Effect of Selected Entomopathogenic Fungal Species on Embryonic Development of Ascaris suum (Nematoda)

Simple Summary Endoparasites such as Ascaris suum can pose a serious threat to the health of livestock and, consequently, humans. One promising way of controlling the threat is the use of natural enemies/antagonistic fungi. In this study, we examined the effects of entomopathogenic fungi (naturally attacking insects) as a bioregulator in the invasive stages of the parasitic nematode A. suum. The conducted study indicates that none of the fungal strains tested have nematocidal activity against A. suum eggs, and they do not meet the criteria required for use in the bioregulation of the parasite’s dispersal stages. Among the strains tested, Isaria fumosorosea and Metarhizium robertsii stood out, combining the highest metabolic activity with nematocidal activity against A. suum. Abstract The aim of the study was to evaluate the potential of using five selected species of entomopathogenic fungi (Beauveria bassiana, B. brongniartii, Conidiobolus coronatus, Isaria fumosorosea, and Metarhizium robertsii) in the bioregulation of the dispersive stages of the parasitic nematode—Ascaris suum. Experimental cultures of each of the selected entomopathogenic fungi, as well as a control culture without fungi, were incubated with A. suum eggs at 26 °C for 28 days. Development of the A. suum eggs was observed using a light microscope on the 7th, 14th, 21st, and 28th days of incubation. The API-ZYM® test was used to determine, semiquantitatively, the activity of 19 hydrolytic enzymes from the entomopathogenic fungi. The cytotoxicity of the fungi was determined using tetrazole salt MTT. It was found that none of the five tested strains of entomopathogenic fungi showed an ovicidal effect, and none of them colonized the A. suum egg shells. However, ovistatic activity was observed mainly until the 14th day of incubation by I. fumosorosea, M. robertsii, and B. bassiana. In the MTT test, M. robertsii showed moderate cytotoxicity, while the other species showed low cytotoxicity. Among the strains tested, I. fumosorosea showed the highest spectrum of hydrolase production (13 out of 19 enzymes gave a positive reaction from 3 to 5; 20–40 nM or more). The absence of morphological changes in the A. suum egg shells suggests that the antagonistic effect of the studied entomopathogenic fungi may be due to their cytotoxicity, associated with the production of secondary metabolites—toxins (M. robertsii) and enzymatic activity (I. fumosorosea).


