultures and the degree of use as sources of nutrients are difficult to clearly determine [42].
Nolan [43] demonstrated, as a result of a long-term study into the development of protoplasts of various Entomophaga aulicae (Reihardt in Bail) Batko strains, that they are autotrophic in vitro to all organic acids and vitamins. From among 20 amino acids added to the medium, whose composition was similar to those discussed in the present study, none was the only source of nitrogen. Most were used in a non-uniform manner by various strains of fungi, for example, in a medium that does not contain any absorption of serine, glutamine, alanine, β-alanine by fungi in its composition of the calf germ serum. The results of the presented studies have simplified the composition of amino acids in the medium used for the cultivation of Entomophaga aulicae protoplasts from 21 in the Grace recipe based medium [44] (with the addition of calf germ serum) to 8 in the final medium enriched with hematine and oleic acid.
Several authors suggested that the composition of the medium may have a significant influence on the pathogenic properties of the strains because they are related to the biological activity of enzymes, formation of toxins, phytohormones and pathogen antibodies. In addition, the physicochemical, physiological properties of amino acids as components of peptide chains may be indirect factors characterizing the pathogenic potential of individual entomopathogenic fungal strains [45][46][47][48][49][50]. Due to the high diversity of qualitative and functional protein components in entomopathogenic fungi, it is important to pay attention to the composition and function of amino acids in the individual development of these pathogens and the disease processes caused by them.

Conclusions
A high-resolution column packed with Ostion LGFA of Amino Acid Analyzer and the system of lithium-cytric buffers is recommended for the separation and determination of the larger number of amino acids −24 in mycelium hydrolysates.
Our preliminary study indicates that there are significant quantitative and qualitative differences of amino acids in the entomopathogenic fungal strains contained in the mycelium between high and low pathogenicity strains. Analyzing the elements characterizing the pathogenicity of individual species of fungi based on infectivity criteria, ranges of infected hosts, and the ability to induce epizootics, these can be ranked in the following order: Isaria farinosa, Isaria tenuipes, Isaria fumosorose, Lecanicillium lecanii, Conidiobolus coronatus, Isaria coleopterorum.
The richest composition of amino acids has been found in the mycelium of Isaria farinosa strain, which most commonly exhibits pathogens in this group of fungi. A wide range of infected hosts and their high frequency recorded in nature are cited as important factors limiting the population of insects. In the mycelium strain of Isaria farinosa (Paecilomyces farinosus), the four compounds: cysteine, methionine, β-alanine and β-aminobutyric acid do not occur in any of the other tested strains. This may suggest that their presence in the composition of the fungus metabolites affects the scope of its affinity for the host pathogenic response because both the number and taxonomic differentiation of arthropods infected by Isaria farinosa (Paecilomyces farinosus) are the greatest of all the tested strains.
In addition, in the mycelium strain of Isaria farinose a very high content of hydroxyproline in comparison with the other species was determined. This amino acid is formed from proline as a result of metabolic pathways in the mycelium.
α-aminobutyric acid, threonine, and 1-methylohistidine not produced by the Isaria farinosa (Paecilomyces farinosus) strain occurred in all other species. α-aminobutyric acid belongs to dominants in three strains of high pathogenicity. Another characteristic feature is the similar and proportionally high share of five amino acids (glycine, alanine, valine, isoleucine, leucine) in hydrolysates of the four most pathogenic fungal strains.
In Isaria farinosa, Isaria tenuipes, Isaria fumosorosea, Leanicillium lecanii, a group of hydrophobic amino acids dominated and their content ranged from 53 to 66%, while hydrophilic amino acids ranged from 34 to 47% in the tested entomapathogenic fungi. With reference to Conidiobolus coronatus and Isaria coleopterorum, the dominant groups in the hydrolysates from their mycelium were generally hydrophilic amino acids, whose content was close to approximately 60% and hydrophobic amino acids at 40%, respectively.
Aspartic acid, hydroksyproline, glycine, alanine, isoleucine, and arginine dominated in all analyzed fungi samples. In the studied entomopathogenic fungi, except Isaria farinosa, the highest concentration of arginine was from 11 to 19%. Its highest content among all identified amino acids was found in Isaria coleopterorum, while the lowest was in Isaria fumosorosea. Arginine is involved in several isoenzymes found in the fungal cell walls.
The results obtained from our study should give a better insight into the content of bounded amino acids in the entomopathogenic fungi and should be helpful in more quantitative characterization of the pathogenicity of this group of fungi.