Introduction
As a result of raising and breeding pigs, a large amount of organic matter is introduced into the environment in the form of animal feces.This leads to the entry of various forms of parasites into the soil.The use of manure from pig farms poses potential risks to human and animal health due to soil-transmitted helminths, such as Ascaris spp.[1].Their eggs can contaminate soils and surface waters, may be transported to wastewater treatment plants, and may be deposited in sludge that is sometimes used as an organic fertilizer [1][2][3][4].
The very high reproductive potential of Ascaris (200,000 eggs/day), the longevity of the invasive eggs, and their resistance to the influence of harmful external factors pose a significant problem in the control of ascariasis, while at the same time resulting in a high extensiveness of infection in humans and pigs [5][6][7].The prevalence of A. suum ranges from a dozen to more than 60% by country, population, husbandry, diet, and age of the carrier pigs [8][9][10][11].The extensiveness of A. suum infection in pigs in Poland is 30-60% [7,8,12] and is also high in other countries.For instance, it ranges from 15-64% in Denmark [10] and is 32.59% in Mumbai, India [9], 25.9% in Ethiopia [13], and 44.5% in South Africa [11].Pigs living outdoors (including organic farms) are at higher risk of Ascaris infection than pigs housed indoors [10,14,15].Ascariasis in pigs is a major economic problem due to detrimental carcass composition, reduced feed conversion efficiency, and losses to the meat industry, but it is also a public health threat [3,14,16,17].
According to Stewart [18], losses in the United States due to swine liver seizures (the effect of A. suum infestation) are estimated at USD 17.5 million per year, and losses associated with reduced feed conversion by animals are an additional USD 60.1 million per year.Some studies have shown that cross-species transmission occurs between pigs and humans living in close proximity or where pig manure is used as fertilizer on vegetables for human consumption.For this reason, A. suum is considered a zoonotic pathogen [14,16,19].Furthermore, a number of researchers have suggested that A. suum and A. lumbricoides are a single species that is only reproductively isolated [20][21][22].
Eggs of the soil-transmitted parasitic nematodes Ascaris, Trichuris, and Toxocara have been considered as indicators in assessing the hygienic status of sewage sludge and organic fertilizers.The eggs of these parasites have been shown to be more resistant to various sanitation methods (liming, pasteurization, composting) than other animal and human endoparasites [2,4,15,23].Due to the economic and health significance of nematode infections (especially ascariasis), as well as some regulations concerning organic farms (especially in Europe where the use of chemical anthelmintics is often prohibited), investigations on natural means active against human and animal parasitic nematodes have been conducted for years [1,24].
One of the methods used to tackle parasitic infestations in humans and animals are fungi, which possess various abilities to reduce helminth populations.Some fungi, such as entomopathogenic fungi, enter the host (insect) through the cuticle and initiate the development of infection, which then occurs in three main phases.The first phase is associated with the adhesion of conidial spores to the host body and their germination in the epicuticle.The second phase involves penetration of the cuticle by mycelial hyphae.This process, according to Mustafa and Kaur [25], is the result of mechanical forces together with enzymes produced by the fungi (proteases, chitinases, lipases, and lipoxygenases).The final stage is the complete destruction of the host and its death.In the course of zoomycosis, fungal metabolites are particularly active, including depsipeptides (e.g., Dsx destructins) produced by members of the genus Metarhizium [26] and Conidiobolus [27], which cause convulsions, loss of motor coordination, and paralysis in insects [28].The nematostatic and nematocidal effects of some insecticidal fungi in reducing helminth populations that cause parasitosis in humans and animals are also known.The ovicidal effects of Pochonia chlamydosporia (H.C.Evans) have been reported in nematode species such as Trichuris vulpis [29], Toxocara vitulorum [30], A. suum [31], and the tapeworm Taenia saginata [32].Penetration of P. chlamydosporia mycelia has also been observed in eggs of the flukes Schistosoma mansonii [33] and Fasciola hepatica [34].Entomopathogenic fungi have also shown inhibitory effects on the development of A. suum and A. lumbricoides eggs.Of note among these species are the following strains: Paecilomyces variotii and P. viridis [35], Isaria fumosorosea (=Paecilomyces fumosoroseus), P. lilacinus, Metarhizium flavoviride, M. anisopliae [36,37], Metacordyceps chlamydosporia (=Verticillium chlamydosporium), and Pochonia chlamydosporia [31,[38][39][40][41].
It seems reasonable to learn about the interaction of other species of entomopathogenic fungi with the dispersal stages of parasitic geohelminths.Therefore, the aim of this study was to evaluate the potential of selected entomopathogenic fungal species (Beauveria bassiana, B. brongniartii, Conidiobolus coronatus, Isaria fumosorosea, and Metarhizium robertsii) for use in the bioregulation of the invasive stages of the parasitic nematode Ascaris suum.
It seems reasonable to learn about the interaction of other species of entomopathogenic fungi with the dispersal stages of parasitic geohelminths.Therefore, the aim of this study was to evaluate the potential of selected entomopathogenic fungal species (Beauveria bassiana, B. brongniartii, Conidiobolus coronatus, Isaria fumosorosea, and Metarhizium robertsii) for use in the bioregulation of the invasive stages of the parasitic nematode Ascaris suum.
Discs (ø 4 mm) were cut from the 3-week-old mycelium of these cultures and placed on PDA medium.Incubation was carried out in the dark at 25 °C for 21 days, after which new discs (ø 4 mm) were cut and transferred to Petri dishes (ø 50 mm) containing an A. suum egg suspension [44,45].Discs (ø 4 mm) were cut from the 3-week-old mycelium of these cultures and placed on PDA medium.Incubation was carried out in the dark at 25 • C for 21 days, after which new discs (ø 4 mm) were cut and transferred to Petri dishes (ø 50 mm) containing an A. suum egg suspension [44,45].
Experimental cultures (with the presence of entomopathogenic fungi) and control culture (without fungi) each contained a 10 mL suspension of A. suum eggs. A. suum eggs were incubated in Petri dishes (ø 50 mm) at 26 °C for 28 days.
A. suum eggs were observed under a light microscope (Olympus CX21, Japan; ×10; 40) on days 7, 14, 21, and 28 of incubation from a collected 0.1 mL of A. suum egg suspension.The following developmental stages were determined in 100 randomly observed eggs: zygote, 2-8 blastomeres, morula/blastula, gastrula, and larva.The evaluation of developmental stages was performed on 100 eggs each time and repeated twice for both experimental and control cultures.
Experimental cultures (with the presence of entomopathogenic fungi) and control culture (without fungi) each contained a 10 mL suspension of A. suum eggs. A. suum eggs were incubated in Petri dishes (ø 50 mm) at 26 • C for 28 days.
A. suum eggs were observed under a light microscope (Olympus CX21, Japan; ×10; 40) on days 7, 14, 21, and 28 of incubation from a collected 0.1 mL of A. suum egg suspension.The following developmental stages were determined in 100 randomly observed eggs: zygote, 2-8 blastomeres, morula/blastula, gastrula, and larva.The evaluation of developmental stages was performed on 100 eggs each time and repeated twice for both experimental and control cultures.

Cytotoxicity of Entomopathogenic Fungi in MTT Assay
Cytotoxicity was determined using tetrazole salt MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma Aldrich, Darmstad, Germany).The study was conducted using the porcine kidney (SK) cell line.The MTT colorimetric assay for cytotoxicity is based on the conversion of yellow tetrazole salts to purple water-insoluble formazan crystals.The salt reduction takes place exclusively in the mitochondria of living and metabolically active cells.If cells are damaged or destroyed by the toxin, the salt does not transform and the yellow color of the tetrazole salts is retained [48].
For the next 48 h, the plates were incubated in a CO 2 -incubator (CB, BINDER GmbH, Tuttlingen, Germany) (5% CO 2 , 37 • C, 98% humidity); then, 20 µL of MTT solution in PBS (Merck, Darmstadt, Germany) was added to the wells and the whole set was incubated for another 4 h.After removing the liquid from the wells, 100 µL of DMSO was added as a solvent for the formazan crystals.After shaking for 5 min (Titramax 101 shaker, Heidolph, Schwabach, Germany), cytotoxicity was quantified using a microplate spectrophotometer (Ledetect 96 Microplate Reader, Labexim Products, Lengau, Austria) coupled with MikroWin 2010 OEM version (Mikrotek Laborsysteme GmbH, Overath, Germany) based on the absorbance measured at a wavelength of 510 nm, which corresponded to the maximum absorption of the formazan derivative.If the absorbance was less than 50% of the cell division activity, all samples analyzed were considered toxic.
The cytotoxicity IC 50 (sample concentration at which cell proliferation is inhibited by 50% compared to control cells) was determined on the basis of serial dilutions according to the scale of Hanelt et al. [48].The cytotoxicity of the analyzed fungal strains was reduced by the IC50 value of the control.Semi-quantitative scale for cytotoxicity grading was adopted: the low cytotoxic effect (+) with a half maximal inhibitory concentration (IC 50 ) value from 31.251 cm 2 /mL to 7.813 cm 2 /mL, the medium cytotoxic effect (++) with a value from 3.906 cm 2 /mL to 0.488 cm 2 /mL, and the high cytotoxic effect (+++) with a value from 0.244 cm 2 /mL to 0.061 cm 2 /mL.No cytotoxic effect was assessed if the extract concentration at 31.251 cm 2 /mL failed to inhibit the growth of the swine kidney cell line (SK) (and if the percentage of extinction with respect to the control group was ≥50%).

Statistical Analyses
Statistical analyses were performed using Statistica version 12 software (StatSoft.Inc., Hamburg Germany).Independent samples t-test was used to determine differences between the two groups (control vs. experimental).Differences at p < 0.05 were considered significant.

Influence of Entomopathogenic Fungi on the Embryonic Development of A. suum
The inhibitory effect of most of the tested fungi on the embryonic development of A. suum was most evident up to the 14th day of incubation (p < 0.05).On the 7th day of incubation, the inhibitory effect on the embryogenesis of A. suum was shown by all tested fungal species except C. coronatus (Figure 3).The most inhibitory effect on the development of A. suum eggs-expressed by a higher percentage of zygotes compared to the control-was shown by the following fungal species, respectively: B. brongniartii, I. fumosorosea, M. robertsii, and B. bassiana (p < 0.05).At the same time, there was a lower percentage of A. suum eggs at the morula/blastula stage incubated with these fungal species (p < 0.05).
of A. suum eggs-expressed by a higher percentage of zygotes compared to the controlwas shown by the following fungal species, respectively: B. brongniartii, I. fumosorosea, M. robertsii, and B. bassiana (p < 0.05).At the same time, there was a lower percentage of A. suum eggs at the morula/blastula stage incubated with these fungal species (p < 0.05).
On day 14 of embryogenesis, a higher percentage of eggs at the morula/blastula stage was found in eggs incubated with M. robertsii and B. bassiana compared to the control, while a lower percentage of eggs at this stage (p < 0.05) was found in eggs incubated with C. coronatus.In the presence of all fungi tested, the percentage of larvae was lower compared to the control, but statistically significant differences were found only for I. fumosorosea.On day 21, a higher percentage of eggs at the zygote stage was found in the presence of B. brongniartii, I. fumosorosea, and B. bassiana, but these differences were not statistically significant compared to the control.On the 28th day of incubation, the highest percentage of eggs at the zygote stage and the lowest percentage of eggs at the larval stage were found only in the presence of I. fumosorosea (p < 0.05).None of the five entomopathogenic fungal strains tested colonized the egg shells of A. suum.

The Enzymatic Activity of Entomopathogenic Fungi
Among the strains tested in the API-ZYM ® test, I. fumosorosea showed the largest spectrum of hydrolase production (positive reaction of 3-5; 20-40 nM or more) (13 out of 19 enzymes).All strains except B. brongniartii were capable of producing leucine arylamidase, while the positive reaction in the production of valine and cysteine arylamidase and trypsin was shown only by the strain I. fumosoresea (Table 1).All strains, except B. On day 14 of embryogenesis, a higher percentage of eggs at the morula/blastula stage was found in eggs incubated with M. robertsii and B. bassiana compared to the control, while a lower percentage of eggs at this stage (p < 0.05) was found in eggs incubated with C. coronatus.In the presence of all fungi tested, the percentage of larvae was lower compared to the control, but statistically significant differences were found only for I. fumosorosea.On day 21, a higher percentage of eggs at the zygote stage was found in the presence of B. brongniartii, I. fumosorosea, and B. bassiana, but these differences were not statistically significant compared to the control.On the 28th day of incubation, the highest percentage of eggs at the zygote stage and the lowest percentage of eggs at the larval stage were found only in the presence of I. fumosorosea (p < 0.05).None of the five entomopathogenic fungal strains tested colonized the egg shells of A. suum.

The Enzymatic Activity of Entomopathogenic Fungi
Among the strains tested in the API-ZYM ® test, I. fumosorosea showed the largest spectrum of hydrolase production (positive reaction of 3-5; 20-40 nM or more) (13 out of 19 enzymes).All strains except B. brongniartii were capable of producing leucine arylamidase, while the positive reaction in the production of valine and cysteine arylamidase and trypsin was shown only by the strain I. fumosoresea (Table 1).All strains, except B. brongniartii, showed C4 esterase activity, while C8 esterase was produced only by strains I. fumosorosea and C. coronatus.

Cytotoxicity of Entomopathogenic Fungi in MTT Test
In the MTT test, only the strain Metarhizium robertsii showed lowest IC ratio (IC 50 3.91 cm 2 /mL) and moderate cytotoxicity among the entomopathogenic fungi tested.For Isaria fumosorosea, the ratio was slightly higher (IC 50 7.81 cm 2 /mL) and corresponded to low cytotoxicity.A similarly low level of cytotoxicity characterized the other tested fungal species (Table 2).

Discussion
This study is part of a series of experiments on the effects of different trophic groups of fungi on A. suum embryogenesis, some of which have been published on soil fungi [45].In both the studies on soil fungi and entomopathogenic fungi, there was a control group in which a surprisingly high percentage of A. suum eggs (more than 50%) remained in the zygote stage until the end of incubation.An explanation for this phenomenon may be that the eggs used in the study (in both the experimental and control cultures) were obtained from 60 female pigs obtained from one organic (N = 30) and one conventional (N = 30) farm.On organic farms, one method of reducing parasite infestations is the use of feed additives in the form of medicinal plants.The active constituents of these plants reduce the level of parasitic infestation and interfere with the physiological functions of the parasite [49].In an organic farm where 50% of the adult forms of A. suum were obtained, a mixture of herbs such as garlic (Allium sativum), pumpkin (Cucurbita pepo), thyme (Thymus vulgaris), mugwort (Artemisia vulgaris), and fennel (Foeniculum vulgare) were added to the feed, with varying nematocidal effects [50][51][52][53][54].The presence of these herb species in the host diet was also most likely responsible for the inhibition of A. suum embryonic development in controls [45].
In recent decades, the fight against parasitic diseases has been based mainly on chemotherapy involving repeated administration of antiparasitic drugs.Research indicating the possibility of a natural, biological reduction of A. suum populations is of great practical importance.The subject of this research is very topical because, in several regions of the world, pig farming has experienced an increase in the extensiveness of A. suum infection, which is associated, among other things, with the use of manure or slurry as fertilizer.In Norway, an increase in the extensiveness of A. suum infection in pigs has been associated with increased humidity in washing pens and mechanical egg transfer [15].
Previous results from studies of antagonistic interactions between entomopathogenic fungi and parasitic geohelminths inspire an evaluation of the potential capabilities of fungi in the bioregulation of invasive stages of parasites, such as the nematode A. suum.
This study showed varying degrees of inhibitory effects of the tested entomopathogenic fungi on the embryonic development of A. suum.Observation of the entire embryogenesis of A. suum in contact with the fungi indicates that the potential for the antagonistic effect of most of the tested strains manifested mainly in the first 14 days of incubation, while at the end of the experiment it persisted only in I. fumosorosea.An exponent of the nematostatic effect of I. fumosorosea was a lower percentage of A. suum larvae on the 7th, 14th, and 28th days of incubation and a higher percentage of eggs at the zygotic stage on the 7th and 28th days of incubation, compared to the control (p < 0.05).The mechanism of this inhibitory effect may have different sources.Many authors point to the importance of the enzymatic activity of entomopathogens in causing zoomycosis [28,55,56], and anthropomycosis [57].The results of the studies conducted also suggest that fungal enzymes may play a major role in the inhibition of A. suum embryogenesis.
In fact, it was found in the most inhibitory strain of A. suum-strain I. fumosorosea, which is associated with the highest number of enzymes detected in the API-ZYM assay (13 enzymes out of 19) and their highest activity, compared to the other fungal strains (Table 1).The most important of these enzymes are probably the peptidases (cysteine and valine arylamidase), whose activity is characterized by I. fumosorosea.This is confirmed by studies linking the virulence of entomopathogenic fungi to the production of the mentioned enzymes [58].In the first phase of insect cuticle degradation, other proteases Pr1 (subtilisinlike peptidase) and Pr2 (trypsin-like peptidase) are also active [25,59].Their presence has been demonstrated in B. bassiana [60][61][62], M. anisopliae [26], and I. fumosorosea [63].Our results confirm trypsin activity only for I. fumosorosea, which seems to warrant a key role for this peptidase in the process of cuticle penetration.However, this does not exclude the involvement of other enzymes in the overall proteolytic activity of fungi [44].
Another group of enzymes important for the pathogenicity of entomopathogenic fungi are lipases and esterases.Their action in the insect epicuticula enables both the hydrolysis of lipoproteins and lipids and the adhesion of fungal spores [29].In addition, these enzymes also affect changes in the permeability of biological membranes.In our study, all strains tested showed the highest activity of acid phosphatase and naphthol-AS-BI phosphohydrolase (concentrations above 40 nmol).As in the case of proteolytic activity, the strain I. fumosorosea stood out among those tested, both in the number and degree of lipase and esterase activity, as confirmed by Ali et al. [63] Individual enzymes from the group of these hydrolases are also produced by B. bassiana, C. coronatus, and M. robertsii (Table 1), which is also confirmed by the studies of other authors [26,29,59,64].Not all enzymatic activity results are so clear, which may indicate differences in strain activity.This suggestion is supported by the study of Bridge et al. [65], who tested 22 strains of M. anisopliae and found that each strain had its own unique enzymatic profile.In their study, M. robertsii produced only six enzymes with moderate to high hydrolytic capacity (above 20 and 40 nmol) (Table 1).This enzymatic activity, combined with the highest degree of cytotoxicity among the fungi tested, may have been responsible for the intensity of the antagonistic effect, which translated into the inhibition of embryogenesis of A. suum.Thus, in further studies, it would be advisable to identify specific toxic compounds produced by entomopathogenic fungi, since the MTT assay is quantitative and not qualitative.

Conclusions
The conducted study indicates that none of the fungal strains tested have nematocidal activity against A. suum eggs and do not meet the criteria required for their use in bioregulation of the parasite's dispersal stages.However, among the strains tested, the I. fumosorosea and robertsii strains stood out, combining the highest metabolic activity with nematostatic activity against A. suum.In the perspective of further research, it is necessary to take into account the method of raising pigs (organic farms vs. traditional farms) because of the possibility of a potential effect of the host diet (i.e., the presence of different types of herbs in the feed) on the embryogenesis of A. suum.

Table 2 .
Cytotoxicity of entomopathogenic fungi in the MTT assay.